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https://openalex.org/W4395096021	http://catalog.liha-pres.eu/index.php/liha-pres/catalog/download/263/7899/17745-1	Ukrainian	null	ПАЛАЦОВО-ПАРКОВІ КОМПЛЕКСИ ПОЛТАВЩИНИ ЯК ОБ’ЄКТ ПІЗНАВАЛЬНОГО ТУРИЗМУ	null	2,024	cc-by	844	Борисова О.В. кандидат географічний наук, доцент, доцент кафедри туризму та готельно-ресторанного бізнесу, Київський національний університет технологій та дизайну ПАЛАЦОВО-ПАРКОВІ КОМПЛЕКСИ ПОЛТАВЩИНИ ЯК ОБ’ЄКТ ПІЗНАВАЛЬНОГО ТУРИЗМУ Пізнавальний туризм є основним напрямком туристичної діяльності як у світі, так і в більшості держав і має безліч форм прояву. Під пізнавальним туризмом розуміються різні види подорожей, які відповідають потребам духовного освоєння та духовного присвоєння культури світу через його відвідування, безпосереднє осягнення та переживання в різних місцях та протікають у формі організованого відпочинку та екскурсійної діяльності. У пізнавальному туризмі особисто побачене стає для подорожуючого особистим надбанням, належністю думки та почуттів. Завдяки екскурсіям та знайомству з культурою інших країн та народів розширюється кругозір туриста, і змінюються горизонти його сприйняття світу та культури. Перспективними об’єктами для організації пізнавального туризму є палацово-паркові комплекси (ППК). Їхній потенціал виступає як можливість для організації нових видів діяльності туристських підприємств, створення тематичних екскурсій та маршрутів. Наявність у регіоні ППК дозволяє організовувати садибний та замковий туризм, використовувати дані об’єкти як засоби розміщення та музейні об’єкти. Одним з перспективних регіонів України з наявними палацово-парковими комплексами є Полтавщина. На Полтавщині, в серці України, знаходяться цікаві старовинні садиби та палаци. Час їхнього процвітання давно 78 минув і сьогодні ці колись величні будинки ніби доживають свій вік, продовжуючи зберігати таємниці у своїх стінах. минув і сьогодні ці колись величні будинки ніби доживають свій вік, продовжуючи зберігати таємниці у своїх стінах. До найбільш відомих палацово-паркових комплексів Полтавщини належить палац Закревських (село Березова Рудка), садиба Котляревської-Родзянко (село Вишняки), реконстру- йована садиба Гоголів – Яновських (село Гоголеве), садиба Муравйових-Апостолів (село Хомутець) [1]. Найбільш відвідуваним палацово-парковим об’єктом Полтавщини є садиба Закревських (у Березовій Рудці). Перша згадка про «Березову рудку» датується 1717-м роком – тоді це була садиба гетьмана Івана Скоропадського. Пізніше її господарем став Григорій Закревський. Основу садиби становив двоповерховий мурований палац у класичному стилі, що пізніше згорів. А в 1838 році на тому місці звели новий двоповерховий палац у стилі необароко, архітектором якого був Є. Червінський [4]. Палац, зведений у стилі необароко, дивує своїми масштабами та деталями архітектури. Над парадним входом збереглися еркери, а простора танцювальна зала нікого не залишить байдужим. Також гості садиби можуть здійснити прогулянку парком площею 45 гектарів з екзотичними рослинами [2]. У селищі Хомутець зберіглася легендарна садиба – старовинний маєток українського гетьмана Данила Апостола, збудований у середині XVIII ст. На початку ХІХ століття новим власником садиби став письменник Муравйов-Апостол, який вирішив удосконалити будівлю та значно реконструював її. Садиба мала ознаки пізнього бароко і раннього класицизму. До складу садиби входили не тільки палац, Троїцька церква й господарські споруди, але й парк з альтанками, містками, штучними озерцями [3]. ПАЛАЦОВО-ПАРКОВІ КОМПЛЕКСИ ПОЛТАВЩИНИ ЯК ОБ’ЄКТ ПІЗНАВАЛЬНОГО ТУРИЗМУ На правому березі Десни, за 40 км північніше Батурина, розкинулося село Вишняки, де частково зберіглася садиба П.А. Рум’янцева-Задунайського. Палац має підковоподібний план та виконаний у стилізованих під «східну» готику барочних формах. Фахівці вважають, що авторами планів ансамблю, завершеного 1787 р., є В. Баженов чи його учні, зокрема М. Мосципанов. 79 Також у 1805 року в селі Вишняки на схилі річки Хорол гадяцький полковник Пламенець збудував садибу за проектом архітектора Миколи Львова. Незважаючи на те, що садибу було збудовано військовим, їй дали ім’я останньої своєї власниці поміщиці Родзянко-Котляревської. Двоповерховий будинок у стилі класицизму з куполом, що увінчується шпилем, раніше потопав у цвітінні фруктового саду. У будівлі є підземна таємна хода, яка з’єднує садибу з церквою, що стоїть навпроти. На жаль, сьогодні потрапити до палацу проблематично, оскільки це територія інтернату, проте розглянути архітектуру можна зовні [2]. За 25 км на схід від Гадяча, на березі річки Псел лежить село Бобрик. Головна памʼятка Бобрика – садиба Масюкових, від якої зберігся гарний класицистичний палац. Його побудував у 1805– 1807 рр., син веприцького сотника, чернігівський губернський предводитель дворянства, поміщик Семен Прокопович Масюков. Це цегляний прямокутний одноповерховий будинок з центральною двоповерховою частиною і поздовжнім плануванням кімнат. Будинок є типовим зразком, побудованим в стилі раннього класицизму. Починаючи з 1922 року в цьому будинку створили місцеву школу, але у 2021 році вона припинила своє існування. Підводячи підсумок, можна стверджувати що палацово- паркові комплекси є перспективними об’єктами для організації пізнавального туризму. Досвід організації спеціалізованих турів у світі накопичений досить значний, тому перспективним напрямком є використання палацово-паркових комплексів Полтавщини для розвитку пізнавального туризму в регіоні. 3. Чиркова М. Доля пам’яток нерухомої культурної спадщини Полтавщини. Краєзнавство. 2017. № 3-4. С. 192–209. 3. Чиркова М. Доля пам’яток нерухомої культурної спадщини Полтавщини. Краєзнавство. 2017. № 3-4. С. 192–209. 4. Шевченко Л.С. Палацово-паркові ансамблі Полтавщини як осередки художньої культури XVIII–ХІХ ст. Українська академія мистецтва: Дослідницькі та науково-методичні праці. 2002. Випуск 9. С. 185–192. Література: 1. Борисова О.В., Степанець І.О., Гринюк Д.Ю. Туристичний потенціал садибної архітектури Полтавщини та садибні комплекси Гадяччини XVIII–XIX ст. Замки та палаци в туризмі. ТОВ «Геопринт», 2023. 1. Борисова О.В., Степанець І.О., Гринюк Д.Ю. Туристичний потенціал садибної архітектури Полтавщини та садибні комплекси Гадяччини XVIII–XIX ст. Замки та палаци в туризмі. ТОВ «Геопринт», 2023. 2. Садиби та палаці Полтавщини: ТОП-3 незвичайних пам'яток. URL: https://pl.vgorode.ua/news/dosuh_y_eda/a1134625-usadby-i-dvortsy- poltavshchiny-top-3-neobychnykh-dostoprimechatelnosti (дата звернення: 25.02.2024). 2. Садиби та палаці Полтавщини: ТОП-3 незвичайних пам'яток. URL: https://pl.vgorode.ua/news/dosuh_y_eda/a1134625-usadby-i-dvortsy- poltavshchiny-top-3-neobychnykh-dostoprimechatelnosti (дата звернення: 25.02.2024). 80 4. Шевченко Л.С. Палацово-паркові ансамблі Полтавщини як осередки художньої культури XVIII–ХІХ ст. Українська академія мистецтва: Дослідницькі та науково-методичні праці. 2002. Випуск 9. С. 185–192. 81 81
https://openalex.org/W2100193578	https://europepmc.org/articles/pmc2481492?pdf=render	English	null	Serum biomarker profiles and response to neoadjuvant chemotherapy for locally advanced breast cancer	Breast cancer research	2,008	cc-by	7,444	Abstract Introduction Neoadjuvant chemotherapy has become the standard of care for the diverse population of women diagnosed with locally advanced breast cancer. Serum biomarker levels are increasingly being investigated for their ability to predict therapy response and aid in the development of individualized treatment regimens. Multianalyte profiles may offer greater predictive power for neoadjuvant treatment response than the individual biomarkers currently in use. Results Biomarker levels were compared retrospectively with clinical and pathologic treatment responses. Univariate analysis of the data identified several groups of biomarkers that differed significantly among treatment outcome groups early in the course of neoadjuvant chemotherapy. Multivariate statistical analysis revealed multibiomarker panels that could differentiate between treatment response groups with high sensitivity and specificity. Methods Serum samples were collected from 44 patients enrolled in a phase I–II, open-label study of liposomal doxorubicin and paclitaxel in combination with whole breast hyperthermia for the neoadjuvant treatment of locally advanced breast cancer (stage IIB or stage III). Samples were collected prior to each of four rounds of treatment and prior to definitive surgery. Samples were assayed by Luminex assay for 55 serum biomarkers, including cancer antigens, growth/angiogenic factors, apoptosis-related molecules, metastasis-related molecules, adhesion molecules, adipokines, cytokines, chemokines, hormones, and other proteins. Conclusion We demonstrate here that serum biomarker profiles may offer predictive power concerning treatment response and outcome in the neoadjuvant setting. The continued development of these findings will be of considerable clinical utility in the design of treatment regimens for individual breast cancer patients. Trial registration #NCT00346229. IIB disease [1]. The clinical definition of LABC continues to evolve and differ among physicians, and now includes nonmet- astatic T3 or T4 tumors as well as N2/N3 disease involving IIB disease [1]. The clinical definition of LABC continues to evolve and differ among physicians, and now includes nonmet- astatic T3 or T4 tumors as well as N2/N3 disease involving Open Access Vol 10 No 3Research article Serum biomarker profiles and response to neoadjuvant chemotherapy for locally advanced breast cancer Brian M Nolen1, Jeffrey R Marks2, Shlomo Ta'san3, Alex Rand3, The Minh Luong4, Yun Wang4, Kimberly Blackwell5,6 and Anna E Lokshin1,7,8,9 Open Access 1University of Pittsburgh Cancer Institute, Hillman Cancer Center, Suite 1.19d, 5117 Centre Avenue, Pittsburgh, PA 15213, USA 2Department of Surgery, Duke University Medical Center, Box 3873 Med Ctr Durham, NC 27710, USA 3Department of Mathematical Sciences, Carnegie Mellon University, Wean Hall, Room 6113, Pittsburgh, PA 15213-3890, USA 4Department of Biostatistics, Graduate School of Public Health, University of Pittsburgh, 130 Desoto Street, 311 Parran Hall, Pittsburgh, PA 15261 USA S 5Department of Radiation Oncology, Duke University Medical Center, Box 3893 Med Ctr Durham, NC 27710, USA 6Department of Medicine, Duke University Medical Center, Box 3893 Med Ctr Durham, NC 27710, USA 7Department of Medicine, School of Medicine, University of Pittsburgh, 1218 Scaife Hall 3550 Terrace Street, Pittsburgh, PA 15213, USA 8Department of Pathology, University of Pittsburgh, S-417 BST, 200 Lothrop Street, Pittsburgh, PA 15261, USA 5Department of Radiation Oncology, Duke University Medical Center, Box 3893 Med Ctr Durham, NC 27710, USA 6Department of Medicine, Duke University Medical Center, Box 3893 Med Ctr Durham, NC 27710, USA 7Department of Medicine, School of Medicine, University of Pittsburgh, 1218 Scaife Hall 3550 Terrace Street, Pittsburgh, PA 15213, USA , y , , , artment of Medicine, School of Medicine, University of Pittsburgh, 1218 Scaife Hall 3550 Terrace Street, Pittsburgh, artment of Pathology University of Pittsburgh S 417 BST 200 Lothrop Street Pittsburgh PA 15261 USA Department of Pathology, University of Pittsburgh, S-417 BST, 200 Lothrop Street, Pittsburgh, PA 15261, USA Department of Obstetrics and Gynecology RS, University of Pittsburgh, 300 Halket Street Pittsburgh, PA 15213, USA 8Department of Pathology, University of Pittsburgh, S-417 BST, 200 Lothrop Street, Pittsburgh, PA 15261, USA 9Department of Obstetrics and Gynecology RS, University of Pittsburgh, 300 Halket Street Pittsburgh, PA 15213, USA 9Department of Obstetrics and Gynecology RS, University of Pittsburgh, 300 Halket Street Pittsburgh, PA 15213, USA Corresponding author: Anna E Lokshin, lokshina@pitt.edu Received: 4 Mar 2008 Revisions requested: 10 Apr 2008 Revisions received: 24 Apr 2008 Accepted: 12 May 2 Breast Cancer Research 2008, 10:R45 (doi:10.1186/bcr2096) This article is online at: http://breast-cancer-research.com/content/10/3/R45 p © 2008 Nolen 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. Available online http://breast-cancer-research.com/content/10/3/R45 Available online http://breast-cancer-research.com/content/10/3/R45 cCR = clinical complete response; EGFR = epidermal growth factor receptor; IL = interleukin; LABC = locally advanced breast cancer; MIF = migra- tion inhibitory factor; MMP = matrix metalloproteinase; NAC = neoadjuvant chemotherapy; pCR = pathologic complete response; TNF = tumor necro- sis factor; tPAI-1 = tissue plasminogen activator inhibitor 1. Introduction Locally advanced breast cancer (LABC) is a generalized diag- nosis that includes all stage III disease and a subset of stage cCR = clinical complete response; EGFR = epidermal growth factor receptor; IL = interleukin; LABC = locally advanced breast cancer; MIF = migra- tion inhibitory factor; MMP = matrix metalloproteinase; NAC = neoadjuvant chemotherapy; pCR = pathologic complete response; TNF = tumor necro- sis factor; tPAI-1 = tissue plasminogen activator inhibitor 1. Page 1 of 9 (page number not for citation purposes) Breast Cancer Research Vol 10 No 3 Nolen et al. limited metastasis [2], thus broadening the already diverse spectrum of LABC presentations. According to the American College of Surgeons Data Base, approximately 6% of all US breast cancer cases present as stage III [3]. This number has declined dramatically over the past decade due to improved screening and detection practices. The 5-year relative survival rate for stage III breast cancer is approximately 50%, com- pared with 87% for stage I. The median survival for women with stage III disease is 4.9 years [1]. duct intreatment analyses of molecular markers that may pre- dict response to therapy. The emergence of new technologies such as transcriptional and proteomic profiling has greatly aided such investigations [18,19]. For instance, it has been reported that mutations in p53 are associated with a lower response rate following NAC [20,21], while coexpression of HER-2/Neu and topoisomerse II is associated with greater response rates [22]. Measurements of the traditional breast cancer markers CA15-3 and HER-2/Neu, however, have dem- onstrated only limited predictive value in the NAC setting [23,24]. Neoadjuvant chemotherapy (NAC), the delivery of systemic chemotherapy prior to surgical resection, has emerged as the preferred initial component of therapy for patients diagnosed with LABC in an effort to enhance the prospect of breast-con- serving surgery and to render inoperable tumors resectable [4,5]. NAC offers the theoretical advantages of early initiation of systemic therapy, delivery of drugs through intact vascula- ture, in vivo assessment of therapy response, and the oppor- tunity to study the biological effects of chemotherapy [6]. Preoperative systemic chemotherapy may also eradicate dis- tant micrometastases and thus improve the overall effective- ness of treatment [7]. Materials and methods Patients Serum samples were collected from patients enrolled in a phase I–II, open-label study of liposomal doxorubicin (Evacet; Elan Corp., Stevenage, UK) and paclitaxel (Bristol Myers Squibb, Princeton, NJ, USA) in combination with whole breast hyperthermia for the neoadjuvant treatment of LABC (stage IIB or stage III). This trial required informed consent and was con- ducted under the approval of the Duke University Institutional Review Board. Protocol-eligible patients were treated with the combination of Evacet, paclitaxel, and hyperthermia every 3 weeks. The hyperthermia procedure has been described pre- viously [25]. A pathologic complete response (pCR) following NAC implies the absence of residual invasive or in situ disease and corre- lates strongly with both prolonged disease-free survival and overall survival [12,13]. A recent review of several randomized clinical trials of NAC for operable breast cancer reported a response rate of 49% to 94% with a pCR rate of 4% to 34% [12]. In patients treated with NAC, 60% to 80% demonstrate some clinical response with 10% to 20% achieving a clinical complete response (cCR) [4]. Clinical response, however, often does not correlate with pathologic response as a full one-third of patients achieving a cCR are found to have path- ologic evidence of residual disease [9,14]. Despite these diffi- culties in assessing response, patients demonstrating complete clinical or pathologic responses to NAC generally achieve improved outcomes to overall treatment [14,15]. Following neoadjuvant therapy, patients received appropriate surgical removal of their primary breast tumor as well as axillary lymph node dissection. Immediately after surgery, patients underwent radiation therapy followed by an additional eight cycles each of 21-day standard dose cyclophosphamide (600 mg/m2), methotrexate (40 mg/m2), 5-fluorouracil (600 mg/m2) and appropriate hormonal therapy. The clinical trial accrued a total of 47 patients. Three patients were deemed nonevaluable because of failure to complete all four cycles of the neoadjuvant portion of the trial. The clinical characteristics of the patient study group are presented in Table 1. Accurate modalities for assessing chemotherapy response are critical to the evaluation and expansion of the use of NAC for breast cancer. Conventional methods including clinical exami- nation, mammogram, and breast ultrasound are incorrect in identifying pCR patients in nearly one-half of all cases [2]. Sev- eral groups have reported promising results in their attempts to predict treatment response utilizing unconventional tech- niques such as diffuse optical spectroscopy and magnetic res- onance imaging [16,17]. Introduction Several studies comparing NAC with more traditional adjuvant chemotherapy have found similar sur- vival rates between the two options [5,8,9], and the use of NAC is further supported by findings that delaying surgery for the administration of chemotherapy does not adversely affect treatment outcome when compared with adjuvant chemother- apy [10,11]. In the present study we examine a diverse panel of serum biomarkers in order to identify individual biomarkers and com- binations that may be useful in predicting treatment response early in the course of NAC for the treatment of LABC. Materials and methods Patients A growing number of investigators have begun to utilize the preoperative nature of NAC to con- Page 2 of 9 (page number not for citation purposes) Pathologic response Within each timepoint of treatment, biomarker expression val- ues – expressed as the median fluorescence intensity – were adjusted by the following procedure. Quantile normalization was performed using the normalize BetweenArrays function (limma package) in R [32], missing values were filled in using k-nearest neighbor imputation, and values were log-trans- formed. Normalized values were filtered according to a univariate, two- sided t test. From this filtered set, values were progressively included and excluded from a stepwise regression model. The final logistic regression model was then subjected to leave- one-out cross-validation to assess the predictive value. Multiplexed bead-based immunoassay Multiplexed bead-based immunoassay The xMAP™ bead-based technology (Luminex Corp., Austin, TX, USA) permits simultaneous analysis of numerous analytes in a single sample. Fifty-five bead-based xMAP™ immu- noassays for a variety of serum biomarkers were utilized in the present study (Table 2). Assays for ErbB2, epidermal growth factor receptor (EGFR), CA 15-3, carcinoembryonic antigen, Cyfra 21-1, CA 19-9, CA 72-4, α-fetoprotein, mesothelin, insulin-like growth factor bind- ing protein 1, human kallikrein 10, and HE4 were developed in the UPCI Luminex Core Facility [26]. The inter-assay variability of each assay was 5% to 11%, and the intra-assay variability was 2% to 9%. Assays for MMP-2 and MMP-3 were obtained from R&D Systems (Minneapolis, MN, USA), assays for MIP- 1β, eotaxin, IP-10, IL-2R, IL-1Rα, IL-6R, DR5, TNF-RI, and TNF-RII were obtained from Invitrogen (Camarillo, CA), and the remaining assays were obtained from Millipore/Linco Research (St Charles, MO, USA). Overall, eight different mul- tiplexed panels were used. Collection and storage of blood serum g Serum samples were obtained prior to the start of neoadjuvant therapy (pretreatment), prior to cycles 2, 3, and 4 of neoadju- vant therapy, and prior to definitive surgery. Blood was drawn using standard phlebotomy procedures and was collected without anticoagulant. Blood was allowed to coagulate for up to 2 hours at room temperature. Sera were separated by cen- trifugation, immediately aliquoted, frozen, and stored at -80°C. No more than two freeze–thaw cycles were allowed for any sample. Page 2 of 9 (page number not for citation purposes) Available online http://breast-cancer-research.com/content/10/3/R45 Available online http://breast-cancer-research.com/content/10/3/R45 Table 1 trations of analytes were quantitated from median fluores- cence intensities using standard curves generated by Bio-Rad five-parameter curve fitting) to the series of known concentra- tions for each analyte. Clinical characteristics of study patients Clinical characteristics of study patients Clinical characteristics of study patients Characteristic Number of patients Patients in study Total enrolled 47 Total completing treatment 44 Clinical stage pretreatment Stage IIB 15 Stage IIIA 12 Stage IIIB 16 Clinical response Complete 12 Partial 20 Stable disease 12 Pathologic response Complete 4 Partial 23 Stable disease 17 Clinical response The Mann–Whitney nonparametric t test was used to evaluate the significance of differences in serum biomarker levels expressed as the median fluorescence intensity between treat- ment response groups separated by treatment timepoints. The level of significance was taken as P < 0.05. For multivariate analysis of biomarker combinations, a CART classification tree [28-30] diagnostic model was created. The Statistical Analy- sis System (SAS version 9: SAS Institute, Inc., Cary, NC, USA) was used to fit the logistic regressions using PROC LOGISTIC. The best subset for each size panel of analytes was identified through the brand and bound algorithm of Fur- nival and Wilson [31]. This algorithm maximizes the score function over all possible combinations of analytes for any given size panel. The Statistical Analysis System was also used to fit the logistic regressions and to identify the best sub- sets for each size panel of biomarkers. Panels were generated from size 1 to size 10. Sensitivities were estimated for specificities of 90%, 95%, and 98% by ranking the predicted fit for each control subject, determining the cutoff points corresponding to these levels of specificity, and applying the cutoff points to the ranked predic- tions for the alternative treatment response group. To minimize overfitting bias, leave-one-out cross-validation was used. The MATLAB routines treefit and treeval were used. For panel selection, markers were selected incrementally. Given an existing subset of the markers, each marker was considered a potential addition to the panel. We began with no markers and added until little additional progress was made. Page 3 of 9 (page number not for citation purposes) Multiplex analysis of serum levels of various biomarkers in LABC patients receiving neoadjuvant chemotherapy Multiplex analysis of serum levels of various biomarkers in LABC patients receiving neoadjuvant chemotherapy Of the 44 evaluable patients enrolled in the clinical trial, 12 demonstrated a cCR, 20 demonstrated a clinical partial response, and 12 had no response. Of the same group, four patients demonstrated a pCR, 23 demonstrated a pathologic partial response, and 17 had no response. One patient out of the total 44 did not have blood drawn prior to the second cycle of NAC and was excluded from the analysis for that timepoint. A CART classification tree analysis of serum biomarker levels from these patients identified a three-biomarker panel consist- ing of α-fetoprotein, soluble vascular cell adhesion molecule 1, and MMP-9 that could distinguish between the response groups with 83% sensitivity and 91% specificity (Table 3). Interestingly, when the 11 patients achieving a cCR were com- bined with 19 patients achieving a clinical partial response and were compared with the nonresponders, tPAI-1 alone could distinguish responders from nonresponders with 75% sensi- tivity and 77% specificity (Table 3 and Figure 1d). Serum lev- els of tPAI-1 were significantly lower in responders (cCR and clinical partial response) compared with nonresponders (P < 0.007, Figure 1b). A bead-based 55-biomarker panel was utilized to screen the sera from patients. The biomarkers included cancer antigens, growth/angiogenic factors, apoptosis-related molecules, metastasis-related molecules, adhesion molecules, adipok- ines, cytokines, chemokines, hormones, proteases, and other proteins (Table 2). Biomarker levels were compared at each treatment timepoint between complete responders, partial responders, and nonresponders within each response classi- fication (clinical or pathologic). Analysis of serum biomarker levels according to clinical response prior to the second cycle of neoadjuvant chemotherapy Combinations of biomarkers were evaluated by multivariate analysis for the ability to predict a particular response. Our analysis was limited to serum samples collected prior to the initiation of NAC and prior to the second cycle of therapy. We did not identify any significant correlations between biomarker levels and pathologic response at the pretreatment timepoint. For this analysis, 11 patients achieving a cCR were compared with 11 patients demonstrated no response. Serum levels of IL-8 were significantly higher and those of insulin-like growth factor binding protein 1 were significantly lower in patients achieving a cCR (P < 0.05, Figure 1c). CART classification tree analysis of serum biomarker levels from these patients identified a three-biomarker panel consisting of MMP-3, luteinizing hormone, and thyroid stimulating hormone that could distinguish between clinical response groups with 82% sensitivity and 73% specificity (Table 3). Table 2 while levels of tissue plasminogen activator inhibitor 1 (tPAI-1) were significantly lower in the same group (P < 0.05, Figure 1a). Higher levels of IL-6 and IL-8 were also observed in patients achieving a cCR, although this observation was not statistically significant (P < 0.07, Figure 1). Multiplex analysis Assays were performed according to the manufacturers' pro- tocols. Luminex Core Facility assays were performed as described previously [27]. Samples were analyzed using the Bio-Plex suspension array system (Bio-Rad Laboratories, Her- cules, CA, USA). Biomarker expression levels were expressed as median fluorescent intensities generated by analyzing 50 to 100 microbeads for each analyte in each sample. The concen- Page 3 of 9 (page number not for citation purposes) Page 3 of 9 Breast Cancer Research Vol 10 No 3 Nolen et al. Breast Cancer Research Vol 10 No 3 Nolen et al. Table 2 Complete list of biomarkers tested Biomarker category Individual biomarkers Cancer antigens/oncogenes α-Fetoprotein, CA 125, CA 19-9, CA 15-3, CA 72-4, carcinoembryonic antigen Cytokines/chemokines/receptors Eotaxin, fractalkine, granulocyte–macrophage colony-stimulating factor, IFNγ, IL-10, IL-12p70, IL-13, IL-1β, IL- 1Rα, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-6R, IL-7, IL-8, IP-10, migration inhibitory factor, MIP-1β, soluble CD40L, TNFα, TNF-R1, TNF-R2 Growth/angiogenic factors Epidermal growth factor receptor, ErbB2, insulin-like growth factor binding protein 1, transforming growth factor alpha Proteases Kallikrein 10, MMP-2, MMP-3, MMP-9 Hormones Adrenocorticotropic hormone, follicle-stimulating hormone, growth hormone, luteinizing hormone, prolactin, thyroid-stimulating hormone Adipokines Adiponectin Apoptosis-related molecules Cyfra 21-1, DR5, soluble Fas, soluble Fas ligand Metastasis-related molecules Myeloperoxidase, tissue plasminogen activator inhibitor 1 Adhesion molecules Soluble intracellular adhesion molecule 1, soluble vascular cell adhesion molecule 1 Other proteins HE4, mesothelin Analysis of pretreatment serum biomarker levels according to clinical response For this analysis, 11 patients achieving a cCR were compared with 12 patients demonstrating no response. Serum levels of MMP-9 were significantly higher in patients achieving a cCR Page 4 of 9 (page number not for citation purposes) Page 4 of 9 (page number not for citation purposes) Available online http://breast-cancer-research.com/content/10/3/R45 Figure 1 g Serum biomarker analysis of neoadjuvant chemotherapy for locally advanced breast cancer according to clinical response Serum biomarker analysis of neoadjuvant chemotherapy for locally advanced breast cancer according to clinical response. (a) Pretreatment serum levels of IL-6, IL-8, MMP-9, and tissue plasminogen activator inhibitor 1 (tPAI-1) were compared between 11 patients achieving a clinical complete response (cCR) and 12 patients demonstrating no response (NR) to neoadjuvant chemotherapy (NAC). (b) Pretreatment serum levels of tPAI-1 were compared between 30 patients achieving a cCR or clinical partial response (cPR) and 12 patients demonstrating NR to NAC. (c) Serum levels of insulin-like growth factor binding protein 1 (IGFBP-1) and IL-8 measured prior to the second round of NAC were compared between 11 patients achieving a cCR and 11 patients demonstrating NR to NAC. (d) Cumulative receiver operating characteristics for responders versus nonresponders based on pretreatment serum levels of tPAI-1. Statistical significance: P < 0.05; P < 0.01. Serum biomarker analysis of neoadjuvant chemotherapy for locally advanced breast cancer according to clinical response Serum biomarker analysis of neoadjuvant chemotherapy for locally advanced breast cancer according to clinical response. (a) Pretreatment serum levels of IL-6, IL-8, MMP-9, and tissue plasminogen activator inhibitor 1 (tPAI-1) were compared between 11 patients achieving a clinical complete response (cCR) and 12 patients demonstrating no response (NR) to neoadjuvant chemotherapy (NAC). (b) Pretreatment serum levels of tPAI-1 were compared between 30 patients achieving a cCR or clinical partial response (cPR) and 12 patients demonstrating NR to NAC. (c) Serum levels of insulin-like growth factor binding protein 1 (IGFBP-1) and IL-8 measured prior to the second round of NAC were compared between 11 patients achieving a cCR and 11 patients demonstrating NR to NAC. (d) Cumulative receiver operating characteristics for responders versus nonresponders based on pretreatment serum levels of tPAI-1. Statistical significance: P < 0.05; *P < 0.01. responders from nonresponders with 85% sensitivity and 69% specificity (Table 3 and Figure 2b). Discussion For this analysis, 27 patients achieving a pCR or pathologic partial response were compared with 16 patients demonstrat- ing no response. Serum levels of EGFR, soluble Fas ligand, migration inhibitory factor (MIF), and MMP-2 were significantly higher in responders compared with non-responders (P < 0.05, Figure 2a). The heterogeneity displayed by patients diagnosed with LABC runs counter to the rationale for generalized treatment regimens. A wide array of treatment options exist for the treat- ment of breast cancer – including adjuvant and NAC, hormone therapy, radiotherapy, and surgery – and the vast majority of these options have been well researched. The ability to dynam- ically tailor the components of a particular treatment regimen on a patient by patient basis would be invaluable. Such an accomplishment will require the identification and develop- A logistic regression analysis of the serum biomarker levels in these patients identified a five-biomarker panel consisting of ErbB2, EGFR, MIF, MMP-2, and CD40L that could distinguish Figure 2 Serum biomarker analysis of neoadjuvant chemotherapy for locally advanced breast cancer according to pathologic response Serum biomarker analysis of neoadjuvant chemotherapy for locally advanced breast cancer according to pathologic response. (a) Serum levels of epidermal growth factor receptor (EGFR), soluble Fas ligand (sFasL), migration inhibitory factor (MIF), and MMP-2 measured prior to the second round of neoadjuvant chemotherapy (NAC) in 27 patients achieving a pathologic complete response (pCR) or partial pathologic response (pPR) and 16 patients demonstrating no response (NR) to NAC. (b) Cumulative receiver operating characteristics for patients achieving a pCR or pPR versus nonresponders to NAC based on serum levels of ErbB2, EGFR, MIF, MMP-2, and CD40 measured prior to the second cycle of NAC. Statistical sig- nificance: P < 0.05. Serum biomarker analysis of neoadjuvant chemotherapy for locally advanced breast cancer according to pathologic response Serum biomarker analysis of neoadjuvant chemotherapy for locally advanced breast cancer according to pathologic response. (a) Serum levels of epidermal growth factor receptor (EGFR), soluble Fas ligand (sFasL), migration inhibitory factor (MIF), and MMP-2 measured prior to the second round of neoadjuvant chemotherapy (NAC) in 27 patients achieving a pathologic complete response (pCR) or partial pathologic response (pPR) and 16 patients demonstrating no response (NR) to NAC. (b) Cumulative receiver operating characteristics for patients achieving a pCR or pPR versus nonresponders to NAC based on serum levels of ErbB2, EGFR, MIF, MMP-2, and CD40 measured prior to the second cycle of NAC. Statistical sig- nificance: P < 0.05. IL-8 have been associated with poor prognosis in women with breast cancer [37,38]. These cytokines have also been impli- cated in the blood-borne response to paclitaxel treatment [39]. The prognostic value of MMP-9 serum levels is currently unclear [33], but several studies have examined extensively the role of matrix metalloproteinases in breast cancer [40,41]. ment of improved prognostic factors on which to base thera- peutic decisions, since currently used measurements of clinical and radiological response lack the necessary preci- sion. In the present article we demonstrate the predictive value of serum biomarkers for the response to NAC for LABC. The diverse nature of the biomarker relationships identified in the study underscores the diversity of the disease characteristics present in the patient population. Our results also demonstrate a correlation between lower pre- treatment serum levels of tPAI-1 and improved clinical response. Table 3 Table 3 Predictive power of multimarker panels Panel Timepoint Response type Sensitivity (%) Specificity (%) α-Fetoprotein, soluble vascular cell adhesion molecule 1, MMP-9 Pretreatment Clinical complete response versus no response 83 91 Tissue plasminogen activator inhibitor 1 Pretreatment Clinical complete response/clinical partial response versus no response 75 77 MMP-3, luteinizing hormone, thyroid-stimulating hormone Pre-cycle 2 Clinical complete response versus no response 82 73 ErbB2, epidermal growth factor receptor, migration inhibitory factor, MMP-2, CD40 ligand Pre-cycle 2 Pathologic complete response/pathologic partial response versus no response 85 69 Predictive power of multimarker panels Breast Cancer Research Vol 10 No 3 Nolen et al. Figure 2 Figure 2 Figure 2 Serum biomarker analysis of neoadjuvant chemotherapy for locally advanced breast cancer according to pathologic response Serum biomarker analysis of neoadjuvant chemotherapy for locally advanced breast cancer according to pathologic response. (a) Serum levels of epidermal growth factor receptor (EGFR), soluble Fas ligand (sFasL), migration inhibitory factor (MIF), and MMP-2 measured prior to the second round of neoadjuvant chemotherapy (NAC) in 27 patients achieving a pathologic complete response (pCR) or partial pathologic response (pPR) and 16 patients demonstrating no response (NR) to NAC. (b) Cumulative receiver operating characteristics for patients achieving a pCR or pPR versus nonresponders to NAC based on serum levels of ErbB2, EGFR, MIF, MMP-2, and CD40 measured prior to the second cycle of NAC. Statistical sig- nificance: P < 0.05. Page 6 of 9 (page number not for citation purposes) References 1. Singletary SE, Allred C, Ashley P, Bassett LW, Berry D, Bland KI, Borgen PI, Clark GM, Edge SB, Hayes DF, Hughes LL, Hutter RV, Morrow M, Page DL, Recht A, Theriault RL, Thor A, Weaver DL, Wieand HS, Greene FL: Staging system for breast cancer: revi- sions for the 6th edition of the AJCC Cancer Staging Manual. Surg Clin N Am 2003, 83:803-819. g , 2. Lee MC, Newman LA: Management of patients with locally advanced breast cancer. Surg Clin N Am 2007, 87:379-398. 3. The American College of Surgeons Commission on Cancer: National Cancer Data Base [http://www.facs.org/cancer/ncdb] g 2. Lee MC, Newman LA: Management of patients with locally advanced breast cancer. Surg Clin N Am 2007, 87:379-398. 3. The American College of Surgeons Commission on Cancer: National Cancer Data Base [http://www.facs.org/cancer/ncdb] 4. Swain SM, Sorace RA, Bagley CS, Danforth DN Jr, Bader J, Wes- ley MN, Steinberg SM, Lippman ME: Neoadjuvant chemotherapy in the combined modality approach of locally advanced non- metastatic breast cancer. Cancer Res 1987, 47:3889-3894. National Cancer Data Base [http://www.facs.org/cancer/ncdb] 4. Swain SM, Sorace RA, Bagley CS, Danforth DN Jr, Bader J, Wes- ley MN, Steinberg SM, Lippman ME: Neoadjuvant chemotherapy in the combined modality approach of locally advanced non- metastatic breast cancer. Cancer Res 1987, 47:3889-3894. In addition to the individual serum biomarkers found to be sig- nificant at each timepoint, our multivariate analysis of the data identified several multimarker panels with predictive value for both clinical response and pathologic response prior to or early in the course of treatment. These multimarker panels demonstrated greater predictive power than any single biomarker and illustrate the potential clinical utility of this type of approach to the design and maintenance of treatment regi- ments. Previous studies from our group have demonstrated the usefulness of multimarker panels in the early detection of ovarian cancer, endometrial cancer, and head and neck can- cers [27,52,53]. , 5. Wolmark N, Wang J, Mamounas E, Bryant J, Fisher B: Preopera- tive chemotherapy in patients with operable breast cancer: nine-year results from National Surgical Adjuvant Breast and Bowel Project B-18. J Natl Cancer Inst Monogr. 2001, 30:96-102. 6. Giordano SH: Update on locally advanced breast cancer. The Oncologist 2003, 8:521-530. g , 7. Hanrahan EO, Hennessy BT, Valero V: Neoadjuvant systemic therapy for breast cancer: an overview and review of recent clinical trials. Expert Opin Pharmacother 2005, 6:1477-1491. p p 8. Authors' contributions BMN participated in the Luminex assays, oversaw the data analysis, and drafted the manuscript. JRM and KB conceived the study and coordination the transfer of patient samples. ST, AR, TML, and YW carried out the statistical analysis. AEL con- ceived the study, participated in the study design, and helped to draft the manuscript. Acknowledgements g The present work was supported by the DAMD17-03-1-0696 DOD Award, the 'Avon–NCI Progress for Patients' Award, and the BCTR0600911 Susan G Komen Foundation Award (AEL). The authors would like to acknowledge Adele Marrangoni, Lyudmila Velikokhatnaya, Matt Winans, and Denise Prosser for their extensive technical support relating to the Luminex analysis. It is noteworthy to mention that our present analysis did not identify any significant relationships between CA 15-3 or HER2/Neu and response to NAC. This observation adds to several sparse and conflicting reports in the literature [23,50,51]. It would appear from this uncertainty that, although these particular biomarkers have shown the most promise in terms of diagnosis and prognosis of breast cancer, they may not offer predictive power for chemotherapy response. Competing interests p g The authors declare that they have no competing interests. With regards to pathologic response, we observed a correla- tion between increased levels of soluble Fas ligand, MMP-2, MIF, and EGFR and improved response. A proapoptotic response following chemotherapy, as evidenced by increased soluble Fas ligand in the sera, is to be expected. Our observa- tion of increased levels of MMP-2 in the sera of responders supports the notion of matrix metalloproteinase involvement in breast cancer. The precise role of macrophage MIF in breast cancer development and treatment response remains unknown, but MIF has been implicated in tumor cell survival pathways [47]. The value of EGFR serum and tissue levels in predicting response to chemotherapy has been examined pre- viously with inconclusive results [43,48]. Increased expression of EGFR has been demonstrated elsewhere to suggest a poor prognosis in breast cancer [49]. References Powles TJ, Hickish TF, Makris A, Ashley SE, O'Brien ME, Tidy VA, Casey S, Nash AG, Sacks N, Cosgrove D, MacVicar D, Fernando I, Ford HT: Randomized trial of chemoendocrine therapy started before or after surgery for treatment of primary breast cancer. J Clin Oncol 1995, 13:547-552. 9. 9. Hage JA van der, Velde CJ van de, Julien JP, Floiras JL, Delozier T, Vandervelden C, Duchateau L: Improved survival after one course of perioperative chemotherapy in early breast cancer patients: long-term results from the European Organization for Research and Treatment of Cancer (EORTC) Trial 10854. Eur J Cancer 2001, 37:2184-2193. Available online http://breast-cancer-research.com/content/10/3/R45 Available online http://breast-cancer-research.com/content/10/3/R45 this will increase immensely. Continuing efforts in line with those presented here should bring us closer to providing effective and efficient individualized treatment to women diag- nosed with this challenging disease. chemotherapy is in line with clinical expectations and is evi- dence that the therapeutic agents are active in the patient's system. The roles of the insulin-like growth factor system and hormones in the response to chemotherapy have not been sig- nificantly evaluated; however, both groups have known roles in the development of breast cancer [45,46]. Figure 2 In fact, tPAI-1 levels alone were able to discriminate responders from nonresponders with 75% sensitivity and 77% specificity prior to the start of NAC. tPAI-1 is a major physiological inhibitor of tissue-type plasminogen activators and has been implicated in tumor growth, invasion, and angio- genesis. The function of tPAI-1 as a regulator of plasminogen activation places it in a position to modulate degradation of the extracellular matrix and antiangiogenic effects mediated by plasmin and angiostatin, respectively. Several groups have illustrated experimentally the role of tPAI-1 in tumor progres- sion and its negative prognostic value in breast cancer [42- 44]. Previous studies have examined the value of biomarkers such as carcinoembryonic antigen, CA 15-3, MMP-2, MMP-9, tis- sue polypeptide antigen (TPA), tissue polypeptide-specific antigen (TPS), EGFR, and HER-2/neu in predicting response to NAC for breast cancer [33-36]. The results of these inves- tigations have been mixed. To our knowledge, the panel of serum biomarkers examined in the present study is the largest and most diverse to date and the majority of the relationships we identify have not been described previously. Our results indicate that elevated serum levels of IL-6, IL-8, and MMP-9 prior to the initiation of treatment correlate with improved clinical response. The observed increases in IL-6 and IL-8 serum levels of 2.74 and 3.13 pg/ml, respectively (Figure 1a), were not significant in our study given the limited 23-patient enrollment. Utilizing these data, we predict that an enrollment of 59 patients would be sufficient to confer signifi- cance (P < 0.05) upon these observations with a power of 0.9. Increased serum levels of the inflammatory cytokines IL-6 and In sera collected after patients had received the initial cycle of chemotherapy we observed that increased levels of IL-4, IL-8, and adrenocorticotropic hormone and decreased levels of insulin-like growth factor binding protein 1 all correlated with improved clinical response at differing levels of significance. The increase in levels of inflammatory cytokines following Page 6 of 9 (page number not for citation purposes) Conclusion Our relatively small study population limits the predictive power of the panels presented here, but the benefits of a serum biomarker and multimarker approach are clearly illus- trated and further studies utilizing larger clinical cohorts, as discussed above, are well warranted. As we increase our knowledge of the biomarkers involved in these panels and continue to identify additional players, our ability to utilize the information we receive from exploratory investigations such as 10. De Lena M, Varini M, Zucali R, Rovini D, Viganotti G, Valagussa P, Veronesi U, Bonadonna G: Multimodal treatment for locally advanced breast cancer. Result of chemotherapy–radiother- apy versus chemotherapy–surgery. Cancer Clin Trials 1981, 4:229-236. 11. Papaioannou A, Lissaios B, Vasilaros S, Miligos S, Papadimitriou G, Kondilis D, Polychronis A, Kozonis J, Papageorgiou G, Platani- otis G, Razis D, Stathopoulos G, Tsiliakos S, Throuvhlas N, Page 7 of 9 (page number not for citation purposes) Page 7 of 9 (page number not for citation purposes) Breast Cancer Research Vol 10 No 3 Nolen et al. Papavasiliou K, Tsarouhas C, Papaevangelou G: Pre- and post- operative chemoendocrine treatment with or without postop- erative radiotherapy for locally advanced breast cancer. Cancer 1983, 51:1284-1290. 29. Hastie T, Tibshirani R, Friedman J: The Elements of Statistical Learning Springer; 233 Spring St., NY 10013, USA; 2001. Papavasiliou K, Tsarouhas C, Papaevangelou G: Pre- and post- operative chemoendocrine treatment with or without postop- erative radiotherapy for locally advanced breast cancer. Cancer 1983, 51:1284-1290. 30. Devroye L, Gyorfi L, Lugosi G: A Probabilistic Theory of Pattern Recognition Springer; 233 Spring St., NY 10013, USA; 1996. , 12. Charfare H, Limongelli S, Purushotham AD: Neoadjuvant chemo- therapy in breast cancer Br J Surg 2005 92:14-23 g p g ; p g , , ; 31. Furnival G, Wilson R: Regressions by leaps and bounds. Tech- nometrics 1974, 16:499-511. g therapy in breast cancer. Br J Surg 2005, 92:14-23 3 H BT H b i GN R i R K H , 32. The R Project for Statistical Computing [http://www.r- project.org/] 13. Hennessy BT, Hortobagyi GN, Rouzier R, Kuerer H, Sneige N, Buzdar AU, Kau SW, Fornage B, Sahin A, Broglio K, Singletary SE, Valero V: Outcome after pathologic complete eradication of cytologically proven breast cancer axillary node metastases following primary chemotherapy. J Clin Oncol 2005, 23:9304-9311. 33. Conclusion Coskun U, Yamac D, Gulbahar O, Sancak B, Karaman N, Ozkan S: Locally advanced breast carcinoma treated with neoadjuvant chemotherapy: are the changes in serum levels of YKL-40, MMP-2 and MMP-9 correlated with tumor response? Neo- plasma 2007, 54:348-352. 14. Hortobagyi GN, Ames FC, Buzdar AU, Kau SW, McNeese MD, Paulus D, Hug V, Holmes FA, Romsdahl MM, Fraschini G, McBride CM, Martin RG, Montague E: Management of stage III primary breast cancer with primary chemotherapy, surgery, and radia- tion therapy. Cancer 1988, 62:2507-2516. p 34. Martinez-Trufero J, de Lobera AR, Lao J, Puertolas T, Artal-Cortes A, Zorrilla M, Alonso V, Pazo R, Valero MI, Rios-Mitchell MJ, Cal- derero V, Herrero A, Antón A: Serum markers and prognosis in locally advanced breast cancer. Tumori 2005, 91:522-530. py 15. Feldman LD, Hortobagyi GN, Buzdar AU, Ames FC, Blumenschein GR: Pathological assessment of response to induction chem- otherapy in breast cancer. Cancer Res 1986, 46:2578-2581. 35. Schippinger W, Dandachi N, Regitnig P, Hofmann G, Balic M, Neumann R, Samonigg H, Bauernhofer T: The predictive value of EGFR and HER-2/neu in tumor tissue and serum for response to anthracycline-based neoadjuvant chemotherapy of breast cancer. Am J Clin Pathol 2007, 128:630-637. 16. Cerussi A, Hsiang D, Shah N, Mehta R, Durkin A, Butler J, Tromb- erg BJ: Predicting response to breast cancer neoadjuvant chemotherapy using diffuse optical spectroscopy. Proc Natl Acad Sci USA 2007, 104:4014-4019. , 36. Sliwowska I, Kopczynski Z, Grodecka-Gazdecka S: Diagnostic value of measuring serum CA 15-3, TPA, and TPS in women with breast cancer. Postepy Hig Med Dosw (Online) 2006, 60:295-299. 17. Segara D, Krop IE, Garber JE, Winer E, Harris L, Bellon JR, Bird- well R, Lester S, Lipsitz S, Iglehart JD, Golshan M: Does MRI pre- dict pathologic tumor response in women with breast cancer undergoing preoperative chemotherapy? J Surg Oncol 2007, 96:474-480. 37. Benoy IH, Salgado R, Van Dam P, Geboers K, Van Marck E, Scharpe S, Vermeulen PB, Dirix LY: Increased serum inter- leukin-8 in patients with early and metastatic breast cancer correlates with early dissemination and survival. Clin Cancer Res 2004, 10:7157-7162. 18. Chang JC, Wooten EC, Tsimelzon A, Hilsenbeck SG, Gutierrez MC, Elledge R, Mohsin S, Osborne CK, Chamness GC, Allred DC, O'Connell P: Gene expression profiling for the prediction of therapeutic response to docetaxel in patients with breast cancer. Lancet 2003, 362:362-369. 38. Conclusion Kozlowski L, Zakrzewska I, Tokajuk P, Wojtukiewicz MZ: Concen- tration of interleukin-6 (IL-6), interleukin-8 (IL-8) and inter- leukin-10 (IL-10) in blood serum of breast cancer patients. Rocz Akad Med Bialymst 2003, 48:82-84. 19. Gianni L, Zambetti M, Clark K, Baker J, Cronin M, Wu J, Mariani G, Rodriguez J, Carcangiu M, Watson D, Valagussa P, Rouzier R, Symmans WF, Ross JS, Hortobagyi GN, Pusztai L, Shak S: Gene expression profiles in paraffin-embedded core biopsy tissue predict response to chemotherapy in women with locally advanced breast cancer. J Clin Oncol 2005, 23:7265-7277. y 39. Pusztai L, Mendoza TR, Reuben JM, Martinez MM, Willey JS, Lara J, Syed A, Fritsche HA, Bruera E, Booser D, Valero V, Arun B, Ibra- him N, Rivera E, Royce M, Cleeland CS, Hortobagyi GN: Changes in plasma levels of inflammatory cytokines in response to paclitaxel chemotherapy. Cytokine 2004, 25:94-102. 40. Jinga DC, Blidaru A, Condrea I, Ardeleanu C, Dragomir C, Szegli G, Stefanescu M, Matache C: MMP-9 and MMP-2 gelatinases and TIMP-1 and TIMP-2 inhibitors in breast cancer: correla- tions with prognostic factors. J Cell Mol Med 2006, 10:499-510. 20. Anelli A, Brentani RR, Gadelha AP, Amorim De Albuquerque A, Soares F: Correlation of p53 status with outcome of neoadju- vant chemotherapy using paclitaxel and doxorubicin in stage IIIB breast cancer. Ann Oncol 2003, 14:428-432. 21. Geisler S, Lonning PE, Aas T, Johnsen H, Fluge O, Haugen DF, Lillehaug JR, Akslen LA, Borresen-Dale AL: Influence of TP53 gene alterations and c-erbB-2 expression on the response to treatment with doxorubicin in locally advanced breast cancer. Cancer Res 2001, 61:2505-2512. 41. Vizoso FJ, Gonzalez LO, Corte MD, Rodriguez JC, Vazquez J, Lamelas ML, Junquera S, Merino AM, Garcia-Muniz JL: Study of matrix metalloproteinases and their inhibitors in breast cancer. Br J Cancer 2007, 96:903-911. , 22. Park K, Kim J, Lim S, Han S: Topoisomerase II-alpha (topoII) and HER2 amplification in breast cancers and response to pre- operative doxorubicin chemotherapy. Eur J Cancer 2003, 39:631-634. 42. Foekens JA, Peters HA, Look MP, Portengen H, Schmitt M, Kramer MD, Brunner N, Janicke F, Meijer-van Gelder ME, Henzen-Log- mans SC, van Putten WL, Klijn JG: The urokinase system of plasminogen activation and prognosis in 2780 breast cancer patients. Cancer Res 2000, 60:636-643. 23. Conclusion Al-azawi D, Kelly G, Myers E, McDermott EW, Hill AD, Duffy MJ, Higgins NO: CA 15-3 is predictive of response and disease recurrence following treatment in locally advanced breast cancer. BMC Cancer 2006, 6:220-226. 43. Harbeck N, Schmitt M, Kates RE, Kiechle M, Zemzoum I, Janicke F, Thomssen C: Clinical utility of urokinase-type plasminogen activator and plasminogen activator inhibitor-1 determination in primary breast cancer tissue for individualized therapy concepts. Clin Breast Cancer 2002, 3:196-200. 24. Formenti SC, Spicer D, Skinner K, Cohen D, Groshen S, Bettini A, Naritoku W, Press M, Salonga D, Tsao-Wei D, Danenberg K, Danenberg P: Low HER2/neu gene expression is associated with pathological response to concurrent paclitaxel and radia- tion therapy in locally advanced breast cancer. Int J Radiat Oncol Biol Phys 2002, 52:397-405. p , 44. Zemzoum I, Kates RE, Ross JS, Dettmar P, Dutta M, Henrichs C, Yurdseven S, Hofler H, Kiechle M, Schmitt M, Harbeck N: Inva- sion factors uPA/PAI-1 and HER2 status provide independent and complementary information on patient outcome in node- negative breast cancer. J Clin Oncol 2003, 21:1022-1028. 45. Ibrahim YH, Yee D: Insulin-like growth factor-I and breast can- cer therapy. Clin Cancer Res 2005, 11(2 Pt 2):944s-950s. 46. Portier CJ: Endocrine dismodulation and cancer. Neuro Endo- crinol Lett 2002, 23(Suppl 2):43-47. S S 44. Zemzoum I, Kates RE, Ross JS, Dettmar P, Dutta M, Henrichs C, Yurdseven S, Hofler H, Kiechle M, Schmitt M, Harbeck N: Inva- sion factors uPA/PAI-1 and HER2 status provide independent and complementary information on patient outcome in node- negative breast cancer. J Clin Oncol 2003, 21:1022-1028. p p and complementary information on patient outcome in node- negative breast cancer. J Clin Oncol 2003, 21:1022-1028. 45. Ibrahim YH, Yee D: Insulin-like growth factor-I and breast can- cer therapy. Clin Cancer Res 2005, 11(2 Pt 2):944s-950s. 46. Portier CJ: Endocrine dismodulation and cancer. Neuro Endo- crinol Lett 2002, 23(Suppl 2):43-47. y 25. Jones EL, Prosnitz LR, Dewhirst MW, Marcom PK, Hardenbergh PH, Marks LB, Brizel DM, Vujaskovic Z: Thermochemoradiother- apy improves oxygenation in locally advanced breast cancer. Clin Cancer Res 2004, 10:4287-4293. g , 45. Ibrahim YH, Yee D: Insulin-like growth factor-I and breast can- cer therapy. Clin Cancer Res 2005, 11(2 Pt 2):944s-950s. 46. Portier CJ: Endocrine dismodulation and cancer. Neuro Endo- crinol Lett 2002, 23(Suppl 2):43-47. , 26. Page 9 of 9 (page number not for citation purposes) Conclusion University of Pittsburgh Cancer Institute Luminex Core Facility [http://www.upci.upmc.edu/facilities/luminex/sources.html] 27 G lik E L d i l DP M i AM M d F V lik kh 47. Lue H, Thiele M, Franz J, Dahl E, Speckgens S, Leng L, Fingerle- Rowson G, Bucala R, Luscher B, Bernhagen J: Macrophage migration inhibitory factor (MIF) promotes cell survival by acti- vation of the Akt pathway and role for CSN5/JAB1 in the con- trol of autocrine MIF activity. Oncogene 2007, 26:5046-5059. 27. Gorelik E, Landsittel DP, Marrangoni AM, Modugno F, Velikokhat- naya L, Winans MT, Bigbee WL, Herberman RB, Lokshin AE: Mul- tiplexed immunobead-based cytokine profiling for early detection of ovarian cancer. Cancer Epidemiol Biomarkers Prev 2005, 14:981-987. y g 48. Tsutsui S, Kataoka A, Ohno S, Murakami S, Kinoshita J, Hachitanda Y: Prognostic and predictive value of epidermal growth factor receptor in recurrent breast cancer. Clin Cancer Res 2002, 8:3454-3460. , 28. Sarkar M, Leong TY: Application of K-nearest neighbors algo- rithm on breast caner diagnosis problem. Proc AMIA Symp 2000:759-763. Page 8 of 9 (page number not for citation purposes) Available online http://breast-cancer-research.com/content/10/3/R45 Available online http://breast-cancer-research.com/content/10/3/R45 49. Klijn JG, Berns PM, Schmitz PI, Foekens JA: The clinical signifi- cance of epidermal growth factor receptor (EGF-R) in human breast cancer: a review on 5232 patients. Endocrine Rev 1992, 13:3-17. 50. Kurebayashi J, Nishimura R, Tanaka K, Kohno N, Kurosumi M, Moriya T, Ogawa Y, Taguchi T: Significance of serum tumor markers in monitoring advanced breast cancer patients treated with systemic therapy: a prospective study. Breast Cancer 2004, 11:389-395. 51. Tiezzi DG, Andrade JM, Ribeiro-Silva A, Zola FE, Marana HR, Tiezzi MG: HER-2, p53, p21 and hormonal receptors proteins expres- sion as predictive factors of response and prognosis in locally advanced breast cancer treated with neoadjuvant docetaxel plus epirubicin combination. BMC Cancer 2007, 7:36-45. 52. Yurkovetsky Z, Ta'asan S, Skates S, Rand A, Lomakin A, Linkov F, Marrangoni A, Velikokhatnaya L, Winans M, Gorelik E, Maxwell GL, Lu K, Lokshin A: Development of multimarker panel for early detection of endometrial cancer. High diagnostic power of prolactin. Gynecol Oncol 2007, 107:58-65. y 53. Linkov F, Lisovich A, Yurkovetsky Z, Marrangoni A, Velikokhatnaya L, Nolen B, Winans M, Bigbee W, Siegfried J, Lokshin A, Ferris RL: Early detection of head and neck cancer: development of a novel screening tool using multiplexed immunobead-based biomarker profiling. Cancer Epidemiol Biomarkers Prev 2007, 16:102-107. Conclusion Page 9 of 9 (page number not for citation purposes)
https://openalex.org/W4293283375	https://zenodo.org/records/7008798/files/Entomophaga_Grylli_Destruction_of_Locust_Oxya-hyla_intricata_in_Vietnam.pdf	English	null	Entomophaga Grylli Destruction of Locust Oxya-hyla intricata in Vietnam	Zenodo (CERN European Organization for Nuclear Research)	1,985	cc-by	1,709	Malawi in 1974. These findings suggest that GM is distributed throughout the Guinea savannas of Africa. Malawi in 1974. These findings suggest that GM is distributed throughout the Guinea savannas of Africa. Malawi in 1974. These findings suggest that GM is distributed throughout the Guinea savannas of Africa. identified as Orseolia oryzivora by the Commonwealth Institute of Entomology, London. This species was identified from the collections in the Lake Chilwa area of was very high in Turners Asbestos Products Projects and at the National Irrigation Research Station. was very high in Turners Asbestos Products Projects and at the National Irrigation Research Station. The GM collected from Zambia was LSD at 5% 7.4 20.9 21.3 28.6 16.8 Grasshopper control with different insecticide dusts. a AARF, Pakistan. Mortality (%) at indicated hours after treatment 1 3 6 12 24 Dust Dosage Cotton dust b BHC 10% grasshoppers 10 13.7 100 100 100 100 10 22.3 100 100 100 100 BHC 10% + DDT 10% 10 18.0 100 100 100 100 1:3 mixture Carbaryl 10% 10 16.3 100 100 100 100 Untreated control – 0.0 a 0.0 a 0.6 a 0.0 a 1.3 a kg ai/ha Feeding on dust-impregnated nursery leaves Cotton dust b 7.20 3.0 42.7 85.0 100 100 BHC 10% 0.75 8.7 49.7 88.3 100 100 BHC 10% + DDT 10% 0.31 5.0 41.0 76.3 100 100 Carbaryl 10% 2.00 8.3 46.3 81.7 100 100 Untreated control – 0.0 a 0.0 a 0.3 a 1.6 a 1.3 a LSD at 5% 6.0 9.3 13.7 15.4 33.8 1:3 mixture. + 0.94 a In each column, averages followed by common letters are significantly similar at 95% level of con- fidence. b Cotton dust is a brand name for the mixture of BHC + DDT + sulfur (3:5:40); the dose of 7.2 kg ai/ha is based on the 48% active ingredient in the formulation. g/100 Direct dusting on grasshoppers Grasshopper control with different insecticide dusts. a AARF, Pakistan. Chemical control of grasshoppers in Pakistan M. A. Zafar, senior subject matter specialist in plant protection, Agricultural Adaptive Research Farm (AARF), Sheikhupura, Pakistan In some years, grasshopper Hieroglyphus spp. cause significant losses to rice nur- series and crops in Pakistan. In 1982 we tested four dusts in doses generally re- commended in treatments for grasshopper control at AARF. Grasshoppers were collected from rice fields and reared at room temperature to achieve a large population of adults of uniform age and size, The dusts were cotton dust (6.3% BHC + 10.4% DDT + 83.3% sulfur), BHC l0%, 1:3 mixture of BHC 10% + DDT l0%, and carbaryl 10%. They were sprinkled on the grasshoppers and rice leaves. In the first trial, 100 grasshoppers were kept in a glass jar without food for 24 h, then were released on fresh food. In the second trial grass- hoppers were caged on dusted leaves of a In each column, averages followed by common letters are significantly similar at 95% level of con- fidence. b Cotton dust is a brand name for the mixture of BHC + DDT + sulfur (3:5:40); the dose of 7.2 kg ai/ha is based on the 48% active ingredient in the formulation. With direct dusting, all grasshoppers died in 3 h. With foliar dusting, they died in 12 h. All the dusts were effective (see table). plants. Insect mortality was recorded 1, 3, 6, 12, and 24 h after treatment. The experiment was in a randomized block design with three replications. 1. Germinating conidia of E. grylli on the wing of O. intricata Scale = 10 µm. 1. Germinating conidia of E. grylli on the wing of O. intricata Scale = 10 µm. Entomophaga grylli destruction of locust Oxya-hyla intricata in Vietnam J. Weiser, V. Matha, N. D. Tryachov, and I. Gelbic, Institute of Entomology, Academy of Science, Prague, Czechos- lova kia 1. Germinating conidia of E. grylli on the wing of O. intricata Scale = 10 µm. 2. Resting spores of E. grylli from the interior of dead O. intricata. Scale = 10 µm. A new record of rice shoot fly in the northwestern hills of India D. K. Garg, Vivekananda Laboratory for Hill Agriculture, Indian Council of Agri- cultural Research, Almora 263601, India Preliminary field observations showed that maximum infestation was 3-4 wk after sowing. Rice planted early, in the first week of May, was more severely damaged than normal plantings in the last week of May. There was no infestation in the transplanted crop. During a survey of rice nurseries in expe- rimental field plots at Hawalbagh in 1983 kharif (May-Oct), we found plants of various entries yellowed and dried in Newly hatched shoot fly maggots bore into the stems of young plants and feed inside the central shoot, severing the Entomophaga grylli destruction of locust Oxya-hyla intricata in Vietnam Damage Grain Planting date grain yield (%) (t/ha) 31 Dec 83 10 Jan 84 20 Jan 84 30 Jan 84 10 Feb 84 20 Feb 84 1 Mar 84 34 3.7 34 42 29 17 5.4 23 25 4.3 3.1 4.4 3.7 4.5 Effect of planting time on rice panicle bug inci- dence Tirur, India. Effect of planting time on rice panicle bug incidence R. Saroja and N. Raju, Paddy Experiment Station, Tirur 602025, Tamil Nadu, India Rice panicle bug Leptocorisa acuta (Thumb) is becoming a major rice pest in Tamil Nadu in the Dec-May and Apr-Aug cropping seasons. Adults and nymphs suck sap from developing grains and cause significant yield loss. Feeding activity was assessed at harvest by counting the insect’s stylet sheaths in the grains obtained from five randomly selected panicles from each plot. Undamaged and damaged grains were counted and damage percentage was calculated. Grain yield also was recorded (see table). We studied the effect of planting time on panicle bug incidence in a field trial with seven planting dates and four repli- cations in 1983-84 Dec-May season. Twenty-five-day-old, short-duration to 4.5 t/ha. When damage exceeded 30%, yield fell to 3.1-3.7 t/ha. Very early and very late crops were prone to panicle bug attack. to 4.5 t/ha. When damage exceeded 30%, yield fell to 3.1-3.7 t/ha. Very early and very late crops were prone to panicle bug attack. ( ) The crop planted on 30 Jan had lowest grain damage and yielded highest. At 20- 30% grain damage, yield ranged from 4.3 apical parts of the plant from the base. The leaf whorl fails to unfold, dries, and falls off. patches. Plants had deadhearts and small, whitish maggots were recovered from the base of the stems. The maggots were reared in petri dishes and flies emerged and were identified as rice shoot fly Athe- rigona oryzae Mall. This is the first recorded occurrence of A. oryzae in the northwestern hills of India. Entomophaga grylli destruction of locust Oxya-hyla intricata in Vietnam J. Weiser, V. Matha, N. D. Tryachov, and I. Gelbic, Institute of Entomology, Academy of Science, Prague, Czechos- lova kia J. Weiser, V. Matha, N. D. Tryachov, and I. Gelbic, Institute of Entomology, Academy of Science, Prague, Czechos- lova kia The fungus E. grylli (Fres.) Batko is the most common Entomophthorales fungus and often is associated with locust and grasshopper outbreaks. Data from South- east Asia describe an outbreak of E. grylli in a Patanga succinata population on maize east of Lopburi Province in Thailand. By the end of the rainy season, the fungus had destroyed 90% of the locust population and was found in 9 other acridid pests in Thailand. In autumn 1983, dense O. intricata populations seriously damaged rice around Hanoi, Vietnam. E. grylli infected 85-90% adults and nymphs, which died on the plants. Masses of conidiophores O. intricata Stål is widely distributed in Southeast Asia — Burma, Thailand, Vietnam, West Malaysia — and some parts of South China. It damages rice and several other crops. 16 IRRN 10:2 (April 1985) with conidia appeared on the interseg- mental membranes (Fig. 1) and on all thin cuticular areas of the head. Conidia of the papillata type of Lakon, 30-40 × 25-36 µm spread over dead insects and plants. Their length-to-breadth ratio varied. Uniform resting pores 30-40 µm in diam developed inside the infected locusts (Fig. 2). E. grylli may be an important natural enemy of this locust in local outbreaks. Effect of planting time on rice panicle bug inci- dence Tirur, India. Damage Grain Planting date grain yield (%) (t/ha) 31 Dec 83 10 Jan 84 20 Jan 84 30 Jan 84 10 Feb 84 20 Feb 84 1 Mar 84 34 3.7 34 42 29 17 5.4 23 25 4.3 3.1 4.4 3.7 4.5 to 4.5 t/ha. When damage exceeded 30%, yield fell to 3.1-3.7 t/ha. Very early and very late crops were prone to panicle bug attack. TM8089 seedlings were planted in 9-m 2 plots at 20- × 10cm spacing. Plots were fertilized with 100 kg N/ha (50% basal and 2 equal splits topdressed) and 22-42 kg PK/ha. No crop protection was practiced. Effect of planting time on rice panicle bug inci- dence Tirur, India. Mortality of adult brown planthoppers (BPH) in different types of cages used for bioassays of entomopathogenic fungi Many hyphomycetous fungal pathogens of the BPH Nilaparvata lugens Stål and related leafhopper species have been collected and isolated on artificial media. A suitab1e bioassay test is necessary to determine the most virulent species and strains of these entomopathogens for use in a biological control program against BPH. In addition, standardized bioassay Many hyphomycetous fungal pathogens of the BPH Nilaparvata lugens Stål and related leafhopper species have been collected and isolated on artificial media. A suitab1e bioassay test is necessary to determine the most virulent species and strains of these entomopathogens for use in a biological control program against BPH. In addition, standardized bioassay techniques that minimize mortality are essential to developing programs for sub- sequent production and field application of the fungi. R. M. Aguda, senior research assistant, IRRI; M. C. Rombach, research associate, Boyce Thompson Institute (BTI) and Collaborative research fellow, IRRI; B. M. Shepard, entomologist, IRRI; and D. W. Roberts, insect pathologist, BTI Entomopathogenic fungi invade the insect host through the cuticle, grow in the body cavity, and eventually kill the host. BPH fungal pathogens normally kill the host in 2-7 d. During this time, IRRN 10:2 (April 1985) 17
https://openalex.org/W3008796205	https://library.oapen.org/bitstream/20.500.12657/50322/1/Poellinger%202017%20Analogy-Based%20Inference%20Patterns%20in%20Pharmacological%20Research%20FINAL.pdf	English	null	Analogy-Based Inference Patterns in Pharmacological Research	Boston studies in the philosophy of science	2,020	cc-by	15,449	Analogy-Based Inference Patterns in Pharmacological Research∗ Roland Poellinger Abstract Analogical arguments are ubiquitous vehicles of knowledge transfer in science and medicine. This paper outlines a Bayesian evidence-amalgamation framework for the purpose of formally exploring different analogy-based infer- ence patterns with respect to their justiﬁcation in pharmacological risk assessment. By relating formal explications of similarity, analogy, and analog simulation, three sources of conﬁrmatory support for a causal hypothesis are distinguished in recon- struction: relevant studies, established causal knowledge, and computational mod- els. Key words: scientiﬁc inference, pharmacology, epistemology, Bayesian conﬁrma- tion, evidence, relevance, similarity, analogy, computer simulation Roland Poellinger Munich Center for Mathematical Philosophy, LMU Munich e-mail: r.poellinger@lmu.de ∗This work is supported by the European Research Council (grant 639276) and the Munich Center for Mathematical Philosophy (MCMP). ∗This work is supported by the European Research Council (grant 639276) and the Munich Center for Mathematical Philosophy (MCMP). Roland Poellinger Munich Center for Mathematical Philosophy, LMU Munich e-mail: r.poellinger@lmu.de 2 In this passage, Hill refers to (i) severe disabilities (even death) among babies linked to the over-the-counter drug thalidomide, prescribed in the 1960s in Germany as Contergan to alleviate morning sickness in pregnant women, and to (ii) miscarriage or children born with the congenital rubella syndrome (CRS) due to infection by the rubella virus during pregnancy. 1 Introduction: scientiﬁc inference in pharmacology Pharmacological research is often driven by many forces at once: Cost effectiveness must be balanced against extensive data-collecting, potential risk against probable beneﬁt, and breadth of applicability against well-documented higher conﬁdence for smaller target groups. Many such decisions must be taken during the development stages of a certain drug before the desired effectiveness and safety level is reached and the drug is allowed to be marketed. A language for expressing both beneﬁt and safety is found in the probabilistic language of expected utilities and dis-utilities. Nevertheless, the formalization of a given decision problem in such vocabulary can only be as informative about future drug users as the evidence it is rooted in. Yet, 1 1 2 2 Roland Poellinger Roland Poellinger whether it is all the evidence about the drug’s effects that is to be taken into con- sideration, or only the best evidence available, is the subject of an ongoing dis- cussion in the philosophy of medicine. A recent paper by Landes, Osimani, and Poellinger (Landes et al, 2017) explores the possibility of amalgamating all avail- able evidence in a Bayesian reconstruction of scientiﬁc inference for the integrated probabilistic assessment of a drug’s causal (side-)effects. Key to this endeavor is the distinction of the conceptual levels involved: (i) the causal hypothesis, (ii) testable indicators of the causal claim, i.e., theoretical consequences of the causal hypothe- sis, and (iii) concrete evidence (actual data) speaking for or against the respective indicators (thereby indirectly supporting the hypothesis, or not). Close to pharmaco- logical practice, it is furthermore useful to introduce an additional meta-evidential level for the purpose of encoding the qualiﬁed assessment of the data at hand. This allows to conceptually distinguish the content of an evidential report from its weight in the evaluation of the hypothesis: Different pieces of evidence may possess differ- ent levels of signiﬁcance (iv). One important justiﬁcation of the conﬁrmatory support a piece of evidence lends to a given hypothesis (by virtue of it being evidence for an indicator of the very hypothesis) is the postulate (or implicit assumption) of analogy between the cir- cumstances generating the evidence and the hypothesis’ intended (future) scope of application. Sir Austin Bradford Hill lists analogy as one of his famous guidelines towards an informed assessment of potential causes in epidemiology: In some circumstances it would be fair to judge by analogy. 1.1 Evidence amalgamation and hypothesis conﬁrmation Causal inference in pharmacology is a difﬁcult task due to sometimes sparse, of- tentimes very heterogeneous evidence, different standards for evidence evaluation and integration, and many sources of random and systematic error. In their plural- ist, conciliatory approach, Landes, Osimani, and Poellinger (Landes et al, 2017) propose a blueprint for tracing the epistemological dynamics of evidence amalga- mation, viewing risk assessment in pharmacology from a meta-perspective. In their framework, causal hypotheses (about adverse drug reactions) and evidential reports (about concrete studies) are related in such a way, that successively cumulating ev- idence allows for probabilistic causal inference. One important point of departure for this approach is Bovens’ and Hartmann’s general reconstruction of scientiﬁc in- ference (Bovens and Hartmann, 2003) in which the epistemic dynamics of all the dependencies between a hypothesis, testable indicators implied by the hypothesis, and evidence for/against such indicators can be visually traced in the graph of a Bayesian network: The conceptual categories are depicted as layers of nodes, with directed edges between the layers marking those paths along which conﬁdence in the hypothesis is boosted (or lowered). The probabilistic model underneath the graphi- cal representation supplements the Bayesian network with quantitative information by encoding (i) conditional degrees of (un)certainty about all variables, and (ii) how these degrees will change under local updates. The example in Fig. 1 illustrates the epistemological structure: The causal hy- pothesis (Hyp) entails n theoretical indicators (i.e., testable consequences Ind1, ..., Indn), which are to be supported by concrete evidence reports Rep1, ..., Repn on the lowest level. Since evidence reports are based on concrete data (e.g., clinical trials, historical studies, or lab experiments), it will be desirable in many cases to modulate their signiﬁcance for the assessment of a particular causal hypothesis for various reasons: The source of information seems undependable, the quality of a study might be doubtful, the reliability of method or measurement device is not guaranteed, test group and target group deviate in relevant details, and so on. To be able to express such levels of signiﬁcance, the report nodes in the graph come with an additional weight node α (for the moment simply a blackbox placeholder for the aforementioned signiﬁcance dimensions, used below to encode evidential relevance in particular, see Sec. 2). 1 Introduction: scientiﬁc inference in pharmacology With the effects of thalidomide and rubella before us we would surely be ready to accept slighter but similar evidence with another drug or another viral disease in pregnancy.2 (Hill, 1965, p. 11) The aim of this paper is to explore different analogy-based inference patterns in the above-sketched layered reconstruction of scientiﬁc reasoning, with respect to their justiﬁcation in pharmacological risk assessment. In particular, different aspects of similarity shall be made transparent in order to compare conceptually different types of evidence in analogical reasoning. Three interrelated questions shall be ad- dressed in the following sections: 1. How can analogy considerations be used to explicate the relevance of evidence for a hypothesis under consideration? (Sec. 2) 2. How can an already well-established hypothesis be used as a supporting analog in conﬁrming a hypothesis about a similar drug? (Sec. 3) 3. By what standards can a mechanistic computational model of a substance’s causal effects as a theoretical analog lend evidential support to a causal hypothe- sis about the actual substance? (Sec. 4) 3 Analogy-Based Inference Patterns in Pharmacological Research Before we can trace the role of analogy in pharmacological research, though, a formal reconstruction of the dynamics underlying scientiﬁc hypothesis testing shall be outlined in the following. Before we can trace the role of analogy in pharmacological research, though, a formal reconstruction of the dynamics underlying scientiﬁc hypothesis testing shall be outlined in the following. 3 As implied by this way of distinguishing reliability and relevance, the relevance weight (attached to a given evidential report) is really meant here to capture the degree of external validity. Of course, there are other ways in which a study can be relevant to the hypothesis – for example, if it is conducted by an acknowledged authority. In the framework of Landes, Osimani, and Poellinger (Landes et al, 2017), this way of being relevant to the hypothesis would be encoded in the reliability weight, which collects all sources of bias. 1.1 Evidence amalgamation and hypothesis conﬁrmation The degree to which a given evidence report supports the hypothesis depends precisely on its “weight”, i.e., its relevance to the hypothesis, the reliability of the source, the error-proneness of the methods used to generate the data, and so on. Such weight nodes might in general be shared between reports – for example in cases where more than one report is based on data generated by the same 4 4 Roland Poellinger measurement device. While Bovens and Hartmann utilize this weighting parameter to explicate the reliability of a report, Landes, Osimani, and Poellinger – aiming at hypothesis conﬁrmation in pharmacology – split this weighting parameter into two variables (reliability and relevance) in order to distinguish the quality of method and information source from questions of external validity (by encoding the degree to which study results can be extrapolated to the target).3 What it means to be an observable consequence of the hypothesis is implicitly stated by the following inequality (see also Bovens and Hartmann 2003, p. 90): P(Indi \|Hyp) = pi > qi = P(Indi \|¬Hyp), (1) P(Indi \|Hyp) = pi > qi = P(Indi \|¬Hyp), (1) where ‘Hyp’ is to be understood as shorthand for ‘Hyp = true’, and analogously for the other variables (in the following, context disambiguates if a variable or its instantiation is referred to). Hyp . . . Ind2 Ind1 Indn−1 Indn α1 Rep1 α2 Rep2 αn Repn−1 Repn Fig. 1: Example of how a causal hypothesis, multiple testable consequences, and weighted evidence reports might be related in a Bayesian network. Fig. 1: Example of how a causal hypothesis, multiple testable consequences, and weighted evidence reports might be related in a Bayesian network. Analogy-Based Inference Patterns in Pharmacological Research 5 The conditional in/dependencies between the epistemic categories can be read off the structure in Fig. 1 by utilizing the graphical d-separation criterion:4 All directed edges mark positive inﬂuence between adjacent nodes (variables, respec- tively), which makes all Hyp, Ind, and Rep variables positively correlated (condi- tional on the empty set). 4 The graphical d-separation criterion (with d for directional) distinguishes conditionally depen- dent (sets of) variables from conditionally independent ones by drawing on structural information, i.e., on how arrows are directed along the paths between the (sets of) variables under consideration; see, e.g., Geiger et al (1990). 5 In this case, independence between the weight variables and the hypothesis “may or may not be a realistic assumption”, as Bovens and Hartmann concede, and they extend their discussion to cases where such weight nodes (reliability of evidence reports) and the hypothesis are made dependent through auxiliary theories (see Bovens and Hartmann 2003, pp. 107ff.). For the purpose of this paper, though, the standard for assigning values to weight variables is assumed to be ﬁxed prior to hypothesis testing. 1.1 Evidence amalgamation and hypothesis conﬁrmation Following Bovens and Hartmann (2003), the weight vari- ables are independent of the theoretical categories (i.e., hypothesis and indicators) and may be used to account for exceptions.5 I will treat the α-weights as binary modulators throughout the paper (with α = 1 indicating signiﬁcance and α = 0 in- dicating insigniﬁcance, respectively); nevertheless, the formal framework can easily be extended to allow for a more ﬁne-grained (in many cases more appropriate) rep- resentation. The in/dependencies visualized in Fig. 1 can be summed up probabilistically as follows, for all i (1 ≤i ≤n): Hyp,Indi ⊥⊥αi (2) P(Repi \| Indi,αi = 1) = 1 (3) P(Repi \|¬Indi,αi = 1) = 0 (4) P(Repi \| Indi,αi = 0) = (5) P(Repi \|¬Indi,αi = 0) = P(Repi \| αi = 0) = qi Hyp,Indi ⊥⊥αi (2) P(Repi \| Indi,αi = 1) = 1 (3) P(Repi \|¬Indi,αi = 1) = 0 (4) P(Repi \| Indi,αi = 0) = (5) P(Repi \|¬Indi,αi = 0) = P(Repi \| αi = 0) = qi Hyp,Indi ⊥⊥αi (2) P(Repi \| Indi,αi = 1) = 1 (3) P(Repi \|¬Indi,αi = 1) = 0 (4) P(Repi \| Indi,αi = 0) = (5) P(Repi \|¬Indi,αi = 0) = P(Repi \| αi = 0) = qi (5) Eq. 2 precisely encodes the independent assignment of the weighting parameter. Eq. 3 and Eq. 4 show that an evidence report marked as “signiﬁcant” to the hypothesis is aligned with the respective indicator. The loose term “signiﬁcance” is meant to be understood in this context as a quite general concept up for interpretation – it does not refer to statistical signiﬁcance of study results, but rather to the quality of evi- dence (as meta-information), e.g., in terms of reliability or relevance, as discussed below. Eq. 5 explicates what happens when an evidence report is qualiﬁed as “ut- terly insigniﬁcant” to the hypothesis: In that case, whether the report speaks for or against the respective indicator becomes probabilistically independent of the truth value of this very indicator variable (with 0 ≤qi ≤1). 4 The graphical d-separation criterion (with d for directional) distinguishes conditionally depen- dent (sets of) variables from conditionally independent ones by drawing on structural information, i.e., on how arrows are directed along the paths between the (sets of) variables under consideration; see, e.g., Geiger et al (1990). 5 In this case, independence between the weight variables and the hypothesis “may or may not be a realistic assumption”, as Bovens and Hartmann concede, and they extend their discussion to cases where such weight nodes (reliability of evidence reports) and the hypothesis are made dependent through auxiliary theories (see Bovens and Hartmann 2003, pp. 107ff.). For the purpose of this paper, though, the standard for assigning values to weight variables is assumed to be ﬁxed prior to hypothesis testing. 6 The Bayes net structure in Fig. 1 for example illustrates that Ind1 is inﬂuenced by (since d- connected to) α1 once we know the value of the “collider variable” Rep1. 1.1 Evidence amalgamation and hypothesis conﬁrmation In particular, an evidence re- port considered irrelevant for the investigated hypothesis should not inﬂuence one’s 5 In this case, independence between the weight variables and the hypothesis “may or may not be a realistic assumption”, as Bovens and Hartmann concede, and they extend their discussion to cases where such weight nodes (reliability of evidence reports) and the hypothesis are made dependent through auxiliary theories (see Bovens and Hartmann 2003, pp. 107ff.). For the purpose of this paper, though, the standard for assigning values to weight variables is assumed to be ﬁxed prior to hypothesis testing. 6 Roland Poellinger conﬁdence in the truth of the hypothesis’ indicators.6 conﬁdence in the truth of the hypothesis’ indicators.6 With this Bayesian framework at hand, explicating what it means for a given piece of “signiﬁcant” evidence to conﬁrm a causal hypothesis is straightforward: An evidence report Repk is said to conﬁrm the hypothesis Hyp iff it raises Hyp’s probability, i.e., iff P(Hyp\|Repk) > P(Hyp). (6) (6) Obviously, this inequality does not hold for αk = 0, i.e., for “insigniﬁcant” evidence reports (unreliable sources of evidence or evidence irrelevant for the hypothesis etc.). Consequently, what it means for a given piece of evidence to be “signiﬁcant” to a hypothesis must be deﬁned before we can start conﬁrming this very hypothesis. Analogical reasoning shall be exploited towards that goal in the following. Obviously, this inequality does not hold for αk = 0, i.e., for “insigniﬁcant” evidence reports (unreliable sources of evidence or evidence irrelevant for the hypothesis etc.). Consequently, what it means for a given piece of evidence to be “signiﬁcant” to a hypothesis must be deﬁned before we can start conﬁrming this very hypothesis. Analogical reasoning shall be exploited towards that goal in the following. 1.2 Analogy as inferential pattern At the heart of analogical arguments lies the assumption of (sufﬁcient) similarity between the two relata (or aspects thereof, respectively). The concept of similarity comes with a mixed bag of notorious epistemological issues of its own, though. For example, if two drugs are to be related in an analogical argument, the following questions come to mind: What does it mean to be sufﬁciently similar in the case under consideration? In what way does the difference between the ﬁrst and the sec- ond drug inﬂuence changes in expected outcome values? How speciﬁc are a drug’s properties? If they are highly speciﬁc – to what extent can this drug be used in an analogical argument, if at all? Despite these conceptual difﬁculties, science and history are full of successful examples of analogical reasoning, even in the case of scientiﬁc discovery: In the nineteenth century, secured knowledge about acoustics was employed in the discovery of spectral lines. Guided by the image of a harmonic oscillator, physicists were able to focus their attention to groups of spectral lines with speciﬁc frequency patterns from the beginning (see Bartha’s in-depth overview of analogical arguments in Bartha 2013, as well as Unruh 2008, Dardashti et al 2017, and Hesse 1952 for discussions of analog arguments in physics). In pharmacology and epidemiology, reasoning by analogy is a key mode of knowledge transfer from study to target population. Indeed, because of the context sensitivity of many causal associations in the biological realm, these associations can hold only in speciﬁc populations, and therefore evidence about causal effects related to one population may not license similar conclusions about another popu- lation, unless the two populations have been established as analogous (in relevant respects). Knowledge about an agent’s mechanisms and about its impact on the bi- ological environment might be sparse and come from quite heterogeneous sources, 7 Analogy-Based Inference Patterns in Pharmacological Research 7 Analogy-Based Inference Patterns in Pharmacological Research however. Yet, already if only little information about the agent’s class of molecules is available, for example, this can justiﬁably be exploited for causal assessment via analogy. As Bartha (2010) points out, analogical arguments fall into a different category than regular evidence as such. Consider the general form of an analogical argument (see Bartha 2013): 1. y is similar to x (in certain known respects), (A) 2. x has additional property A, 3. therefore: y has property A′ (similar to A). 7 Landes et al (2017) contains a non-exclusive list of six causal indicators derived from Hill’s guidelines in Hill (1965). See also Poellinger (n.d.) for a discussion of the conceptual relationships of these causal indicators and the ramiﬁcations of theory choice in causal assessment. 1.2 Analogy as inferential pattern The investigated causal hypothesis Hyp may be related to a second causal hy- pothesis Hyp∗which has already been established in previous studies. Now, if scientists have sufﬁcient reason to postulate analogy between Hyp and Hyp∗ (e.g., a high degree of chemical or functional similarity), knowledge about this second causal hypothesis supports the ﬁrst hypothesis Hyp via analogy. This case requires an extension of the layered network introduced above and will be treated in Sec. 3. 2. The investigated causal hypothesis Hyp may be related to a second causal hy- pothesis Hyp∗which has already been established in previous studies. Now, if scientists have sufﬁcient reason to postulate analogy between Hyp and Hyp∗ (e.g., a high degree of chemical or functional similarity), knowledge about this second causal hypothesis supports the ﬁrst hypothesis Hyp via analogy. This case requires an extension of the layered network introduced above and will be treated in Sec. 3. 3. As a special sub-case of 2., I will discuss how Hyp can possibly be conﬁrmed by way of computational modeling (instead of by relating it to a second causal hypothesis Hyp∗about a biological system): In cases where scientists want to conﬁrm mechanistic assumptions, computer simulation has become a viable al- ternative to costly, unethical, or otherwise inaccessible experimental studies. As- sumptions about the efﬁcacious mechanistic relationships may be modeled in a virtual analog to ﬁnd out more about the protein a drug binds to or to simulate an agent’s interaction with the biological system, for instance, w.r.t. dosage (see, e.g., Britton et al 2013 and Carusi et al 2012). In order to treat this special case I will tweak the network structure even further in Sec. 4. My aim in this paper is to exploit the concept of analogy in explicating these dif- ferent knowledge transfer strategies. To this end, I will investigate the conﬁrmatory dynamics of analogical reasoning in a formal way: All three abovementioned cases shall successively be located in the evidence-amalgamating framework for the pur- pose of unifying the inferential patterns and emphasizing their structural differences at the same time. I will start by discussing ways of designing possible (compara- tive or numerical) distance measures for expressing similarity (between substances, populations, etc.). 1.2 Analogy as inferential pattern (A) 1. y is similar to x (in certain known respects), 1. y is similar to x (in certain known respects), (A) 2. x has additional property A, 3. therefore: y has property A′ (similar to A). This argument obviously encodes basic evidence about x which is then used to make inferences about y. It is unclear, as Bartha points out, how such a structured argu- ment could be encapsulated in a single evidential proposition to ﬁgure as a condition in the scheme of Bayesian conﬁrmation, stated in Eq. 6 above. Moreover, according to Bartha, even if we were to condense a full analogical argument into one such ev- idential proposition A, the postulated similarity between source and target domain must have been established before A can be used in conﬁrmation, which makes A “old evidence” and useless for conﬁrmation (since it does not boost the degree of belief in the hypothesis). This becomes obvious once Eq. 6 is relativized to ﬁxed background knowledge K. With the analogical argument A entailed by K, we get P(Hyp\|A,K) = P(Hyp\|K). With the above-sketched Bayesian reconstruction of scientiﬁc hypothesis testing at hand, we already have a structure-rich framework utilizable for a different formal- ization of analogical reasoning: The key idea is to understand analogy not as single nodes, but to trace conﬁrmation by analogy along the edges in the graph. In other words, analogical arguments shall be understood and expressed rather as inferential patterns than as “evidence nodes”. The evidence-amalgamation framework can be operationalized for this purpose in the following ways: 1. The question of applicability of a study’s ﬁndings is best phrased in terms of cor- respondence between study and target populations, where correspondence shall be understood as similarity (above a certain threshold): If study and target pop- ulations are sufﬁciently similar, researchers are licensed to reason about causal links in the target population by analogy with their test cases. The extent to which this kind of transfer is licensed is encoded in the framework as relevance of avail- able reports (such that independent reports are each assigned a different degree of relevance with respect to the investigated hypothesis). The more characteris- tics are shared between study and target, the higher the relevance of evidence obtained in this study for the hypothesis under investigation. This case is treated in Sec. 2. 8 Roland Poellinger Roland Poellinger 2. 8 LaFollette and Shanks (1995) argue, e.g., that animal studies are limited to hypothesis generation. 9 In particular, the question must be answered whether the partial result can be combined in an additive fashion with information about further sub-mechanisms, or whether complex inter- dependencies forbid partitioning the full mechanism into stand-alone modules. 2.1 Heterogeneous evidence The evidence-amalgamating framework, as introduced above, presents itself as a tool for describing speciﬁc cases in that it can be adapted to accommodate speciﬁc pieces of evidence for (or against) speciﬁc theoretical consequences of the causal hypothesis under consideration. At the same time, it is meant to be understood as a normative statement about the quality of the causal assessment: The more infor- mation about the indicator variables is available, the more reliable the assessment of the hypothesis. This is especially true in the case of causal hypotheses, given the variety of methodological approaches towards discovering causal associations in the sciences.7 9 Analogy-Based Inference Patterns in Pharmacological Research The multi-layered framework introduces report nodes as placeholders for hetero- geneous pieces of evidence. The reports can come from all levels of the “evidence hierarchy” – the framework treats all these levels as relevant in causal assessment: Systematic reviews, meta-analyses of randomized clinical trials or observational studies, comparative non-randomized studies (cohort or case-control studies), ev- idence of (sub-)mechanisms from basic science (lab experiments etc.), as well as expert judgment. Relevant information may come also from single case reports, case series, and animal studies. Amalgamating these different types of evidence has not only epistemological value (since it traces the epistemological dynamics from observed signals of Nature to the establishment of hypotheses) but also methodolog- ical value (since it readily accommodates different approaches towards evidence as- sessment and related disputes like the seeming tension between paradigms such as best-evidence vs. pluralistic approaches). 8 LaFollette and Shanks (1995) argue, e.g., that animal studies are limited to hypothesis generation. 9 In particular, the question must be answered whether the partial result can be combined in 2.2 Relevance However heterogeneous the evidence, different evidence reports (understood as data interpreted relative to the hypothesis under consideration) will be attributed different levels of signiﬁcance for the hypothesis: Virtually no pharmacological study allows for straightforwardly transferring shown results from studied population to target population of possible future drug users – population size, inherent structure, spe- ciﬁc circumstances, possible interactions with uncontrolled substances, etc. might be similar, but will virtually never be the exact same (see Chan and Altman 2005, p. 1180, Doll and Peto 1980, Worrall 2007, p. 992). Furthermore, patient inclusion cri- teria for participation in RCTs will skew the inference transfer from study to target population even more (see, e.g., Revicki and Frank 1999 and Upshur 1995, p. 483). When the results of animal studies are to be applied to a population of future hu- man drug users it becomes quite clear that questions of applicability must be settled ﬁrst before such transfer is licensed.8 Moreover, in their recent analysis of the role of evidence about a substance’s mechanisms in risk assessment, Luj´an, Todt, and Bengoetxea (Luj´an et al, 2016) point towards the lack of guarantee that similarity of modes of action may warrant extrapolation of phenotypic effects from one chemical to another – chemicals considered similar in important respects might not necessar- ily produce similar effects (for a given population). And ﬁnally, when laboratory ex- periments yield information about some component of the causal mechanism (e.g., at the molecular level or in terms of cell behavior), the signiﬁcance of this (partial) result for the hypothesis about the entire mechanism must be determined ﬁrst.9 10 Roland Poellinger Roland Poellinger In all these cases scheme (A) is applied implicitly or explicitly: The hypothesis is conﬁrmed by analogy, i.e., its degree of conﬁdence is raised by a given evidence report, once similarity between studied subjects (or objects) and intended applica- tion is established. In the framework of Landes, Osimani, and Poellinger, this type of signiﬁcance of a given evidence report for a hypothesis to be tested is expressed as evidential relevance factor (whose value is to be determined outside the model), cf. Landes et al 2017, Sec. 3.3:10 Ideally, pharmacological studies would license the same inferences for the studied popula- tion and the target population of future drug users. 10 Note that I am distinguishing evidential relevance (as a property of evidence relevant in causal assessment) from causal relevance (as a property of a variable causally relevant to a second variable in a causal model), cf. footnote 18. Also see footnote 3 above for a remark on other interpretations of relevance. 12 This suggests that the distinction between changeable and unchanged aspects of the study pop- ulation will be a static one prior to modeling. Nevertheless, Paul and Healy discuss cases in which the modeler is forced to revisit her model because a clinical trial impacts on relevant character- istics of the population in a sort of feedback loop (see Paul and Healy 2016 on Transformative Treatments). For the present purpose it is uncritical to assume that the initial model can be reﬁned at later stages to accommodate previously exogenous assumptions into the model as endogenous parameters/relations. 11 Note that for such an interpretation the prior of the network is required to be set up in such a way that the αs render the Reps independent of (i.e., irrelevant to) the hypothesis Hyp in the extreme case. Formally: P(Hyp\|Repk = true,αk = irrelevant) = P(Hyp\|αk = irrelevant) = P(Hyp). See also my discussion of the prior in Sec. 1.1 above. 2.2 Relevance In reality, studies are not conducted on the entire population of future drug users but on a much smaller number of patients, see Chan and Altman 2005, p. 1180, Button et al 2013 for this problem in neuro science, Doll and Peto 1980 in cancer research and a philosophical discussion of this problem in Worrall 2007, p. 992. Additionally, studied populations, in particular in RCTs, often fail to be rep- resentative for the target population due to strict patient inclusion criteria, see Revicki and Frank 1999 and Upshur 1995, p. 483. Therefore, there is a need to reason by analogy from the studied population to the population of interest [. . . ] The relevance pertaining to an item of evidence measures how well the observed results in a study population can be transferred to the target population of future drug users. Hyp Ind1 Indn Rep1 α1 Repn αn . . . . . . . . . support by analogy Fig. 2: Support by analogy from the level of evidence reports (Rep1,...,Repn) up- wards to the hypothesis under consideration. Hyp Fig. 2: Support by analogy from the level of evidence reports (Rep1,...,Repn) up- wards to the hypothesis under consideration. Fig. 2 illustrates this relationship between an item of evidence and the target: When hypothesis Hyp is on the test bench, testable consequences Ind1,...,Indn (indicators of causation) are supported (or not) by evidence reports Rep1,...,Repn. Since the framework is set up in Bayesian fashion, once the value of Repk is ﬁxed 11 Analogy-Based Inference Patterns in Pharmacological Research (e.g., to Repk = true, indicating that there is evidence in support of Indk = true), the respective signiﬁcance variable αk will determine the relative strength of Repk’s contribution to Hyp (since – in Bayesian terms – knowing a collider’s value opens the ﬂow of information along the path the collider lies on). Note, that – in principle – two (or more) report nodes might share the same signiﬁcance, as exempliﬁed in Fig. 1, when inter-dependencies are to be indicated (in terms of either reliability or extrapolability, i.e., relevance). – two (or more) report nodes might share the same signiﬁcance, as exempliﬁed in Fig. 1, when inter-dependencies are to be indicated (in terms of either reliability or extrapolability, i.e., relevance). 2.2 Relevance If we now interpret the α-layer as encoding information about the relevance of our evidence reports, we can try to make the relationship between αk and Hyp trans- parent in terms of similarity.11 13 If the causal hypothesis is thought of as a causal graph, D, E, and U are meant to represent designated (sets of) variables with token values in a causally interpreted structure M (possibly encoding the speciﬁcs of direct causal relations and assumptions about causal in/dependencies on type level). Note that, more generally, M and Mk can be thought of as sets of structural constraints, i.e., as classes of causal graphs. 1. ⟨D,M,U⟩is similar to ⟨Dk,Mk,Uk⟩, (A) therefore, by stipulation: Repk is relevant for Hyp, i.e., αk is high, 2.3 Measuring distance In order to be able to compare the hypothesis and incoming evidence reports, the structural ingredients of these categories shall be made explicit in more detail. In risk assessment, Hyp is meant to be a causal hypothesis about a speciﬁc (harmful) side-effect E of a certain drug D – to be abbreviated as ‘D c⃝E’ in the follow- ing. Since the evidence-amalgamating framework is intended to yield quantitative (probabilistic) information for decision-making (e.g., in terms of expected utilities), D c⃝E must be evaluated in a quantitative framework that allows for the speciﬁca- tion of all relevant causal relations in a suitable formal model M. One important modeling decision will be the separation of parameters inﬂuenced within the model (i.e., ‘observed’ or ‘endogenous’ variables – in particular, the modeled effects and side-effects of interest, along with further variables on the causal path from drug to potentially harmful outcome) from ceteris paribus conditions (‘context’ or ‘world’ or ‘circumstances’, encoded as ‘exogenous’ variables in a causal model). A fourth parameter shall be added to our language to express precisely these contextual con- ditions, i.e., the ceteris-paribus aspects of the population for which the causal mech- anism is to be tested. The parameter U shall sum up those characteristics of pop- ulation plus environment that will not be changed by, e.g., a clinical trial (as, e.g., average age, nutrition habits, aspects of the medical history, socio-economic status, etc.).12 We can accordingly expand Hyp to encode the aforementioned ingredients: 12 Roland Poellinger Hyp : D c⃝E in model M with context U (7) (7) Hyp : D c⃝E in model M with context U Explicating the causal hypothesis in this way is not meant here to express a spe- ciﬁc commitment as to how c⃝is to be interpreted. In particular, it is not meant to conﬁne the investigation to an epistemic, model-relative theory of causality. This speciﬁc way of encoding the causal hypothesis is rather meant to be as informa- tive as possible about the intended scope of the causal claim and to mark deviations between studies and target with respect to population characteristics (information about age, multimorbidity, and so on, encoded in U(k)) and structural assumptions (confounders, causal history, possible disablers, etc., encoded in M(k)).13 Explicating the causal hypothesis in this way is not meant here to express a spe- ciﬁc commitment as to how c⃝is to be interpreted. 14 These accounts of similarity share the intuition that comparing two things means (i) comparing certain aspects of those things and (ii) aggregating one’s evaluation of those aspects in a certain manner. Lewis’ idea of comparative similarity is tightly connected to his concept of causation, where a cause–effect relation is evaluated in terms of the corresponding counterfactuals. The cause reveals its power in the effect event where the rest of the world remains unperturbed, i.e., as similar as possible to the state of world prior to the cause event. Lewis suggests a priority ordering for the assessment of similarity, where local changes in physical facts are understood as a lesser deviation from actuality than far-reaching global changes in natural laws, see Lewis (1973b). The geometric account locates an object’s properties (deemed relevant for comparison) in a multi- dimensional space by assigning a speciﬁc value to each of those properties. Similarity is then spelled out in terms of vector distance from a reference object. The question of how to assign such values is circumvented in the contrast approach which deals well with similarity as partial identity, since in this approach degrees of similarity are assessed by assigning weights to co-instantiated identical properties (which might make the approach suit- able rather for comparing different states of one and the same object than for comparing different objects). 2.3 Measuring distance Repk: Φ[Dk,Ek,Mk,Uk] with Ek similar to the hypothesized E, with Ek similar to the hypothesized E, 3. therefore: Φ′[D,E,M,U] with Φ′ similar to Φ, where Φ is a quantitative or qualitative statement about (the components of) the study (and Φ′ a statement about the target, respectively). For example, Φ might encode the conditional probability P(Ek \|Dk) = q; therefore, by analogy, Φ′: P(E \|D) ≈q (for high αk). Note that, by design, Repk will inﬂuence Hyp on a path through a suitable (set of) mediating indicator variable(s). If now Repk makes a positive contribution to the indicator level, it will boost conﬁdence in Hyp, thereby conﬁrming D c⃝E: Lewis’ idea of comparative similarity is tightly connected to his concept of causation, where a cause–effect relation is evaluated in terms of the corresponding counterfactuals. The cause reveals its power in the effect event where the rest of the world remains unperturbed, i.e., as similar as possible to the state of world prior to the cause event. Lewis suggests a priority ordering for the assessment of similarity, where local changes in physical facts are understood as a lesser deviation from actuality than far-reaching global changes in natural laws, see Lewis (1973b). 2.3 Measuring distance In particular, it is not meant to conﬁne the investigation to an epistemic, model-relative theory of causality. This speciﬁc way of encoding the causal hypothesis is rather meant to be as informa- tive as possible about the intended scope of the causal claim and to mark deviations between studies and target with respect to population characteristics (information about age, multimorbidity, and so on, encoded in U(k)) and structural assumptions (confounders, causal history, possible disablers, etc., encoded in M(k)).13 Now, using a piece of evidence for the conﬁrmation of a hypothetical causal effect E by analogy may be licensed for different reasons: 1. Similar substance D: The substance used on the study population is similar to the substance to be applied to the target population. Of course, if an experimental setup is designed with D c⃝E in mind, the experiment will generally test the causal effects of D itself. Nevertheless, what may vary is the administered dosage or indeed the ﬁnal formula with respect to additional components. 2. Similar causal model M: Causal efﬁcacy is judged with respect to similar mod- eling assumptions (variables and their order, causal in/dependencies, and so on) in the study and for the intended application. For example, when cohort studies point to the existence of a causal path between paracetamol and asthma, knowl- edge about these studies can only be transferred to a population of future drug users if the core causal assumptions are kept ﬁxed. Postulating a deviating causal structure for the target, e.g., a common cause of paracetamol use and asthma in- stead of a causal path, would prevent using evidence from such cohort studies for predicting the effects of certain policy interventions towards decreasing asthma morbidity. 3. Similar context U: Contexts, i.e., study and target population plus respective en- vironment, are similar in relevant respects. For example, when drugs are tested on animals ﬁrst, pigs are one of the preferred species, because they share much of the human genetic make-up and consequently many of the complex genetic diseases, such that a drug’s causal effects in pigs can to some extent be used in inference about human biology. Scheme (A) above can now be adjusted for learning from relevant evidence in the evidence-amalgamating layered reconstruction of scientiﬁc inference: (A) 13 Analogy-Based Inference Patterns in Pharmacological Research Analogy-Based Inference Patterns in Pharmacological Research 2. Repk: Φ[Dk,Ek,Mk,Uk] with Ek similar to the hypothesized E, 2. 14 These accounts of similarity share the intuition that comparing two things means (i) comparing certain aspects of those things and (ii) aggregating one’s evaluation of those aspects in a certain manner. 15 In the formal notation used here, ∼denotes some (reﬂexive, transitive, and symmetric) equiv- alence relation (equivalence w.r.t. a given property) such that for a domain A, some object a ∈A, and an equivalence relation ∼on A: [a]∼:= {x ∈A\|x ∼a}. The expressions [D]∼, [M]∼, and [U]∼ are to be understood as encoding each a speciﬁc equivalence relation, since – to be precise – each category comes with its own standards for how equivalence classes are to be generated. If standards are set high, e.g., Dk might be in the class [D]∼only if it is identical with D, while comparing U and Uk will naturally demand ﬂexibility for possibly very different populations. (I will not add a further index, though, to avoid notational clutter.) P(D c⃝E \|Repk,αk) ≥P(D c⃝E). P(D c⃝E \|Repk,αk) ≥P(D c⃝E). The inference pattern (A) crucially relies on a high relevance parameter αk which is determined in assessing the similarity of the triples ⟨D,M,U⟩and ⟨Dk,Mk,Uk⟩ The literature on similarity conceptualizations and measures is vast, spanning from Lewis’ lexicographic comparative similarity of possible worlds (Lewis, 1973a) to Shepard’s geometric account in terms of vector distances (Shepard, 1980) and to contrast approaches evaluating similarity by weighting and comparing properties (see, e.g., Tversky 1977, Weisberg 2012, and Weisberg 2013).14 Some are formu- lated in a symmetric way, others encode asymmetric relations. All approaches seem tied to speciﬁc practices and purposes with seemingly none universally applicable. Nevertheless, with analogical arguments employed successfully in pharmacological research, general features of the similarity concept needed for (A) shall be outlined in the following. Claiming that ⟨D,M,U⟩is similar to ⟨Dk,Mk,Uk⟩(as in line 1 of the inference scheme for relevant evidence) quite generally means that the individual components are in the same equivalence class (by pairs), formally: Dk ∈[D]∼, Mk ∈[M]∼, and 14 These accounts of similarity share the intuition that comparing two things means (i) comparing certain aspects of those things and (ii) aggregating one’s evaluation of those aspects in a certain manner. 14 Roland Poellinger Roland Poellinger Uk ∈[U]∼.15 These equivalence classes are induced by (i) the components of the hy- pothesis, D, M, U, and (ii) highly purpose-oriented considerations as to balancing the properties to be compared (i.e., as to how properties are to be ordered in Lewis- style approaches, as to how which properties are metrized in geometric approaches, and as to how properties are to be weighed in contrast comparisons). Presenting sim- ilarity this way allows a research community to ﬁll in a meaningful, informative un- derstanding of similarity, and it separates purpose-driven considerations concerning how to suitably deﬁne an equivalence relation from the decision about if and when two components actually are equivalent (relative to a speciﬁc deﬁnition). Note that however dissimilar study and target, if some Hyp’s components ⟨D,M,U⟩and some Repk’s components ⟨Dk,Mk,Uk⟩were not understood to be comparable whatsoever, Repk would not be considered evidence for Hyp in the ﬁrst place. The threshold between sufﬁcient similarity and unacceptable deviation is straight- forwardly encoded as a component’s membership of the respective equivalence class. Line 1 in (A) can thus be understood as follows: ⟨D,M,U⟩is similar to ⟨Dk,Mk,Uk⟩:⇔ (8) Dk ∈[D]∼and Mk ∈[M]∼and Uk ∈[U]∼. P(D c⃝E \|Repk,αk) ≥P(D c⃝E). (8) Meaningfully evaluating the different components’ membership of a given equiv- alence class calls for the distinction between similarity of numeric properties and similarity of structural properties. In the following I will outline possible paths one might take towards assessing similarity both of the numeric and the structural type. 2.3.1 Similarity of numeric properties I will ﬁrst consider the question of how to possibly compare arrays of numeric prop- erties (or properties represented numerically, respectively). What it means to be a member of an equivalence class in that case can be expressed in a weighted geomet- ric approach as the following criterion (with X schematically representing D, M, U): Xk ∈[X]∼:⇔d(−→ β ◦−→ xk,−→ β ◦−→x ) ≤σX. (9) (9) Eq. 9 states that component Xk (e.g., some study population Uk) is similar to X (e.g., the target population U) for a purpose-oriented similarity evaluation ∼, explicated as distance between vectors of m properties x1 k,...,xm k (of Xk) and x1,...,xm (of X) Analogy-Based Inference Patterns in Pharmacological Research 15 above a certain distance threshold σ (relative to component X). To keep the criterion as generally applicable as possible, the elements of −→ xk and −→x should represent nor- malized values of the properties measured for comparison. When comparing two populations, for example, their relevant characteristics, such as, e.g., average age and population size, might be mapped onto scales of equal magnitude, to arrive at a balanced aggregate assessment. Additionally, the vectors to be compared are weighted by componentwise multiplication (denoted by ‘◦’) with relative weights β 1,...,β m, indicating the relative importance of each property for comparison, i.e., its contribution to the similarity measure.16 Of course, the distance measure d must be speciﬁed (Euclidean distance, Man- hattan distance, etc.) and the set of properties to be compared chosen carefully – in particular, a simple distance measure might not be straightforwardly applicable if the selected properties are dependent. What counts as independent and what not is, again, ultimately to be decided in a purpose-oriented, context-sensitive way. In par- ticular, different similarity standards could lead to different in/dependence require- ments. A toy example may serve as an illustration of such dependencies: Consider the task of comparing different apples. If shape and color are to be compared, then a big red apple is more similar to a big green apple than to a small green apple. If, however, “naturalness” is used as similarity standard, then a big red apple might be considered more similar to a small green apple than to a big green apple, since the natural apple naturally ripens from small and green to big and red, and not to “unnatural” big and green. 16 Componentwise multiplication of two vectors (also referred to as “Hadamard Product”) multi- plies vectors A and B (both of length n) element by element and returns a vector C (also of length n). Example: ⟨a,a,a⟩◦⟨0,a,b,⟩= ⟨0,2a,ab⟩. 2.3.1 Similarity of numeric properties The latter case precisely illustrates the dependency be- tween properties, and how such dependency is relative to the similarity standard to be employed. Example. The following simpliﬁed case equally shows advantages and shortcomings of such a geometric approach. Consider the assessment of a potential side-effect of a drug targeted at elderly people, who are likely to be using multiple medicines and likely to be affected by multiple diseases (note that those properties are dependent to a certain degree). Similarity between this speciﬁc target population and a test group (associated with report Repk) might be determined in comparison of property triples −→u = ⟨u1,u2,u3⟩and −→ uk = ⟨u1 k,u2 k,u3 k⟩where the properties to be compared are all numeric and partitioned into meaningful classes w.r.t. the current investigation. For example, it might be meaningful to distinguish the class of people who take 1 drug from the class of people who take 2 drugs, but it might not be particularly useful 16 Roland Poellinger Roland Poellinger to draw the line between 11 and 12 multiple drugs. In our example, the selected characteristics u(k) of U(k) are partitioned as follows:17 to draw the line between 11 and 12 multiple drugs. In our example, the selected characteristics u(k) of U(k) are partitioned as follows:17 u1 (k): (Average) age with integer values between 0 and 100 years of age; ( ) u2 (k): (number of) multiple medicines in ﬁve classes: ( ) u2 (k): (number of) multiple medicines in ﬁve classes: (k) 0, 1, 2, 3-5, > 5 medicines; 3 (k) 0, 1, 2, 3-5, > 5 medicines; u3 (k): (number of) multiple diseases in four classes: u3 (k): (number of) multiple diseases in four classes: (k) 0, 1, 2, > 2 diseases. To be able to attach numbers to the vectors, we have to translate the description of U and Uk into concrete values step by step: 1. Suppose that the target population is described as 70 years old (on average), as taking 1 more medicine besides the tested drug (i.e., 2 in total), and as having more than 2 diseases considered relevant for the investigation (one targeted, the other possibly interacting). p y g 2. The study is conducted on a population of 55 years on average with the same amount of medicines taken but with only one disease (the targeted one). 3. To be able to aggregate those properties on the same level, those characteristics are mapped onto the unit interval with respect to the partitions stated above. Example. Moreover, for the current purpose, each class is simply represented by its central value (e.g., if the unit interval is partitioned into four intervals of equal length, the second interval, ranging from .25 to .5 is represented by its central value .375.). 4. Suppose further that – for the current investigation – average age is considered somewhat important (50%), whether multiple drugs are taken or not is consid- ered highly important (100%), and the number of diseases is negligible (with importance of 0%). The following tables show the step-wise calculation of the difference between weighted components needed for the determination of the distance between vectors −→u and −→ uk: study population target population class normalized quantized class normalized quantized u1 k: 55 .55 u1: 70 .7 u2 k: 2 .5 u2: 2 .5 u3 k: 1 .375 u3: > 2 .625 β study target ∆ u1 (k): .5 .275 .35 .075 u2 (k): 1 .5 .5 0 u3 (k): 0 0 0 0 17 In this example, study and target are compared merely in terms of population characteristics −→ u(k). In general, though, differences between the substances and between the causal structures will also play a role in assessing the weight of a report, as explicated in Eq. 8. For sake of illustration it might be assumed here that substances and causal structures have been found equivalent w.r.t. the present purpose. Analogy-Based Inference Patterns in Pharmacological Research 17 Analogy-Based Inference Patterns in Pharmacological Research The upper tables show each property’s class and its normalization in the unit in- terval (graphically with class partitions, and quantized using those classes’ central values). The lower table shows the absolute differences between the individual com- ponents, weighted by corresponding β-values (relative importance). In our example, only age ﬁgures in the calculation of similarity between the two populations (since both groups take two medicines in total and the number of diseases is considered unimportant for the current comparison). Example. If a Euclidean measure is to be used for calculating the distance, we arrive at the following value: d(−→ β ◦−→ xk,−→ β ◦−→x ) = qβ 1 ·(u1 −u1 k) 2 = q.5·(.35−.275) 2 = .053 (10) (10) If, for comparison, the number of diseases were indeed to be taken into consideration with a relative importance weight of .25, the dissimilarity increases: d( −→ β ′ ◦−→ xk, −→ β ′ ◦−→x ) = (11) qβ 1 ·(u1 −u1 k) 2 + β 3 ·(u3 −u3 k) 2 = q.5·(.35−.275) 2 + .25·(.625−.375) 2 = p .03752 +.06252 = .073 (11) A σU benchmark value for testing sufﬁcient similarity with the target population (as formulated in Eq. 9) will have to be deﬁned with respect to the theoretical max- imum distance and to allowable deviations from the target characteristics. Once σU is ﬁxed and sufﬁcient similarity between study and target properties is shown, the report Repk can be ﬂagged as relevant (with a high degree of conﬁdence in αk, i.e., 1 in the binary formulation), and (A) will allow inference by analogy from relevant evidence to the hypothesis under consideration. There are various ways to reﬁne the distance measure and adjust it to one’s needs. Two remarks on the difﬁcult task of choosing a suitable measure shall conclude this section: 1. The example above illustrates that the chosen Euclidean distance measure com- pletely disregards identical properties, even such with high relative importance, since it is deﬁned to simply collect deviations. In the example, the identical prop- erties u2 and u2 k could be missing from the vectors −→u and −→ uk and could receive high or low relative importance – the Euclidean distance would return the same value in all these cases. This goes against the intuition “the more identical prop- erties two property vectors share, the lower their distance”. If this intuition is to be encoded formally, though, an additional weighting factor must be added to the distance measure. Each component xi (0 < i < m) of −→x might additionally be multiplied by its relative β-contribution, e.g., in the following way: 18 Roland Poellinger Roland Poellinger d′(−→ β ◦−→ xk,−→ β ◦−→x ) = s m ∑ i=1 β i ·(xi k −xi) 2 · β i ∑m j=1 β j . Example. (12) (12) Using d′ on our example case above returns a lower dissimilarity, thus reﬂecting the intuition that identical properties contribute to our similarity assessment (in decreasing dissimilarity). With ∑m j=1 β j = 1.5, we have: d′(−→ β ◦−→ xk,−→ β ◦−→x ) = (13) r β 1 ·(u1 −u1 k) 2 · β 1 1.5 + β 2 ·(u2 −u2 k) 2 · β 2 1.5 = r.5·(.35−.275) 2 · .5 1.5 +0 = r .03752 · 1 3 = .022 (13) And again, for comparison – for d′ with relative importance of .25 for u3 k, we get: d′( −→ β ′ ◦−→ xk, −→ β ′ ◦−→x ) = (14) r.5·(.35−.275) 2 · .5 1.75 + .25·(.625−.375) 2 · .25 1.75 = .031 (14) 2. If, e.g., a certain property is required to be identical in study and target popula- tion in order to grant transferability of study results, partial identity, being central to the contrast account of similarity, can be expressed in a straightforward gen- eralization of the geometric approach exempliﬁed above: Instead of determining sufﬁcient similarity for full vectors −→u and −→ uk, those vectors are partitioned into sub-vectors. −→u and −→ uk are then considered sufﬁciently similar iff all distances of corresponding sub-vectors are lower than associated σ-values. For example, average age, number of medicines, and number of diseases (as in the case above) might be partitioned into sub-vector 1, average age plus number of diseases, and sub-vector 2 containing number of medicines only. If now the number of medicines taken is to be identical in target and study population (e.g., it might be required to be only the investigated drug), then the σ-value for sub-vector 2 is set to 0, while the σ-value for sub-vector 1 might be higher to allow for approximate correspondence. 18 See Pearl 2000, Sec. 7.3.3 for a discussion of causal relevance. Note, that I am distinguishing causal relevance as part of the causal knowledge (encoded as report nodes) from epistemic or inferential relevance (encoded as attributional Rlv weight nodes). 2.3.2 Similarity of structural properties When it comes to determining whether two structures (or, alternatively, sets of struc- tural constraints) Mk and M are sufﬁciently similar, things are different: There is no straightforward way of encoding topology on a numeric scale. In particular, it de- pends very much on the given case what the comparison criteria are, and how they Analogy-Based Inference Patterns in Pharmacological Research 19 possibly interact to jointly suggest a similarity ranking over a set of causal struc- tures. Nevertheless, the relevance of a given study for the investigated hypothesis cru- cially depends also on structural knowledge about the (hypothesized) causal associ- ations in both study and target. For example, when an RCT on animals supports a dose-response relationship between drug D and adverse drug reaction E, this report can only be considered relevant if assuming a causal path from D to E is compatible with knowledge about the target’s causal structure. And conversely, as soon as fur- ther studies show the association to be spurious in human drug users, the relevance of the animal study for the current investigation will be downgraded. The following compilation presents a (non-exhaustive) list of structural proper- ties to consider when comparing causal topologies. The respective questions can be understood as a heuristic survey for assessing structural similarity: 1. Shared variables: How many and which variables are shared between study and target? How inﬂuential are the ones that are not shared? 2. Causal in/dependencies of shared variables: Are the causal (conditional) in/dependenc of the variables shared by study and target in agreement? In other words, are the causal ir/relevance relations in study and target the same?18 If not, is the dis- agreement resolvable (e.g., by including suspected confounders)? 3. Presence of co-factors: If a causal structure marks contributing causes or nec- essary pre-conditions – can these be identiﬁed both in study in target? Are the co-factors shared between study and target? How weighty are those that are not shared? 4. Distance between D and E: How many mediating variables lie on the path be- tween D and E? If one structure contains more such mediators, does this reﬂect temporally or spatially higher actual distance? And do such mediators mark pos- sible points for disruption by disablers, such that the causal process must be considered “less stable”? 5. 19 The way it is presented here, one’s assessment of such similarity between study and target is obviously relative to the set of aspects included in one’s considerations. There is an argument to be made here that possible further differences not considered should lower one’s conﬁdence in the similarity assessment. In principle, in the Bayesian framework employed, it is possible to add an unspeciﬁed counter-weight (much like an error term) in order to encode one’s uncertainty about potentially neglected, though relevant differences between study and target. Yet, assigning a number to this weight is a subjective task again. Indeed, I would like to argue that such analogy- based arguments are inherently perspectival: They rest on a speciﬁc choice of relevant aspects (reasonably motivated) and a speciﬁc way of relating those aspects (non-arbitrarily). Thus, making the ingredients of such arguments explicit helps reﬁning or potentially also refuting them. 2.3.2 Similarity of structural properties Interacting inﬂuences: If E is inﬂuenced through multiple paths which jointly (but non-additively) produce E, then identifying these inﬂuences (and their inter- action) in study and target is crucial for transferring predictions about the effect of interventions on D – this is particularly important in the case of masked back- up mechanisms present in the target. 5. Interacting inﬂuences: If E is inﬂuenced through multiple paths which jointly (but non-additively) produce E, then identifying these inﬂuences (and their inter- action) in study and target is crucial for transferring predictions about the effect of interventions on D – this is particularly important in the case of masked back- up mechanisms present in the target. 6. Differences between study and target: Going beyond the points above, one might also utilize knowledge about how and where two causal structures differ. Infor- mation about differences at key stages along the causal path from D to E will be most helpful if those stages screen off inﬂuences of minor stages “upstream” along the causal ﬂow (i.e., above or before). This idea is exploited in comparative process tracing, where two processes are investigated in stepwise comparison of their entities. Steel relates this method to external validity in Steel 2008, p. 89: 20 Roland Poellinger Roland Poellinger [T]he greater the similarity of conﬁguration and behavior of entities involved in the mechanism at [. . . ] key stages, the stronger the basis for the extrapolation. The reliability of comparative process tracing depends on correctly identifying the points at which signiﬁcant differences between the model and the target are likely to arise. Signiﬁcant differences are those that would make a difference to whether the causal generalization to be extrapolated is true in the target. The strategy summarized here is especially suitable for causal chains with such key variables – identifying precisely those variables (and describing deviating behavior in study and target) will help in deciding whether given study results provide an insight into the target. One of the virtues of comparative process trac- ing is indeed that it points to the portions of a causal structure that allow for an effective assessment of the similarity between study and target. This list of structural properties is meant to provide a guide in making implicit assumptions about study and target explicit. 2.3.2 Similarity of structural properties Just like in the case of comparing nu- meric properties above, all these considerations feed into the purpose-oriented expli- cation of a similarity measure which ultimately determines what sufﬁcient similarity means in light of the investigated hypothesis.19 As above, once sufﬁcient similarity between structural properties of study and target is shown, a particular report Repk about Mk can be ﬂagged as relevant (with a high degree of conﬁdence in αk, i.e., 1 in the binary formulation), and (A*) will allow inference by analogy from relevant evidence, summarized in Repk, to the hypothesized causal association, D c⃝E in the causal structure M, thereby facilitating Bayesian conﬁrmation: P(D c⃝E \|Repk,αk) ≥P(D c⃝E). P(D c⃝E \|Repk,αk) ≥P(D c⃝E). P(D c⃝E \|Repk,αk) ≥P(D c⃝E). 2.4.1 What can make RCT evidence relevant? In her discussion of predictions about the effectiveness of policy interventions in the context of evidence-based policy (EBP), Cartwright points out that “[t]he EBP liter- ature rates positive outcomes in well-conducted RCTs as gold standard evidence for effectiveness predictions” (Cartwright, 2011, p. 221). And she critically continues: Conventionally cited facts, like similarity between target and experimental situations, are then supposed to make this likely. But this is the wrong way to look at the relation between experimental results and the claims whose truth they bear on. Experimental results can help justify conﬁdence that the same result – or that some different result – will hold elsewhere; i.e., they can be evidence for one of these claims. Whether they are evidence depends on whether they play the right kind of role in a good argument for that claim. Similarity, or just the right kind of dissimilarity, might play a role, but if so, only by ﬁtting into a good argument. What then might a good argument look like that makes RCT results evidence for effective- ness predictions? In this passage, Cartwright expresses precisely the worry that prompted Landes, Osimani, and Poellinger (Landes et al, 2017) to disentangle reliability and rele- vance as meta-evidential attributes. In order to address the question “What can make RCT evidence relevant?”, Cartwright presents the following argumentative template (Cartwright 2011, p. 222; formatting adjusted): A1 x plays a causal role in the principle that governs y’s production there. A1 x plays a causal role in the principle that governs y’s production there A2 x plays the same causal role here as there. A3 The support factors necessary for x to operate are present for some individuals here. Therefore: x plays a causal role here and the support factors necessary for it to operate are present for some individuals. Therefore: x plays a causal role here and the support factors necessary for it to operate are present for some individuals. In this argument, x is to be understood as a cause of y, ‘here’ indicates the tar- get, ‘there’ the study. The four lines evidently resemble scheme (A) above: A2 and A3 establish similarity in the relevant (numeric, structural) aspects, and A1 encodes the RCT evidence (e.g., some effect size) to be transferred to the target in the con- clusion. 2.4 Extrapolating with good arguments and breaking the extrapolator’s circle I want to conclude Sec. 2 with a discussion of two points in the current debate on analogical reasoning in causal assessment. The ﬁrst is Nancy Cartwright’s worry about external validity, namely that even highly reliable RCT methodology only provides an insufﬁcient warrant for the transferability of evidence from study to target (Cartwright, 2011; Cartwright and Stegenga, 2011). The second is a much- 21 Analogy-Based Inference Patterns in Pharmacological Research discussed riddle about the soundness of analogical arguments – the ‘extrapolator’s circle’ (Guala, 2010; Steel, 2008). 2.4.2 The extrapolator’s circle In his discussion of mechanistic reasoning for the purpose of extrapolation, Daniel Steel (see Steel 2008, p. 78) presents the following challenge any viable account of extrapolation ought to address (see also Guala’s comments in Guala 2010): In his discussion of mechanistic reasoning for the purpose of extrapolation, Daniel Steel (see Steel 2008, p. 78) presents the following challenge any viable account of extrapolation ought to address (see also Guala’s comments in Guala 2010): [A]dditional information about the similarity between the model and the target – for in- stance, that the relevant mechanisms are the same in both – is needed to justify the extrap- olation. The extrapolator’s circle is the challenge of explaining how we could acquire this additional information, given the limitations on what we can know about the target. In other words, it needs to be explained how we could know that the model and the target are similar in causally relevant respects without already knowing the causal relationship in the target. In the account of Landes, Osimani, and Poellinger (Landes et al, 2017), this circle is broken since the proposed Bayesian framework can be used to probabilistically model successive evidence accumulation and amalgamation. At the beginning of the process, experimental results from basic science might contribute causal knowledge about a drug’s metabolization – and even if this knowledge only illuminates part of the target mechanism, it can often be considered robust and highly relevant. Once animal studies come into play to answer more speciﬁc research questions, knowl- edge from previous unrelated studies (done on the same animals, maybe with simi- lar substances) ﬁgures in forming a relevance estimate for the current investigation. Step by step, a picture of the target’s causal structure emerges: The careful extrapo- lator can thus utilize the previous stage for his assessment of the next. While Steel proposes mechanism-based comparative process tracing (see also Sec. 2.3.2 above) as a solution to the extrapolator’s circle, the Bayesian evidence-amalgamation ap- proach presents a different type of ‘process tracing’ in providing a toolbox for trac- ing the dynamic process of evidence synthesis from a higher-level epistemological perspective. The focus of this section lay on the conﬁrmatory support of relevant pieces of evi- dence for a causal hypothesis (“upwards” in the evidence-amalgamating framework) – the next section will consider cases where an independently conﬁrmed causal link serves for boosting one’s conﬁdence in a hypothesized relation. 2.4.1 What can make RCT evidence relevant? In the Bayesian framework presented above, the existence of such a good argument would be encoded as high relevance, consequently making the RCT re- sult evidence for effectiveness predictions by boosting the weight of the respective evidential report. 22 Roland Poellinger be known about the mechanisms of substances D∗and/or D, Hyp is considered to share relevant theoretical consequences with Hyp∗(or variants thereof). be known about the mechanisms of substances D∗and/or D, Hyp is considered to share relevant theoretical consequences with Hyp∗(or variants thereof). In order to formalize knowledge transfer across theoretical networks, our concep- tual frame needs to be widened slightly. In a recent paper on analogical inference in physics, Dardashti et al (2017) discuss analogies between experimentally accessible test setups and potentially less accessible target systems we want to gain insights about. The authors introduce the formal concept of analog simulation for this pur- pose which shall be introduced brieﬂy in the following before it is applied in the context of pharmacology. Analog simulation bridges two basic frames: The source system is prepared, ma- nipulated, and observed to make inferences about the target system. Let us introduce some terminology ﬁrst to relate all concepts in a formal way:20 1. The target system T (a class of situations of interest) is to be modeled as MT in a suitably chosen modeling framework LT; 2. MT is constrained by certain background assumptions AT, summarizing theo- retical and empirical knowledge as well as the domain of conditions DT to which the model is intended to apply; 3. MT can be used to predict phenomena PT and will in turn be validated by evi- dence in accordance with PT; 4. The accessible source system S is to be modeled as MS in a suitably chosen modeling framework LS; 5. MS is constrained by background assumptions AS, containing the domain of conditions DS to which the model is intended to apply; 6. Just as on the T side, MS can be used to predict phenomena PS and will in turn be validated by evidence in accordance with PS. be validated by evidence in accordance with PS. The source system S will now allow analog simulation of target T’s behavior if (i) there exist exploitable structural similarities between MS and MT sufﬁcient to deﬁne a syntactic isomorphism robust within the domains DS and DT, respectively, and if (ii) this isomorphism is prompted by and based on a set of model-external empirically grounded arguments, abbreviated as MEEGA. Figure 3 relates these elements in a conceptual graph: The rounded box on the left side contains all elements of the target frame, while the right box contains all el- ements of the source system. 20 In the following, I deviate from Dardashti et al (2017) in notational details. 3 Transferring knowledge from conﬁrmed causal links When Hill states that “with the effects of thalidomide and rubella before us we would surely be ready to accept slighter but similar evidence with another drug or another disease in pregnancy” (Hill, 1965), he refers to the case of an independently conﬁrmed causal link providing support to a hypothesis still unsettled. Unlike in the case of learning from relevant evidence along the vertical upward arrow in Fig. 2, it is not an evidence report about an indicator of Hyp itself, but instead a well- tested second hypothesis Hyp∗that boosts belief in Hyp. Even though little might Analogy-Based Inference Patterns in Pharmacological Research 23 21 As an illustration, consider the following: In many cases, evidence for similarity of the drug’s causal effects comes from mechanistic knowledge, maybe in relating the molecular structure of the substances to known classes of biochemical processes. So, if D∗is known to be harmful because of its capacity to block some speciﬁc mechanism, and if this capacity is judged to be relevant in com- paring D and D∗, then such blocking behavior should be part of Hyp’s testable consequences Indk. Owing to differences in the investigated substances, the testable consequences of Hyp and Hyp∗ are in general not identical, but can be related non-arbitrarily in motivating a speciﬁc theoretical mapping, i.e., some isomorphism at a suitably chosen level of description. be known about the mechanisms of substances D∗and/or D, Hyp is considered to share relevant theoretical consequences with Hyp∗(or variants thereof). MEEGA prompt the establishment of a bridge between theoretical networks in the form of a syntactic isomorphism as translation between the systems’ components. In Dardashti et al (2017), the terminology is illustrated with an example from physics, where observations of phenomena PS in table-top ﬂuid systems boost con- ﬁdence in theoretical assumptions AT about gravitational phenomena described in 24 Roland Poellinger AT MT PT constrains predicts AS MS PS constrains predicts syntactic isomorphism MEEGA prompt Fig. 3: The analog simulation scheme: Framework LT (left box) is used to model target system T in model MT; source system S is accordingly treated in framework LS (right box). MEEGA Fig. 3: The analog simulation scheme: Framework LT (left box) is used to model target system T in model MT; source system S is accordingly treated in framework LS (right box). framework LT. The syntactic isomorphism (motivated by additional knowledge about the underlying physics of both frames) allows for the transfer of knowledge about acoustic Hawking radiation in the ﬂuid system to Hawking radiation in black holes. Now, the three-layered reconstruction of scientiﬁc domains on both sides of the syntactic bridge essentially represents the same conceptual categories as our lay- ered reconstruction of inference in pharmacological research in Fig. 2: Scientiﬁc hypotheses entail system constraints which in turn predict (and are tested by) real- world phenomena. The syntactic isomorphism can be understood as a formal expres- sion of similarity in terms of partial identity under translation. The set MEEGA can be understood to empirically, pragmatically, and semantically prompt the choice of relevant theoretical elements to be mapped onto each other. Syntactic isomorphism alone (unaware of semantic context) would be too weak a requirement for analogy since it can be used to translate far more models into each other than one would like to call analog. With the analog simulation scheme at hand, we can now trace the conﬁrmatory boost of a well-tested causal link to an unsettled hypothesis in phar- macological research. Fig. 4 shows the independently conﬁrmed hypothesis Hyp∗on the right side, e.g., as in Hill’s example, the conﬁrmed link between thalidomide (D∗), also known Analogy-Based Inference Patterns in Pharmacological Research 22 This topic is a subject of current discussion in the philosophy of science with some authors re- garding computational models as simply an implemented variant of scientiﬁc models as such (e.g., Frigg and Reiss 2009), while others emphasize as a special feature of computer simulations the possibility to experiment with such models as virtual test objects (e.g., Parker 2009 and Morrison 2015). Analogy-Based Inference Patterns in Pharmacological Research 25 Hyp Ind1 Indn Rep1 α1 Repn αn . . . . . . . . . Hyp∗ Ind∗ 1 Ind∗ n Rep∗ 1 α∗ 1 Rep∗ n α∗ n . . . . . . . . . theoretical mapping similarity assumption support by analogy Fig. 4: Support by analogy from a second drug (i.e., from established knowledge about hypothesis Hyp∗) to the hypothesis under consideration. Fig. 4: Support by analogy from a second drug (i.e., from established knowledge about hypothesis Hyp∗) to the hypothesis under consideration. as Contergan, and phocomelia (E∗). Frame-external similarity assumptions (e.g., about the population of pregnant women, or even new evidence for relevant relations shared by both M and M∗) suggest an understanding of theoretical variables Indk in terms of Ind∗ k, e.g., a characteristic dose-response curve hinting at functional properties shared by D and D∗. Having established such a mapping between relevant theoretical elements, updating one’s beliefs about the variables Ind∗ k amounts to updating one’s beliefs about the variables Indk and, moreover, to updating one’s belief about Hyp (quite in the Bayesian sense).21 Consequently, old evidence −−→ Rep∗ about Hyp∗can indeed provide novel support for Hyp across theoretical networks, with P(Hyp\|−−→ Rep∗) > P(Hyp). (15) (15) How exactly this theoretical bridge between two frames might look and how a relevance ﬁlter is to ﬁlter out precisely those properties and those indicator variables relevant for the posited analogy, is a subject of current discussion in the philosophy of science; see, e.g., Dardashti et al (n.d) on analog simulation in Bayesian terms, 26 Roland Poellinger or Beebe and Poellinger (n.d.) on conﬁrmation from analog models in formal exten- sions of Bayesian networks. 4 Conﬁrmatory support from in silico simulation When insights are to be gained about mechanistic workings of biochemical phenom- ena that are not directly observable in vivo, one strategy of choice is computational modeling and simulation. As in the cases of learning from relevant evidence (Sec. 2) and transferring knowledge from conﬁrmed causal links (Sec. 3), using computa- tional models to learn about biological, medical, or pharmacological facts is based on analogy between simulation and target system. The argumentative strength of analog simulation in physics is based on the fact that two physical systems are related on the grounds of model-external background assumptions about common underlying physical principles. In the case of computa- tional simulation, we seemingly loose two important aspects: (M) Computational simulation does not link two physical systems but rather a physical and a virtual system; and (M) Computational simulation does not link two physical systems but rather a physical and a virtual system; and (N) the set of model-external, empirically grounded assumptions (MEEGA) is replaced by model-internal, theoretical principles in the implementation phase: The symbolic system MS is constructed directly from MT’s background assump- tions. (N) the set of model-external, empirically grounded assumptions (MEEGA) is replaced by model-internal, theoretical principles in the implementation phase: The symbolic system MS is constructed directly from MT’s background assump- tions. These aspects raise questions about virtues of materiality (M) and novelty of virtual outcomes (N). Both shall be addressed in the following. Computer simulations follow their source code and will behave like programmed as virtual, artiﬁcial environments, lacking the material link (M).22 The position of the skeptic about computer simulation is pointedly summarized by Diez Roux in her discussion of the distinction between observation-based and simulation-based causal inference in epidemiology (Diez Roux, 2015, p. 101): [. . . ] there is a fundamental distinction between causal inference based on observations (as in traditional epidemiology) and causal inference based on simulation modeling. The tra- ditional tools of epidemiology are used to extract (hopefully) reasonable conclusions from necessarily partial and incomplete (often messy) observations of the real world. [. . . ] In contrast, when we use the tools of complex systems, we create a virtual world (based on prior knowledge or intuition) and then explore hypotheses about causes under the assump- tions encoded in this virtual world. 23 In the context of modeling with Bayesian networks, this demand is captured in the requirement that all variables represent distinct events. 4 Conﬁrmatory support from in silico simulation In the simulation model, we cannot directly determine 22 This topic is a subject of current discussion in the philosophy of science with some authors re- garding computational models as simply an implemented variant of scientiﬁc models as such (e.g., Frigg and Reiss 2009), while others emphasize as a special feature of computer simulations the possibility to experiment with such models as virtual test objects (e.g., Parker 2009 and Morrison 2015). 27 Analogy-Based Inference Patterns in Pharmacological Research whether X causes Y in the real world (because the world in which we are working is of our own creation); we can only explore the plausible implications of changing X on levels of Y under the conditions encoded in the model. In the real world, we have fact (what we observe) and we try to infer the counterfactual condition (what we would have observed if the treatment had been different). In the simulated world, everything is counterfactual in the sense that the world and all possible scenarios are artiﬁcially created by the scientist. Nevertheless, even though computational models are virtual constructs, if they are to be employed for inference about our world they are required to be anchored in reality just as any classical scientiﬁc model. In a systematic review of successful agent-based computational models, Casini and Manzo (2016) aim to address Diez Roux’s worries. In particular, they pin down various trends which have shown to increase the fruitfulness of such models. In the following I derive from their dis- cussion three conditions that might even be understood as applicability criteria for causal inference from computational, agent-based models (ABMs) in general: 24 This strategy introduces a secondary set of model-external, empirically grounded arguments in the picture which is ﬁrst motivated by the logical cross-link between the two frames and later guided by anchoring considerations. See Osimani and Poellinger (n.d.) for a detailed reconstruction of model creation, veriﬁcation, and validation for computer simulation in systems biology. “Ideal” ABMs are to be “Ideal” ABMs are to be 1. based on problem-related theoretical knowledge (rather than merely common- sense, mathematical, topological etc. assumptions), 1. based on problem-related theoretical knowledge (rather than merely common- sense, mathematical, topological etc. assumptions), 2. shaped by data, 2. shaped by data, 3. and iteratively calibrated by more data where the model shows weaknesses. 3. and iteratively calibrated by more data where the model shows weaknesses Fig. 5 illustrates the relation between target system and computational model in the analog simulation scheme, which I modify here to accommodate the anchor- ing concept: The source system (our computational model) inherits all theoretical background assumptions from the target side. The arrow from AT directly to MS represents the preparation of the simulation system as logical cross-dependency, which can be called model-internal in a sense: Without model-external motivation provided by MEEGA, the rigid link from AT to MS expresses the instantiation of the theoretical assumptions AT in a concrete (computational) model MS within the constraints of AT. Whenever incoming data alters the background assumptions AT, MS (the implementation) can (must) be revised accordingly. What this picture reveals, though, is that – as formulated above in (N) – the prediction of PS now turns into plain inference from AT. Consequently, it would not make sense to use PS for the conﬁrmation of hypothesis AT. Whatever theory of conﬁrmation is to be employed, analog reasoning will only be justiﬁed if source and target systems are kept sufﬁciently independent: The computational model must not be determined by the target system in order to leave room for the possibility of disconﬁrmation.23 What are our options? – The direct, logical dependence between AT and MS may be disrupted by a reintroduced AS in different ways: (i) AS might be built 28 Roland Poellinger AT MT PT constrains predicts MS PS constrains predicts syntactic isomorphism Fig. 5: The virtual source system inherits the target’s theoretical background as- sumptions; the arrow from AT to MS represents the preparation of MS as logical cross-dependency. 28 Roland Poellinger Fig. 5: The virtual source system inherits the target’s theoretical background as- sumptions; the arrow from AT to MS represents the preparation of MS as logical cross-dependency. upon a lower-level/ﬁner-grained theory than AT, (ii) AS might draw on a different set of parameters, (iii) AS might incorporate theories from different domains, (iv) AS might utilize databases generated from previously conducted material simula- tions. 25 For a discussion of surprise in computer simulation see Parke (2014). “Ideal” ABMs are to be Of course, the list is certainly not exhaustive, and strategies might possibly be combined. Once the cross-link from AT to MS is disrupted by integrating external assumptions, PS regains its conﬁrmatory support towards AT.24 In a case study on computational modeling of cell proliferation mechanisms in systems biology, Osi- mani and Poellinger (n.d.) identify different ways in which a computer simulation can provide novel insights about the object of interest and conﬁrmatory support to a scientiﬁc hypothesis: 1. The bottom-up combination of independently secured pieces of knowledge may produce unexpected results precisely because the components’ interaction might inﬂuence the functioning of the whole mechanism. 2. Combining mechanistic knowledge from different sources might reveal surpris- ing insights about hidden mechanisms in mismatches between virtual measure- ments and expectation.25 Analogy-Based Inference Patterns in Pharmacological Research 29 Analogy-Based Inference Patterns in Pharmacological Research 29 3. Moreover, novel insights might be generated when phenomena (potentially not predictable from a small set of basic rules) emerge in iterations of a complex computer simulation. Hyp Ind1 Indn Rep1 α1 Repn αn . . . . . . . . . Ind∗ 1 Ind∗ n Rep∗ 1 α∗ 1 Rep∗ n α∗ n . . . . . . . . . implementation theoretical mapping support by analogy Fig. 6: Support by analogy from a computational model of the target system to the hypothesis about the target. support by analogy implementation theoretical mapping Fig. 6: Support by analogy from a computational model of the target system to the hypothesis about the target. Fig. 6 illustrates the conceptual dependencies in conﬁrmation by analogy in our layered reconstruction of pharmacological research: Hyp is implemented by us- ing the theoretical background assumptions about D c⃝E in the ﬁrst version of the computational model. By reﬁning and enriching this model during veriﬁcation and validation, the logical cross-dependency between Hyp and its implementation is dis- rupted to an extent that allows for novel ‘observations’ during runs of the simulation, such that analogy can be used for the conﬁrmation of Hyp once more. 5 Conclusions The evidence-amalgamation framework, introduced in Sec. 1, opens the possibil- ity of tracing the dynamics of analogical reasoning across distinct epistemological categories: theoretical hypothesis (Hyp), testable indicators (Ind), and evidence re- ports (Rep). Within this layered reconstruction of scientiﬁc inference, the concept of Bayesian conﬁrmation can be utilized to formulate precisely how a causal hypoth- esis about a drug’s potentially harmful side-effects is conﬁrmed or disconﬁrmed. 30 Roland Poellinger Roland Poellinger This paper distinguishes three analogy-based inference patterns signiﬁcant in phar- macological research: This paper distinguishes three analogy-based inference patterns signiﬁcant in phar- macological research: 1. Inference from relevant reports: When the conditions of a given study corre- spond to the intended application of the investigated hypothesis, reports about the study are marked as relevant for the hypothesis, thus facilitating knowledge transfer from evidence to hypothesis. In Sec. 2, study and target conditions are broken into three components: the drug itself (D), the causal model implicit in the hypothesis (M), and the respective population (U). Pair-wise comparison is based on a similarity measure to be chosen w.r.t. the nature of the investigation; e.g., the components might be compared using a geometric measure of similarity as given by the distance between property vectors like in the example case. Obvi- ously, some important decisions are to be taken outside the framework presented, though: Questions as to how to arrive at selection criteria for the properties to be compared are left for another paper. 2. Inference from established causal knowledge: Sec. 3 discusses analogical in- ference from a second well-tested hypothesis Hyp∗. In this case, the connection between source and target is not established via similarity but across a syntactic isomorphism between the hypotheses’ consequence sets, i.e., a theoretical map- ping on the indicator level. Once this bridge is deﬁned (motivated and justiﬁed by model-external empirically grounded arguments), evidence for Hyp∗(the es- tablished hypothesis) will also boost conﬁdence in Hyp (the hypothesis under investigation). 3. Inference from computational models: If the analog system is a virtual, compu- tational model of the investigated hypothesis, the bridge between source and tar- get is not motivated by model-external considerations but much rather by model- internal constraints. Sec. 4 discusses how an artiﬁcial system can possibly pro- vide support for a causal hypothesis about an actual drug with real risk. 5 Conclusions While the paper focuses on hypothesis testing for the purpose of risk assess- ment in pharmacology, the second and third pattern in the list above make the role of analogical reasoning in the formulation of a hypothesis obvious. In particular, a computer simulation will support an investigated hypothesis if the codebase is not merely constructed from theoretical assumptions about the target hypothesis, but infused with further theoretical considerations or additional sources of knowledge, thereby breaking the logical dependency between source and target (see Sec. 4). Once a theoretical bridge is established on the indicator level, unexpected, surpris- ing, possibly unpredictable evidence reports about an analog system will propel hy- pothesis discovery and theory revision. Indeed, this extends beyond computational modeling: When Otto Schaumann created meperidine, the ﬁrst fully synthetic opi- oid pain medication, in 1937, he observed that meperidine and morphine produced similar physiological signs when administered to rats in lab experiments. In addi- tion, meperidine was known to share chemical structural properties with morphine. Schaumann consequently (and rightly) hypothesized that meperidine also shares morphine’s narcotic effects (see Bartha 2013). This further episode of successful inference by analogy illustrates the peculiar nature of pharmacology, integrating Analogy-Based Inference Patterns in Pharmacological Research 31 different levels of description from chemical structure to clinical observation. Jus- tifying analogical arguments by reconstruction calls for a framework capable of accommodating heterogeneous sources of evidence that allows tracing conﬁrma- tory support across distinct epistemological categories – possibly also from analog systems. The collection of modules presented in this paper may serve as a toolbox for such justiﬁcation in the scientiﬁc dialog between hypothesis testing and theory revision. Acknowledgements This paper was presented at workshops and conferences in Munich, Sydney, Groningen, Bologna, Bochum, and Exeter. I greatly beneﬁted from the comments and sugges- tions made by the audiences, and I am particularly thankful for personal discussions with Cameron Beebe, Lorenzo Casini, Radin Dardashti, Stephan Hartmann, Adam La Caze, J¨urgen Landes, Bar- bara Osimani, Jan-Willem Romeijn, Karim Th´ebault, Naftali Weinberger, and Michael Wilde, whose valuable comments helped me clarify my aims and shape the ﬁnal version of this paper. References Bartha P (2010) By Parallel Reasoning: The Construction and Evaluation of Ana- logical Arguments. Oxford University Press Bartha P (2013) Analogy and analogical reasoning. In: Zalta EN (ed) The Stanford Encyclopedia of Philosophy, fall 2013 edn Beebe C, Poellinger R (n.d.) Bayesian Conﬁrmation from Analog Models, forth- coming. Bovens L, Hartmann S (2003) Bayesian Epistemology. Oxford University Press Britton OBOA, Van Ammel K, Lu H, Towart R, Gallacher D, Rodriguez B (2013) Exeperimentally calibrated population of models predicts and explains intersub- ject variability in cardiac cellular electrophysiology. Proceedings of the National Academy of Sciences of the United States of America (110):E2098–105 Button KS, Ioannidis JPA, Mokrysz C, Nosek BA, Flint J, Robinson ESJ, Munafo MR (2013) Power failure: why small sample size undermines the reliability of neuroscience. Nature Reviews Neuroscience 14:365–376, URL http://dx. doi.org/10.1038/nrn3475 Cartwright N (2011) Predicting what will happen when we act. what counts for warrant? Preventive Medicine 53(4):221 – 224, DOI https://doi.org/ 10.1016/j.ypmed.2011.08.011, URL http://www.sciencedirect.com/ science/article/pii/S0091743511003008 special Section: Epi Cartwright N (2011) Predicting what will happen when we act. what counts for warrant? Preventive Medicine 53(4):221 – 224, DOI https://doi.org/ 10.1016/j.ypmed.2011.08.011, URL http://www.sciencedirect.com/ science/article/pii/S0091743511003008, special Section: Epi- demiology, Risk, and Causation Cartwright N, Stegenga J (2011) A Theory of Evidence for Evidence-Based Pol- icy. In: Dawid P, Twinning M William Vasilaki (eds) Evidence, Inference and Enquiry, OUP, chap 11, pp 291–322 Carusi A, Burrage K, Rodriguez B (2012) Bridging experiments, models and sim- ulations : an integrative approach to validation in computational cardiac elec- trophysiology. American Journal of Physiology - Heart and Circulatory Physi- 32 Roland Poellinger gy eprints.qut.edu.au/75632/ Casini L, Manzo G (2016) Agent-based models and causality: A methodologi- cal appraisal. The IAS Working Paper Series (Link¨oping University Electronic Press) (7), URL http://liu.diva-portal.org/smash/get/diva2: 1058813/FULLTEXT01.pdf 1058813/FULLTEXT01.pdf Chan AW, Altman DG (2005) Epidemiology and reporting of randomised trials published in PubMed journals. The Lancet 365(9465):1159–1162, URL http: Chan AW, Altman DG (2005) Epidemiology and reporting of randomised trials published in PubMed journals. The Lancet 365(9465):1159–1162, URL http: //dx.doi.org/10.1016/S0140-6736(05)71879-1 Dardashti R, Th´ebault K, Winsberg E (2017) Conﬁrmation via analogue simulation: What dumb holes could tell us about gravity. The British Journal for the Philoso- phy of Science 68(1):55, DOI 10.1093/bjps/axv010, URL http://dx.doi. Dardashti R, Th´ebault K, Winsberg E (2017) Conﬁrmation via analogue simulation: What dumb holes could tell us about gravity. The British Journal for the Philoso- phy of Science 68(1):55, DOI 10.1093/bjps/axv010, URL http://dx.doi. References org/10.1093/bjps/axv010, /oup/backfile/content_public/ journal/bjps/68/1/10.1093_bjps_axv010/2/axv010.pdf Dardashti R, Hartmann S, Th´ebault K, Winsberg E (n.d) Conﬁrmation via analogue simulation: A bayesian analysis, forthcoming. Diez Roux AV (2015) The virtual epidemiologist – promise and peril. American Journal of Epidemiology 181(2):100–102 Doll R, Peto R (1980) Randomised controlled trials and retrospective controls. British Medical Journal 280:44, URL http://www.ncbi.nlm.nih.gov/ pmc/articles/PMC1600504/ Frigg R, Reiss J (2009) The philosophy of simulation: hot new issues or same old stew? Synthese 169(3):593–613 Geiger D, Verma T, Pearl J (1990) Identifying independence in bayesian networks. Networks 20(5):507–534 Guala F (2010) Extrapolation, analogy, and comparative process tracing. Philosophy of Science 77(5):1070–1082 Hesse MB (1952) Operational Deﬁnition and Analogy in Physical Theories. British Journal for the Philosophy of Science 2(8):281–294, URL http://www. jstor.org/stable/686017 Hill AB (1965) The Environment and Disease: Association or Causation? Proceed- ings of the Royal Society of Medicine 58(5):295–300 LaFollette H, Shanks N (1995) Two Models of Models in Biomedical Research. Philosophical Quarterly 45(179):141–160, URL http://www.jstor.org/ stable/2220412 Landes J, Osimani B, Poellinger R (2017) Epistemology of Causal In- ference in Pharmacology. Towards an Epistemological Framework for the Assessment of Harms. European Journal for Philosophy of Science DOI 10.1007/s13194-017-0169-1, URL http://dx.doi.org/10.1007/ s13194-017-0169-1 Lewis D (1973a) Causation 70(17):556–567 Lewis D (1973b) Counterfactuals, 2nd edn. Wiley-Blackwell Luj´an JL, Todt O, Bengoetxea JB (2016) Mechanistic Information as Ev- idence in Decision-Oriented Science. Journal for General Philosophy 33 Analogy-Based Inference Patterns in Pharmacological Research of Science 47(2):293–306, URL http://dx.doi.org/10.1007/ s10838-015-9306-8 Morrison M (2015) Reconstructing Reality: Models, Mathematics, and Simulations. Oxford University Press Osimani B, Poellinger R (n.d.) A Protocol for Model Validation and Causal Infer- ence from Computer Simulation, forthcoming. Parke EC (2014) Experiments, simulations, and epistemic privilege. Philosophy of Science 81(4):516–536 Parker WS (2009) Does matter really matter? computer simulations, experiments, and materiality. Synthese 169(3):483–496 Paul LA, Healy K (2016) Transformative treatments. Noˆus 50(4) Pearl J (2000) Causality: Models, Reasoning, and Inference, 1st edn. Cambridge University Press Poellinger R (n.d.) On the Ramiﬁcations of Theory Choice in Causal Assessment: Indicators of Causation and Their Conceptual Relationships, forthcoming. Revicki DA, Frank L (1999) Pharmacoeconomic Evaluation in the Real World. PharmacoEconomics 15(5):423–434, URL http://dx.doi.org/ 10.2165/00019053-199915050-00001 Shepard RN (1980) Multidimensional scaling, tree-ﬁtting, and clustering. Sci- ence 210(4468):390–398, DOI 10.1126/science.210.4468.390, URL http: //science.sciencemag.org/content/210/4468/390, http:// science.sciencemag.org/content/210/4468/390.full.pdf Steel D (2008) Across the Boundaries. Extrapolation in Biology and Social Sci- ences. Oxford University Press Tversky A (1977) Features of similarity. References Psychological Review 84(4):327–352 Unruh WG (2008) Dumb holes: analogues for black holes. Philosophical Transac- tions of The Royal Society A 366:2905–2913, URL http://dx.doi.org/ 10.1098/rsta.2008.0062 Upshur R (1995) Looking for Rules in a World of Exceptions: reﬂections on evidence-based practice. Perspectives in Biology and Medicine 48(4):477–489, URL http://dx.doi.org/10.1353/pbm.2005.0098 Weisberg M (2012) Getting Serious about Similarity. Philosophy of Science 79(5):785–794 Weisberg M (2013) Simulation and Similarity: Using Models to Understand the World. Oxford University Press Worrall J (2007) Evidence in Medicine and Evidence-Based Medicine. Philos- ophy Compass 2(6):981–1022, URL http://dx.doi.org/10.1111/j. 1747-9991.2007.00106.x
https://openalex.org/W2979850911	https://europepmc.org/articles/pmc6786614?pdf=render	English	null	Coherent diversification in corporate technological portfolios	PloS one	2,019	cc-by	13,723	RESEARCH ARTICLE Emanuele Pugliese1,2,3, Lorenzo NapolitanoID1,4, Andrea ZaccariaID1,2, Luciano Pietronero1,2,5 1 Istituto dei Sistemi Complessi (ISC)-CNR, UOS Sapienza, Rome, Italy, 2 International Finance Corporation, World Bank Group, 20433 Washington, United States of America, 3 European Commission, Joint Research Centre (JRC), Seville, Spain, 4 Istituto di Economia, Scuola Universitaria Superiore Sant’Anna, Pisa, Italy, 5 Dipartimento di Fisica, Sapienza Università di Roma, Rome, Italy and.zaccaria@gmail.com * and.zaccaria@gmail.com a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Pugliese E, Napolitano L, Zaccaria A, Pietronero L (2019) Coherent diversification in corporate technological portfolios. PLoS ONE 14(10): e0223403. https://doi.org/10.1371/journal. pone.0223403 Editor: Stefan Cristian Gherghina, The Bucharest University of Economic Studies, ROMANIA Editor: Stefan Cristian Gherghina, The Bucharest University of Economic Studies, ROMANIA Copyright: © 2019 Pugliese 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: The data underlying the results presented in the study are available from EPO-PATSTAT (www.epo.org/searching-for- patents/business/patstat.html) and Bureau van Dijk (www.bvdinfo.com/amadeus). Abstract We study the relationship between the performance of firms and their technological portfo- lios using tools borrowed from complexity science. In particular, we ask whether the accu- mulation of knowledge and capabilities associated with a coherent set of technologies leads firms to experience advantages in terms of productive efficiency. To this end, we analyze both the balance sheets and the patenting activity of about 70 thousand firms that have filed at least one patent over the period 2004-2013. We define a measure of corporate coherent diversification, based on the bipartite network linking companies with the technological fields in which they patent, and relate it to firm performance in terms of labor productivity. Our measure favors technological portfolios that can be decomposed into large blocks of closely related fields over portfolios with the same breadth of scope, but a more scattered diversifi- cation structure. We find that the coherent diversification of firms is quantitatively related with their economic performance and captures relevant information about their productive structure. In particular, we prove on a statistical basis that a naive definition of technological diversification can explain labor productivity only as a proxy of size and coherent diversifica- tion. This approach can be used to investigate possible synergies within firms and to recom- mend viable partners for mergers and acquisitions. OPEN ACCESS Citation: Pugliese E, Napolitano L, Zaccaria A, Pietronero L (2019) Coherent diversification in corporate technological portfolios. PLoS ONE 14(10): e0223403. https://doi.org/10.1371/journal. pone.0223403 Editor: Stefan Cristian Gherghina, The Bucharest University of Economic Studies, ROMANIA Received: June 14, 2019 Accepted: September 20, 2019 Published: October 10, 2019 Copyright: © 2019 Pugliese 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. Coherent diversification in corporate technological portfolios Emanuele Pugliese1,2,3, Lorenzo NapolitanoID1,4, Andrea ZaccariaID1,2*, Luciano Pietronero1,2,5 We claim that CTD is particularly suited to study firms or small geographic aggregations which are natu- rally constrained by their size in the total amount of capabilities they can acquire. In fact, while countries are large to always benefit from a more diversified product basket and always absorb new capabilities [8, 14], firms are by necessity far more specialized and face a trade-off between the opportunity of increasing their scope and the need to maintain a cohesive core business. To draw a naturalistic analogy, the fact that an ecosystem becomes richer through diversity does not imply that all the species it hosts will occupy all the available niches, since each species takes a different path to strike a balance between adaptability and specialization in order to maximize survival probability. the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. Competing interests: E.P., A.Z., and L.P. are affiliated with the International Finance Corporation, World Bank Group. This does not alter our adherence to PLOS ONE policies on sharing data and materials. These authors have declared that no competing interests exist. the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. Competing interests: E.P., A.Z., and L.P. are affiliated with the International Finance Corporation, World Bank Group. This does not alter our adherence to PLOS ONE policies on sharing data and materials. These authors have declared that no competing interests exist. Our contribution builds on a capabilities-based view of the firm [15], which allows to model the potential returns to scope associated with pursuing innovation in complementary fields of technology. In this view, capabilities are intangible assets relating to the necessary know-how for the effective development of production and other internal organizational pro- cesses [16]. For our purposes, a capability-based model of the firm can be seen as a network connecting specific technological or organizational capabilities to one or more products, thus highlighting heterogeneous and non trivial interactions between specific technological fields. Starting with [17], many studies have tried to take advantage of firm- or product-level data to understand the possible synergies between different products. Coherent diversification in corporate technological portfolios to measure the degree of coherence that goes into the technological portfolios of innovating firms and its association with some measure of their performance. Establishing this connection is useful to advance our understanding of the importance of generating technological know- how for economic agents. In fact, it is known that firms, in a sense, can know more than they make [5–7]; however, uncovering the structure of such knowledge is another important piece of the same puzzle. In this view, it is intuitively appealing to think that innovators’ efforts to diversify their knowledge base should focus on adding domains that are functionally adjacent to their current knowledge stock rather than on taking blind leaps through the technology space. We show that benefits for patenting companies accrue not so much from the number of technologies in which they perform R&D, but rather from the overall coherence of the fields in which their research activities concentrate. To this aim, we adopt the Economic Complexity approach [8–10], an innovative methodology that leverages tools taken from complexity sci- ence [11, 12] to investigate economic development which has recently started addressing the interplay of technological, scientific, and industrial production [13]. In this paper, we propose a network-based measure of coherence that allows us to decompose corporate patent baskets into clusters of functionally related technological fields. We thus measure not only the number of such knowledge blocks, but more importantly their average size. In this sense, our proposed measure—Coherent Technological Diversification (CTD)—is an intensive measure of diversi- fication. We further show that the benefits of a more coherent assembly of corporate technol- ogy portfolios are reflected in a higher productive efficiency. This finding is consistent with a representation of production in which coherent knowledge blocks map to internally consistent production processes (or perhaps, product lines). CTD significantly differs from a simple (extensive) measure of technological diversification (TD): while the latter simply counts the number of technological fields in which a company is active, the former allows, given the same breadth of scope, to tell apart companies with a diversification structure comprising blocks of closely related fields from companies with more scattered technological portfolios. Our metric is designed to test the hypothesis that a broader knowledge stock can be leveraged more effec- tively by (or within) a productive business unit the higher the internal consistency. Introduction Innovation and technological change are driven by an intricate web of capabilities that evolve thanks to the continuous cross-fertilization between fields of knowledge. For instance, the importance of heterogeneous inputs in knowledge creation has been widely recognized in recent methodological contributions [1] as well as empirical studies concerning the nexus between interdisciplinarity and innovativeness e.g. in R&D teams [2]. Relatedly, recent investi- gations have focused on the effect of technological recombination in driving innovative impact [3] and the spill-overs generated by inventions based on similar vis à vis relatively unrelated technologies [4]. Though there is consensus around the fact that successful diversification strategies cannot be based on randomly assembling different technologies, it is far from trivial Funding: The authors acknowledge funding from the Italian Ministry of University and Research (www.miur.gov.it) within the project CRISIS LAB PNR 2011-2013. E.P., A.Z., and L.P are affiliated to the International Finance Corporation, World Bank Group. IFC provided support in the form of salaries for authors. The funders did not have any role in PLOS ONE \| https://doi.org/10.1371/journal.pone.0223403 October 10, 2019 1 / 22 PLOS ONE \| https://doi.org/10.1371/journal.pone.0223403 October 10, 2019 the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. Competing interests: E.P., A.Z., and L.P. are affiliated with the International Finance Corporation, World Bank Group. This does not alter our adherence to PLOS ONE policies on sharing data and materials. These authors have declared that no competing interests exist. Literature review This paper aims to build a bridge between two seemingly distant fields by applying a method- ology inspired by the Economic Complexity approach to the study corporate technological diversification and its link to innovation. In a way, the innovation-diversification nexus is well established in the economic and managerial literature. This is particularly the case for the recombinant perspective on innovation, according to which innovations emerge from piecing together existing knowledge. This view, which can be traced back to [21], has been embraced by many notable scholars over the years [22–24]. As pointed out by [25], a large body of empirical studies has uncovered abundant evidence of knowledge recombination in several manufacturing industries (e.g. biotech, semiconductors, automotive) as well as cul- tural and creative industries; moreover, novelty through recombination can arise through a variety of mechanisms that are still not fully understood. For instance, serendipity can play an important role in discoveries [26]. Nevertheless, only a small role in successful innovation are ascribable to chance. Furthermore, the sheer size of the landscape would rule out any pos- sibility for organizations to take a brute force approach toward recombination. For this rea- son, the search process leading to discoveries is crucially hinges on the ability of firms and organizations to accumulate knowledge and on exploit it via their combinative capabilities [27]. Though a clear understanding of the dynamics underlying an effective search process remains an open question, the strategic management literature has identified several poten- tial avenues for knowledge-enhancement within the firm [25]. Some such mechanisms (e.g. building social capital, promoting social relations among co-workers, and mixing work groups) take place within the boundaries of the firm; other mechanisms instead rely on tech- nological cross-fertilization following the targeted acquisition of external technologies [28, 29] and interaction with the external environment in the form of e.g. partnerships or alliances [30–33]. Parallel to the above organizational perspective on recombinant innovation, a fur- ther stream of literature exists which has gained relevance in recent years thanks to the increasing availability of comprehensive collections of patent data [34–36]. On one hand, the firm-centered and the patent-based literatures share the theoretical basis and the hypothesis that combining new and existing technological capabilities plays a central role in generating novelty. A different perspective on the same problem has been championed in recent years by the literature on economic complexity, which has modeled capabilities as an invisible layer linking economic agents with the outcome of their activities [18, 19]. This approach has also successfully extended the notion outside the PLOS ONE \| https://doi.org/10.1371/journal.pone.0223403 October 10, 2019 2 / 22 Coherent diversification in corporate technological portfolios corporate domain by applying it to nations and geographical regions in general [8, 9]. In a way, the present work lies in-between the traditional interpretation of capabilities and the complexity view, in that it models capabilities as a hidden layer and at the same time interprets them as mediators between firms and their productive efforts. However, differently from both the above approaches, our analysis focuses on the production of technological innovation (and its relation with performance), thus applying a notion of capabilities that is close in spirit to the technological competencies proposed by [20]. This paper is organized as follows. First, we review the relevant literature on the topic of corporate diversification, presenting an overview of prominent diversification measures, and then we discuss the contribution of this work with respect to the existing economic complexity approach. We then briefly describe the data employed for the study. At this point we introduce our metric of coherent diversification, discuss its economical meaning and relevance also with respect to the existing literature as well as the originality of the findings it yields. PLOS ONE \| https://doi.org/10.1371/journal.pone.0223403 October 10, 2019 Literature review On the other hand, the latter takes a more data-driven approach to the empirical analysis and concentrates on the patterns of technological combinations that characterize the global landscape of patented inventions. The success of patent data in large-scale empirical analyses has certainly benefitted from its comprehensive geographical and longitudinal cov- erage as well as the rich information that it provides about inventions (e.g. bibliographic PLOS ONE \| https://doi.org/10.1371/journal.pone.0223403 October 10, 2019 3 / 22 Coherent diversification in corporate technological portfolios data, citations, claims, technological fields impacted by the patents) [37, 38]. Of course, pat- ent data also has limitations. In particular, not all inventions are eligible for protection under current intellectual property legislation and not all industries have the same incentive to bear the cost of patenting. Consequently, any patent data set will present a partial view of innova- tion as a whole. Nevertheless, for our purposes the advantages of using patents as a source of data outweigh the drawbacks. The present paper lies in-between the firm-centered and the patent-centered views on recombinant innovation by taking firms as units of analysis and evaluating their innovative efforts based on information derived by the entire technological landscape. In fact, by decomposing corporate patent portfolios into their constituent technol- ogies, we are able to construct a network of technological knowledge in which the proximity between fields grows with the number of times they co-occur in the same firm. This way, we can analyze corporate technological portfolios against the background of the global network and ask whether having a more diverse technological portfolio (and thus more elements for potential new combinations) is always better or whether some connections can be predicted to be more valuble. Before getting into the details of our proposed methodology, we briefly review how the standard economic approach and the tools of Economic Complexity have been applied to the study of diversification in the past. The standard economic approach Technology has come to prominence in the economic literature more recently than produc- tion, so it is not surprising that the tools developed over time to study the latter have inspired the later effort addressing the former. For this reason, though this paper is concerned with technologies, it makes sense to start our discussion about previous measures of diversification by first addressing corporate productive scope before moving on to the literature about tech- nological diversification. Moreover, though the technological and productive dimensions are very different, they are also strongly interconnected and complement each other in driving the evolution of firms. An exhaustive review of the literature, would be beyond the scope of the paper (but see e.g. [39] for a comprehensive review of the diversification measures adopted in the economics and management literature); here, we provide a concise overview of some of the indexes of diversification that have been proposed over time and use them as the starting point to trace the path in the literature connecting diversification to the concepts of relatedness and coherence, the building blocks of our proposed approach to measuring technological diversification and its meaning for innovative firms. Diversification. The drivers and implications of firm diversification have interested scholars at least since [40], which has pioneered the idea that the “firm is not confined to ‘given’ products, but the kind of activity it moves into is usually related in some way to its exist- ing resources [and] pools of unused productive services [which,] together with the changing knowledge of management, create a productive opportunity which is unique for each firm.” [41]. Several scholars [42, 43] have built upon this intuition and re-framed the general problem in quantitative terms, extending the analysis to collected data about firms across different industries. In particular, early quantitative studies concerning diversification, which have attempted to explain the rise of industrial conglomerates in manufacturing [42, 44] have con- centrated mainly on the productive scope of manufacturing firms as measured the number of sectors encompassed by their activities. In addition to the wealth of theoretical contributions spurred by the widespread interest in understanding the determinants of corporate product diversification (for an interesting discussion, see e.g. [45]), a great deal of empirical work has also been devoted to understanding the relation between the performance of firms and the number of activities or markets in which they engage e.g. [46–48]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0223403 October 10, 2019 The standard economic approach PLOS ONE \| https://doi.org/10.1371/journal.pone.0223403 October 10, 2019 4 / 22 Coherent diversification in corporate technological portfolios However, products are not the only area in which companies diversify and it has not escaped scholarly attention that the drivers of corporate technological scope are a meaningful area of investigation. This line of inquiry has gained prominence especially in the last decades of the twentieth century, which have witnessed the emergence of rising complexity in products and production processes [49, 50]; increasing specialization in knowledge production [51]; and an accelerated pace of innovation in many industries. All of the above have contributed to making “diversity particularly across technologies . . . no longer a choice” [52]. g y p y g g Relatedness. Early attempts to tie diversification with relatedness [43, 53] have aimed to establish a link between corporate strategy and profitability. Including relatedness in the pic- ture implies changing the perspective from measuring simply the observed breadth in scope of business activities and requires new tools capable of measuring the distance between the activi- ties in which firms diversify. For instance, [53] has tested the hypothesis, formulated based on anecdotal evidence from US manufacturing that amidst diversified firms “the highest levels of profitability were exhibited by those having a strategy of diversifying primarily into those areas that drew on some common core skill or resource”. This was accomplished by developing a classification (not an index) of diversification strategies based on the share of revenues due to single product lines in a sample of large US firms. In this view, relatedness between business units—i.e. the “existence of shared facilities [and of] attempts to exploit common factors of production”—is a function of both product diversification and the contribution to the com- pany’s revenues of the largest group of closely related products. The intuition behind [43] has been expanded upon by [17], which has embraced the view according to which the implica- tions of scope for the evolution of companies and industrial structure can be better understood by including in the analysis an assessment of the overall coherence of corporate activities. This approach reflects the idea that the strategic motives behind diversification should be accounted for in order to build a taxonomy of corporate types which, in turn, can be usefully incorpo- rated in a theory of their evolution. PLOS ONE \| https://doi.org/10.1371/journal.pone.0223403 October 10, 2019 The standard economic approach To this end, and because of their reliance on a much larger data sample than the ones available to its predecessors, the measure of relatedness of [17] is based on the survivor principle, i.e. the assumption that economic competition eventually drives inefficient organizational forms out of the market, thus promoting the co-occurrence of activities that are well integrated with one another through the reliance on complementary technological capabilities. In virtue of the survivor principle, the data can be trusted to reveal efficient combinations of activities to occur with a significantly higher frequency than one would expect as a consequence of sheer randomness. Consequently, it is possible to summarize the activity portfolios of firms in a binary matrix and use it to derive a matrix of co-occur- rences between products; statistically significant combinations of activities can be uncovered through a statistic (τ) based on a standard t-test comparing the values of the cells of the empiri- cal co-occurrences matrix to their expected value under the null hypothesis of random diversi- fication. This leads to different measures of coherence whose dynamics in time show that as firm scope increases the average distance between all the activities grows with diversification, while the link between more highly related activities grows stronger. E h h h d f h h i i ll f d li i i h d Even though the study of corporate coherence has originally found application in the prod- uct domain, it has been shown to be extremely meaningful also to understand the technological performance and evolution of firms [54–58]. Of course, coherence in the realm of technologies is not the same thing as coherence in the product domain and arguably has different implica- tions. Nevertheless, the concepts are complementary in understanding firm evolution, so it is not surprising that scholars interested in technological coherence have drawn from the exist- ing analytical toolbox. For instance, Breschi et al. [59] have built on the methodology proposed by [17] to investigate whether firms patent in fields that share a common knowledge base with those in which they innovated in the past; the analysis of the technological diversification of PLOS ONE \| https://doi.org/10.1371/journal.pone.0223403 October 10, 2019 5 / 22 Coherent diversification in corporate technological portfolios firms employs a matrix of co-occurrences between technological fields and rejected the null hypothesis of random diversification. In a similar vein, Nesta et al. The standard economic approach [60] have studied corporate knowledge coherence in the US pharmaceutical industry and showed that both the scope and the coherence of the knowledge base “contribute positively and significantly to the firm’s inno- vative performance”, as measured by the number of patents it produces weighted by the num- ber of citations received. Balland et al. [61] have investigated the possibility to build smart specialization strategies by using technological portfolios at regional level. An interesting dis- cussion about the statistical properties of these approaches can be found in [62]. Napolitano et al. [63] have shown how these relatedness-based approaches can be used to investigate inno- vation dynamics. firms employs a matrix of co-occurrences between technological fields and rejected the null hypothesis of random diversification. In a similar vein, Nesta et al. [60] have studied corporate knowledge coherence in the US pharmaceutical industry and showed that both the scope and the coherence of the knowledge base “contribute positively and significantly to the firm’s inno- vative performance”, as measured by the number of patents it produces weighted by the num- ber of citations received. Balland et al. [61] have investigated the possibility to build smart specialization strategies by using technological portfolios at regional level. An interesting dis- cussion about the statistical properties of these approaches can be found in [62]. Napolitano et al. [63] have shown how these relatedness-based approaches can be used to investigate inno- vation dynamics. The economic complexity approach The intuition behind the survivor principle is also central in the literature on economic com- plexity, which in recent years has focused on explaining the composition and evolution of the export baskets of nations engaging in international trade (e.g. the product space [64] the taxon- omy network [9]) as well as predicting their future growth trajectories [8, 64]. The assumption underlying economic complexity is that the patterns of competitive advantage observed in national export baskets are the result of intangible country-specific endowments called capa- bilities (see Fig 1), which countries must acquire and combine effectively in order to thrive in global competition [9, 65, 66]. On one hand, this implies that it is possible to build a network in which products are closer the greater the overlap between the capabilities needed to produce them. On the other hand, if a nation alone has a competitive advantage in exporting a given good, we can infer that it pos- sesses an adequate combination of capabilities. Practically, we can define a binary matrix in which the generic element Mcp takes value one if country c has a Revealed Comparative Advan- tage [67] in exporting product p. Thus defined, M is the key ingredient to define product proximity within the product space [64] by counting the co-occurrences of products and nor- malizing them using the ubiquity of products, i.e the number of countries which export them. In turn, proximity represents the empirical counterpart of a kind of symmetrized conditioned probability to export a product, given the export of another product. In a similar fashion, M enters the definition of the taxonomy network proposed by [9], the adjacency matrix of which is B 2 RPP Bpp0 ¼ 1 maxðup; up0Þ X c McpMcp0 dc ; ð1Þ ð1Þ where dc ∑p Mcp is the diversification of country c, i.e the number of products it exports, and up ∑c Mcp is the ubiquity of product p. Differently from the product space approach, in Eq 1, the frequency of a product is not only conditioned to the presence of another prod- uct but also evaluated with respect to a random binomial case; the latter would imply an expected frequency of dc/P (the constant factor P is usually neglected). Firm data We aim to investigate the relation between the structure of the technological portfolios of firms and their productive efficiency, which we measure with a simple labor productivity met- ric. To extract this information, as in [70], we rely on AMADEUS, a commercial database maintained by Bureau van Dijk Electronic Publishing (BvD), which specializes in providing financial, administrative, and balance sheet information about (generally private) companies based in Europe. The database accounts for over 20 million companies for which public data is collected and harmonized sourced from several providers using a multitude of data typically collected by public institutions [71]. A notable advantage of AMADEUS is its straightforward connection with the Worldwide Patent Statistical Database (PATSTAT) of the European Pat- ent Office (EPO), which we describe below. Joining the two databases leaves us with detailed information about almost 70 thousand firms that have filed at least one patent over the period covered by our AMADEUS edition (2004-2013) and for which the balance sheet information is available firm size and productivity. Note that the focus of AMADEUS on Europe warrants a caveat about the representativity of our sample for non-European firms since only their European subsidiaries are considered. It is possible that the data is a bit skewed towards rela- tively large non-European corporate entities. Nevertheless, the quality of the data and the ease of cleanly merging it with PATSTAT is worth the trade off. Moreover, potential biases are mit- igated by the fact the larger companies have much higher probability of being active in patent- ing with respect to small firms. The economic complexity approach Left: Capabilities mediate between countries and their export baskets Right: Since capabilities are not observable, their role must be inferred from the bipartite network connecting countries to products. https://doi.org/10.1371/journal.pone.0223403.g001 https://doi.org/10.1371/journal.pone.0223403.g001 a Product Progression Network that considers the time evolution of countries in a space defined by both products and services [69]. The economic complexity approach Following [68], Eq 1 can be also interpreted in terms of the probability to go from a product to the other perform- ing a random walk defined on the tripartite product-country-product network. A similar approach has been also used in [13] with the aim of building a network of human activities spanning from technological innovation, to scientific research to industrial production. That paper introduces two noteworthy methodological additions with respect to the previous approaches: the presence of a statistical validation for each link of the resulting network, and an explicit time dependence that takes into account the diffusion of innovation in the various countries. The same methodology has been also applied to a novel database to build where dc ∑p Mcp is the diversification of country c, i.e the number of products it exports, and up ∑c Mcp is the ubiquity of product p. Differently from the product space approach, in Eq 1, the frequency of a product is not only conditioned to the presence of another prod- uct but also evaluated with respect to a random binomial case; the latter would imply an expected frequency of dc/P (the constant factor P is usually neglected). Following [68], Eq 1 can be also interpreted in terms of the probability to go from a product to the other perform- ing a random walk defined on the tripartite product-country-product network. A similar approach has been also used in [13] with the aim of building a network of human activities spanning from technological innovation, to scientific research to industrial production. That paper introduces two noteworthy methodological additions with respect to the previous approaches: the presence of a statistical validation for each link of the resulting network, and an explicit time dependence that takes into account the diffusion of innovation in the various countries. The same methodology has been also applied to a novel database to build PLOS ONE \| https://doi.org/10.1371/journal.pone.0223403 October 10, 2019 6 / 22 Coherent diversification in corporate technological portfolios Fig 1. Relationship between countries, capabilities, and products. Left: Capabilities mediate between countries and their export baskets Right: Since capabilities are not observable, their role must be inferred from the bipartite network connecting countries to products. Fig 1. Relationship between countries, capabilities, and products. PLOS ONE \| https://doi.org/10.1371/journal.pone.0223403 October 10, 2019 Patents and technology codes Following an established tradition in the economic literature on innovation [35, 37, 38], we proxy innovative activity with patents, a rich and growing source of information, which over the past years has benefited from cumulative data collection efforts of scholars as well as public agencies. Though it is well-known that patents are not a perfect tool to study all aspects of innovation [37] and that indeed there are valuable alternative methodological approaches like e.g. surveys [72, 73], patents have desirable properties for the kind of large-scale analysis we perform about the combinations of technological fields in which firms innovate. In particular, we concentrate on information concerning the set of technological fields to which inventions pertain; each field is represented by a standard code defined within the International Patent Classification (IPC), an internationally recognized hierarchical classification system main- tained and constantly updated by the World International Patent Organization (WIPO). The PLOS ONE \| https://doi.org/10.1371/journal.pone.0223403 October 10, 2019 7 / 22 Coherent diversification in corporate technological portfolios IPC codes are organized in different aggregation levels. For instance the most aggregated class counts 8 sections, while we will compute our measures at a very disaggregated level, that com- prises about 7000 groups. Apart from the obvious practical advantages of relying on standard- ized definitions, decomposing patents into their constituent technologies allows us to consider inventions as the product of a successful recombination of variously related preexisting tech- nologies and knowledge. The heart of patent applications are the claims, i.e. the part of the patent document that describes the novel aspects of the invention with respect to the relevant prior art and justifies the request for intellectual property protection and exclusive commercial rights. Claims undergo individual examination by patent office experts and, if approved, are assigned one or more IPC codes relating to the technologies touched upon by the correspond- ing claim. As mentioned above, our source of data about patents and the technologies embedded therein is PATSTAT, which aggregates data collected from national and regional patent offices; among other things, it also collects applications that have been filed at different times or in different countries but refer to the same invention into so-called patent families [74]. Since institutional and procedural differences between patent offices could introduce a bias in the sample, we take into account only so-called triadic patent families [75], i.e. Patents and technology codes families includ- ing at least one application filed at the EPO, one filed at the Japanese Patent Office (JPO), and one granted by the United States Patent Office (USPTO). This way, we select high-value inter- national inventions, but we also ensure that the patented inventions we consider have under- gone similar scrutiny processes. For each year of data, we start by decomposing the patent families with at least one applica- tion into the set of associated IPC codes and attributing the codes to patenting firms. We then assign each active family one unit of weight and divide it into equal shares between all the observed (company, technology) pairs excluding double counts. Every such pair maps to a cell of a matrix, the value of which is the sum of the shares that point to that pair; the above matrix is binarized to obtain M (the procedure is fully described in the S1 File). To summarize, M defines the technological portfolio embedded in the patents filed by all active firms in a specific year; it thus allows to look into the structure of such portfolios and investigate its relation to firm efficiency. PLOS ONE \| https://doi.org/10.1371/journal.pone.0223403 October 10, 2019 Coherent diversification The contribution of this paper lies at the intersection between the literature on corporate coherence [60] and the contributions to the economic complexity literature. In particular, our aim is to transpose the definition of relatedness proposed in [9] at the firm level and apply this measure (Eq 1) to corporate patent portfolios in order to uncover the structure of the underly- ing network of technologies. This serves as a stepping stone to define a measure of the coherent technological diversification and examine its relation to firm performance. Fig 2 illustrates our view of what defines a coherent technological portfolio; each tree repre- sents a firm (circle) that branches out into its products (squares) via the embedded technolo- gies (triangles). Analogously to Fig 1, where the hidden layer of capabilities enable countries to export products in a competitive way [65, 76], here technologies act as mediators between companies and their production lines. Following the idea that the more capabilities are needed for a product, the more complex it is [9, 66], we assume that a single production line that bene- fits from a large set of dedicated technologies will be more efficient. Since many companies have more than one production line, we will need i) to identify clusters of technological blocks as proxy for products, and ii) to measure the average number of technologies a company has for each cluster. This will be our measure of Coherent Technological Diversification (CTD), to PLOS ONE \| https://doi.org/10.1371/journal.pone.0223403 October 10, 2019 8 / 22 Coherent diversification in corporate technological portfolios Fig 2. Corporate technological portfolios conceal information about feasible output baskets. Technological portfolios can be used to infer the coherence of companies’ production lines. With respect to Fig 1, where capabilities are the actual, but hidden mediators between economic agents and their output, here the (known) technologies can be seen as enablers for more efficient (but hidden) products. https://doi.org/10.1371/journal.pone.0223403.g002 Fig 2. Corporate technological portfolios conceal information about feasible output baskets. Technological portfolios can be used to infer the coherence of companies’ production lines. With respect to Fig 1, where capabilities are the actual, but hidden mediators between economic agents and their output, here the (known) technologies can be seen as enablers for more efficient (but hidden) products. https://doi.org/10.1371/journal.pone.0223403.g002 https://doi.org/10.1371/journal.pone.0223403.g002 https://doi.org/10.1371/journal.pone.0223403.g002 be introduced in this section using the stylized example depicted in Fig 2. The company on the left, labeled x, produces computers and smartphones. PLOS ONE \| https://doi.org/10.1371/journal.pone.0223403 October 10, 2019 Coherent diversification The net- work B represented in Fig 3 (right), on the contrary, can be seen as a filtered one, in which the links are computed by considering more companies, and in which only the heavier links are kept. In this case, it is reasonable to expect to find the technologies related to cars to form a sin- gle disconnected component, and the technologies related to computers and smartphones to be relatively closer. where df is the diversification of firm f and ut is the ubiquity of technology t. B can be interpreted as the adjacency matrix of a monopartite network of technologies like the one represented in Fig 3 (right). Each of the triangular nodes in the figure corresponds to a techno- logical field and is colored to highlight its proximity to the more frequently co-occurring (thus more related) technologies to which it is linked. The figure shows that B embeds the notion that specific combinations of technologies concur to generate products, even though it is not possible to establish the correspondence between the technology and the production domains. We point out that this representation is purely illustrative and does not represent the applica- tion of Eq 1 to the matrix M defined in Fig 3 (left). Indeed, in this case the orange technologies would have been linked to the black ones thanks to the co-occurrence in company z. The net- work B represented in Fig 3 (right), on the contrary, can be seen as a filtered one, in which the links are computed by considering more companies, and in which only the heavier links are kept. In this case, it is reasonable to expect to find the technologies related to cars to form a sin- gle disconnected component, and the technologies related to computers and smartphones to be relatively closer. Fig 4 shows a filtered representation of the network of technologies at a high level of aggre- gation. We use the previously introduced empirical data to compute B and then we filter the adjacency matrix employing the minimal spanning forest algorithm [9, 77]. By construction, each node represents a technological field and it is connected with the field with which it shares the heaviest link. Coherent diversification In order to PLOS ONE \| https://doi.org/10.1371/journal.pone.0223403 October 10, 2019 9 / 22 Coherent diversification in corporate technological portfolios Fig 3. Matrices m and B. Left: the circles represent firms and the triangles represent the technological fields included in their technological portfolios; this is our starting database. Right: the triangular nodes in the graph correspond to a technological fields and are colored to highlight proximity between more frequently co-occurring (and thus more related) technologies. Fig 3. Matrices m and B. Left: the circles represent firms and the triangles represent the technological fields included in their technological portfolios; this is our starting database. Right: the triangular nodes in the graph correspond to a technological fields and are colored to highlight proximity between more frequently co-occurring (and thus more related) technologies. https://doi.org/10.1371/journal.pone.0223403.g003 https://doi.org/10.1371/journal.pone.0223403.g003 define the coherent diversification we first need a measure of technological relatedness. To this end, we redefine the matrix B of Eq 1 as follows to account for firms and technological fields (instead of products) define the coherent diversification we first need a measure of technological relatedness. To this end, we redefine the matrix B of Eq 1 as follows to account for firms and technological fields (instead of products) Btt0 ¼ 1 maxðut; ut0Þ X f MftMft0 df : ð2Þ ð2Þ where df is the diversification of firm f and ut is the ubiquity of technology t. B can be interpreted as the adjacency matrix of a monopartite network of technologies like the one represented in Fig 3 (right). Each of the triangular nodes in the figure corresponds to a techno- logical field and is colored to highlight its proximity to the more frequently co-occurring (thus more related) technologies to which it is linked. The figure shows that B embeds the notion that specific combinations of technologies concur to generate products, even though it is not possible to establish the correspondence between the technology and the production domains. We point out that this representation is purely illustrative and does not represent the applica- tion of Eq 1 to the matrix M defined in Fig 3 (left). Indeed, in this case the orange technologies would have been linked to the black ones thanks to the co-occurrence in company z. Coherent diversification The technologies that are specific to a single product are colored either in solid orange or light blue. However, since the two products are highly related from a technological point of view, we can assume that some of the capabili- ties needed to manufacture both products overlap and, consequently, some technologies are shared; this is illustrated by the striped triangles. In a sense, the coherent company par excel- lence is the firm in the center, labeled y in Fig 2, which is specialized in a single product and thus needs to master only the capabilities (and the technological fields) related with its business activity. On the contrary, firm z produces unrelated products and this results in an incoherent technological portfolio. In what follows, we test the hypothesis that the performance of a firm is related not only with its technological diversification (i.e. the number of technology codes in its patent portfo- lio), but also with the coherence of its technological capabilities. Comparing Fig 2 with Fig 1 shows that modeling technological portfolios to get a glimpse of the structure underlying prod- uct baskets is operationally similar to attempting to understand the relevance of intangible capabilities from the composition of the output mix produced by agents. Conceptually, how- ever, the two endeavors are quite different. While in Fig 1 capabilities are the actual mediators between economic agents and their output, Fig 2 depicts products as the hidden layer. How- ever, it would be wrong to deduce from the latter picture that products mediate between agents and technological fields, because it would be like assuming that production is instrumental to R&D, while it seems more plausible to assume that the relation flows in the opposite direction. The basic data we need to define Coherent Diversification in corporate technological port- folios is the matrix M defined in the Data section. This matrix represents a bipartite network linking companies to the technological fields in which they actively innovate. For the sake of exposition, the results presented below refer to the data for 2011, the most recent year for which we trust the data coverage to be reasonably complete; the results are however robust and hold also for previous time periods. A stylized graphical representation of the bipartite compa- nies-technologies network, whose adjacency matrix is M, is depicted in Fig 3 (left). PLOS ONE \| https://doi.org/10.1371/journal.pone.0223403 October 10, 2019 Coherent diversification The nodes in the graph represent IPC subsections, a highly aggregated level of classification in which all technological codes are grouped in 23 subsections; for the analysis we consider a much more detailed classification, that counts about 7000 technological sectors. The nodes are colored according to the class corresponding to the immediate higher PLOS ONE \| https://doi.org/10.1371/journal.pone.0223403 October 10, 2019 10 / 22 Coherent diversification in corporate technological portfolios Fig 4. Minimal spanning forest of B. The nodes in the graph represent IPC subsections and are colored according to the section they belong to. Each node is connected to the technological field which is linked to it with the highest weight. The color pattern shows that the driver for the co- occurrence of technologies is not technologies themselves, but a hidden layer (products). https://doi.org/10.1371/journal.pone.0223403.g004 Fig 4. Minimal spanning forest of B. The nodes in the graph represent IPC subsections and are colored according to the section they belong to. Each node is connected to the technological field which is linked to it with the highest weight. The color pattern shows that the driver for the co- occurrence of technologies is not technologies themselves, but a hidden layer (products). https://doi.org/10.1371/journal.pone.0223403.g004 https://doi.org/10.1371/journal.pone.0223403.g004 https://doi.org/10.1371/journal.pone.0223403.g004 aggregation: each subsection, defined by a letter and a number, is colored according to the sec- tion it belongs to (see legend). Quite remarkably, the color pattern of the graph suggests that the hierarchical structure of the IPC classification does not play a role in identifying the stron- gest connections between technological fields. If this were the case, we would observe nodes of the same color attached to one another; instead, nodes of the same color are generally not adja- cent. This is at odds with what one usually observes e.g. in similar representations of co-occur- ring products brought to market by countries [9, 69]. This lack of proximity among similar technological codes points out that co-occurrences of technologies are driven not by technolo- gies themselves, but by what technologies are for: products. As depicted in Fig 2, products are in our case a hidden layer between companies and the technologies they need for their produc- tion lines. In order to combine the general structure of technology relatedness with firm-specific information, we first need to measure, for each company, the coherence between all of the technologies in which it holds patents. PLOS ONE \| https://doi.org/10.1371/journal.pone.0223403 October 10, 2019 Coherent diversification Fig 5 qualitatively illustrates such measure for a generic technology t1 and two toy companies—1 and 2—depicted respectively in the left and right pan- els. In both panels, the network structure connecting the triangles in the background repre- sents a simplified (binary) illustration of B; the opaque triangles stand for technological fields contained in the patent portfolio of each firm, while the transparent triangles represent tech- nological fields in which the firm has not filed patents. Notice that both firms are equally diver- sified, having patents covering the same number (eight) of technological fields. The glaring difference between firm 1 and firm 2 resides in their diversification structure. In particular, the PLOS ONE \| https://doi.org/10.1371/journal.pone.0223403 October 10, 2019 11 / 22 Coherent diversification in corporate technological portfolios Fig 5. Illustration of γ for a generic technology t1 and two firms (1 and 2), depicted respectively in the left and right panels. In both panels, the graph represents the binary B of Fig 3 (right): the opaque triangles stand for technological fields in which the associated firm holds patents. Both firms are diversified in the same number of technological fields. However, those of firm 1 are connected within B forming a unique block; on the contrary, those of firm 2 are scattered through the graph. As a consequence, technology t1 is highly coherent in firm 1 but not in firm 2. https://doi.org/10.1371/journal.pone.0223403.g005 Fig 5. Illustration of γ for a generic technology t1 and two firms (1 and 2), depicted respectively in the left and right panels. In both panels, the graph represents the binary B of Fig 3 (right): the opaque triangles stand for technological fields in which the associated firm holds patents. Both firms are diversified in the same number of technological fields. However, those of firm 1 are connected within B forming a unique block; on the contrary, those of firm 2 are scattered through the graph. As a consequence, technology t1 is highly coherent in firm 1 but not in firm 2. https://doi.org/10.1371/journal.pone.0223403.g005 https://doi.org/10.1371/journal.pone.0223403.g005 technological fields of the first company are all connected within B and form a connected block, whereas the technologies of the second are scattered throughout the network. As a con- sequence, technology t1, which is owned by both firms, has a high intra-firm coherence within firm 1, but attains a low score in firm 2. PLOS ONE \| https://doi.org/10.1371/journal.pone.0223403 October 10, 2019 Toy examples Example 1. Let us first focus on how our framework rewards diversification only if it defines a coherent portfolio. Suppose that company f owns in total two technologies (t and t0), which implies that Mft = Mft0 = 1, and that these technologies are connected in B, i.e. Btt0 = 1 8(t, t0) = 1, 2 (note that by definition any technology is connected to itself in B, so that Btt = 1 8t). A straightforward application of Eq 3 yields γft = BttMft + Btt0Mft0 = 1 + 1 = 2 and, by the same argument, γft0 = 2. Plugging the above values for γft and γft0 into Eq 4 yields Gf ¼ 2þ2 2 ¼ 2, so that in this case the CTD of firm f is equal to its TD, because the closeness of the two tech- nologies in B suggests that they could be employed by firm f to develop the same product line. g gg y p y y f p p Consider instead what happens to the CTD of firm f if we assume that t and t0 are not con- nected in B. Notice that, since the number of technological fields contained in the portfolio of f has not changed, its TD is still 2. However, now Btt0 = 0 8t 6¼ t0, which implies that the intra- firm coherence of both the technologies owned by f is lower than before. In particular, γft BttMft + Btt0Mft0 = 1 + 0 = 1. The same is necessarily true for γft0, because t and t0 are the only two technological fields in which we are assuming f to be active, therefore this basic example is symmetric by construction. Plugging the values of γft and γft0 into Eq 4 yields Gf ¼ 1þ1 2 ¼ 1, less than the TD of f. In this case, the composition of the firm’s technological portfolio suggests that its knowledge stock is structured around two smaller subsets of non-complementary capa- bilities (e.g. two distinct product lines) rather than around one larger, more homogeneous, set of capabilities. Example 2. In order to further clarify the economic interpretation behind CTD and its relation with production lines, we now take one step forward and proceed to a slightly more involved calculation based on Fig 2. Coherent diversification In reality, the linkages we measure at each step of the analysis between companies and technology fields are not binary but, rather, weighted and it is important to keep this into account in our analytical definition of the coherence. We thus define the intra-firm Coherence of the technological field t with respect to the technological basket of firm f ð3Þ gft ¼ X t0 Btt0Mft0 : ð3Þ ð3Þ The rectangular matrix g 2 RFT, whose elements are defined above, represents the analyti- cal counterpart of Fig 5. The intuition behind Eq 3 is the following. For each technological field t and for each firm f we count how many of the technologies t0 owned by f are connected with t, using Btt0 as a weight. If the technological portfolio of f is such that t is sorrounded by a large number of strongly connected technologies f owns, then t will be very coherent with respect to f, i.e. γft will be high. On the contrary, if t belongs to a portion of the network of tech- nologies far from the patenting activity of f, γft will be low. Finally, we can the derive the corporate coherent diversification of technologies by aggregating, within each firm, the information about the intra-firm Coherence of all the tech- nological fields in which it holds patents. This can be interpreted as a reweighing of the diversi- fication structure of firms, which highlights the connected technologies and in principle has a correspondence with the corporate product basket, though the the explicit map connecting what a firm knows with what it produces remains hidden beneath the surface. We define firm- specific Coherent Technological Diversification (CTD) G 2 RF as Gf ¼ P tMftgft df ; ð4Þ ð4Þ where df ∑t Mft is the Technological Diversification (TD) of firm f. In practice, Γ computes where df ∑t Mft is the Technological Diversification (TD) of firm f. In practice, Γ computes where df ∑t Mft is the Technological Diversification (TD) of firm f. In practice, Γ computes PLOS ONE \| https://doi.org/10.1371/journal.pone.0223403 October 10, 2019 12 / 22 Coherent diversification in corporate technological portfolios the average coherence γ of the technologies in which f is patenting. Coherent diversification In the limit in which B is a binary matrix, γft simply counts how many technologies of f are connected with t and, as a con- sequence, Γf will be equal to the average size of the clusters owned by f. This means that the CTD computes the average number of technological fields included in coherent blocks within the technological portfolio of each company. Since our idea is that each one of these coherent blocks corresponds to a production line, Γ will be a proxy of the number of technologies each firms adopts for each product. In the following section we discuss two simple to models that help clarify the features of both Coherence γ and CTD Γ and their interpretation. PLOS ONE \| https://doi.org/10.1371/journal.pone.0223403 October 10, 2019 Toy examples Consider three companies: the first one (company x) has two product lines (computers and smartphones) and its portfolio contains eight technological fields, of which three are purely related with computers, three are necessary for smart-phones, and two are useful for both products; the second company, y, is instead specialized in cars and controls three technological fields related with this single product line; and finally, the third company, z, has two unrelated production lines, computers and cars, relying respectively on groups of three and two technological fields. The associated M matrix is depicted at the center of Fig 6. In order to compute the coherence of these technological portfolios, we need a mea- sure of distance between technological fields, B (note that for ease of exposition, in this exam- ple we do not compute B from M like we do for the real data; on the contrary, we suppose that the three companies operate within the technological space defined by a larger set of compa- nies that are not individually considered in the example). In particular, we take the technologi- cal network depicted in Fig 3 (right), whose adjacency matrix B is represented in the top left of Fig 6. The technologies related with cars (black squares) are homogeneous (i.e., fully con- nected) and independent of the technologies used for their product lines (i.e., there are no off diagonal elements connecting them to other technologies), forming a single unitary block. On PLOS ONE \| https://doi.org/10.1371/journal.pone.0223403 October 10, 2019 13 / 22 Coherent diversification in corporate technological portfolios Fig 6. Example 2. A graphical representation of two stylized B and M matrices alongside the Coherent Diversification of the associated firms. See text for a detailed calculations. https://doi org/10 1371/journal pone 0223403 g006 Fig 6. Example 2. A graphical representation of two stylized B and M matrices alongside the Coherent Diversification of the associated firms. See text for a detailed calculations. Fig 6. Example 2. A graphical representation of two stylized B and M matrices alongside the Coherent Diversification of the associated firms. See text for a detailed calculations. https://doi.org/10.1371/journal.pone.0223403.g006 https://doi.org/10.1371/journal.pone.0223403.g006 https://doi.org/10.1371/journal.pone.0223403.g006 the contrary, computer and smartphone technologies are homogeneous but mildly related through two off-diagonal technologies (the fourth and the fifth rows of B). Toy examples Note that for sim- plicity in this example we still assume that B is a binary matrix, meaning that technological fields are either related or totally unrelated, but in general the elements of B can take any con- tinuous value. Let us now compute the intra-firm Coherence of technologies, i.e. the enhancement that technology t gets by belonging to the portfolio of company f. Applying Eq 3 we obtain the bot- tom matrix γ of Fig 6. In this simple case, the matrix just counts the neighbors of a technology that are owned by the company. Notice that the block of car technologies is more coherent in firm y than in firm z, since they own 3 and 2 technologies in that block, respectively. Finally, using Eq 4, we can compute the CTD of the three companies. For company y we obtain Γ = 3. Under the simplifying assumptions we introduced for this toy model, the CTD is simply the average number of technologies used for each production line. Such interpretation is a zero order approximation, which turns to be exact only for independent and homogeneous produc- tion lines. Let us now consider company x. In this case, the enhancement due to the close tech- nologies is stronger, as one can notice looking at the first row of the γ matrix; averaging over the owned technologies, one obtains Γ = 3.5. Finally, company z has Γ = 2.6, which can be interpreted as a weighted average over the production lines: the first production line (comput- ers) has three technologies, all with an intra-firm coherence equal to three, while the second production line (cars) can use only two technologies, and this implies a lower coherence, equal to two. To compute Γ we employ Eq 4: we weigh the Coherence value of each technological field within the firm with the relative number of technologies used for each product, yielding 1 5 3 þ 3 þ 3 þ 2 þ 2 ð Þ ¼ 2:6. Notice that some of the values of the last row appearing of matrix γ of Fig 6 are contained in cells with lighter background color (fourth, fifth and last column). PLOS ONE \| https://doi.org/10.1371/journal.pone.0223403 October 10, 2019 14 / 22 Coherent diversification in corporate technological portfolios Fig 7. Coherent diversification VS labor productivity. Toy examples The graph plots the binned values of Coherent Diversification (Γ) of the firms in our sample against the intra-bin quantiles of labor productivity. The clear positive association between Γ and labor productivity suggests that the Coherent Diversification of technological portfolios captures relevant information about the corporate productive structure. https://doi.org/10.1371/journal.pone.0223403.g007 Fig 7. Coherent diversification VS labor productivity. The graph plots the binned values of Coherent Diversification (Γ) of the firms in our sample against the intra-bin quantiles of labor productivity. The clear positive association between Γ and labor productivity suggests that the Coherent Diversification of technological portfolios captures relevant information about the corporate productive structure. Fig 7. Coherent diversification VS labor productivity. The graph plots the binned values of Coherent Diversification (Γ) of the firms in our sample against the intra-bin quantiles of labor productivity. The clear positive association between Γ and labor productivity suggests that the Coherent Diversification of technological portfolios captures relevant information about the corporate productive structure. https://doi.org/10.1371/journal.pone.0223403.g007 https://doi.org/10.1371/journal.pone.0223403.g007 https://doi.org/10.1371/journal.pone.0223403.g007 These technologies would contribute to Γz if firm z had them in its technological portfolio, i.e. if the corresponding cells in matrix M were colored. These technologies would contribute to Γz if firm z had them in its technological portfolio, i.e. if the corresponding cells in matrix M were colored. PLOS ONE \| https://doi.org/10.1371/journal.pone.0223403 October 10, 2019 Coherent diversification in corporate technological portfolios Table 1. Statistical significance of the Coherent Technological Diversification. Regressors Model 1 Model 2 Model 3 Model 4 Size 0.079 (0.023) 0.079 (0.008) 0.081 (0.008) TD 0.010 (0.045) 0.074 (0.009) CTD 0.136 (0.045) 0.154 (0.017) 0.200 (0.016) R2 0.063 0.062 0.040 0.060 Regressions of labor productivity against Coherent Technological Diversification (CTD), Technological Diversification (TD), and Size. CTD is always statistically significant. 1% l h h ld Table 1. Statistical significance of the Coherent Technological Diversification. Regressions of labor productivity against Coherent Technological Diversification (CTD), Technological Diversification (TD), and Size. CTD is always statistically significant. of technologies comprising corporate technological portfolios) vanishes once CTD is added to the set of regressors suggests that the former can be considered a proxy for the latter. Our find- ings thus suggest that what firms know is relevant to what they produce and that the internal consistency of their knowledge stock is even more relevant than its the sheer scope. Notice that we did not include any control, as we are only looking at correlations here, without any claim of causation. We are not claiming that a firm increasing the coherence of its patent portfolio will increase their labor productivity. We are only noticing that a firm efficiency is correlated with its CTD, while any correlation with between firm efficiency and TD is due to the correla- tion of both with CTD. One could think that the vanishing significance of the coefficient of TD is due to the instability of the estimate related to the possible collinearity between TD and CTD. To make it more evident to the reader the relationship between the three variables we can visualize it by means of a three dimensional plot, in which we consider labor productivity as a function of both TD and CTD. In Fig 8 we use the two variables to aggregate the firms into areas colored on the basis of their ranking in terms of labor productivity. As expected, there is a strong correlation between coherent and not coherent diversifica- tion, which leads to the presence of white (empty) spaces away from the main diagonal (see the S1 File for the empirical distribution of these quantities). Regarding the relationship between these two measures of diversification and labor produc- tivity, it is clear from the picture that CTD has more explanatory power with respect to TD. Results This section tests the measure of firm coherence Γ by correlating it with an index of firm effi- ciency. If our hypothesis that innovating in related technological fields is conducive to the development of an effective mix of firm-level capabilities, which is in turn reflected in produc- tion, CTD should correlate with firm performance. The first test is illustrated in Fig 7, which plots the binned values of Γ against the intra-bin quantiles of labor productivity (measured as value added over employees) for the firms in our sample. The plot shows a clear positive asso- ciation, providing preliminary evidence that our measure of the coherence of technological portfolios captures relevant information about the productive structure of the firms. As a further test of the ability of Γ to capture a relevant aspect of corporate productive effi- ciency, we regress it against labor productivity. The results of the least squares regressions, which are summarized in Table 1, further confirm the intuition conveyed by Fig 7. The coefficient associated to CTD remains positive and significant in all regressions, even when we add firm size (measured by total assets) and TD as controls. Moreover, though TD is statistically significant if used alone, it loses explanatory power when used in the same model as CTD. This is particularly interesting, because it suggests that the number of connected tech- nologies within the technological knowledge portfolio of a company, as quantified by our mea- sure of coherence, is more relevant than the raw number of technological fields in which the company innovates. In particular, the fact that the statistical significance of TD (the number PLOS ONE \| https://doi.org/10.1371/journal.pone.0223403 October 10, 2019 15 / 22 PLOS ONE \| https://doi.org/10.1371/journal.pone.0223403 October 10, 2019 https://doi.org/10.1371/journal.pone.0223403.t001 https://doi.org/10.1371/journal.pone.0223403.g008 for this kind of studies. This is in line with the evidence that firms are naturally more special- ized than the geographical regions in which they operate and with the conjecture that the co- occurrence of technologies in firms or regions have different implications. for this kind of studies. This is in line with the evidence that firms are naturally more special- ized than the geographical regions in which they operate and with the conjecture that the co- occurrence of technologies in firms or regions have different implications. In fact, on average, moving horizontally from left to right the colored area of the plot shows a strong gradient in labor productivity that cannot instead be observed moving vertically from the bottom towards the top. Interestingly, labor productivity does not vary randomly along the vertical direction, but rather it tends to be negative. This lends itself to a stronger interpreta- tion of the regression results presented in Table 1, according to which, if CTD is kept fixed (i.e. if one scrolls vertically through the plot), labor productivity and TD are often negatively associ- ated. In this view, the significantly positive effect of TD in the regressions that do not include CTD among the explanatory variables is mostly due to its strong correlation with the latter. Notice in fact that the colored feather-shaped area in Fig 8 is concentrated along the diagonal and that labor productivity clearly increases moving from the bottom left to the top right. The Supporting Information contains a comparative analysis of the structure of the techno- logical space when countries, and not firms, are considered as patenting entities showing that the level of aggregation at which the analysis is performed plays a relevant role in shaping the empirical results. In particular, we find that the explanatory power of Coherent Technological Diversification on firm performance is higher if significant co-occurrences between technolog- ical fields are observed at the firm level, which therefore represents a more representative scale PLOS ONE \| https://doi.org/10.1371/journal.pone.0223403 October 10, 2019 16 / 22 Coherent diversification in corporate technological portfolios Fig 8. Labor productivity as a function of diversification and coherent diversification. Technological Diversification loses its explanatory power in favor of Coherent Technological Diversification when both are considered, in agreement with the regressions shown in Table 1. Notice that, given a fixed value of Technological Diversification, labor productivity tends to increase with Coherent Technological Diversification (i.e., from left to right, considering horizontal slices), while the opposite does not hold. https://doi org/10 1371/journal pone 0223403 g008 Fig 8. Labor productivity as a function of diversification and coherent diversification. Technological Diversification loses its explanatory power in favor of Coherent Technological Diversification when both are considered, in agreement with the regressions shown in Table 1. Notice that, given a fixed value of Technological Diversification, labor productivity tends to increase with Coherent Technological Diversification (i.e., from left to right, considering horizontal slices), while the opposite does not hold. PLOS ONE \| https://doi.org/10.1371/journal.pone.0223403 October 10, 2019 Conclusions In this work we have presented a quantitative assessment of the coherence of the patenting activity of firms and a study of its relationship with performance. The idea is that successful companies shape their technological portfolios on the basis of well defined production lines, and that this strategic behavior can be understood by looking at the technologies to which their patents belong. In particular, we introduce a methodology to reconstruct an estimate of both the size and number of the coherent blocks of knowledge a firm owns, and we show that their average size is correlated with firms performance. From a practical point of view, we use a database of about 70 thousand firms and followed their patenting activity in about 7000 technological sectors for ten years. This activity defines a bipartite companies-technologies network in which a link is present if a firm patents in a given technological field, as reported by the IPC codes in their submitted patents. We have then built a monopartite network of technologies by adapting a measure of relatedness originally con- ceived to uncover the common capabilities that countries should have to export specific prod- uct pairs. In this network the nodes are technologies, and they are connected by links whose weight is given by the (suitably normalized) co-occurrences in different firms. The idea is that the resulting clusters of technologies should correspond to the respective production lines. We PLOS ONE \| https://doi.org/10.1371/journal.pone.0223403 October 10, 2019 17 / 22 Coherent diversification in corporate technological portfolios are checking the correctness of this intuition in a quantitative way, and this will be the subject of a future publication. In this work we used this network to assess the relative integration of technological activities within firms. In particular, we define the Coherence of each technology with respect to each firm’s portfolio as the number of related technologies the firm owns, and we weight them using the network. By considering the mean of these coherence values over each firm, we obtain the Coherent Technological Diversification (CTD), a weighted average of the relatedness of the technological fields included in the portfolio of a firm. According to our interpretation, that is illustrated using two toy models, the CTD can be seen as a proxy of the average size of the coherent blocks of technological knowledge controlled by a company. Supporting information S1 File. In the Supplementary Information pdf file we discuss a number of issues, namely: • In Section 1, we describe in detail the database and how the technological portfolios of firms are defined • In Section 2, we discuss our measure of Coherent Diversification in comparison with the simple diversification • In Section 3, we show that the scale (i.e. if we perform our exercise at firm or country level) plays a major role • In Section 4, we discuss the role of firms’ size (PDF) Acknowledgments We thank Alex Coad, Matteo Chinazzi, Giovanni Dosi, and Francesca Tria for the useful sugges- tions and discussions. Disclaimer: The findings, interpretations, and conclusions expressed in this paper are entirely those of the authors. They do not necessarily represent the views of the International Bank for Reconstruction and Development/World Bank and its affiliated organiza- tions, or those of the Executive Directors of the World Bank or the governments they represent. Conclusions We have then compared our measure of the coherence of technological portfolios with firm per- formance. We have empirically found that the CTD explains labor productivity, in a statisti- cally significant way, and even after controlling for simple technological diversification (TD) and firm size. In particular we have found that when both CTD and TD are used as regressors TD loses its explanatory power. This finding has remarkable practical consequences; for instance, it points out that CTD, and not TD, should be taken into account in concrete applica- tions such as the evaluation of firms’ techological portfolios or in the analyses of merger and acquisitions between companies. This work opens up a number of possible further studies. For instance, in our analysis prod- uct lines represent a hidden layer that can be proxied by pinpointing coherent blocks in corpo- rate technological portfolios. When one analyzes products directly, these blocks should clearly emerge, giving rise to well defined clusters possibly in agreement with the standard classifica- tion—while we expect this to be not true for technological sectors. The study of the different clustering behavior of product and technologies will be the subject of a future paper. Author Contributions Conceptualization: Emanuele Pugliese, Lorenzo Napolitano, Andrea Zaccaria, Luciano Pietronero. Pietronero. PLOS ONE \| https://doi.org/10.1371/journal.pone.0223403 October 10, 2019 18 / 22 Coherent diversification in corporate technological portfolios Data curation: Emanuele Pugliese, Lorenzo Napolitano, Andrea Zaccaria. Formal analysis: Emanuele Pugliese, Lorenzo Napolitano, Andrea Zaccaria. Funding acquisition: Luciano Pietronero. Investigation: Emanuele Pugliese, Lorenzo Napolitano, Andrea Zaccaria, Luciano Pietronero. Methodology: Emanuele Pugliese, Lorenzo Napolitano, Andrea Zaccaria, Luciano Pietronero. Resources: Luciano Pietronero. Software: Emanuele Pugliese, Lorenzo Napolitano, Andrea Zaccaria. Supervision: Luciano Pietronero. Validation: Emanuele Pugliese, Lorenzo Napolitano, Andrea Zaccaria. Visualization: Emanuele Pugliese, Lorenzo Napolitano, Andrea Zaccaria. Writing – original draft: Emanuele Pugliese, Lorenzo Napolitano, Andrea Zaccaria. Writing – review & editing: Emanuele Pugliese, Lorenzo Napolitano, Andrea Zaccaria, Luciano Pietronero. Data curation: Emanuele Pugliese, Lorenzo Napolitano, Andrea Zaccaria. Formal analysis: Emanuele Pugliese, Lorenzo Napolitano, Andrea Zaccaria. Investigation: Emanuele Pugliese, Lorenzo Napolitano, Andrea Zaccaria, Luciano Pietronero. Methodology: Emanuele Pugliese, Lorenzo Napolitano, Andrea Zaccaria, Luciano Pietronero. Resources: Luciano Pietronero. Software: Emanuele Pugliese, Lorenzo Napolitano, Andrea Zaccaria. Validation: Emanuele Pugliese, Lorenzo Napolitano, Andrea Zaccaria. Visualization: Emanuele Pugliese, Lorenzo Napolitano, Andrea Zaccaria. Writing – original draft: Emanuele Pugliese, Lorenzo Napolitano, Andrea Zaccaria. Writing – review & editing: Emanuele Pugliese, Lorenzo Napolitano, Andrea Zaccaria, Luciano Pietronero. References Technological paradigms and technological trajectories: a suggested interpretation of the deter- minants and directions of technical change. Research policy. 1982; 11(3):147–162. https://doi.org/10. 1016/0048-7333(82)90016-6 16. Dosi G, Faillo M, Marengo L. Organizational capabilities, patterns of knowledge accumulation and gov- ernance structures in business firms: an introduction. Organization Studies. 2008; 29(8-9):1165–1185. https://doi.org/10.1177/0170840608094775 17. Teece DJ, Rumelt R, Dosi G, Winter S. Understanding corporate coherence. Journal of Economic Behavior & Organization. 1994; 23(1):1–30. http://dx.doi.org/10.1016/0167-2681(94)90094-9. 18. Hausmann R, Klinger B. Structural transformation and patterns of comparative advantage in the product space. 2006. 19. Hausmann R, Hidalgo CA. The network structure of economic output. Journal of Economic Growth. 2011; 16(4):309–342. https://doi.org/10.1007/s10887-011-9071-4 20. Granstrand O, Patel P, Pavitt K. Multi-Technology Corporations: Why They Have Distributed Rather Than Distinctive Core Competencies. California Management Review. 1997; 39(4):8–25. https://doi. org/10.2307/41165908 21. Schumpeter JA. Business cycles: a theoretical, historical, and statistical analysis of the capitalist pro- cess. McGraw-Hill New York; 1939. 22. Nelson RR, Winter SG. An evolutionary theory of economic change. harvard university press; 1982. 23. Henderson RM, Clark KB. Architectural innovation: The reconfiguration of existing. Administrative sci- ence quarterly. 1990; 35(1):9–30. https://doi.org/10.2307/2393549 24. Arthur WB. The structure of invention. Research policy. 2007; 36(2):274–287. https://doi.org/10.1016/j. respol.2006.11.005 25. Savino T, Messeni Petruzzelli A, Albino V. Search and recombination process to innovate: a review of the empirical evidence and a research agenda. International Journal of Management Reviews. 2017; 19(1):54–75. https://doi.org/10.1111/ijmr.12081 26. Fleming L. Recombinant uncertainty in technological search. Management science. 2001; 47(1):117– 132. https://doi.org/10.1287/mnsc.47.1.117.10671 27. Kogut B, Zander U. Knowledge of the firm, combinative capabilities, and the replication of technology. Organization science. 1992; 3(3):383–397. https://doi.org/10.1287/orsc.3.3.383 28. Lichtenthaler U. Open innovation in practice: an analysis of strategic approaches to technology transac- tions. IEEE transactions on engineering management. 2008; 55(1):148–157. https://doi.org/10.1109/ TEM.2007.912932 29. Ardito L, Natalicchio A, Messeni Petruzzelli A, Garavelli AC. Organizing for continuous technology acquisition: The role of R&D geographic dispersion. R&D Management. 2018; 48(2):165–176. https:// doi.org/10.1111/radm.12270 30. Rothaermel FT, Deeds DL. Exploration and exploitation alliances in biotechnology: A system of new product development. Strategic management journal. 2004; 25(3):201–221. https://doi.org/10.1002/ smj.376 31. Sampson RC. R&D alliances and firm performance: The impact of technological diversity and alliance organization on innovation. Academy of management journal. 2007; 50(2):364–386. https://doi.org/10. 5465/amj.2007.24634443 32. Capaldo A, Messeni Petruzzelli A. Origins of knowledge and innovation in R&D alliances: a contingency approach. Technology Analysis & Strategic Management. 2015; 27(4):461–483. https://doi.org/10. 1080/09537325.2015.1011612 33. Elia S, Petruzzelli AM, Piscitello L. References 1. McNamee RC. Can’t see the forest for the leaves: Similarity and distance measures for hierarchical tax- onomies with a patent classification example. Research Policy. 2013; 42(4):855–873. https://doi.org/10. 1016/j.respol.2013.01.006 2. Lee YN, Walsh JP, Wang J. Creativity in scientific teams: Unpacking novelty and impact. Research Pol- icy. 2015; 44(3):684–697. https://doi.org/10.1016/j.respol.2014.10.007 3. Keijl S, Gilsing V, Knoben J, Duysters G. The two faces of inventions: The relationship between recom- bination and impact in pharmaceutical biotechnology. Research Policy. 2016; 45(5):1061–1074. https:// doi.org/10.1016/j.respol.2016.02.008 4. Battke B, Schmidt TS, Stollenwerk S, Hoffmann VH. Internal or external spillovers—Which kind of knowledge is more likely to flow within or across technologies. Research policy. 2016; 45(1):27–41. https://doi.org/10.1016/j.respol.2015.06.014 5. Patel P, Pavitt K. The technological competencies of the world’s largest firms: complex and path-depen- dent, but not much variety. Research policy. 1997; 26(2):141–156. https://doi.org/10.1016/S0048-7333 (97)00005-X 6. Brusoni S, Prencipe A, Pavitt K. Knowledge specialization, organizational coupling, and the boundaries of the firm: why do firms know more than they make? Administrative science quarterly. 2001; 46 (4):597–621. https://doi.org/10.2307/3094825 7. Dosi G, Grazzi M, Moschella D. What do firms know? What do they produce? A new look at the relation- ship between patenting profiles and patterns of product diversification. Small Business Economics. 2017; 48(2):413–429. https://doi.org/10.1007/s11187-016-9783-0 8. Tacchella A, Cristelli M, Caldarelli G, Gabrielli A, Pietronero L. A new metrics for countries’ fitness and products’ complexity. Scientific reports. 2012; 2. https://doi.org/10.1038/srep00723 PMID: 23056915 9. Zaccaria A, Cristelli M, Tacchella A, Pietronero L. How the Taxonomy of Products Drives the Economic Development of Countries. PLoS ONE. 2014; 9(12):1–17. https://doi.org/10.1371/journal.pone. 0113770 10. Tacchella A, Mazzilli D, Pietronero L. A dynamical systems approach to gross domestic product fore- casting. Nature Physics. 2018; 14(8):861. https://doi.org/10.1038/s41567-018-0204-y 11. Anderson PW. More is different. Science. 1972; 177(4047):393–396. https://doi.org/10.1126/science. 177.4047.393 PMID: 17796623 12. Pietronero L. Complexity ideas from condensed matter and statistical physics. Europhysics news. 2008; 39(6):26–29. https://doi.org/10.1051/epn:2008603 13. Pugliese E, Cimini G, Patelli A, Zaccaria A, Pietronero L, Gabrielli A. Unfolding the innovation system for the development of countries: co-evolution of Science, Technology and Production. arXiv preprint arXiv:170705146. 2017;. PLOS ONE \| https://doi.org/10.1371/journal.pone.0223403 October 10, 2019 19 / 22 Coherent diversification in corporate technological portfolios 14. Pugliese E, Chiarotti GL, Zaccaria A, Pietronero L. Complex economies have a lateral escape from the poverty trap. PloS one. 2017; 12(1):e0168540. https://doi.org/10.1371/journal.pone.0168540 PMID: 28072867 15. Dosi G. References The impact of cultural diversity on innovation performance of MNC subsidiaries in strategic alliances. Journal of Business Research. 2019; 98:204–213. https://doi.org/10. 1016/j.jbusres.2019.01.062 34. Strumsky D, Lobo J, Van der Leeuw S. Measuring the relative importance of reusing, recombining and creating technologies in the process of invention. Working Paper; 2011. 35. Strumsky D, Lobo J, van der Leeuw S. Using patent technology codes to study technological change. Economics of Innovation and New Technology. 2012; 21(3):267–286. https://doi.org/10.1080/ 10438599.2011.578709 36. Youn H, Strumsky D, Bettencourt LMA, Lobo J. Invention as a combinatorial process: evidence from US patents. Journal of The Royal Society Interface. 2015; 12(106):20150272. https://doi.org/10.1098/ rsif.2015.0272 20 / 22 PLOS ONE \| https://doi.org/10.1371/journal.pone.0223403 October 10, 2019 Coherent diversification in corporate technological portfolios 37. Griliches Z. Patent statistics as economic indicators: a survey. Journal of Economic Literature. 1990; 28 (4):1661–1707. 38. Hall BH, Jaffe AB, Trajtenberg M. The NBER patent citation data file: Lessons, insights and methodo- logical tools. National Bureau of Economic Research; 2001. 39. Knecht M. Diversification, Industry Dynamism, and Economic Performance: The Impact of Dynamic- related Diversification on the Multi-business Firm. Springer Science & Business Media; 2013. 40. Penrose E. The theory of the growth of the firm. New York: John Wiley; 1959. 41. Penrose ET. The growth of the firm—a case study: the Hercules Powder Company. Business History Review. 1960; 34(01):1–23. https://doi.org/10.2307/3111776 42. Gort M. Diversification and Integration in American Industry. National Bureau of Economic Research, Inc; 1962. Available from: http://EconPapers.repec.org/RePEc:nbr:nberbk:gort62-1. 43. Rumelt RP. Strategy, structure, and economic performance. 1974. 44. Berry CH. Corporate Growth and Diversification. The Journal of Law & Economics. 1971; 14(2):371– 383. https://doi.org/10.1086/466714 45. Montgomery CA. Corporate diversification. The Journal of Economic Perspectives. 1994; 8(3):163– 178. https://doi.org/10.1257/jep.8.3.163 46. Palepu K. Diversification strategy, profit performance and the entropy measure. Strategic management journal. 1985; 6(3):239–255. https://doi.org/10.1002/smj.4250060305 47. Palich LE, Cardinal LB, Miller CC. Curvilinearity in the diversification–performance linkage: an examina- tion of over three decades of research. Strategic management journal. 2000; 21(2):155–174. https:// doi.org/10.1002/(SICI)1097-0266(200002)21:2%3C155::AID-SMJ82%3E3.0.CO;2-2 48. Miller DJ. Firms’ technological resources and the performance effects of diversification: a longitudinal study. Strategic Management Journal. 2004; 25(11):1097–1119. https://doi.org/10.1002/smj.411 49. Rycroft RW, Kash DE. The complexity challenge: Technological innovation for the 21st century. Cen- gage Learning EMEA; 1999. 50. Cohen WM, Nelson RR, Walsh JP. Protecting their intellectual assets: Appropriability conditions and why US manufacturing firms patent (or not). National Bureau of Economic Research; 2000. 51. Pavitt K. References Technologies, products and organization in the innovating firm: what Adam Smith tells us and Joseph Schumpeter doesn’t. Industrial and Corporate change. 1998; 7(3):433–452. https://doi.org/10. 1093/icc/7.3.433 52. Fai F. Technological Diversification, its Relation to Product Diversification and the Organisation of the Firm. Unitversity of Bath School of Management Working Paper Series. 2004. 53. Rumelt RP. Diversification strategy and profitability. Strategic management journal. 1982; 3(4):359– 369. https://doi.org/10.1002/smj.4250030407 54. Engelsman EC, van Raan AF. A patent-based cartography of technology. Research Policy. 1994; 23 (1):1–26. https://doi.org/10.1016/0048-7333(94)90024-8 55. Piscitello L. Relatedness and coherence in technological and product diversification of the world’s larg- est firms. Structural Change and Economic Dynamics. 2000; 11(3):295–315. https://doi.org/10.1016/ S0954-349X(00)00019-9 56. Leten B, Belderbos R, Van Looy B. Technological diversification, coherence, and performance of firms. Journal of Product Innovation Management. 2007; 24(6):567–579. https://doi.org/10.1111/j.1540-5885. 2007.00272.x 57. Joo SH, Kim Y. Measuring relatedness between technological fields. Scientometrics. 2010; 83(2):435– 454. https://doi.org/10.1007/s11192-009-0108-9 58. Rigby DL. Technological relatedness and knowledge space: entry and exit of US cities from patent clas- ses. Regional Studies. 2015; 49(11):1922–1937. https://doi.org/10.1080/00343404.2013.854878 59. Breschi S, Lissoni F, Malerba F. Knowledge-relatedness in firm technological diversification. Research Policy. 2003; 32(1):69–87. https://doi.org/10.1016/S0048-7333(02)00004-5 60. Nesta L, Saviotti PP. Firm knowledge and market value in biotechnology. Industrial and Corporate Change. 2006; 15(4):625–652. https://doi.org/10.1093/icc/dtl007 61. Balland PA, Boschma R, Crespo J, Rigby DL. Smart specialization policy in the European Union: relat- edness, knowledge complexity and regional diversification. Regional Studies. 2018; p. 1–17. 62. Bottazzi G, Pirino D. Measuring industry relatedness and corporate coherence. Available at SSRN 1831479. 2010. 63. Napolitano L, Evangelou E, Pugliese E, Zeppini P, Room G. Technology networks: the autocatalytic ori- gins of innovation. Royal Society open science. 2018; 5(6):172445. https://doi.org/10.1098/rsos. 172445 PMID: 30110482 21 / 22 PLOS ONE \| https://doi.org/10.1371/journal.pone.0223403 October 10, 2019 Coherent diversification in corporate technological portfolios 64. Hidalgo CA, Klinger B, Baraba´si AL, Hausmann R. The product space conditions the development of nations. Science. 2007; 317(5837):482–487. https://doi.org/10.1126/science.1144581 PMID: 17656717 65. Hidalgo CA, Hausmann R. The building blocks of economic complexity. Proceedings of the National Academy of Sciences. 2009; 106(26):10570–10575. https://doi.org/10.1073/pnas.0900943106 66. Saracco F, Di Clemente R, Gabrielli A, Pietronero L. From innovation to diversification: a simple com- petitive model. PloS one. 2015; 10(11):e0140420. https://doi.org/10.1371/journal.pone.0140420 PMID: 26544685 67. Balassa B. Trade liberalisation and “revealed” comparative advantage. The Manchester School. 1965; 33(2):99–123. https://doi.org/10.1111/j.1467-9957.1965.tb00050.x 68. Zhou T, Ren J, Medo M, Zhang YC. Bipartite network projection and personal recommendation. Physi- cal Review E. PLOS ONE \| https://doi.org/10.1371/journal.pone.0223403 October 10, 2019 References 2007; 76(4):046115. https://doi.org/10.1103/PhysRevE.76.046115 69. Zaccaria A, Mishra S, Cader M, Pietronero L. Integrating services in the economic fitness approach. World Bank Policy Research Working Paper No 8485. 2018;. 70. Di Guilmi C, Clementi F, Di Matteo T, Gallegati M. Social networks and labour productivity in Europe: an empirical investigation. Journal of Economic Interaction and Coordination. 2008; 3(1):43. https://doi. org/10.1007/s11403-008-0034-6 71. Ribeiro SP, Menghinello S, De Backer K. The OECD ORBIS Database. 2010. 72. Archibugi D, Planta M. Measuring technological change through patents and innovation surveys. Tech- novation. 1996; 16(9):451519–468. https://doi.org/10.1016/0166-4972(96)00031-4 73. Bogliacino F, Perani G, Pianta M, Supino S. Innovation and development: The evidence from innovation surveys. Latin American Business Review. 2012; 13(3):219–261. https://doi.org/10.1080/10978526. 2012.730023 74. Martinez C. Insight into different types of patent families. 2010. 75. Dernis H, Khan M. Triadic patent families methodology. 2004;. 76. Cristelli M, Gabrielli A, Tacchella A, Caldarelli G, Pietronero L. Measuring the intangibles: A metrics for the economic complexity of countries and products. PloS one. 2013; 8(8):e70726. https://doi.org/10. 1371/journal.pone.0070726 PMID: 23940633 77. Caldarelli G, Cristelli M, Gabrielli A, Pietronero L, Scala A, Tacchella A. A network analysis of countries’ export flows: firm grounds for the building blocks of the economy. PloS one. 2012; 7(10):e47278. https://doi.org/10.1371/journal.pone.0047278 PMID: 23094044 PLOS ONE \| https://doi.org/10.1371/journal.pone.0223403 October 10, 2019 22 / 22
https://openalex.org/W1968624861	https://zenodo.org/records/1809305/files/article.pdf	English	null	BOYLE'S LAW	School science and mathematics	1,906	public-domain	1,402	SCHOOL SCIENCE AND MATHEMATICS SCHOOL SCIENCE AND MATHEMATICS 566 only. covering the ground of the regular course, but, so far as possible, supplementing this course. As a relief from mere problem-work in this laboratory exer- cise, I should strongly recommend the investigation by the class of mathematical fallacies. Many illustrations of these fallacies may be found,’of course, in the works devoted to such subjects. DeMorgan and Ball are within reach of most teachers. But it is very easy to invent others, so that the. class may be kept con- stantly on the alert. The detection of the error in a false course of reasoning is quite as good exercise as the solution of the ordi- nary problem, and while not always as easy, is often more inter- esting. The laboratory time should be so divided between the subjects that a fairly complete supplementary course may be worked out in each, but that, in any event, the interest of the class should not be allowed to flag. In all work whether in laboratory or regular class, neatness of form, and accuracy of expression should be absolutely insisted upon. Clarity of thought cannot exist without accuracy of ex- pression, whether oral or written, whether expressed in symbols or diagrams. Harmony and order in ideas should carry with them symmetry and elegance in the expression of those ideas. (To be continued) Michigan State Normal College. The fact that the product of the volume of a given mass of gas, at a constant temperature, and the pressure under which it is placed is a constant, is known as Boyle’s or Mariotte’s law. It seems that Robert Boyle of England formulated the law in 1662, and Mariotte of France, without any knowledge of Boyle’s dis- covery verified the same law in 1676. Thus in France it is known as Mariotte’s law. Nearly every pupil knows that a gas varies in volume accord- ing as there is a greater or less pressure upon it; but he does not know that there is a definite relation existing between the amount of pressure applied and the volume of the gas until he encounters the law just stated. For the proof of this law many pieces of apparatus have been made, simple in operation and BOYLE’S LAW 567 fairly accurate. It is my purpose to present an apparatus simple in form and differing little from some pieces already in use, but perhaps with some merit which will be brought out in a description of it and an explanation of its use.* The apparatus is a U shaped tube, with a long and a short arm as shown in Fig. I. It is similar to the J- shaped one in common use, differing in that the short arm is about 90 cm long with a glass stopcock C at the end ^ ’"[ while the long arm about 150 cm long opens with a small, fun- nel into which mercury may be poured. Near the end of the long, arm is a stopcock D by which the mercury may be drawn off. The extra length of the short arm enables the in- strument to be used as a barometer. With the stopcocks C and D, the volume of the gas and the height of the mer- cury may be easily adjusted. The heights of the mercury col- umns are read by moving the detachable slide up or down on FinJ FiaTI the graduated stick E. The <-d glass tube and graduated stick are mounted upon a base 64 inches long as shown in the figure. The instrument may be, sup- ported by a hole at the top or it may be fastened permanently by means of screws. The slide F, as shown in Fig. *The instrument was made in the laboratory at the Michigan State Normal College and tested giving very satisfactory results. In making the instrument aid was given by the pro- fes&ors in the departmentProfessors Strong, Peet, and Gorton. Michigan State Normal College. II, mounted upon the scale stick A, has two arms d, d, made of brass, which extend over the glass tubes and may be made with a vernier at- tachment as indicated at a. The slide is held in place by clips c, c, made of spring brass. The apparatus is a U shaped tube, with a long and a short arm as shown in Fig. I. It is similar to the J- shaped one in common use, differing in that the short arm is about 90 cm long with a glass stopcock C at the end ^ ’"[ while the long arm about 150 cm long opens with a small, fun- nel into which mercury may be poured. Near the end of the long, arm is a stopcock D by which the mercury may be drawn off. The extra length of the short arm enables the in- strument to be used as a barometer. With the stopcocks C and D, the volume of the gas and the height of the mer- cury may be easily adjusted. The heights of the mercury col- umns are read by moving the detachable slide up or down on FinJ FiaTI the graduated stick E. The <-d glass tube and graduated stick are mounted upon a base 64 inches long as shown in the figure. The instrument may be, sup- ported by a hole at the top or it may be fastened permanently by means of screws. The slide F, as shown in Fig. II, mounted upon the scale stick A, has two arms d, d, made of brass, which extend over the glass tubes and may be made with a vernier at- tachment as indicated at a. The slide is held in place by clips c, c, made of spring brass. I ^ ’"[ FinJ FiaTI <-d inJ FiaTI <-d FiaTI FinJ In experimenting the stopcock D is closed and the one at C opened. Mercury is then poured in at B until ,it rises on the level in both arms just above C. C is then closed and by opening D mercury is drawn off until it begins to come away from C. HOOL SCIENCE AND MATHEMATICS 5G The heights of the mercury in the tubes are then taken and the difference recorded as the reading of the barometer. Michigan State Normal College. If air is the gas used the stopcock C is then opened and mercury added or drawn off until the desired air column is obtained. C is again closed. If the original air column is about 30 cm then readings may be taken for air under pressure of more or less than one atmosphere. Mercury is then poured into the tube at B until full and the height of the mercury in each arm noted. Mercury may then be drawn off a little at a time by means of stopcock D and several readings made. From the readings the following data may be found. The sum of the barometer reading and the height of the mercury in the long arm minus the height of the mercury in the short arm may always be used as the pressure, while the length of the short arm minus the height of mercury in it may always be regarded as the volume of the gas. Then if, H==Height of mercury in the long arm H==Height of mercury in the long arm H’==Height of mercury in the short arm H ==Height of mercury in the short arm B==Barometer reading L=The length of the short arm B==Barometer reading g L=The length of the short arm L=The length of the short arm Pressure (P)==B+HH’ Volume (V^LW and PXV==a constant. Results may be recorded as follows: BAROMETER READING 75. BAROMETER READING 75. H 150 60 10 H’ 75 60 40 L 90 90 90 Pressure B + H - H’ To + 150 - 75 75 + 60 - 60 75 + 10 40 Volume L - H’ 90 - 75 90 - 60 90 40 Constant (B+H-H’)(L-H’) lp0 X 15 = 2250 75 X 30 == 2250 45 X 50 = 2250 The same precautions as to pureness and dryness of the mer- cury and gas used must be taken as in any other instrument. The instrument being made of glass, saves the contamination of the mercury by metallic or rubber connections. With proper precautions very good results may be obtained.
https://openalex.org/W2158316849	https://dash.harvard.edu/bitstream/1/41275565/1/50611%20aam%20nihms-275815.pdf	English	null	The relationship between social network, social support and periodontal disease among older Americans	Journal of clinical periodontology	2,011	cc-by	6,988	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:41275565 http://nrs.harvard.edu/urn-3:HUL.InstRepos:41275565 Citation Sabbah, Wael, Georgios Tsakos, Tarani Chandola, Tim Newton, Ichiro Kawachi, Aubrey Sheiham, Michael G. Marmot, and Richard G. Watt. 2011. “The Relationship between Social Network, Social Support and Periodontal Disease among Older Americans.” Journal of Clinical Periodontology 38 (6): 547–52. https://doi.org/10.1111/j.1600-051x.2011.01713.x. NIH Public Access Author Manuscript NIH-PA Author Manuscript Published in final edited form as: blished in final edited form as: Clin Periodontol. 2011 June ; 38(6): 547–552. doi:10.1111/j.1600-051X.2011.01713.x. J Clin Periodontol. 2011 June ; 38(6): 547–552. doi:10.1111/j.1600-051X.2011.01713.x. 1Corresponding Author: w.sabbah@ucl.ac.uk, Phone: +44 20 7679 5671, Fax: +44 20 7813 0280, Department of Epidemiology and Public Health, University College London, 1-19 Torrington Place, WC1E-6BT, London, UK.. The authors declare no conflict of interest. Abstract Aim—The objectives of this study were to examine the relationship between social network, social support and periodontal disease among older American adults and to test whether social network and support mediates socioeconomic inequality in periodontal disease. Materials and Methods—Data pertaining to participants aged 60 years and over from the National Health and Nutrition Examination Survey 2001-2004 was used. Periodontal disease variables were extent loss of periodontal attachment ≥ 3mm, and moderate periodontitis. Social support and networks were indicated by need for emotional support, number of close friends and marital status. Results—Widowed and those with lowest number of friends had higher rates of the extent of loss of periodontal attachment (1.27,95%CI:1.03,1.58) and (1.22,95%CI:1.03,1.45), respectively. Marital status and number of friends were not significantly associated with moderate periodontitis after adjusting for behavioural factors. The need for more emotional support was not related to periodontal disease in this analysis. Social networks and support had no impact on socioeconomic inequality in periodontal disease. NIH-PA Author Manuscript Conclusion—Certain aspects of social network, namely being widowed and having fewer friends were linked to the extent of loss of periodontal attachment but not to the definition of moderate periodontitis, in older adults. Share Your Story The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . Accessibility NIH-PA Author Manuscript Keywords Periodontal disease; social network; social support; older adults The relationship between social network, social support and periodontal disease among older Americans or Manuscript NIH-PA Author Manuscript Wael Sabbah1,2, Georgios Tsakos2, Tarani Chandola3, Tim Newton4, Ichiro Kawachi5, Aubrey Sheiham2, Michael G Marmot2, and Richard G Watt2 2Department of Epidemiology and Public Health, University College London 3University of Manchester, School of Social Sciences, Manchester, United Kingdom 4Kings College London, Dental Institute, London, United Kingdom 5Harvard School of Public Health, Department of Society, Human Development, and Health, Boston, Massachusetts, United States The authors declare no conflict of interest. Introduction Several theories have been postulated about the mechanism linking social network and support to health and mortality (Berkman 1985; Stansfeld 2006). They include direct financial and physical support to engage in health enhancing behaviours and to access care, gaining information from the social network pertaining to innovative health promoting behaviours and health hazards. Furthermore, social and emotional support could help mitigate the consequences of stressful events and coping with diseases, risk factors, and the negative effect of social isolation (Berkman 1985; Kawachi et al 1996) through alterations in neuroendocrine and immunological control systems (Brunner and Marmot 2006). As there are common pathways affecting general and oral health, (Sheiham & Nicolau 2005; Sabbah et al 2008 Borrell & Crawford 2010), some dental studies have examined the relationship between social network and support and other societal characteristics with oral health in children and adolescents (Hanson et al 1994; Pattussi et al 2007; 2008; Aida et al 2008; 2009) and in adults (McGrath & Bedi 2002; Merchant et al 2003; Monteira da Silva 1996). One study examined the relationship between social networks and periodontitis among male health professionals in the USA and found that individuals having friends and who participated in social activities were less likely to have peridontitis (Merchant et al 2003). Another study reported an association between social isolation and periodontitis (Monteiro da Silva 1996). However, these two studies on the relationship between periodontitis and social network and support were either conducted in a specific occupational group or on a relatively small sample. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Auth While social support and networks were suggested to mediate the socioeconomic inequalities in general health (Stansfeld 2006), no study has examined this possible role of social support and networks in relation to oral health. Therefore, we set out to examine the relationship between social network and support and periodontal disease in a nationally representative sample of American older adults, and to assess whether social network and support mediated the known socioeconomic inequality in periodontal diseases (Stansfeld 2006). We postulated that higher levels of social network and support were inversely related to periodontal disease among older American adults. Considering the different mechanisms through which social network and support influences health (Berkman 1985; Kawachi et al 1996), our second hypothesis was that social network and support mediates the relationship between socioeconomic position and periodontal disease in older adults. Introduction The objectives of the study were to test: (1) the relationship between three markers of social network and support (Cohen et al 2000), namely number of close friends, need for emotional support and marital status, with periodontal disease; (2) whether social network and support mediated the socioeconomic disparities in periodontal disease, in a national representative sample of Americans aged 60 years and older. NIH-PA Author Manuscript Introduction Lower levels of social network, support and isolation are related to various aspects of physical and mental disease, and to rates of mortality (Berkman & Kawachi 2000; Stansfeld 2006). There is an inverse relationship between higher levels of social network and support on the one hand and morbidity, coronary heart disease and cause specific mortality rates, on Page 2 Sabbah et al. Sabbah et al. Page 2 NIH-PA Author Manuscript the other hand (Ruberman et al 1984; Orth-Gomer & Johnson 1987; Berkman et al 1992; Kawachi et al 1996; Eng et al 2002; Stansfeld 2006; Hoppmann & Gerstorf 2009; Mead et al 2010). Several theories have been postulated about the mechanism linking social network and support to health and mortality (Berkman 1985; Stansfeld 2006). They include direct financial and physical support to engage in health enhancing behaviours and to access care, gaining information from the social network pertaining to innovative health promoting behaviours and health hazards. Furthermore, social and emotional support could help mitigate the consequences of stressful events and coping with diseases, risk factors, and the negative effect of social isolation (Berkman 1985; Kawachi et al 1996) through alterations in neuroendocrine and immunological control systems (Brunner and Marmot 2006). As there are common pathways affecting general and oral health, (Sheiham & Nicolau 2005; Sabbah et al 2008 Borrell & Crawford 2010), some dental studies have examined the relationship between social network and support and other societal characteristics with oral health in children and adolescents (Hanson et al 1994; Pattussi et al 2007; 2008; Aida et al 2008; 2009) and in adults (McGrath & Bedi 2002; Merchant et al 2003; Monteira da Silva 1996). One study examined the relationship between social networks and periodontitis among male health professionals in the USA and found that individuals having friends and who participated in social activities were less likely to have peridontitis (Merchant et al 2003). Another study reported an association between social isolation and periodontitis (Monteiro da Silva 1996). However, these two studies on the relationship between periodontitis and social network and support were either conducted in a specific occupational group or on a relatively small sample. the other hand (Ruberman et al 1984; Orth-Gomer & Johnson 1987; Berkman et al 1992; Kawachi et al 1996; Eng et al 2002; Stansfeld 2006; Hoppmann & Gerstorf 2009; Mead et al 2010). J Clin Periodontol. Author manuscript; available in PMC 2012 June 1. Periodontal disease variables Periodontal assessments in NHANES 2001-2004 were made at three facial sites (mesio-, disto-, and mid-facial) on each fully erupted permanent tooth, except third molars, in two randomly selected quadrants (one maxillary and one mandibular). Detailed information on the NHANES dental examinations for the survey periods are published elsewhere (Drury et al 1996; Dye et al 2007a;b). Periodontal data were used from the surveys 2001-2002 and 2003-2004 for consistency in the number of sites diagnosed for periodontal disease. Two variables were created: (1) extent of loss of periodontal attachment of 3mm+ (expressed as the percentage of affected sites), and (2) moderate periodontitis was defined by the presence of 2 or more inter-proximal sites with loss of attachment of 4mm or more (not on same tooth), or 2 or more inter-proximal sites with pocket depth 5mm or more (not on same tooth) (Page & Eke 2007). NIH-PA Author Manuscript Socio-demographic variables These included age (continuous variable), gender, and race/ ethnicity (White, African Americans, Mexican Americans, other Hispanic and other ethnicities). Income was indicated by family poverty-income ratio, which is the ratio between house hold income and poverty threshold, and is used to account for inflation throughout the years of the survey. Poverty income ratio was categorised into quartiles. Education reflects whether the participants had less than high school diploma, high school, or more than high school. Behavioural variables NIH-PA Author Manuscript These included smoking and dental visits. Smoking indicates whether the person is a current smoker, a former smoker who smoked at least 100 cigarettes, or never smoked. Dental visit indicates whether the participant visited a dentist at least once within the past 2 years. J Clin Periodontol. Author manuscript; available in PMC 2012 June 1. Materials and Methods Data were on participants aged 60 years and over who participated in the National Health and Nutrition Examination Survey (NHANES) 2001 to 2004. NHANES 2001-2004 collected information on a nationally representative sample in each year using a stratified, multistage design to obtain a representative probability sample of the civilian noninstutionalized population of the United States (CDC, 2005).. The survey collected data on individual characteristics such as gender, age, race/ ethnicity, education, income and marital status. The interviewer also asked questions about selected behaviours, including smoking and dental visits. Furthermore, the survey included questions on social network and support providing data on emotional, material, and network (the number of members in a Sabbah et al. Page 3 network) support (CDC, 2005). The questions were selected from the Yale Health and Aging Study and the Social Network Index – Alameda County Study (Ruberman et al 1984). network) support (CDC, 2005). The questions were selected from the Yale Health and Aging Study and the Social Network Index – Alameda County Study (Ruberman et al 1984). NIH-PA Author Manuscript The MEC examination included a detailed oral examination (Dye et al 2007b). Participation in the oral health component was limited to age groups considered most critical for monitoring oral health (13 years and older for periodontal examination). Information was also obtained from the home interview covering assessments on perceived oral health status (CDC, 2005). The NHANES periodontal examination protocol was based upon the (NIDCR) criteria with minor modifications (Dye et al 2007b). Social network and support variables Three variables indicating social network and support were used. Whether the participants needed more emotional help during the past year was used as a measure of social support. Number of close friends, and marital status were used as indicators of social networks. These three markers of social network and support have been repeatedly used in medical (Manzoli et al 2007; Hoppmann & Gerstorf 2009; Mead et al 2010), and dental studies (Marcenes & Sheiham 1996; Merchant et al 2003; Aida et al 2009). The need for emotional support was a dichotomous variable (yes, no), the number of close friends was categorised into tertiles. Marital status was grouped to indicate whether the participants were (1) married or live with a partner, (2) widowed, (3) divorced, separated or single. Statistical analysis First we assessed the distribution of all explanatory variables by periodontal status (moderate periodontitis). A series of regression models were conducted for both periodontal disease variables alternatively adjusting for markers of social network and support. The first Sabbah et al. Page 4 Sabbah et al. model was adjusted for gender, age and race/ ethnicity, the second model additionally adjusted for education, income, number of teeth and diabetes, the third model additionally adjusted for dental visits and smoking. The sequence of adjustment is based on the hypothesis that social network and support influence health through an impact on health- related behaviours (Berkman 1985). This method was used in medical research to account for direct and indirect effect of explanatory factors (van Oort et al. 2005). Logistic regression was employed in the analysis pertaining to moderate periodontitis, and negative binomial regression in the analysis pertaining to extent of loss of periodontal attachment. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Aut Other studies have indicated that men benefit more than women from social network and support (Berkman 1985; Berkman & Kawachi 2000), hence we tested for interaction between social support and networks variables and gender. We also tested interaction between social networks and each of age, smoking and dental visits. To demonstrate that the relationship between social support and networks with periodontal disease operates via a behavioural pathway, we also examined the association between the three markers of social support and networks with each of dental visits and smoking, adjusting for sex and age. Manuscript NIH-PA Author Manuscript Regression models were conducted to measure the variation in income and education disparities in periodontal disease and the impact of adjusting for social support and network on the socioeconomic disparities in periodontal disease. The first model adjusted for age, gender, race/ ethnicity, number of teeth, diabetes, smoking status and dental visits, in addition to income and education. The second model additionally adjusted for marital status, number of friends and needing more emotional support, in order to assess whether the relationship between income and education and periodontal disease is attenuated after adjustment for social networks and support. A four year weight variable based on the 2- years mobile examination weight was created and used to adjust for the complexity of the survey. Stata survey command was used throughout the analysis. NIH-PA Author Manuscript J Clin Periodontol. Author manuscript; available in PMC 2012 June 1. Results The analysis was conducted for 1632 older adults aged 60 years and over who had complete social network and support and periodontal disease data in NHANES 2001-2004. There was no significant difference in periodontal disease between the 1632 individuals included in the analysis and the 134 individuals excluded for having incomplete data in this age group. The mean age was 69.7 years. The prevalence of moderate periodontitis was 23.1% and 17.1% among participants aged 60 to 70 years and 71 and older, respectively. Married and cohabiting individuals had lower prevalence of moderate periodontitis (18.6%) than widowed (23.7%), and divorced (19.1%). Similarly, individuals with the highest number of friends had the lowest prevalence of periodontitis (Table 1). NIH-PA Author Manuscript NIH-PA Author Manuscript The mean age of widowed individuals (74.8 years) was higher than that of married and cohabiting (68.9 years) and divorced and single (67.5). On the other hand, more women were widowed (37.5%) than men (10.8%). The mean age across tertiles of number of close friends was 69.7, 69.8 and 70.5 years in the highest, middle and lowest friend group. There was a relationship between the three markers of social support and networks, and smoking and dental visits. Smoking was highest among divorced (22%) than widowed (8%) and married (7%). Similarly, 13% of participants with least number of friends were smokers, compared to 8% among participants with more friends. Individuals who needed emotional support had higher prevalence of smoking (10%) than those who did not need emotional support (9%). Married participants reported more visits to a dentist (82%) than widowed (73%) and divorced (72%). Individuals with highest number of friends reported more dental visits (83%) than those in the middle (81%) and lowest (70%) friend groups. Participants Page 5 Sabbah et al. Page 5 Sabbah et al. NIH-PA Author Manuscript who did not need emotional support reported more dental visits (80%) than those who needed emotional support (71%). NIH-PA Author Manuscript N Widowed and divorced participants were more likely to be a current or former smoker compared to married participants, with odds ratios of 1.58 (95%CI 1.16, 2.14) and 1.79 (95%CI 1.41, 2.27), respectively in a regression model adjusting for sex and age. Widowed and divorced individuals were also less likely to visit a dentist with odds ratios of 0.50 (95%CI 0.38, 0.65), and 0.62 (95%CI 0.49, 0.78) respectively. Results On the other hand, participants in the middle and lowest number of friends groups had insignificant odds ratios of 1.07 (95%CI 0.86, 1.34) and 1.12 (95%CI 0.92, 1.39) respectively for being current or former smokers in a regression model adjusting for sex and age. Participants with the lowest number of friends were significantly less likely to visit a dentist than those with highest number of friends with odds ratios of 0.52 (95%CI 0.42, 0.64). The odds ratios for the associations between need for support and being a current or former smoker, and dental visits were not significant with odds ratios of 0.93 (95%CI 0.74, 1.16) and 0.82 (95%CI 0.63, 1.05), respectively. Table 2 shows the association between the three markers of social network and support (marital status, needing more emotional support and number of friends) with extent of loss of periodontal attachment of 3mm or more and case definition of moderate periodontitis (Page & Eke 2007). Widowed and divorced had higher levels of loss of periodontal attachment than married persons, with rate ratios 1.60 (CI 1.27, 2.01) and 1.33 (CI 1.09, 1.63), respectively. After adjusting for income, education, smoking and dental visits, widowed still had significantly higher levels of loss of periodontal attachment (Table 2). Widowed individuals were significantly more likely to have moderate periodontitis than married individuals (OR 1.65, CI 1.09, 2.05). However, this relationship lost significance after adjusting for income, education, smoking and dental visits. Participants with the least number of friends had significantly higher levels of loss of periodontal attachment than those with the highest number of friends. This relationship remained significant even after adjusting for socioeconomic and behavioural factors (rate ratio 1.22, CI 1.03, 1.45) (Table 2). Similarly, persons who had the lowest number of friends were significantly more likely to have moderate periodontitis (OR 1.53, CI 1.15, 2.04). The relationship was eliminated after adjusting for behaviours. Needing more emotional support did not appear to have an association with periodontitis or the extent of loss of periodontal attachment (Table 2). While there was clear income gradients in moderate periodontitis, education did not seem to have a great impact on moderate periodontitis for this age group (Table 3). Overall, social network and support had a very modest impact on the association between income and education, and moderate periodontitis. Individuals in the lowest income quartile had odds ratios of 2.17 (CI 1.26, 3.73) for having moderate periodontitis. Results After adjusting for social network and support, this odds ratio hardly changed (Table 3). On the other hand, participants in the lowest education group had higher levels of loss of periodontal attachment (rate ratio 1.42, CI 1.15, 1.76). After adjusting for social network and support the relationship was slightly attenuated and remained significant (Table 3). There was no interaction between social network variables with neither of age, gender, smoking nor dental visits. NIH-PA Author Manuscript J Clin Periodontol. Author manuscript; available in PMC 2012 June 1. Discussion Marital status and numbers of friends were significantly associated with two indicators of periodontal disease measuring case definition and severity of the disease among older Americans aged 60 years and over. Considering the postulated mechanism for the relationship between social network, support and oral health through health behaviours Sabbah et al. Sabbah et al. Page 6 NIH-PA Author Manuscript (Berkman 1985), the aforementioned relationships lost significance after adjustment for behaviours, with the exception of the relationships between being widowed and having lowest number of friends with extent of loss of periodontal attachments. Furthermore, social network and support variables did not seem to have an impact on socioeconomic disparities in periodontal disease. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Auth In the current study we used number of friends, needing more emotional support and marital status to indicate social network and support (Cohen et al 2000). Numbers of friends and the need for emotional support have both been frequently used as markers of social network and support (Ruberman et al 1984; Orth-Gomer & Johnson 1987; Berkman et al 1992; Kawachi et al 1996; Eng et al 2002; Stansfeld 2006; Hoppmann & Gerstorf 2009; Mead et al 2010; Mead et al 2010). Furthermore, two dental studies found that having a greater number of friends was inversely related to periodontal disease (Monteiro da Silva 1996; Merchant et al 2003). The current study supports these findings by demonstrating them in a nationally representative sample of American older adults. On the other hand, the need for emotional support was not associated with periodontal disease in this study. Marital status was an important determinant of health and mortality, especially among older adults (Manzoli et al 2007; Hoppmann & Gerstorf 2009). Additionally, Marcenes and Sheiham (1996) demonstrated the importance of the quality of marriage for oral health. In the current study, marital status appeared to be the most important social networks factor in relation to periodontal diseases among older adults with being widowed maintaining its significant relationship with the extent of loss of periodontal attachment even after adjusting for smoking and dental visits. It is worth noting that while widowed persons were generally older than the rest of the sample, which would imply that the observed relationship with periodontal disease is due to the accumulative effect of age. However, there was no interaction between marital status and age. J Clin Periodontol. Author manuscript; available in PMC 2012 June 1. Discussion Furthermore, the majority of widowed were women, who generally have lower levels of periodontal disease than men. This is a cross-sectional study which cannot provide evidence on causal inferences. Being widowed had a stronger relationship with periodontal disease than being divorced or single. As the death of a spouse is very likely to be a stressful life event, this finding is consistent with psychological pathways affecting periodontal disease by reducing host resistance and the ability to cope with stressors (Sheiham & Nicolau 2005). Being divorced may also be a stressful life event, though in this age group it is reasonable to speculate that becoming widowed was probably a relatively more recent life event than being divorced, and this may partly contribute to the stronger association with current periodontal status. On the other hand, periodontitis is a progressive, cumulative disease; hence the importance of more recent stressful events is questionable. The relatively stronger relationship between being widowed and periodontal disease could also be attributed to the older age of widowed participants, potentially indicating the possibility of residual confounding by age; however the analysis adjusted for the effect of age group. NIH-PA Author Manuscript NIH-PA Author Manuscript In this study we also examined income and education gradients in periodontal disease and the impact of controlling for social network and support on the aforementioned relationship. There were income and education gradients in periodontal disease. However social network and support appeared to have no impact on the social gradients in periodontal disease. Stansfeld (2006) found that social network and support had a limited effect on the social gradients in general health. Other studies have indicated that men benefit more than women from social network and support (Berkman 1985; Berkman & Kawachi 2000), in this study there was no interaction between gender and social network and support. J Clin Periodontol. Author manuscript; available in PMC 2012 June 1. Page 7 Sabbah et al. NIH-PA Author Manuscript The effect of social network and support on health was postulated to occur through four pathways (Berkman 1985; Kawachi et al 1996): first, an instrumental support, for example financial support; second, informational support where individuals acquire knowledge about health promoting habits and hazards to health from their social network; third, emotional support which help individuals to cope with the disease and with health hazards; fourth, through an influence on host resistance. Acknowledgments Conflict of Interest and Source of Funding This study was supported by the National Institute of Dental and Craniofacial Research/ NIH grant number: 1 R21 DE019535-01. Discussion It is highly likely that similar pathways affect periodontal disease in older adults (Sheiham and Nicolau 2005). For example, social network and support could influence oral health through a change in behaviours (McGrath & Bedi 2002), or by buffering the negative effect of stressful events on periodontal disease (Sheiham and Nicolau 2005). These pathways linking social network and support and oral health could not be tested in this study due to data limitations. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Auth This study demonstrated that social networks, indicated by number of friends and marital status, were significantly related to severity of loss of attachment in a nationally representative sample of older Americans aged 60 years and over. The importance of certain aspects of social networks, namely being widowed, and having few friends were further demonstrated as such individuals remained more likely to have greater levels of loss of periodontal attachment even after adjusting for behavioural determinants of periodontitis such as smoking and dental visits. Social network and support appeared to have no impact on the social gradients in periodontal disease in this age group. NIH-PA Author Manuscript The current study has the advantages of using a nationally representative sample of American older adults and assessing the impact of social network and support on the social gradients in periodontitis. However, considering the cross-sectional nature of the study, no causal association could be concluded from this study. Additionally, the dataset lacked a number of important behavioural factors, such as tooth-brushing, which could have influenced the results. Furthermore, the variable on the need for emotional support is a crude measure of social support as it indicates individuals’ perception of the need for emotional support. Others have used a question asking whether an individual received any emotional support (Berkman & Kawachi 2000). In this study, it is possible that even individuals who reported the need for emotional support still received some support. Social networks indicated by number of friends and marital status was significantly related to the extent of loss of periodontal attachment, but not the definition of moderate periodontitis, after adjusting for socioeconomic and behavioural factors in a nationally representative sample of older Americans aged 60 years and over. Social network and support appeared to have no impact on the social gradients in periodontal disease in this age group. NIH-PA Author Manuscript J Clin Periodontol. Author manuscript; available in PMC 2012 June 1. Aida J, Hanibuchi T, Nakade M, Hirai H, Osaka K, Kondo K. The different effects of vertical social capital and horizontal social capital on dental status: A multilevel analysis. Social Science & Medicine. 2009; 69:512–8. [PubMed: 19573968] Aida J, Ando Y, Oosaka M, Niimi K, Morita M. Contributions of social context to inequality in dental caries: a multilevel analysis of Japanese 3-year-old children. Community Dentistry and Oral Epidemiology. 2008; 36:149–56. [PubMed: 18333879] Aida J, Ando Y, Oosaka M, Niimi K, Morita M. Contributions of social context to inequality in dental caries: a multilevel analysis of Japanese 3-year-old children. Community Dentistry and Oral Epidemiology. 2008; 36:149–56. [PubMed: 18333879] Aida J, Hanibuchi T, Nakade M, Hirai H, Osaka K, Kondo K. The different effects of vertical social capital and horizontal social capital on dental status: A multilevel analysis. Social Science & Medicine. 2009; 69:512–8. [PubMed: 19573968] References Aida J, Ando Y, Oosaka M, Niimi K, Morita M. Contributions of social context to inequality in dental caries: a multilevel analysis of Japanese 3-year-old children. Community Dentistry and Oral Epidemiology. 2008; 36:149–56. [PubMed: 18333879] Aida J, Ando Y, Oosaka M, Niimi K, Morita M. Contributions of social context to inequality in dental caries: a multilevel analysis of Japanese 3-year-old children. Community Dentistry and Oral Epidemiology. 2008; 36:149–56. [PubMed: 18333879] Aida J, Hanibuchi T, Nakade M, Hirai H, Osaka K, Kondo K. The different effects of vertical social capital and horizontal social capital on dental status: A multilevel analysis. Social Science & Medicine. 2009; 69:512–8. [PubMed: 19573968] J Clin Periodontol. Author manuscript; available in PMC 2012 June 1. J Clin Periodontol. Author manuscript; available in PMC 2012 June 1. Sabbah et al. Page 8 Page 8 NIH-PA Author Manuscript Berkman, LF.; Kawachi, I. Social epidemiology. Oxford University Press; New York: 2000. Berkman LF, Leo-Summers L, Horwitz RI. Emotional support and survival after myocardial infarction. Annals of Intern Medicine. 1992; 117:1003–9. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Auth Berkman, LF. The relationship of social networks and social support to morbidity and mortality. In: Cohen, S.; Syme, SL., editors. Social support and health. Academic Press; New York: 1985. p. 241-62. Borrell LN, Crawford ND. Social disparities in periodontitis among US adults: the effect of allostatic load. Journal of Epidemiology and Community Health. 2010 Equb ahead of print PMID 19996354. Brunner, E.; Marmot, M. Social organization, stress and health. In: Marmot, M.; Wilkinson, R., editors. Social determinants of health. Oxford University Press; Oxford: 2006. p. 6-30. Centers for Disease Control and Prevention (CDC). National Center for Health Statistics (NCHS). National Health and Nutrition Examination Survey. Questionnaires, datasets and related documentation, 1999-2004. 2005. http://www.cdc.gov/nchs/nhanes/nhanes_questionnaires.htm Cohen, S.; Underwood, LG.; Gottlieb, BH. Social support measurement and intervention: a guide for health and social scientists. Oxford University Press; New York: 2000. Drury T, Winn D, Snowden C, Kingman A, Kleinman D, Lewis B. An overview of the oral health component of the 1988–91 National Health and Nutrition Examination Survey. Journal of Dental Research. 1996; 75:620–30. Spec Issue. [PubMed: 8594086] Dye BA, Barker LK, Selwitz RH, Lewis BG, Wu T, Fryar CD, et al. Overview and quality assurance for the National Health and Nutrition Examination Survey (NHANES) oral health component, 1999–2002. Community Dentistry and Oral Epidemiology. 2007a; 35:140–51. References [PubMed: 17331155] Dye BA, Tan S, Smith V, Lewis BG, Barker LK, Thornton-Evans G, et al. Trends in oral health status: United States, 1988–1994 and 1999–2004. National Center for Health Statistics. Vital and Health Statistics Series. 2007b; 11:248. Eng PM, Rimm EB, Fitzmaurice G, Kawachi I. Social ties and change in social ties in relation to subsequent total and cause-specific mortality and coronary heart disease incidence in men. American Journal of Epidemiology. 2002; 155:700–9. [PubMed: 11943687] Hanson BS, Liedberg B, Owall B. Social network, social support and dental status in elderly Swedish men. Community Dentistry and Oral Epidemiology. 1994; 22:331–337. [PubMed: 7813188] Hoppmann C, Gerstorf D. Spousal interrelations in old age--a mini-review. Gerontology. 2009; 55:449–59. [PubMed: 19346740] Kawachi I, Colditz GA, Ascherio A, Rimm EB, Giovannucci E, Stampfer MJ, et al. A prospective study of social networks in relation to total mortality and cardiovascular disease in men in the USA. Journal of Epidemiology and Community Health. 1996; 50:245–251. [PubMed: 8935453] NIH-PA Author Manuscript Manzoli L, Villari P, Pirone G, Boccia A. Marital status and mortality in the elderly: a systematic review and meta-analysis. Social Science & Medicine. 2007; 64:77–94. [PubMed: 17011690] NIH-PA Author Manuscript Marcenes W, Sheiham A. The relationship between marital quality and oral health status. Psychology & Health. 1996; 11:357–369. McGrath C, Bedi R. Influences of social support on the oral health of older people in Britain. Journal of Oral Rehabilitation. 2002; 29:918–922. [PubMed: 12421323] Mead N, Lester H, Chew-Graham C, Gask L, Bower P. Effects of befriending on depressive symptoms and distress: systematic review and meta-analysis. British Journal of Psychiatry. 2010; 196:96– 101. [PubMed: 20118451] Merchant AT, Pitiphat W, Ahmed B, Kawachi I, Joshipura K. A prospective study of social support, anger expression and risk of periodontitis in men. Journal of the American Dental Association. 2003; 134:1591–6. [PubMed: 14719755] Monteiro da Silva AM, Oakley DA, Newman HN, Nohl FS, Lloyd HM. Psychosocial factors and adult onset rapidly progressive periodontitis. Journal of Clinical Periodontology. 1996; 23:789–94. [PubMed: 8877667] van Oort F, van Lenthe F, Mackenbach J. Material, psychosocial, and behavioural factors in the explanation of educational inequalities in mortality in the Netherlands. Journal of Epidemiology and Community Health. 2005; 59:214–220. [PubMed: 15710599] J Clin Periodontol. Author manuscript; available in PMC 2012 June 1. Sabbah et al. Sabbah et al. Page 9 Page 9 NIH-PA Author Manuscript Orth-Gomer K, Johnson JV. Social network interaction and mortality. J Clin Periodontol. Author manuscript; available in PMC 2012 June 1. References Journal of Chronic Diseases. 1987; 40:949–957. [PubMed: 3611293] NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Au Page RC, Eke PI. Case definitions for use in population-based surveillance of periodontitis. Journal of Periodontology. 2007; 78:1387–99. [PubMed: 17608611] Pattussi MP, Lalloo R, Bassani DG, Olinto MTA. The role of psychosocial, behavioural and emotional factors on self-reported major injuries in Brazilian adolescents: A case-control study. Injury. 2008; 39:561–569. [PubMed: 18339390] Pattussi MP, Olinto MTA, Hardy R, Sheiham A. Clinical, social and psychosocial factors associated with self-rated oral health in Brazilian adolescents. Community Dentistry and Oral Epidemiology. 2007; 35:377–386. [PubMed: 17822486] Ruberman W, Weinblatt E, Goldberg ID, Chaudhary BS. Psychosocial influences on mortality after myocardial infarction. New England Journal of Medicine. 1984; 311:552–559. [PubMed: 6749228] Manuscript NIH-PA Author Manuscript Sabbah W, Watt RG, Sheiham A, Tsakos G. Effects of allostatic load on the social gradient in ischaemic heart disease and periodontal disease: evidence from the third national health and nutrition examination survey. Journal of Epidemiology and Community health. 2008; 62:415–20. [PubMed: 18413454] Sheiham A, Nicolau B. Evaluation of social and psychological factors in periodontal disease. Periodontology 2000. 2005; 39:118–31. [PubMed: 16135067] NIH-PA Author Manuscript Stansfeld, SA. Social support and social cohesion. In: Marmot, M.; Wilkinson, R., editors. Social determinants of health. Oxford University Press; Oxford: 2006. p. 148-171.2eds NIH-PA Author Manuscript NIH-PA Au NIH-PA Author Manuscript NIH-PA Author Manuscript Page 10 Page 10 NIH-PA Author Manuscript Sabbah et al. Sabbah et al. J Clin Periodontol. Author manuscript; available in PMC 2012 June 1. J Clin Periodontol. Author manuscript; available in PMC 2012 June 1. Table 1 NIH-PA Author Manuscript Mean extent of loss of periodontal attachment and percentage of moderate periodontitis within groups of explanatory variables among older Americans (n=1632) Mean extent of loss of periodontal attachment and percentage of moderate periodontitis within groups of explanatory variables among older Americans (n=1632) explanatory variables among older Americans (n=1632) Explanatory factors (N) Percentage with moderate peridontitis (95%CI) Mean extent of loss of periodontal attachment (95%CI) Gender Males (832) 24.6% (21.3, 28.3) 0.24 (0.21, 0.26) Females (800) 15.7% (13.5, 18.2) 0.16 (0.14, 0.17) Age group 60-70 years (884) 17.1% (14.2, 20.4) 0.17 (0.15, 0.19) 71 and over (748) 23.1% (20.4, 26.1) 0.22 (0.20, 0.25) Race/ethnicity White Americans (948) 17.5% (15.2, 20.0) 0.18 (0.16, 0.20) African Americans (252) 29.9% (24.7, 35.6) 0.25 (0.22, 0.28) Mexican Americans (353) 31.6% (24.3, 40.0) 0.25 (0.20, 0.29) Other Hispanic (40) 37.2% (22.7, 54.3) 0.30 (0.23, 0.37) Other ethnicities (39) 21.8% (11.0, 38.8) 0.21 (0.13, 0.29) Education More than high school (701) 16.0% (13.2, 19.2) 0.16 (0.14, 0.17) High school (384) 18.8% (15.2, 23.0) 0.18 (0.16, 0.21) Less than high school (547) 30.2% (24.9, 36.0) 0.29 (0.25, 0.33) Poverty-income ratio Highest quartile (410) 14.8% (11.7, 18.6) 0.15 (0.13, 0.16) Second highest (407) 16.5% (13.2, 20.4) 0.18 (0.15, 0.20) Second lowest (406) 25.6% (20.9, 30.9) 0.23 (0.20, 0.26) Lowest quartile (409) 34.7% (27.7, 42.4) 0.32 (0.28, 0.36) Marital status Married/ cohabiting (1056) 18.6% (16.2, 21.2) 0.18 (0.16, 0.20) Widowed (341) 23.7% (18.9, 29.4) 0.23 (0.19, 0.26) Divorced, separated, single (253) 19.1% (14.0, 25.4) 0.22 (0.18, 0.25) Number of close friends Highest tertile (621) 16.2% (13.7, 19.1) 0.16 (0.15, 0.18) Middle tertile (519) 19.4% (16.7, 22.4) 0.19 (0.17, 0.21) Lowest tertile (492) 26.2% (21.9, 31.1) 0.25 (0.22, 0.28) Need more emotional help Yes (235) 18.5% (12.9, 25.9) 0.20 (0.16, 0.24) No (1397) 19.9% (17.6, 22.3) 0.19 (0.18, 0.21) Dental visits Within past 2 years (1197) 17.4% (15.1, 20.1) 0.17 (0.15, 0.18) Longer than 2 years (435) 28.2% (23.8, 33.2) 0.29 (0.26, 0.32) Smoking Current smoker (168) 34.8% (27.1, 43.5) 0.37 (0.32, 0.42) Former smoker (656) 22.9% (19.9, 26.3) 0.21 (0.18, 0.23) J Clin Periodontol. Author manuscript; available in PMC 2012 June 1. NIH-PA Author Manuscript Page 11 Page 11 Sabbah et al. Table 1 NIH-PA Author Manuscript Table 3 Income and education gradients in periodontal disease for Americans aged 60 years and over (n=1632) (NHANES 2001-2004) Education (Reference group: > high school) Income (quartiles of poverty-income ratio, reference group: highest quartile) High school < high school 2nd highest quartile 2nd lowest quartile Lowest quartile Rate ratios for extent loss of attachment Model 1 1.16NS (0.94, 1.42) 1.42 (1.15, 1.76) 1.05NS (0.86, 1.29) 1.49 (1.19, 1.87) 1.58 (1.23, 2.04) Model 2 1.11NS (0.89, 1.38) 1.38 (1.12, 1.70) 1.03NS (0.84, 1.27) 1.44 (1.15, 1.79) 1.52 (1.18, 1.96) Odds ratio for moderate periodontitis Model 1 1.18NS (0.87, 1.62) 1.58* (1.03, 2.44) 1.10NS (0.76, 1.59) 1.80 (1.22, 2.67) 2.17 (1.26, 3.73) Model 2 1.13NS (0.82, 1.55) 1.51NS (0.97, 2.37) 1.10NS (0.76, 1.59) 1.76 (1.18, 2.64) 2.19 (1.25, 3.84) Model 1 adjusted for age, gender, race/ ethnicity, number of teeth, diabetes, smoking status (current, former smoker, never-smoker) and dental visits, in addition to income and education. Model 2 additionally adjusted for marital status, number of friends and needing more emotional support. *p<0.001 p<0.01 p<0.05 NSinsignificant J Clin Periodontol. Author manuscript; available in PMC 2012 J Table 3 Income and education gradients in periodontal disease for Americans aged 60 years and over (n=1632) (NHANES 2001-2004) Education (Reference group: > high school) Income (quartiles of poverty-income ratio, reference group: highest quartile) High school < high school 2nd highest quartile 2nd lowest quartile Lowest quartile Rate ratios for extent loss of attachment Model 1 1.16NS (0.94, 1.42) 1.42* (1.15, 1.76) 1.05NS (0.86, 1.29) 1.49 (1.19, 1.87) 1.58 (1.23, 2.04) Model 2 1.11NS (0.89, 1.38) 1.38 (1.12, 1.70) 1.03NS (0.84, 1.27) 1.44 (1.15, 1.79) 1.52** (1.18, 1.96) Odds ratio for moderate periodontitis Model 1 1.18NS (0.87, 1.62) 1.58* (1.03, 2.44) 1.10NS (0.76, 1.59) 1.80 (1.22, 2.67) 2.17 (1.26, 3.73) Model 2 1.13NS (0.82, 1.55) 1.51NS (0.97, 2.37) 1.10NS (0.76, 1.59) 1.76 (1.18, 2.64) 2.19 (1.25, 3.84) Model 1 adjusted for age, gender, race/ ethnicity, number of teeth, diabetes, smoking status (current, former smoker, never-smoker) and dental visits, in addition to income and education. Model 2 additionally adjusted for marital status, number of friends and needing more emotional support. *p<0.001 p<0.01 p<0.05 NSinsignificant J Clin Periodontol. Author manuscript; available in PMC 2012 June 1. Table 1 Explanatory factors (N) Percentage with moderate peridontitis (95%CI) Mean extent of loss of periodontal attachment (95%CI) Never smoked (808) 14.3% (12.0, 17.0) 0.15 (0.13, 0.17) diabetes Yes (274) 21.9% (16.8, 28.2) 0.23 (0.19, 0.27) No (1358) 19.3% (17.5, 21.3) 0.19 (0.17, 0.20) NIH-PA Author Manuscript NIH-PA Author ript NIH-PA Author Manuscript NIH-PA Author Manuscript Page 12 Page 12 NIH-PA Author Manuscript Sabbah et al. Sabb Table 2 ween social network and support indicators and two measures of periodontal disease among Americans aged 60 years and over (n=1632) -2004) Table 2 Association between social network and support indicators and two measures of periodontal disease among Americans aged 60 years and over (n=1632) (NHANES 2001-2004) Marital status (Reference group: married) Need more emotional support Number of friends (Reference group: highest Tertile) widowed Divorced/ separated Middle Tertile Lowest Tertile Rate ratio for extent loss of attachment Model 1 1.60 (1.27, 2.01) 1.33 (1.09, 1.63) 0.98NS (0.74, 1.30) 1.05NS (0.88, 1.26) 1.43* (1.21, 1.68) Model 2 1.33 (1.09, 1.63) 1.14NS (0.90, 1.44) 0.86NS (0.69, 1.07) 1.02NS (0.85, 1.22) 1.23* (1.05, 1.46) Model 3 1.27* (1.03, 1.58) 1.07NS (0.86, 1.34) 0.87NS (0.72, 1.-6) 0.99NS (0.83, 1.19) 1.22* (1.03, 1.45) Odds ratio for moderate periodontitis Model 1 1.65* (1.09, 2.50) 1.07NS (0.70, 1.61) 0.86NS (0.51, 1.44) 1.19NS (0.93, 1.53) 1.53 (1.15, 2.04) Model 2 1.36NS (0.88, 2.10) 0.88NS (0.56, 1.37) 0.73NS (0.45, 1.20) 1.14NS (0.87, 1.78) 1.32NS (0.97, 1.78) Model 3 1.28NS (0.82, 2.00) 0.80NS (0.52, 1.24) 0.76NS (0.46, 1.25) 1.09NS (0.84, 1.40) 1.29NS (0.95, 1.77) Model 1 adjusted for age, gender and race/ ethnicity, in addition to the variables of social network/ support. Model 2 additionally adjusted for income, education, diabetes and number of teeth. Model 3 additionally adjusted for smoking status (current, former smoker, never-smoker) and dental visits. J Clin Periodontol. Author manusc es of periodontal disease among Americans aged 60 years and over (n=1632) NIH-PA Author Manuscript NIH-PA Author Manuscript J Clin Periodontol. Author manuscript; available in PMC 2012 June 1. NIH-PA Author Manuscript Page 13 Page 13 Sabbah et al. J Clin Periodontol. Author manuscript; available in PMC 2012 June 1. Table 1 Table 3 Income and education gradients in periodontal disease for Americans aged 60 years and over (n=1632) (NHANES 2001-2004) Education (Reference group: > high school) Income (quartiles of poverty-income ratio, reference group: highest quartile) High school < high school 2nd highest quartile 2nd lowest quartile Lowest quartile Rate ratios for extent loss of attachment Model 1 1.16NS (0.94, 1.42) 1.42 (1.15, 1.76) 1.05NS (0.86, 1.29) 1.49 (1.19, 1.87) 1.58 (1.23, 2.04) Model 2 1.11NS (0.89, 1.38) 1.38 (1.12, 1.70) 1.03NS (0.84, 1.27) 1.44 (1.15, 1.79) 1.52** (1.18, 1.96) Odds ratio for moderate periodontitis Model 1 1.18NS (0.87, 1.62) 1.58* (1.03, 2.44) 1.10NS (0.76, 1.59) 1.80 (1.22, 2.67) 2.17 (1.26, 3.73) Model 2 1.13NS (0.82, 1.55) 1.51NS (0.97, 2.37) 1.10NS (0.76, 1.59) 1.76 (1.18, 2.64) 2.19 (1.25, 3.84) Model 1 adjusted for age, gender, race/ ethnicity, number of teeth, diabetes, smoking status (current, former smoker, never-smoker) and dental visits, in addition to income and education. Model 2 additionally adjusted for marital status number of friends and needing more emotional support J Clin Periodontol NIH-PA Author Manuscript J Clin Periodontol. Author manuscript; available in PMC 2012 June 1.
https://openalex.org/W3164081297	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/B57E5023601F1D38CDA3D68827775413/S0020589320000469a.pdf/div-class-title-polycentricity-and-polyphony-in-international-law-interpreting-the-corporate-responsibility-to-respect-human-rights-div.pdf	English	null	Polycentricity and Polyphony in International Law. Interpreting the Corporate Responsibility to Respect Human Rights	Social Science Research Network	2,020	cc-by	18,993	© The Author(s), 2020. Published by Cambridge University Press on behalf of the British Institute of International and Comparative Law. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited * Assistant Professor of Transnational Regulation and Governance, Tilburg University; Associate Researcher, T.M.C. Asser Institute, e.d.partiti@tilburguniversity.edu. The author would like to thank Nadia Bernaz, Stephanie Bijlmakers, Nikolas Rajkovic, the participants of Tilburg University’s International Legal Incubator series and the two anonymous reviewers. All errors remain responsibility of the author alone. This contribution was written in the context of a research project that received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (Grant Agreement No 725798). [ICLQ vol 70, January 2021 pp 133–164] https://doi.org/10.1017/S0020589320000469 Published online by Cambridge University Press 2 Human Rights Council, ‘Guiding Principles on Business and Human Rights: Implementing the United Nations’ “Protect, Respect and Remedy” Framework’, A/HRC/17/31 (21 March 2011). 4 A Peters, L Koechlin, T Förster and GF Zinkernagel (eds), Non-State Actors as Standard Setters (Cambridge University Press 2009). On corporations implementing and enforcing international law: J Butler, ‘The Corporate Keepers of International Law’ (2020) 114 AJIL 189. 5 p p ( ) 5 JG Ruggie ‘Global Governance and “New Governance Theory”: Lessons from Business and Human Rights’ 20 Global Governance 8. 6 9 H Krieger and A Peters (eds), Due Diligence in International Law (Oxford University Press 2020) forthcoming; N McDonald, ‘The Role of Due Diligence in International Law’ (2019) 68 ICLQ 1041. 3 J Pauwelyn, R Wessel and J Wouters (eds), Informal International Lawmaking (Oxford University Press 2012). 4 1 Human Rights Council Res. 8/7, UN Doc A/C/RES/8/7 (18 June 2008). 2 g ( g y ) 7 J Van Erp, M Faure, A Nollkaemper and N Philipsten (eds), Smart Mixes for Transboundary Environmental Harm (Cambridge University Press 2019) 9. 6 See generally KW Abbott, P Genschel, D Snidal and B Zangl (eds), International Organisations as Orchestrators (Cambridge University Press 2015). International and Comparative Law Quarterly International and Comparative Law Quarterly POLYCENTRICITY AND POLYPHONY IN INTERNATIONAL LAW: INTERPRETING THE CORPORATE RESPONSIBILITY TO RESPECT HUMAN RIGHTS ENRICO PARTITI* Abstract Complex multi-actors and multi-level governance structures have emerged in areas that were traditionally exclusively the preserve of the State and treaty-making. The adoption of the United Nations Guiding Principles on Business and Human Rights (UNGP) aﬃrmed a corporate responsibility to respect human rights to be implemented through human rights due diligence (HRDD), ie via management processes. The open- ended character of the UNGP generated the emergence of other soft instruments oﬀering guidance to corporations in structuring HRDD. This contribution conceptualises the UNGP from the perspective of regulation as a principles-based exercise in polycentric governance reliant on regulatory intermediaries for interpretation. It then assesses the role of various sui generis normative instruments in providing interpretation to the UNGP and, how the presence of an additional layer of interpretative material contributes to the institutionalisation of responsible corporate conduct. The analysis of instruments drafted by international, non- governmental and business organisations reveals both a decentralising tension between diﬀerent intermediaries due to disagreements and divergence concerning the precise extent of corporate human rights responsibilities, as well as attempts to centralise the interpretation of the UNGP. The article concludes by recommending some caution towards the employment of polycentric governance regimes and their lack of centralised interpretive authority in this domain of international law and suggests possible ways to formally establish centralised interpretation. Keywords: human rights, human rights due diligence, interpretation, polycentricity, United Nations Guiding Principles on Business and Human Rights, corporate responsibility to respect. * Assistant Professor of Transnational Regulation and Governance, Tilburg University; Associate Researcher, T.M.C. Asser Institute, e.d.partiti@tilburguniversity.edu. The author would like to thank Nadia Bernaz, Stephanie Bijlmakers, Nikolas Rajkovic, the participants of Tilburg University’s International Legal Incubator series and the two anonymous reviewers. All errors remain responsibility of the author alone. This contribution was written in the context of a research project that received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (Grant Agreement No 725798). doi:10.1017/S0020589320000469 doi:10.1017/S0020589320000469 [ICLQ vol 70, January 2021 pp 133–164] 134 1 Human Rights Council Res. 8/7, UN Doc A/C/RES/8/7 (18 June 2008). 2 Human Rights Council, ‘Guiding Principles on Business and Human Rights: Implementing the United Nations’ “Protect, Respect and Remedy” Framework’, A/HRC/17/31 (21 March 2011). 3 J Pauwelyn, R Wessel and J Wouters (eds), Informal International Lawmaking (Oxford University Press 2012). 4 A Peters, L Koechlin, T Förster and GF Zinkernagel (eds), Non-State Actors as Standard Setters (Cambridge University Press 2009). On corporations implementing and enforcing international law: J Butler, ‘The Corporate Keepers of International Law’ (2020) 114 AJIL 189. 5 JG Ruggie ‘Global Governance and “New Governance Theory”: Lessons from Business and Human Rights’ 20 Global Governance 8. 6 See generally KW Abbott, P Genschel, D Snidal and B Zangl (eds), International Organisations as Orchestrators (Cambridge University Press 2015). 7 J Van Erp, M Faure, A Nollkaemper and N Philipsten (eds), Smart Mixes for Transboundary Environmental Harm (Cambridge University Press 2019) 9. 8 G De Búrca ‘Human Rights Experimentalism’ (2017) 111 AJIL 277. 9 H Krieger and A Peters (eds), Due Diligence in International Law (Oxford University Press 2020) forthcoming; N McDonald, ‘The Role of Due Diligence in International Law’ (2019) 68 ICLQ 1041. g y , , g ( ), Organisations as Orchestrators (Cambridge University Press 2015). 7 J Van Erp, M Faure, A Nollkaemper and N Philipsten (eds), Smart Mixes for Transboundary E i l H (C b id U i it P 2019) 9 ( g y ) 8 G De Búrca ‘Human Rights Experimentalism’ (2017) 111 AJIL 277. Organisations as Orchestrators (Cambridge University Press 2015). 7 J Van Erp M Faure A Nollkaemper and N Philipsten (eds) Smart Mixes for Transboundary J Van Erp, M Faure, A Nollkaemper and N Philipsten (eds), Smart Mixes Environmental Harm (Cambridge University Press 2019) 9. See generally KW Abbott, P Genschel, D Snidal and B Zangl (eds), International Organisations as Orchestrators (Cambridge University Press 2015). 7 J Van Erp M Faure A Nollkaemper and N Philipsten (eds) Smart Mixes for Transboundary I. INTRODUCTION The unanimous adoption by the United Nations’ Human Rights Council of the Respect, Protect, Remedy Framework1 designed by Harvard Professor John Ruggie, and its consequent operationalisation in the almost universally acclaimed United Nations (UN) Guiding Principles on Business and Human Rights (UNGP),2 illustrate the pivotal role that both informal norm-making processes3 and private actors, including single corporations, have acquired in international law.4 While recognising and stigmatising the role of business in human rights violations across the globe, the UNGP framework seeks to leverage traditional internal corporate approaches to manage risks arising from the intersection of transnational corporate activities, regulation of global value chains, and human rights. Ruggie described the UNGP as a ‘conceptual and normative framework establishing the parameters and perimeters of business and human rights as an international policy domain’,5 with the aim of translating human rights into the processes that corporations employ to manage their risks. The UNGP therefore constitutes a testament to the increasingly frequent formal and informal use by public authorities of private actors, and their rules and standards, in the transnational pursuit of public goals6 and in the quest for an eﬀective ‘smart mix’ of regulatory actors, instruments and techniques7—especially in the ﬁeld of human rights.8 The UNGP framework rests on three pillars establishing a State duty to protect against human rights abuses, matched by corporate responsibility to respect human rights, and the right of victims to have access to remedies. Human rights due diligence (HRDD) represents the most original feature of the UNGP. Part of a growing trend in international law focusing on procedural rather than substantive norms,9 the UNGP urges business to use HRDD and incorporate respect for human rights at the core of internal management processes and practice. 15 Ruggie (n 5); JG Ruggie, ‘The Social Construction of the UN Guiding Principles on Business and Human Rights’ (2017) Corporate Responsibility Initiative Working Paper No 67. Cambridge, MA: John F. Kennedy School of Government, Harvard University. 14 S Bijlmakers, Corporate Social Responsibility, Human Rights and the Law (Routledge 2018) 48–52. 15 p 12 For a discussion and critique on the actual inclusiveness in the UNGP drafting: S Bijlmakers, ‘Business and Human Rights Governance and Democratic Legitimacy: The UN ‘‘Protect, Respect and Remedy’’ Framework and the Guiding Principles’ (2013) 26 Innovation 288. 13 10 Oﬃce of the High Commissioner on Human Rights (2012) The Corporate Responsibility to Respect Human Rights. An Interpretative Guide. HR/PUB/12/02, 6. 11 y g p ( ) 13 S Deva and D Bilchitz ‘The Human Rights Obligations of Business: A Critical Framework for the Future’ in S Deva and D Bilchitz (eds), Human Rights Obligations of Business: Beyond the Corporate Responsibility to Respect? (Cambridge University Press 2013). 14 p g 11 UNGP Principle 17. 12 https://doi.org/10.1017/S0020589320000469 Published online by Cambridge University Press p 12 For a discussion and critique on the actual inclusiveness in the UNGP drafting: S Bijlmakers, ‘Business and Human Rights Governance and Democratic Legitimacy: The UN ‘‘Protect, Respect and Remedy’’ Framework and the Guiding Principles’ (2013) 26 Innovation 288. 13 S Deva and D Bilchitz ‘The Human Rights Obligations of Business: A Critical Framework for the Future’ in S Deva and D Bilchitz (eds), Human Rights Obligations of Business: Beyond the Corporate Responsibility to Respect? (Cambridge University Press 2013). 14 S Bijlmakers, Corporate Social Responsibility, Human Rights and the Law (Routledge 2018) 48–52. 15 Ruggie (n 5); JG Ruggie, ‘The Social Construction of the UN Guiding Principles on Business and Human Rights’ (2017) Corporate Responsibility Initiative Working Paper No 67. Cambridge, MA: John F. Kennedy School of Government, Harvard University. ‘Business and Human Rights Governance and Democratic Legitimacy: The UN ‘‘Protect, Respect and Remedy’’ Framework and the Guiding Principles’ (2013) 26 Innovation 288. 13 S Deva and D Bilchitz ‘The Human Rights Obligations of Business: A Critical Framework for the Future’ in S Deva and D Bilchitz (eds), Human Rights Obligations of Business: Beyond the Corporate Responsibility to Respect? (Cambridge University Press 2013). 14 S Bijlmakers, Corporate Social Responsibility, Human Rights and the Law (Routledge 2018) 48–52. 16 C Rodríguez-Garavito, ‘Business and Human Rights: Beyond the End of the Beginning’ in C Rodríguez-Garavito (ed), Business and Human Rights: Beyond the End of the Beginning (Cambridge University Press 2017) 22. 17 JG Ruggie, ‘Business and Human Rights: The Evolving International Agenda’ (2007) 101 AJIL 819; Ruggie (n 5). 18 HH Koh, ‘Is There a ‘‘New’’ New Haven School of International Law?’ (2007) 32 YaleJIntlL 567–8. 19 G Shaﬀer and T Ginsburg, ‘The Empirical Turn in International Legal Scholarship’ (2012) 106 AJIL 1. I. INTRODUCTION HRDD ‘comprises an ongoing https://doi.org/10.1017/S0020589320000469 Published online by Cambridge University Press 135 Polycentricity and Polyphony in International Law management process that a reasonable and prudent enterprise needs to undertake, in the light of its circumstances (including sector, operating context, size and similar factors) to meet its responsibility to respect human rights’.10 In addition to the publication of a policy commitment and ensuring internal remedial mechanisms, HRDD should include human rights impact assessments, the integration of their outcomes in corporate procedures, the tracking of responses, and external communication.11 One of the peculiarities of the UNGP Framework is its consensual multi- stakeholder ‘production process’,12 it being the ﬁrst instrument not drafted by States to be adopted by the UN Human Rights Council. In light of the need to obtain broad support—including from corporations—the UNGP are a framework not just characterised by limited options for enforcement, but also by indeterminacy in its substance.13 An approach characterised by ‘principled pragmatism’ and a pragmatic conception of the corporate responsibility to respect have led to broad principles being distilled on the basis of existing business practices through studies and multi-stakeholder engagement. Broad principles enhance ﬂexibility for companies when giving meaning to HRDD and may facilitate compliance by stimulating the adoption of measures commensurate to the capabilities of each company and appropriate to the circumstances.14 The open-ended nature of the UNGP leaves corporations with a wide margin of discretion when deciding how to operationalise HRDD. On the one hand, the UNGP create, foster, and legitimise the activity of private actors such as corporations in the protection and enforcement of human rights. On the other, the UNGP provide only limited opportunities to constrain and guide their implementation by ﬁrms in practical contexts. Given the lack of clear guidance, corporations can exploit the open framework by making unsubstantiated claims to respect human rights or to dilute their responsibility. I. INTRODUCTION By design, the UNGP were established as an exercise in transnational polycentric governance, bringing together public governance, civil governance and corporate governance.15 Such a polycentric structure reﬂects not just the inherently pluralistic nature of the legal regulation of https://doi.org/10.1017/S0020589320000469 Published online by Cambridge University Press 136 International and Comparative Law Quarterly business and human rights which derives from a multiplicity of national, international, transnational hard and soft law, including private regulation.16 Ruggie hoped that the UNGP would mark the beginning of an orchestrating process between these three governance systems, in which the various institutions would align their respective instruments, further develop and bring about the interpretation and implementation of the UNGP in order to contribute to the fulﬁlment of human rights.17 The UNGP thus aims at instigating and supporting a process of bottom-up emergence and convergence of transnational regulatory regimes establishing businesses’ human rights responsibility. They are just the starting point for a transnational process of assertion and reﬁnement of the boundaries of corporate human rights responsibility by governmental organisations, private actors, transnational networks and interpretative communities.18 A global conversation over responsible business conduct is thus expected to remedy the indeterminacy of the UNGP. Many public and non-public actors are called on to help guide the application of the Principles to speciﬁc sectors, contexts of operation, and factual situations. The operationalisation of HRDD by corporations is mediated and inﬂuenced by the interpretive and implementing work of many actors, with many and varying goals. This article analyses how selected actors, such as international organisations, non-governmental organisations (NGOs) and business associations, that have been formally and informally called on to implement and give meaning to the general principles in the UNGP and the boundaries of HRDD, have interpreted and transposed them into standards, guidelines and best practices. It explores the conditions and the processes through which the corporate responsibility to respect human rights is formed.19 It begins by sketching a framework for understanding the UNGP based on polycentric regulation, principles-based regulation and management-based regulation. The approach links the ‘macro’ level of international law and global governance structures with the ‘micro’ level of corporations, their practices and incentives. Drawing on literature concerning regulatory intermediaries, the article discusses the way in which key actors interpret, and to a lesser extent implement, the UNGP. 20 PS Berman, ‘Global Legal Pluralism’ (2007) 80 SCalLRev 1155. 21 G Auld and S Renckens ‘Rule-making Feedbacks through Intermediation and Evaluation in Transnational Private Governance’ (2017) 670 Annals of the American Academy of Political and Social Science 93. 22 D De Felice ‘Banks and Human Rights Due Diligence: A Critical Analysis of the Thun Group’s Discussion Paper on the UN Guiding Principles on Business and Human Rights’ (2015) 19 IJHR 321. 23 Other crucial issues include which human rights corporations have to respect—a complex questions where human rights originally established as oﬀering protection from States are involved—and what precise actions are required by corporations in ‘enforcing’ human rights with respect to other actors in the value chain to which they are linked. 24 These are conceptually diﬀerent from those under the Articles on State Responsibilities for attribution of private conduct to the State. Draft Articles on Responsibility of States for Internationally Wrongful Acts, with commentaries. Yearbook of the International Law Commission (2001) vol II, Pt Two, 31, arts 5, 8 and 11. 24 These are conceptually diﬀerent from those under the Articles on State Responsibilities for attribution of private conduct to the State. Draft Articles on Responsibility of States for Internationally Wrongful Acts, with commentaries. Yearbook of the International Law Commission (2001) vol II, Pt Two, 31, arts 5, 8 and 11. https://doi.org/10.1017/S0020589320000469 Published online by Cambridge University Press 22 D De Felice ‘Banks and Human Rights Due Diligence: A Critical Analysis of the Thun Group’s Discussion Paper on the UN Guiding Principles on Business and Human Rights’ (2015) 19 IJHR 321. 23 23 Other crucial issues include which human rights corporations have to respect—a complex questions where human rights originally established as oﬀering protection from States are involved—and what precise actions are required by corporations in ‘enforcing’ human rights with respect to other actors in the value chain to which they are linked. 24 20 PS Berman, ‘Global Legal Pluralism’ (2007) 80 SCalLRev 1155. I. INTRODUCTION Studying the consequences of intermediation for the interpretation of the UNGP, and in particular the boundaries of HRDD, is of key importance when seeking to understand the corporate responsibility to respect human rights as set out in the UNGP, and its potential eﬀectiveness. Norms are https://doi.org/10.1017/S0020589320000469 Published online by Cambridge University Press 137 Polycentricity and Polyphony in International Law generated and reﬁned through the interactions and practices of diﬀerent normative communities.20 At the early stages of the development of any regime actors interpreting general and broad rules have a signiﬁcant impact on its evolution and on how it is to be applied.21 Deviations or misinterpretation of the UNGP have long-lasting repercussions for how corporations structure their HRDD and manage their human rights risks.22 Among the points left open in the UNGP Framework,23 this article focuses on the three categories for the ‘attribution’24 of negative human rights impacts to corporations which are identiﬁed in the UNGP: ie causation, contribution and direct linkage. These concepts are not straightforward to apply to the wide variety of contractual and factual situations characterising contemporary global value chains. In line with the polycentric nature of the UNGP, standards, guidelines and best practices drafted by international organisations, non-governmental organisations (NGOs) and business associations oﬀer more precise frameworks for HRDD. However, as this article will show, the interpretations oﬀered may also considerably deviate from the UNGP. This divergence exposes coordination challenges and a number of unconventional strategies have been adopted in order to ‘steer’ intermediaries’ interpretation. This contribution reﬂects on the appropriateness of the polycentric design of the UNGP and argues that polycentricity, an indispensable governance feature in the UNGP according to Ruggie, may hinder business responsibility to respect human rights. Polycentricity risks degenerating into polyphony, ie unavoidable divergences between equally valid interpretative approaches. While the absence of central authority, textual uncertainty and divergence in interpretation are well-known issues in international law, the UNGP have been deliberately designed to rely on, and embrace, broad principles and ‘decentralised’ interpretation via polycentricity as key vehicles through which to establish and advance a corporate responsibility to respect. This article argues that such an approach has its drawbacks, as it is structurally open to divergence in interpretation. I. INTRODUCTION Divergence is particularly problematic where corporations have to operationalise provisions originating https://doi.org/10.1017/S0020589320000469 Published online by Cambridge University Press International and Comparative Law Quarterly 138 in soft law, and it threatens the establishment of a clear and uncontested corporate responsibility to respect human rights, and the boundaries thereof. This article suggests that ‘centralised’ forms of interpretation—more in line with international law instruments—are necessary for the establishment of a corporate responsibility to respect. It will consider whether more centralised interpretative approaches are already emerging as a result of the process to adopt a binding treaty on Business and Human Rights which is currently taking place. This article is structured as follows. Section II explores the transnational regulatory governance resulting from the UNGP through a theoretical framework synthesising polycentric governance, principles-based regulation, management-based regulation and the regulator-intermediary-target (RIT) model. It discusses HRDD and its ongoing interpretation, stressing the importance of there being substantive clarity concerning the ‘attribution’ of impacts to a corporation. This framework allows the identiﬁcation of challenges connected to the governance structures informally established around the UNGP concerning interpretive functions, and their consequences for the operationalisation of HRDD by businesses. Section III discusses the interpretative work found in public and private standards and guidelines concerning attribution. It assesses the International Organisation for Standardisation (ISO) 26000 standard, relevant sectoral guidance documents by the Organisation for Economic Cooperation and Development (OECD), the International Finance Corporation’s (IFC) Performance Criteria, the International Labour Organisation (ILO) tripartite Declaration of Principles concerning Multinational Enterprises and Social Policy, and guidance provided by the Thun Group of Banks, an association of private ﬁnancial entities. The section identiﬁes a number of tensions and divergences with the UNGP, explicit attempts to dilute their obligations, and attempts by some of these bodies to retain interpretive control. Section IV reﬂects upon the challenges of a polycentric approach for the development of a corporate responsibility to respect human rights, as well as possible alternatives. Section V concludes. 25 United Nations, Oﬃce of the High Commissioner on Human Rights, ‘Frequently Asked Questions about the Guiding Principles on Business and Human Rights’ (2014) 9–10. 26 R Mares, ‘Global Corporate Social Responsibility, Human Rights and The Law: An Interactive Regulatory Perspective on the Voluntary-Mandatory Dichotomy’ (2010) 1 TLT 221. 27 UNGP Principle 11. 28 B Santoso, ‘‘‘Just Business’’: Is the Current Regulatory Framework an Adequate Solution to Human Rights Abuses by Transnational Corporations?’ (2017) 18 German Law Journal 549. Polycentricity and Polyphony in International Law 139 A. The Regulatory Eﬀects of HRDD As described in the UNGP, HRDD diﬀers from corporate social responsibility (CSR) because it constitutes a global and overarching expectation of all corporations, rather than being a voluntary undertaking in a speciﬁc ﬁeld and in respect of a restricted number of beneﬁciaries.25 In contrast to a voluntary understanding of CSR, reﬂecting various economic and non-economic motivations, the conceptualisation of corporate responsibility advanced by the UNGP is grounded in case law and business practices which accepts and institutionalises a ‘duty of care’ towards aﬃliates.26 This responsibility, however, does not just encompass the (self) regulation of corporate activities between a mother company and its subsidiaries, but extends to all human rights impacts stemming from all commercial and non-commercial relations that a corporation has, or may have, with other business entities (including governments), employees, customers, and human rights holders aﬀected by its operations.27 Companies have a considerable margin when determining how to structure their HRDD. However, the manner in which it is operationalised identiﬁes the scope of its human rights responsibility, and determines its response to human rights risks in a way that not only aﬀects human rights holders but also other business entities, and (at least in theory) forms the basis for future remediation.28 The UNGP require corporations to seek to identify, prevent, and use their leverage to mitigate human rights impacts directly linked to their operations, as well as requiring remediation in speciﬁc situations. UNGP 13 acknowledges that corporations can be associated with adverse human rights impacts, both through their own activities, or as a result of their business relationships. In the ﬁrst case, corporations are considered to have caused negative impacts, and therefore have to take the necessary steps to cease or prevent such impacts through HRDD and to oﬀer remediation. Where a corporation generates adverse human rights impacts through its commercial relations, two scenarios are possible. A corporation contributes to negative human rights impacts where impacts are co-generated either with another entity, or via a third entity with which it has a business relationship. In this case, the enterprise should take the necessary steps to cease or prevent its contribution, and in addition use its leverage to mitigate any remaining impacts. In the second scenario it is possible to trace a direct link between adverse impacts and a corporation’s operations, products or services through its commercial relations with other companies. 25 United Nations, Oﬃce of the High Commissioner on Human Rights, ‘Frequently Asked Questions about the Guiding Principles on Business and Human Rights’ (2014) 9–10. g y 27 UNGP Principle 11. 28 B Santoso, ‘‘‘Just Business’’: Is the Current Regulatory Framework an Adequate Solution to Human Rights Abuses by Transnational Corporations?’ (2017) 18 German Law Journal 549. Q g p g ( ) 26 R Mares, ‘Global Corporate Social Responsibility, Human Rights and The Law: An Interactive Regulatory Perspective on the Voluntary-Mandatory Dichotomy’ (2010) 1 TLT 221. 27 UNGP Principle 11 II. THE REGULATORY GOVERNANCE OF THE UNGP AND HRDD This section explores the conceptual and theoretical background to both the UNGP framework and HRDD with reference to scholarship on transnational regulation. From a regulation perspective, studying an international (soft) law instrument and the complex governance structures it has generated exposes potential challenges resulting from the architecture of the UNGP and the characteristics of HRDD that have so far been overlooked. At the same time, it allows an appraisal from a diﬀerent perspective to that of international law, of the implications of replacing traditional international law instruments with complex multi-level governance structures reliant on soft law. https://doi.org/10.1017/S0020589320000469 Published online by Cambridge University Press Polycentricity and Polyphony in International Law 26 R Mares, ‘Global Corporate Social Responsibility, Human Rights and The Law: An Interactive Regulatory Perspective on the Voluntary-Mandatory Dichotomy’ (2010) 1 TLT 221. 27 UNGP Principle 11. 28 B Santoso, ‘‘‘Just Business’’: Is the Current Regulatory Framework an Adequate Solution to Human Rights Abuses by Transnational Corporations?’ (2017) 18 German Law Journal 549. 29 UNGP Principle 19 and commentary. 30 GA Sarfaty, ‘Shining Light on Global Supply Chains’ (2015) 56 HarvIntlLJ 419. 31 UNGP Principle 17. 32 UNGP Principle 19. 33 E Bardach and R Kagan, Going by the Book: The Problem of Regulatory Unreasonableness (Temple University Press 1982) 224. 34 J Black, ‘Paradoxes and Failures: New Governance Techniques and the Financial Crisis’ (2012) 75 MLR 1037. 35 J Black, ‘Forms and Paradoxes of Principles-Based Regulation’ (2008) 3 CMLJ 430. 36 V Di Lorenzo, ‘Principles-Based Regulation and Legislative Congruence’ (2012) 45 NYUJLeg&Pol 46. A. The Regulatory Eﬀects of HRDD The UNGP acknowledge that https://doi.org/10.1017/S0020589320000469 Published online by Cambridge University Press 140 International and Comparative Law Quarterly this scenario is the most complex both in terms of identiﬁcation and in terms of determining appropriate action, as it may involve entities with which the company has no direct contractual relationship, such as the supplier of a supplier. Generally, no remediation is required, and the company should use its leverage to prevent or mitigate adverse impacts.29 This scenario is the most far reaching, since it potentially stretches upstream into a company’s value chain. Through the concepts of ‘contribution’ and, especially, of ‘direct linkage’, corporations are required to act as a transnational value chain (self)regulator/ enforcer in the ﬁeld of human rights.30 Corporations are called on to design procedures to monitor their human rights impacts and ensure that international human rights norms are respected in their activities and business relations throughout the entirety of their supply chain. As regards ‘contribution’ and ‘direct link’, this includes strategies to engage with other entities, to exercise leverage, and ultimately to ‘sanction’ non-complying suppliers through disengagement. Internally, HRDD is operationalised by assessing actual and potential human rights impacts, integrating and acting upon ﬁndings, tracking responses, and communicating how such impacts are addressed.31 Such processes should be fully integrated.32 HRDD has, therefore, many similarities with procedural and management-based approaches to regulation, requiring corporations to engage in their own planning, information-collection and internal rule-making.33 29 UNGP Principle 19 and commentary. 30 B. Principles-Based Regulation The operationalisation of HRDD by corporations must be discussed in the light of its origins, being embedded within the UNGP which has a principles-based nature. Principles-based regulation (PBR) encompasses a variety of techniques.34 PBR involves regulators communicating their goals via broad principles, while regulatees are required to engage in self-reﬂection and develop internal management-based regulation which ensures that the desired goals are met.35 Proponents of PBR argue that broad principles are more successful than detailed rules in achieving eﬀectiveness and congruence with overarching legislative purposes.36 The transposition of these ‘new governance’ techniques to the transnational stage of regulation of the global https://doi.org/10.1017/S0020589320000469 Published online by Cambridge University Press 141 Polycentricity and Polyphony in International Law commons is not a new phenomenon.37 As noted by Ruggie, the UNGP extend this approach to the ﬁeld of business and human rights.38 Similarly to PBR, the UNGP are grounded on a broad normative, qualitative, and purposive principle that corporations should generally respect human rights in their activities.39 To meet this goal, corporations are required to adopt, design and continuously evaluate and update managerial risk-based processes.40 This approach shifts the responsibility to design regulatory solutions to the regulated entities, which are required to determine what needs to be done in order to meet the objectives provided by the norms in question.41 As has been said, the UNGP do not provide concrete guidance for corporations concerning the implementation of their HRDD responsibilities, leaving considerable leeway as regards their practical application. However, requiring corporations to engage in critical learning—such as acquiring information concerning human rights impacts, and to ‘know and show’ that compliance with human rights occurs42—is not suﬃcient to meet the goals of the regime. Regardless of the tools of enforcement, and to minimise recourse to sanctions and litigation, it is necessary to ensure that the correct tasks are performed, and in the proper manner. 37 KW Abbott and D Snidal, ‘The Governance Triangle: Regulatory Standards Institutions and the Shadow of the State’ in W Mattli and N Woods (eds), The Politics of Global Regulation (Princeton University Press 2009) 44–88. 38 Ruggie (n 5). 39 UNGP Principle 11. 42 JG Ruggie and JF Sherman ‘The Concept of ‘‘Due Diligence’’ in the UN Guiding Principles on Business and Human Rights: Reply to Professors Bonnitcha and McCorquodale’ (2017) 28 EJIL 921–8. 43 Black (n 34) 1062–3. 44 Black (n 35) 426. 45 N Mazar, O Amir and D Ariely, ‘The Dishonesty of Honest People: A Theory of Self-Concept Maintenance’ (2008) 45 Journal of Marketing Research 633. 46 37 KW Abbott and D Snidal, ‘The Governance Triangle: Regulatory Standards Institutions and the Shadow of the State’ in W Mattli and N Woods (eds), The Politics of Global Regulation (Princeton University Press 2009) 44–88. 38 Ruggie (n 5). 39 UNGP Principle 11. 40 UNGP Principles 15 and 17. 41 Black (n 35) 443. 42 JG Ruggie and JF Sherman ‘The Concept of ‘‘Due Diligence’’ in the UN Guiding Principles on Business and Human Rights: Reply to Professors Bonnitcha and McCorquodale’ (2017) 28 EJIL 921–8. 43 Black (n 34) 1062–3. 44 Black (n 35) 426. 45 N Mazar, O Amir and D Ariely, ‘The Dishonesty of Honest People: A Theory of Self-Concept Maintenance’ (2008) 45 Journal of Marketing Research 633. 46 C Parker ‘The ‘‘Compliance’’ Trap: The Moral Message in Regulatory Enforcement’ (2006) 40 L&Soc’yRev 609. 47 CL Ford, ‘New Governance, Compliance and Principles-Based Securities Regulation’ (2008) AmBusLJ 37. 48 J Zeitlin, ‘Introduction’ in J Zeitlin (ed), Extending Experimentalist Governance? The European Union and Transnational Regulation (Oxford University Press 2015) 1–19. ( y ) 40 UNGP Principles 15 and 17. 47 CL Ford, ‘New Governance, Compliance and Principles-Based Securities Regulation’ (2008) AmBusLJ 37. ( y ) gg ( ) p 40 UNGP Principles 15 and 17. 41 Black (n 35) 443. 42 B. Principles-Based Regulation When engaging in PBR, structures should be established which do not rely on the assumption that corporations have both the incentives and the capacities to properly implement the principles and manage their risks.43 It is of fundamental importance that clear goals and outcomes are communicated.44 Ambiguity can provide justiﬁcations for undesirable behaviour.45 The lack of a precise and shared understanding of how principles should be interpreted and operationalised hinders the achievement of the goals of the regime and compliance with those goals.46 Crucial elements which determine the eﬀectiveness of PBR are the mechanisms and actors that add content to the principles via interpretation,47 and further the understanding of both regulators and regulated entities.48 42 JG Ruggie and JF Sherman ‘The Concept of ‘‘Due Diligence’’ in the UN Guiding Principles on Business and Human Rights: Reply to Professors Bonnitcha and McCorquodale’ (2017) 28 EJIL 921–8. 43 Black (n 34) 1062–3. 44 Black (n 35) 426. ( ) ( ) 45 N Mazar, O Amir and D Ariely, ‘The Dishonesty of Honest People: A Theory of Self-Concept Maintenance’ (2008) 45 Journal of Marketing Research 633. 46 46 C Parker ‘The ‘‘Compliance’’ Trap: The Moral Message in Regulatory Enforcement’ (2006) 40 L&Soc’yRev 609. 47 47 CL Ford, ‘New Governance, Compliance and Principles-Based Securities Regulation’ (2008) AmBusLJ 37. 48 48 J Zeitlin, ‘Introduction’ in J Zeitlin (ed), Extending Experimentalist Governance? The European Union and Transnational Regulation (Oxford University Press 2015) 1–19. https://doi.org/10.1017/S0020589320000469 Published online by Cambridge University Press 142 International and Comparative Law Quarterly 49 LB Solum, ‘The Interpretation-Construction Distinction’ (2010) 27 Constitutional Commentary 95. 50 Black (n 35) 428. 51 49 LB Solum, ‘The Interpretation-Construction Distinction’ (2010) 27 Constitutional Commentary 95. 50 Black (n 35) 428. 51 Resolution A/HRC/RES/35/7 Adopted by the Human Rights Council on 22 June 2017. 52 K Buhmann, ‘Business and Human Rights: Understanding the UN Guiding Principles from the Perspective of Transnational Business Governance Interactions’ (2015) 6 TLT 399. 53 JG Ruggie and T Nelson, ‘Human Rights and the OECD Guidelines for Multinational Enterprises: Normative Innovations and Implementation Challenges’ (2015) Corporate Social Responsibility Initiative Working Paper No 66, Cambridge, MA: John F. Kennedy School of Government, Harvard University. 54 KW Abbott, D Levi-Faur and D Snidal, ‘Theorising Regulatory Intermediaries: The RIT Model’ (2017) 660 AnnalsAmAcadPol&SocSci 19. 54 KW Abbott, D Levi-Faur and D Snidal, ‘Theorising Regulatory Intermediaries: The RIT Model’ (2017) 660 AnnalsAmAcadPol&SocSci 19. p ( ) 53 JG Ruggie and T Nelson, ‘Human Rights and the OECD Guidelines for Multinational Enterprises: Normative Innovations and Implementation Challenges’ (2015) Corporate Social Responsibility Initiative Working Paper No 66, Cambridge, MA: John F. Kennedy School of Government, Harvard University. 54 51 Resolution A/HRC/RES/35/7 Adopted by the Human Rights Council on 22 June 2017. 52 K Buhmann, ‘Business and Human Rights: Understanding the UN Guiding Principles from the Perspective of Transnational Business Governance Interactions’ (2015) 6 TLT 399. 51 Resolution A/HRC/RES/35/7 Adopted by the Human Rights Council on 22 June 2017. 52 K Buhmann, ‘Business and Human Rights: Understanding the UN Guiding Principles from C. Intermediaries as (More Than) Interpretative Gap-Fillers in a Polycentric Governance Structure In principles-based regimes, interpretation and implementation are blurred.49 When broad principles are involved, giving meaning and giving eﬀect to them overlap considerably, but remain separate functions. In this article ‘interpretation’ refers to providing meaning to constructs in the UNGP, whereas ‘implementation’ refers to their transposition into standards and procedures for corporations to use. This latter function arguably has fewer normative implications than the former. Corporate ‘operationalisation’ refers to the actions and choices taken by businesses when integrating due diligence into their daily processes and operations. PBR may de-centralise interpretation to several public and private actors, soft law and non-soft law instruments. These features characterise a ‘polycentric’ PBR structure, where many actors participate in an evolutionary and iterative manner in the co-constitution of a legal regime.50 Since the Working Group on Business and Human Rights in the Oﬃce of the High Commissioner for Human Rights does not have the mandate to adopt authoritative interpretations of the UNGP,51 the open nature of the Principles has spurred a number of private and public actors to elaborate on their meaning and their speciﬁc application.52 This accords with Ruggie’s strategy of dissemination and implementation in cooperation with international and national bodies and stakeholder groups, including business itself, in the hope of achieving a high degree of coherence and alignment with the UNGP.53 The interpretation of key HRDD provisions takes place through soft-law instruments drafted by a variety of organisations and entities in the form of standards, guidance, and best practices. These actors can be considered as intermediaries, ie entities acting, directly or indirectly, in conjunction with a regulator or a regulatory regime to aﬀect the behaviour of those targeted by the UNGP.54 Intermediaries are needed because of their expertise. They interpret and clarify the principles, as well as adapting them to the speciﬁc circumstances of a given industry, country, or sector. Intermediaries may be created or activated by the rule-maker in order to expand regulatory capacity—such as providing interpretation in a PBR regime. 55 A Marx and J Wouters, ‘Rule Intermediaries in Global Labour Governance’ (2017) 660 AnnalsAmAcadPol&SocSci 189. 56 T Havinga and P Verbruggen, ‘Understanding Complex Governance Relationships in Food Safety Regulation: The RIT Model as a Theoretical Lens’ (2017) 660 AnnalsAmAcadPol&SocSci 73. 57 KW Abbott, D Levi-Faur and D Snidal, ‘Enriching the RIT Framework’ (2017) 660 AnnalsAmAcadPol&SocSci 280–8. 58 Auld and Renckens (n 21). 59 J Black, ‘Constructing and Contesting Legitimacy and Accountability in Polycentric Regulatory Regimes’ (2008) 2 Regulation & Governance 140. 60 J Van Zeben, ‘Polycentricity as a Theory of Governance’ in J Van Zeben and A Bobic (eds), Polycentricity in the European Union (Cambridge University Press 2019) 24. C. Intermediaries as (More Than) Interpretative Gap-Fillers in a Polycentric Governance Structure They may also emerge spontaneously, with the express purpose of pursuing organisational https://doi.org/10.1017/S0020589320000469 Published online by Cambridge University Press 143 Polycentricity and Polyphony in International Law goals, or even of inﬂuencing policy goals to their advantage,55 and operate independently of each other.56 goals, or even of inﬂuencing policy goals to their advantage,55 and operate independently of each other.56 Open-ended Guiding Principles with a broad scope of application have motivated other actors to contribute to their interpretation. One of the overarching purposes of the UNGP is to coordinate and align existing tools. Organisations whose work intersects with that of business and human rights, such as the OECD and ISO, have amended their standards, and other actors—including entities not necessarily previously involved with human rights impacts, such as business organisations—have also oﬀered interpretation. A sort of ‘spontaneous interpretation’ has been stimulated under the UNGP in which: i) there are no barriers to the provision of interpretation, and ii) no consequences stem from following one interpretative approach over another. Regardless of whether it originates from international organisations such as the OECD, ISO, IFC or ILO, or from business associations and NGOs, these interpretative functions can involve drafting new instruments or amending existing instruments to reﬂect the UNGP. Ideally, intermediaries enhance trust and dialogue between participants to a legal regime. They may, however, operate according to goals which are not aligned to those of the legislator or the regime at hand. This occurs where intermediaries are linked to targets, or where the targets act as intermediaries. Deliberative processes within intermediaries may also spontaneously lead to diﬀerent outcomes than those achieved by other institutions. While all activities exercised by the intermediaries are not merely technical but have considerable normative implications,57 normativity becomes most noticeable when interpreting broad principles. Intermediaries play a vital function in the initial establishment and further development of regimes based on open- ended principles.58 However, the polycentric approach of the UNGP fails to recognise that a typical functional challenge of polycentric regimes is ensuring coordination between the intermediaries at the ‘poles’ of the regime. A central focus of authority is lacking, and the organisations within the regime (in our case those providing interpretation) whilst connected, act independently of each other.59 Particular challenges are faced by regimes lacking a clearly identiﬁed interpreter of the principles. 55 A Marx and J Wouters, ‘Rule Intermediaries in Global Labour Governance’ (2017) 660 AnnalsAmAcadPol&SocSci 189. 56 61 W Mattli and J Seddon, ‘New Organisational Leadership: Nonstate Actors in Global Economic Governance’ (2015) 6 Global Policy 266. 62 TD Lytton, ‘The Taming of the Stew: Regulatory Intermediaries in Food Safety Governance’ (2017) 660 AnnalsAmAcadPol&SocSci 89. See also N Krisch, ‘Liquid Authority in Global Governance’ (2017) 9 International Theory 237. 63 R Schmidt, Regulatory Integration across Borders: Public–Private Cooperation in Transnational Regulation (Cambridge University Press 2018) 149. 64 O Perez and O Stegmann, ‘Transnational Networked Constitutionalism’ (2018) 45 JL&Soc’y S135. 65 LJ Obara, ‘“What Does This Mean?” How UK Companies Make Sense of Human Rights’ (2017) 2 BHRJ 249. 66 UNHRC, The Corporate Responsibility to Respect Human Rights: An Interpretative Guide (2012) 13. C. Intermediaries as (More Than) Interpretative Gap-Fillers in a Polycentric Governance Structure For a polycentric regime to be eﬀective, an overarching shared system for conﬂict resolution between the poles and disputes over interpretation must be present.60 In spite of its formal https://doi.org/10.1017/S0020589320000469 Published online by Cambridge University Press 144 International and Comparative Law Quarterly nature as international soft law, a formalised process deﬁning how, and by whom the UNGP should be interpreted is still lacking. The actors providing interpretation, however informal their relation with the UNGP is, enjoy considerable power in deﬁning the rules of responsible business conduct.61 In polycentric regimes, the eﬀects of intermediaries’ work are considerable and it becomes diﬃcult to identify the principal rule-maker. Rules emerge, are reﬁned, and solidify from interactions between diﬀerent actors in various institutional settings.62 If actors embrace and spread a consistent understanding of human rights business responsibility, a limited process of constitutionalisation emerges, in so far as it becomes harder for corporations to evade certain norms or to engage in certain behaviour.63 In this context, the authority of a rule is not linked to a single rule-maker or legislator; rather it increases over time on the basis of its general acceptance.64 The notion of network authority matters for the UNGP and the intermediaries around them because it addresses the lack of a single and formally binding interpretive and implementing mechanism. The intermediaries at the ‘poles’ of a polycentric system, regardless of their capacity to draft binding norms, increase over time the acceptance and authority of both the general principles in the UNGP and the responsibility to respect. This process takes place through the acceptance of their rule interpretation by network participants and, ultimately, the companies adopting HRDD. 64 O Perez and O Stegmann, ‘Transnational Networked Constitutionalism’ (2018) 45 JL&Soc’y S135. 65 63 R Schmidt, Regulatory Integration across Borders: Public–Private Cooperation in Transnational Regulation (Cambridge University Press 2018) 149. 64 ( ) y 62 TD Lytton, ‘The Taming of the Stew: Regulatory Intermediaries in Food Safety Governance’ (2017) 660 AnnalsAmAcadPol&SocSci 89. See also N Krisch, ‘Liquid Authority in Global Governance’ (2017) 9 International Theory 237. 63 (2017) 2 BHRJ 249. 66 UNHRC, The Corporate Responsibility to Respect Human Rights: An Interpretative Guide (2012) 13. 61 W Mattli and J Seddon, ‘New Organisational Leadership: Nonstate Actors in Global Economic Governance’ (2015) 6 Global Policy 266. 62 65 LJ Obara, ‘“What Does This Mean?” How UK Companies Make Sense of Human Rights’ (2017) 2 BHRJ 249. 66 UNHRC, The Corporate Responsibility to Respect Human Rights: An Interpretative Guide (2012) 13. D. Substantive Indeterminacy in the UNGP and Its Consequences D. Substantive Indeterminacy in the UNGP and Its Consequences Reﬁning the meaning of the UNGP, and especially HRDD, is particularly important since current corporate understandings of human rights, and the consequent scope of responsibility, vary greatly.65 Arguably, this diﬃculty in understanding possible human rights impacts is traceable to the UNGP’ reference to international human rights instruments which were drafted with States in mind as the primary duty-bearers. In principle, any recognised international human right can be negatively impacted by corporations.66 The transposition of these instruments to economic actors results in https://doi.org/10.1017/S0020589320000469 Published online by Cambridge University Press 145 Polycentricity and Polyphony in International Law extremely complex conceptual problems which should not be glossed over.67 In order to eﬀectively assign to corporations the responsibility to respect human rights, the boundaries of such responsibilities should be clear and entail as little discretion as possible.68 Even where human rights responsibilities can be easily transplanted into other contexts, there are many situations where it is diﬃcult to determine whether corporations contribute, are directly linked, or are not linked to human rights impacts. A key element in need of reﬁnement concerns the ‘attribution’ of negative impacts to a corporation, and in particular impacts brought about by the activities of other economic entities. When is a corporation connected to a negative human rights impact? While the category of causing human rights impacts is relatively straightforward, the concepts of contribution and direct link are less clear. Would a bank, through a loan to a client, cause human rights violations, contribute, or be directly linked to negative impacts brought about by the actions of its client? The limits of a direct link to a negative impact also need to be interpreted. Would a corporation be directly linked to human rights violations committed by a second-tier supplier or, even more remotely, by those who supply its second- tier supplier? Would a social network be contributing or be directly linked to bullying and child abuse committed by its users through its platforms? These interpretative challenges in the operationalisation of the UNGP are far from theoretical. The UNGP provide that human rights expectations should be set out in a corporate statement of policy. 67 S Deva, ‘Treating Human Rights Lightly: A Critique of the Consensus Rhetoric and the Language Employed by the Guiding Principles’ in Deva and Bilchitz (n 13) 88. 68 DJ Karp, Responsibility for Human Rights, Transnational Corporations in Imperfect States. (Cambridge University Press 2014) 54; LL Fuller, The Morality of Law (Yale University Press 1969). 69 UNGP Commentary to Principle 13. 70 Oﬃce of the High Commissioner for Human Rights, The Corporate Responsibility to Respect Human Rights: An Interpretative Guide (2012) HR/PUB/12/02. 71 Corporate Human Rights Benchmark, ‘Key Findings 2017. Available at <https://www. corporatebenchmark.org/sites/default/ﬁles/2017-03/CHRB_Findings_web_pages.pdf>. 72 R Maher, ‘De-contextualized Corporate Human Rights Benchmarks: Whose Perspective Counts? See Disclaimer’ 5 BHRJ 156. 67 S Deva, ‘Treating Human Rights Lightly: A Critique of the Consensus Rhetoric and the Language Employed by the Guiding Principles’ in Deva and Bilchitz (n 13) 88. 68 g g p y y g p ( ) 68 DJ Karp, Responsibility for Human Rights, Transnational Corporations in Imperfect States. (Cambridge University Press 2014) 54; LL Fuller, The Morality of Law (Yale University Press 1969). 69 UNGP Commentary to Principle 13. 70 70 Oﬃce of the High Commissioner for Human Rights, The Corporate Responsibility to Respect Human Rights: An Interpretative Guide (2012) HR/PUB/12/02. 72 R Maher, ‘De-contextualized Corporate Human Rights Benchmarks: Whose Perspective Counts? See Disclaimer’ 5 BHRJ 156. g p ( ) 71 Corporate Human Rights Benchmark, ‘Key Findings 2017. Available at <https://www. corporatebenchmark.org/sites/default/ﬁles/2017-03/CHRB_Findings_web_pages.pdf>. 72 Corporate Human Rights Benchmark, Key Findings 2017. Available at <https://www. corporatebenchmark.org/sites/default/ﬁles/2017-03/CHRB_Findings_web_pages.pdf>. 72 R Maher, ‘De-contextualized Corporate Human Rights Benchmarks: Whose Perspective Counts? See Disclaimer’ 5 BHRJ 156. D. Substantive Indeterminacy in the UNGP and Its Consequences This should also stipulate the enterprise’s human rights expectations of its business partners and other parties directly linked to its operations, products or services.69 This provides the starting point from which the enterprise deﬁnes and leverages respect for human rights in these relationships, should this be necessary.70 If it is unclear that these expectations regarding human rights are a ﬁrm policy of the enterprise, they can easily become ‘negotiable’ and be sidelined. Recent benchmarking in corporate human rights practices has highlighted that the operationalisation of HRDD proceeds very slowly due to limited capacity to establish appropriate internal processes in the area of human rights.71 Even benchmarking initiatives themselves structurally fail to include the voices of those impacted.72 Empirical studies also indicate that companies face https://doi.org/10.1017/S0020589320000469 Published online by Cambridge University Press International and Comparative Law Quarterly 146 diﬃculties in identifying human rights aﬀected by their activities and in structuring speciﬁc components of HRDD.73 The continuing lack of guidance on how to interpret the UNGP remains a key challenge in their implementation.74 Corporations may thus exploit their open-ended nature in HRDD processes to strategically dilute their human rights responsibility,75 or approach it as a box-ticking exercise.76 Similar to other forms of management-based regulation, HRDD is exposed to the risk of resulting in empty proceduralisation lacking clear deﬁnitions, boundaries and goals.77 Without an unequivocal framework for interpretation, minimalistic and watered-down implementation of HRDD is a tangible risk. Corporate conceptualisations of compliance, even if misaligned with the UNGP, may then be accepted as fully lawful by courts and judges.78 73 K Salcito and M Wielga, ‘What Does Human Rights Due Diligence for Business Relationships Really Look Like on the Ground?’ (2018) 3 BHRJ 113. 74 S f t ( 30) 448 9 75 S t ( 28) p y 74 Sarfaty (n 30) 448–9. 6 y ( ) ( ) 76 Working Group on the issue of human rights and transnational corporations and other business enterprises, ‘The Report of the Working Group to the General Assembly on the issue of human rights and transnational corporations and other business enterprises’ A/73/163 (16 July 2018) 8ﬀ. 77 , g g ( ) 78 LB Edelman, SR Fuller and I Mara-Drita, ‘Diversity Rhetoric and the Managerialisation of Law’ (2001) 106 AmJSoc 1589. 79 Ruggie (n 5). 80 73 K Salcito and M Wielga, ‘What Does Human Rights Due Diligence for Business Relationships Really Look Like on the Ground?’ (2018) 3 BHRJ 113. 74 Sarfaty (n 30) 448–9. 75 Santoso (n 28). 76 Working Group on the issue of human rights and transnational corporations and other business enterprises, ‘The Report of the Working Group to the General Assembly on the issue of human rights and transnational corporations and other business enterprises’ A/73/163 (16 July 2018) 8ﬀ. 77 J Black, ‘Proceduralising Regulation: Part 1’ (2000) 20 OJLS 598. 78 LB Edelman, SR Fuller and I Mara-Drita, ‘Diversity Rhetoric and the Managerialisation of Law’ (2001) 106 AmJSoc 1589. 79 Ruggie (n 5). 80 J Black, ‘Regulatory Conversations’ (2002) 29 JL&Society 163. 80 J Black, ‘Regulatory Conversations’ (2002) 29 JL&Society 163. III. POLYCENTRIC INTERPRETATIONS OF THE UNGP The adoption of the UNGP stimulated a proliferation of standards, best practices and guidance documents. International organisations active in international standardisation such as the OECD, ISO and IFC, have all enacted instruments incorporating the UNGP and in particular the concept of HRDD, or have amended previous instruments in order to accommodate the framework.79 NGOs, think tanks, business organisations and even management consultancies have also contributed to this global regulatory conversation80 by drafting best practices and guidance documents oﬀering interpretations of the UNGP. The majority of these instruments provide operational guidance by focusing on the procedural elements of HRDD, therefore addressing only marginally the substantive issues in need of interpretation ﬂagged above. This section addresses the discrepancies between the interpretations of the categories of attribution in the UNGP in key standards drafted by three leading international organisations: the OECD Guidelines for Multinational Corporations, and in particular some of its Due Diligence Guidance documents; ISO 26000 standard, and the IFC Sustainability Principle and Performance Standards. The section also touches upon the ILO Declaration of Principles concerning Multinational Enterprises and Social Policy. It then addresses explicit challenges to the content and approach of the UNGP such as that raised by the Thun Group, an association https://doi.org/10.1017/S0020589320000469 Published online by Cambridge University Press Polycentricity and Polyphony in International Law 147 of ﬁnancial entities. Finally, the section highlights attempts to inﬂuence the interpretative poles by actors as diverse as the UN Human Rights Council, Ruggie himself, and Shift, an NGO established by Ruggie with the purpose of contributing to the implementation of the UNGP. 81 See art 2.4 of the World Trade Organisation’s Technical Barriers to Trade (TBT) Agreement. 82 Buhman (n 52) 429. 83 G Schuler, ‘Eﬀective Governance through Decentralised Soft Implementation: The OECD Guidelines for Multinational Enterprises’ in A von Bogdandy et al. (eds), The Exercise of Public Authority by International Institutions (Springer Verlag 2008) 197. 84 OECD, OECD Guidelines for Multinational Enterprises (OECD Publishing 2011) 83. 82 Buhman (n 52) 429. 83 81 See art 2.4 of the World Trade Organisation’s Technical Barriers to Trade (TBT) Agreement. 82 B h ( 52) 429 81 See art 2.4 of the World Trade Organisation’s Technical Barriers to Trade (TBT) Agreement. 82 Buhman (n 52) 429. 83 83 G Schuler, ‘Eﬀective Governance through Decentralised Soft Implementation: The OECD Guidelines for Multinational Enterprises’ in A von Bogdandy et al. (eds), The Exercise of Public Authority by International Institutions (Springer Verlag 2008) 197. 85 Employment and industrial relations, environment, bribery, consumer interest, technology transfer, competition and taxation. 86 OECD/FAO, OECD-FAO Guidance for Responsible Agricultural Supply Chains (OECD Publishing 2016). 87 OECD, OECD Due Diligence Guidance for Responsible Supply Chains of Minerals from Conﬂict-Aﬀected and High-Risk Areas (3rd edn, OECD Publishing 2016). 88 OECD, OECD Due Diligence Guidance for Responsible Supply Chains in the Garment and Footwear Sector (OECD Publishing 2017). 89 OECD, Due Diligence Guidance for Responsible Business Conduct (OECD Publishing 2018). 90 OECD, OECD Due Diligence Guidance for Responsible Supply Chains in the Garment and Footwear Sector (OECD Publishing 2017) Foreword, 3. 91 ISO, ISO 26000 Guidance on Social Responsibility (2010) ISO/FDIS 26000:2010(E). 92 Schmidt (n 63) 105–53. A. Divergence between International Standards and the UNGP The OECD, ISO, IFC and ILO are four crucial international organisations whose standard-setting work is essential for interpreting the UNGP and their successful embedding in business practices. Remarkably, standards drafted by these organisations may qualify as ‘international standards’ under international economic law, a status that grants them a strong normative claim and requires States to use them in regulation where necessary and possible.81 While operating independently and with distinct goals from those pursued by the UNGP, the work of these organisations has obvious intersections and synergies with corporate responsibility. To varying extents, however, their standards display discrepancies with the UNGP approach to the attribution of human rights impacts. This should not detract from the overall successful acceptance of the broader ideas behind the UNGP, their transposition in several instruments, and the progress made in their implementation and clariﬁcation.82 These diﬀerences, nonetheless, expose the practical diﬃculty in aligning approaches to key elements which are crucial to their full eﬀectiveness. The OECD was the ﬁrst multilateral organisation concerned with the regulation of multinational corporations. Its 1976 Guidelines for Multinational Corporations (OECD Guidelines) are among the oldest set of international standards for business conduct. The Guidelines are a key instrument in the governance of transnational corporate conduct through a multi-level and mediation-based dispute resolution mechanism between corporations and human rights victims, through a system of National Contact Points (NCPs).83 Even if focusing on mediation and good oﬃces, the NCPs are an important mechanism through which a ‘jurisprudence’ on business and human rights and HRDD can be developed globally based on their interpretation of the Guidelines in so-called ‘speciﬁc instances’, ie disputes concerning a corporation’s implementation of the Guidelines.84 It is therefore particularly important that the Guidelines themselves are aligned to the UNGP. Their 2011 revision took place in parallel with the development of the UNGP and, on the basis of input from the UN Special Representative for https://doi.org/10.1017/S0020589320000469 Published online by Cambridge University Press 148 International and Comparative Law Quarterly Business and Human Rights, incorporates a revised human rights chapter which the OECD considers to be in line with the UNGP. The OECD Guidelines adopt the concept of due diligence and apply it to human rights and other areas which they cover.85 The most elaborate discussion of HRDD is, however, not in the OECD Guidelines, but in their implementing Guidance documents transposing due diligence to speciﬁc sectors and their risks. A. Divergence between International Standards and the UNGP Examples include the agricultural,86 mineral,87 and garment and footwear88 supply chains. In May 2018 the OECD also published a general Guidance focusing on human rights due diligence across sectors.89 These standards contribute positively to the implementation of HRDD by oﬀering a common understanding of due diligence processes and related terminology.90 In addition, they make recommendations to companies on how to implement due diligence in their value chains and incorporate HRDD in management systems and procedures. Guidance documents are drafted following a multi-stakeholder approach overseen by the OECD Working Party on Responsible Business Conduct, with the involvement of non-OECD countries and representatives from business, trade and civil society. A second important organisation providing interpretation and implementation of the UNGP, and in particular in embedding HRDD in corporate management standards is the ISO. The ISO is a non-State industry- driven organisation made up of national (mostly private) standard-setting bodies. Its core-business standard-setting work relates to technical and interoperability standards. An increasing number of standards drafted by the ISO are in the form of audible management systems standards. Management systems standards integrate public policy goals, such as environmental protection and health and safety, into corporate procedures. ISO 26000 is the ISO’s ﬁrst foray in the ﬁeld of sustainability, human rights and corporate responsibility.91 It was the product of extensive consultation with other organisations and stakeholders not routinely involved in ISO work, including the ILO, the OECD and the UN Global Compact.92 However, ISO 26000 does not extend the management systems standards approach to the domain of human rights and sustainability, both by not being certiﬁable and by not https://doi.org/10.1017/S0020589320000469 Published online by Cambridge University Press Polycentricity and Polyphony in International Law 149 identifying actual management processes, as do other ISO management standards.93 The third key document is the IFC Performance Standards on Environmental and Social Sustainability.94 This adopts the IFC’s (the private-sector lending arm of the World Bank) risk management approach to project ﬁnancing.95 The relevance of the IFC standards is magniﬁed exponentially through their reference in the Equator Principles,96 the instrument most frequently used by ﬁnancial corporations to ensure responsibility in project ﬁnancing. 93 S Bijlmakers and G van Calster, ‘“You’d be Surprised How Much It Costs to Look This Cheap!” A Case Study of ISO 26000 on Social Responsibility’ in P Delimatsis (ed), The Law, Economics and Politics of International Standardisation (Cambridge University Press 2015) 309. 94 IFC, Performance Standards on Environmental and Social Sustainability (2012). 95 ibid 2. 96 The Equator Principles, ‘A Financial Industry Benchmark for Determining, Assessing and Managing Environmental and Social Risk in Projects’ (2020) Available at <https://equator- principles.com/wp-content/uploads/2020/05/The-Equator-Principles-July-2020-v2.pdf>. 97 IFC Performance Standard 1, at 7. 98 International Labour Organisation, Tripartite Declaration of Principles concerning Multinational Enterprises and Social Policy (5th edn, March 2017). 99 <https://www.ilo.org/empent/areas/mne-declaration/lang--en/index.htm>. 93 S Bijlmakers and G van Calster, ‘“You’d be Surprised How Much It Costs to Look This Cheap!” A Case Study of ISO 26000 on Social Responsibility’ in P Delimatsis (ed), The Law, Economics and Politics of International Standardisation (Cambridge University Press 2015) 309. 94 IFC, Performance Standards on Environmental and Social Sustainability (2012). 95 ibid 2. 96 The Equator Principles, ‘A Financial Industry Benchmark for Determining, Assessing and Managing Environmental and Social Risk in Projects’ (2020) Available at <https://equator https://doi.org/10.1017/S0020589320000469 Published online by Cambridge University Press p y ( , ) 99 <https://www.ilo.org/empent/areas/mne-declaration/lang--en/index.htm>. 97 IFC Performance Standard 1, at 7. 98 93 S Bijlmakers and G van Calster, ‘“You’d be Surprised How Much It Costs to Look This Cheap!” A Case Study of ISO 26000 on Social Responsibility’ in P Delimatsis (ed), The Law, A. Divergence between International Standards and the UNGP Project ﬁnancing presents many opportunities to embed human rights considerations into corporate operations, such as the adoption of environmental and social management systems identifying and mitigating risks and improving performance that incorporate features of HRDD as deﬁned in the UNGP.97 Finally, the ILO’s work concerning labour rights also touches upon the subject matter of the UNGP. The ILO tripartite Declaration of Principles concerning Multinational Enterprises and Social Policy98 is a prime example. The Declaration sets out principles for governments, employers, workers and multinationals for measures furthering social progress and responsible workplace practices. The Declaration is particularly relevant as it is the only ILO instrument providing readily implementable guidance to enterprises on matters connected to social policy and inclusive, responsible and sustainable practices in the workplace.99 It is thus an additional vehicle through which HRDD concepts can be interpreted in the context of labour rights. While many aspects of these instruments are in line, if not with the language, at least with the broad spirit of the UNGP, a careful analysis shows that they do not always fully align. While not all Guidance documents at the OECD level explicitly address the issue of attribution, the Garment and Footwear Guidance seems to tackle it by introducing variations to the UNGP approach. According to the Guidance, an enterprise contributes to negative human rights impact if the actions of the enterprise cause, facilitate or incentivise another entity to cause adverse impacts. However, a signiﬁcant qualiﬁcation is introduced, ie that such contribution must be substantial. Neither the UNGP nor other UN oﬃcial publications on the UNGP mention the concept of substantial contribution. The subject is only touched upon in discussions https://doi.org/10.1017/S0020589320000469 Published online by Cambridge University Press 150 International and Comparative Law Quarterly concerning legal complicity in the context of international criminal law, relating to the standard of aiding and abetting.100 While questions of legal complicity may arise if an enterprise contributes to human rights breaches, the test for contribution is separate from, and broader than questions of legal complicity.101 That a contribution must be substantial was reiterated in the 2018 OECD Due Diligence Guidance for Responsible Business Conduct, promoting a common understanding of due diligence generally, ie across economic sectors. The Guidance aims to become the primary reference point for companies implementing HRDD.102 The Guidance establishes that a contribution must be substantial; this means that it does not include minor or trivial contributions. 100 ie, ‘knowingly providing practical assistance or encouragement that has a substantial eﬀect on the commission of a crime’. United Nations Human Rights Oﬃce of the High Commissioner, ‘The Corporate Responsibility to Respect Human Rights: An Interpretative Guide (2012) HR/PUB/12/ 02, at 5. 101 See UNGP, commentary to Principle 17. 102 103 OECD, Due Diligence Guidance for Responsible Business Conduct (2018) 70. 104 OECD ‘Global Forum on Responsible Business Conduct: Expert Letters and Statements on the Application of the OECD Guidelines for Multinational Enterprises and UN Guiding Principles on Business and Human Rights in the Context of the Financial Sector’ (26–7 June 2014, OECD Conference Centre, Paris). 105 102 C Shavin, ‘Unlocking the Potential of the New OECD Due Diligence Guidance on Responsible Business Conduct’ (2019) 4 BHRJ 139. 105 Draft Norms on the Responsibilities of Transnational Corporations and Other Business Enterprises with regard to Human Rights: Draft Norms/ submitted by the Working Group on the Working Methods and Activities of Transnational Corporations pursuant to resolution 2002/8, E/CN.4/Sub.2/2003/12. 106 O de Schutter, ‘The Challenge of Imposing Human Rights Norms on Corporate Actors’ in O de Schutter (ed), Transnational Corporations and Human Rights (Hart Publishing 2006) 12. 100 ie, ‘knowingly providing practical assistance or encouragement that has a substantial eﬀect on the commission of a crime’. United Nations Human Rights Oﬃce of the High Commissioner, ‘The Corporate Responsibility to Respect Human Rights: An Interpretative Guide (2012) HR/PUB/12/ 02, at 5. 101 See UNGP, commentary to Principle 17. 102 C Shavin, ‘Unlocking the Potential of the New OECD Due Diligence Guidance on Responsible Business Conduct’ (2019) 4 BHRJ 139. 103 OECD, Due Diligence Guidance for Responsible Business Conduct (2018) 70. 104 OECD ‘Global Forum on Responsible Business Conduct: Expert Letters and Statements on the Application of the OECD Guidelines for Multinational Enterprises and UN Guiding Principles on Business and Human Rights in the Context of the Financial Sector’ (26–7 June 2014, OECD Conference Centre, Paris). 105 Draft Norms on the Responsibilities of Transnational Corporations and Other Business Enterprises with regard to Human Rights: Draft Norms/ submitted by the Working Group on the Working Methods and Activities of Transnational Corporations pursuant to resolution 2002/8, E/CN.4/Sub.2/2003/12. 106 O de Schutter, ‘The Challenge of Imposing Human Rights Norms on Corporate Actors’ in O de Schutter (ed), Transnational Corporations and Human Rights (Hart Publishing 2006) 12. A. Divergence between International Standards and the UNGP To assess whether a contribution is substantial, several factors matter, including the degree to which the activity increased the risk of the impact occurring; the degree of foreseeability; and the degree to which any of the enterprise’s activities actually mitigated the adverse impact or decreased the risk of impact occurring.103 Despite previous eﬀorts by the OECD and the OHCHR to coordinate deﬁnitions for the terms relating to attribution,104 it seems that both the OECD Garment Guidance and the general Due Diligence Guidance embrace a narrower approach to the identiﬁcation of contribution. According to the UNGP, companies may prioritise and address substantial human rights impacts, but that does not aﬀect their involvement. The OECD approach towards substantial contribution, and what seems like a de minimis test, could result in more limited grounds for the involvement of corporations in human rights impact under the OECD instruments. ISO 26000 departs even more from the UNGP main features. As regards attribution, ISO 26000 at several critical junctures embraces a so-called ‘sphere-of-inﬂuence’ approach. The notion of sphere of inﬂuence originates in the failed attempt to adopt the Draft Norms on the Responsibilities of Transnational Corporations.105 It recognises that corporations have the ability to inﬂuence actions outside their own organisations through their relationships with other actors.106 Such inﬂuence would be greater in relation to actors in 106 O de Schutter, ‘The Challenge of Imposing Human Rights Norms on Corporate Actors’ in O de Schutter (ed), Transnational Corporations and Human Rights (Hart Publishing 2006) 12. https://doi.org/10.1017/S0020589320000469 Published online by Cambridge University Press Polycentricity and Polyphony in International Law 151 close proximity to the business entity in question.107 Under the Draft Norms, within their sphere of inﬂuence corporations have the obligation to promote, secure the fulﬁlment of, respect, ensure respect of, and protect human rights.108 This approach was expressly rejected by Ruggie on the ground that corporations need clearer and more precise guidance concerning their responsibilities. In Ruggie’s view, the concept was problematic because imposing responsibility whenever a company has leverage would entail the assumption that ‘can implies ought’, and made it diﬃcult to separate the duties and responsibilities of States and enterprises. 107 Special Representative of the Secretary General on Business and Human Rights, John Ruggie, ‘Clarifying the Concepts of ‘‘Sphere of Inﬂuence’’ and ‘‘Complicity’’: Report of the Special Representative of the Secretary-General on the Issue of Human Rights and Transnational Corporations and other Business Enterprises’ (15 May 2008) UN Doc A/HRC/8/16. 108 Draft Norms, art A.1. 109 See (n 107) UN Doc A/HRC/8/16 at 5. 110 S Wood, ‘The Case for Leverage-based Corporate Human Rights Responsibility’ (2012) 22 Business Ethics Quarterly 70. 111 ISO 26000, Clause 4.8. 112 ibid, Clause 5.2.3. 113 Wood (n 110) 71. 107 Special Representative of the Secretary General on Business and Human Rights, John Ruggie, ‘Clarifying the Concepts of ‘‘Sphere of Inﬂuence’’ and ‘‘Complicity’’: Report of the Special Representative of the Secretary-General on the Issue of Human Rights and Transnational Corporations and other Business Enterprises’ (15 May 2008) UN Doc A/HRC/8/16. A. Divergence between International Standards and the UNGP Under their sphere of inﬂuence, companies would be responsible for human rights impacts of entities over which they may have some leverage, including cases where they are not contributing to, nor are a causal agent of, the negative human rights impact at hand.109 The rejection of this approach led to the adoption of the three categories discussed above, which centre on a corporation’s network of business activities and relations, and its involvement in actual and potential impacts, rather than on the possibility of exerting leverage. While elements of ISO 26000, despite not adopting the same terminology, are aligned with the spirit of the UNGP,110 the standard also embraces the concept of sphere of inﬂuence as well as taking a positive approach to business human rights responsibility in which corporations are expected to promote human rights where they have the inﬂuence to do so.111 Speciﬁcally concerning attribution, ISO 26000 provides that an organisation will be responsible for negative impacts committed by entities over which it has formal or factual control. In addition, it provides that a corporation may be capable of aﬀecting the behaviour of entities within and beyond a value chain over which it has inﬂuence. However, the standard does not clarify what would be expected when there was such inﬂuence: the deﬁnition of sphere of inﬂuence does not imply a responsibility to exercise it. Furthermore, ISO 26000 does not necessarily consider that the entirety of an organisation’s value chain falls within its sphere of inﬂuence.112 This stands in sharp contrast to the far- reaching notion of direct linkage in the UNGP, which spans the entirety of a value chain. All in all, ISO 26000 resulted in considerable confusion concerning the actual responsibility of businesses and the attribution to businesses of human rights impacts committed by other entities. By mixing negative, positive, impact-based and leverage-based conceptions of responsibility,113 ISO does not clarify situations in which a corporation is https://doi.org/10.1017/S0020589320000469 Published online by Cambridge University Press 152 International and Comparative Law Quarterly responsible for human rights impacts, and when inﬂuence/leverage should be exercised.114 In the IFC Performance Standards, the treatment of direct linkage to human rights impact also displays certain discrepancies from the UNGP. A. Divergence between International Standards and the UNGP The IFC Performance Standards start by providing that clients ‘should respect human rights, which means to avoid infringing on the human rights of others and address adverse human rights impacts business may cause or contribute to’,115 with no mention of direct link. The standard then reduces the scope of application of due diligence to the speciﬁc projects ﬁnanced, to be deﬁned on a case-by-case basis with the lender,116 and its impacts as may be identiﬁed by the area of inﬂuence of the project. These include: the areas aﬀected by the project and the client’s activities and facilities that are directly owned, operated or managed and that are a component of the project; impacts from unplanned but predictable developments caused by the project that may occur later or at a diﬀerent location; and indirect project impacts on biodiversity or on ecosystem services upon which aﬀected communities’ livelihoods are dependent.117 With respect to human rights impacts resulting from third party actions or omissions, the client is expected to address such impacts ‘in a manner commensurate with the client’s control and inﬂuence’. This seems in line with the idea that control and inﬂuence do not determine the existence of responsibility, but only how the company should discharge it. However, the Performance Standards provide that where control can be exercised, risks and impacts covered by management systems and due diligence also extends to those in the primary supply chain, ie that of goods or materials essential for the core business processes of the project.118 As the wording employed seems to suggests that control determines the scope of application and of responsibility, this approach seems incompatible with the UNGP. The IFC Performance Standards also retain elements of the sphere of inﬂuence approach which was rejected by the UNGP, and they are not exclusively based on the presence of a business relationship. In addition, the Performance Standards limit the scope of due diligence in the value chains in two ways. Firstly, due diligence, and in particular human rights risk assessment, only covers suppliers and entities in the chain over which the client can exercise reasonable control. The concept of control establishes a rather high threshold, certainly higher than ‘inﬂuence’ and is diﬀerent from ‘business relations’. The concept itself represents an addition to the language in the UNGP. 114 K Webb, ‘ISO 26000 Social Responsibility Standard as “Proto Law” and a New Form of Global Custom: Positioning ISO 26000 in the Emerging Transnational Regulatory Governance Rule Instrument Architecture’ (2015) 6 Transnational Legal Theory 466. ( ) g y 115 IFC Performance Standards, 6. 116 ibid. 117 ibid 8. 118 ibid 17 119 ibid 2. 120 International Labour Organisation, ILO Tripartite Declaration of Principles concerning Multinational Enterprises and Social Policy (5th edn, March 2017). 121 ibid, para 10. 122 ibid, para 65. 123 ibid, Annex 3. 124 For another example: International Bar Association, ‘Practical Guide on Business and Human Rights for Business Lawyers’ (2016) available at <https://www.ibanet.org/Conferences/Practical- Guide-Launch.aspx>. A. Divergence between International Standards and the UNGP Secondly, the only value chain to be covered by HRDD only relates to products essential for the core business process of the project. This means that for business relations in other value chains there would be no https://doi.org/10.1017/S0020589320000469 Published online by Cambridge University Press 153 Polycentricity and Polyphony in International Law HRDD obligation. On the one hand, this narrower scope reﬂects the fact that the IFC Performance Standards also relate to the due diligence responsibilities of the lender in relation to projects being ﬁnanced. Impacts outside such projects should not concern the lender as they would fall outside the scope of its business relationship with the client and thus lack a direct link. On the other hand, if the rationale behind the IFC Performance Standards is to manage environmental and social risks and impacts so that development opportunities are enhanced,119 singling out the value chains of the products essential for the core business process of the project is artiﬁcial and narrows the possible positive impacts of ensuring due diligence in the context of a loan. Finally, the ILO tripartite Declaration of Principles concerning Multinational Enterprises and Social Policy was also revised in 2017 to incorporate the UNGP.120 As the instrument applies also to corporations, the latest revision introduced a general reference to the UNGP and to HRDD. These include the main features of HRDD (although no mention is made of reporting), and the three categories of attribution.121 Reference to the UNGP is then reiterated in the more detailed parts of the Declaration concerning areas such as employment and working conditions. For example, companies shall take action against child labour ‘in their operations’ in line with the notion of direct linkage, with, however, a qualiﬁcation that such actions must fall ‘within their competence’. It is possible that this phrasing could reduce the range of actions taken when exerting leverage. Leverage is not mentioned in the Declaration, except where it provides, rather restrictively, that multinationals should use their leverage to persuade their partners to provide remedies.122 We know from the UNGP that leverage should be exercised to address and mitigate impacts. As with the OECD Guidelines, the ILO Declaration has a mechanism for disputes concerning its application and interpretation and this also could contribute to diﬀering interpretations of HRDD.123 B. A. Divergence between International Standards and the UNGP Explicit Resistance by Corporations: The Case of the Thun Group of Banks In addition to the work of international organisations, sectoral associations have also released documents and guidance for their members which seek to translate the UNGP into standards and practices within their sectors.124 The ﬁnancial sector is crucial for the successful operationalisation of the corporate responsibility to respect human rights, in light of both the size of the sector, https://doi.org/10.1017/S0020589320000469 Published online by Cambridge University Press 154 International and Comparative Law Quarterly and its potential role as ‘enabler’ of human rights violations. The performance of banks with respect to human rights has considerable scope for improvement.125 However, given the various commercial relationships between banks and clients, as well as the variety and complexity of many ﬁnancial instruments, transposing the UNGP to the ﬁnancial sector is challenging. The ﬁnancial sectors’ attempts to do so have also resulted in the most explicit deviation from the concepts found in the UNGP, and an open challenge to them has been made. The Thun Group of Banks,126 in a series of discussion papers published between 2013 and 2017, set out their common understanding of Principles 13 and 17, in particular focusing on the concept of ‘direct link’ to human rights risk. There has been some uncertainty in deﬁning the boundaries of ﬁnancial institutions’ complicity (both in legal and non-legal terms) in human rights violations.127 The position taken by the banks limits the possibility of their being considered as causing or contributing to negative human rights impacts as a result of their commercial relations. The 2017 discussion paper clearly states that banks would generally not be considered to cause or contribute to adverse human rights impact arising from the operations of their clients, since such impact would not occur as part of the banks’ own activities.128 The position of the Thun Group replaces two independent categories of attribution in the UNGP with their own speciﬁc requirements. Even more importantly, this approach means that banks can only be ‘directly linked’, and never be ‘contributing’ to human rights impacts.129 However, it is clear from the UNGP that situations where a company enables, encourages or facilitates other entities to harm human rights would be considered as a contribution. In the Thun Group framework, direct linkage would occur only when speciﬁc ﬁnancial products or services are delivered. 127 M Bordignon, ‘The Compliance to Human Rights in Business Sector: Focusing on Banks’ (2013) 1 Journal of Global Policy and Governance 217. 128 128 Thun Group of Banks, ‘Paper on the Implications of UN Guiding Principles 13b and 17 in a Corporate and Investment Banking Context’ 6. Available at <https://www.business-humanrights. org/sites/default/ﬁles/documents/2017_12_Thun%20Group%20of%20Banks_Paper_UNGP% 2013b%20and %2017.pdf>. 126 The Thun Group is an informal group of banks cooperating with the purpose of furthering understanding of the UNGP in the banking sector and how the UNGP may be applied across diﬀerent banking activities. See Thun Group, Statement on the ‘Guiding Principles for the Implementation of the United Nations “Protect, Respect and Remedy” Framework on Human Rights’ (19 October 2011). 125 BankTrack, ‘Banking with Principles? Benchmarking Banks Against the UN Guiding Principles on Business and Human Rights’ (2016) available at <https://www.banktrack.org/ download/banking_with_principles>. 126 The Thun Group is an informal group of banks cooperating with the purpose of furthering understanding of the UNGP in the banking sector and how the UNGP may be applied across diﬀerent banking activities. See Thun Group, Statement on the ‘Guiding Principles for the Implementation of the United Nations “Protect, Respect and Remedy” Framework on Human Rights’ (19 October 2011). 127 M Bordignon, ‘The Compliance to Human Rights in Business Sector: Focusing on Banks’ (2013) 1 Journal of Global Policy and Governance 217. 128 Thun Group of Banks, ‘Paper on the Implications of UN Guiding Principles 13b and 17 in a Corporate and Investment Banking Context’ 6. Available at <https://www.business-humanrights. org/sites/default/ﬁles/documents/2017_12_Thun%20Group%20of%20Banks_Paper_UNGP% 2013b%20and %2017.pdf>. 129 It should not be forgotten that, in case of direct link, no remediation is required. 125 BankTrack, ‘Banking with Principles? Benchmarking Banks Against the UN Guiding Principles on Business and Human Rights’ (2016) available at <https://www.banktrack.org/ download/banking_with_principles>. 126 https://doi.org/10.1017/S0020589320000469 Published online by Cambridge University Press p 129 It should not be forgotten that, in case of direct link, no remediation is required. 125 BankTrack, ‘Banking with Principles? Benchmarking Banks Against the UN Guiding Principles on Business and Human Rights’ (2016) available at <https://www.banktrack.org/ download/banking_with_principles>. 126 The Thun Group is an informal group of banks cooperating with the purpose of furthering understanding of the UNGP in the banking sector and how the UNGP may be applied across diﬀerent banking activities. See Thun Group, Statement on the ‘Guiding Principles for the Implementation of the United Nations “Protect, Respect and Remedy” Framework on Human Rights’ (19 October 2011). 127 di h li i h i i i k 130 ibid 3. 131 ibid 7–8. 132 UNGP Principle 17(b). 133 H Ward, ‘The ISO 26000 International Guidance Standard on Social Responsibility: Implications for Public Policy and Transnational Democracy’ (2011) 12 Theoretical Inquires in Law 695. 134 JG Ruggie, ‘Note on ISO 26,000 Guidance Draft Document’ (2009) Available at <https:// www.business-humanrights.org/sites/default/ﬁles/media/documents/ruggie-note-re-iso-26000- nov-2009.pdf>. 135 Clarifying the concepts of ‘sphere of inﬂuence’ and ‘complicity’, A/HRC/8/16. A. Divergence between International Standards and the UNGP Direct linkage is further limited by the introduction of the novel idea of a ‘suﬃcient degree of linkage’, or ‘proximity’, between the ﬁnancial products and services oﬀered and the 128 Thun Group of Banks, ‘Paper on the Implications of UN Guiding Principles 13b and 17 in a Corporate and Investment Banking Context’ 6. Available at <https://www.business-humanrights. org/sites/default/ﬁles/documents/2017_12_Thun%20Group%20of%20Banks_Paper_UNGP% 2013b%20and %2017.pdf>. 129 It should not be forgotten that, in case of direct link, no remediation is required. https://doi.org/10.1017/S0020589320000469 Published online by Cambridge University Press 155 Polycentricity and Polyphony in International Law adverse impact.130 Proximity would allow a bank to assess and manage human rights risk. The 2017 discussion paper also introduces the concept of a unit of analysis, which would set the boundaries of the due diligence exercise and the emergence of a direct link. Asset-speciﬁc ﬁnancing would require determining human rights impacts resulting from the use of that asset. More general corporate ﬁnancing would require undertaking due diligence assessment of all the activities of the client. If human rights impacts were to occur within the appropriate unit of analysis, a bank would be directly linked to these impacts.131 This would remove the need to perform due diligence in relation to subsidiaries of parent companies, or parent companies of subsidiaries. However, the human rights risk ﬂows from the relationship, rather than the type of product or service oﬀered.132 Regardless of the type of ﬁnancial product or service oﬀered, ﬁnancing the activities of an entity which is a high risk in terms of its likelihood of committing human rights violations granted in the absence of due diligence and strict conditionality, may result in a ‘contribution’, and not merely direct linkage. C. The Role of Ruggie, the Oﬃce of the High Commissioner for Human Rights, and Shift in Overseeing Interpretation It is important that when implementing and interpreting the UNGP, decentralised networks adhere to the spirit of the UNGP. When this does not happen, the interpretation oﬀered by the networks must be ‘steered’ in the right direction. This steering function has been exercised by various actors such as the UN Oﬃce of the High Commissioner for Human Rights (OHCHR) and the UN Working Group on Business and Human Rights, but also the Shift Project—an NGO created by Ruggie to support the implementation and adoption of the UNGP—and by Ruggie himself. A. Divergence between International Standards and the UNGP He has intervened in the global conversation concerning the UNGP on several occasions, both in his capacity as Special Representative and after his mandate formally ended in 2011—in his personal capacity. It is important that when implementing and interpreting the UNGP, decentralised networks adhere to the spirit of the UNGP. When this does not happen, the interpretation oﬀered by the networks must be ‘steered’ in the right direction. This steering function has been exercised by various actors such as the UN Oﬃce of the High Commissioner for Human Rights (OHCHR) and the UN Working Group on Business and Human Rights, but also the Shift Project—an NGO created by Ruggie to support the implementation and adoption of the UNGP—and by Ruggie himself. He has intervened in the global conversation concerning the UNGP on several occasions, both in his capacity as Special Representative and after his mandate formally ended in 2011—in his personal capacity. During his mandate, Ruggie became involved in the drafting of ISO 26000. Informal contacts between his team and the ISO Strategic Advisory Group took place133 before Ruggie, more formally, voiced his concerns about the inclusion of the concept of ‘sphere of inﬂuence’ in the standard and the consequent inconsistency with the UNGP that it would generate. In a note,134 Ruggie highlighted the problems in embracing that approach and drew attention to his previous criticism of the concept in a preparatory report of the UNGP.135 https://doi.org/10.1017/S0020589320000469 Published online by Cambridge University Press 156 International and Comparative Law Quarterly The ISO Working Group on Social Responsibility redrafted the deﬁnition of sphere of inﬂuence and relevant articles in close cooperation with Ruggie’s team, downplaying the connection between the possibility to inﬂuence and responsibility and stressing the concept of ‘impact’.136 However, as discussed above, the idea of sphere of inﬂuence still permeates ISO 26000, and even suggests that business responsibility is not just negative but also positive, in express contradiction to the UNGP.137 As with other notes submitted by civil society and NGOs, such as WWF, the note submitted by Ruggie was formally accepted but only minimally reﬂected in the ﬁnal text of ISO 26000.138 Ruggie was also in contact with the IFC and the OECD at various points.139 His contact with the OECD during the drafting of the 2018 Due Diligence Guidance for Responsible Business Conduct was particularly signiﬁcant. 136 Wood (n 110) 70. 137 ISO 26000, clause 4.1. 138 A Aseeva, ‘Global Trade Governance and Informal Voluntary Standards: The Socio- normative Analysis of Legitimacy of the ISO’ in A Tutumlu and G Güngör (eds), Multilateralism in Global Governance; Formal and Informal Institutions (Peter Lang 2016) 91. 139 Buhmann (n 52) 426. 140 For Ruggie’s letter see <https://www.business-humanrights.org/ sites/default/ﬁles/documents/OECD%20Workshop%20Ruggie%20letter%20-%20Mar%202017_0. pdf>. 141 Debevoise & Plimpton and Enodo Rights. ‘Practical Deﬁnitions of Cause, Contribute and Directly Linked to Inform Business Respect for Human Rights’ (2017) Available at <https://www. business-humanrights.org/sites/default/ﬁles/documents/Debevoise-Enodo-Practical-Meaning-of- Involvement-Draft-2017-02-09.pdf>. 142 ibid 7–8. 143 JG Ruggie, ‘Comments on Thun Group of Banks Discussion Paper on the Implications of UN Guiding Principles 13 & 17 in a Corporate and Investment Banking Context’ (2017) Available at <https://www.business-humanrights.org/sites/default/ﬁles/documents/Thun%20Final.pdf>. 136 Wood (n 110) 70. 138 p 143 JG Ruggie, ‘Comments on Thun Group of Banks Discussion Paper on the Implications of UN Guiding Principles 13 & 17 in a Corporate and Investment Banking Context’ (2017) Available at <https://www.business-humanrights.org/sites/default/ﬁles/documents/Thun%20Final.pdf>. p 141 Debevoise & Plimpton and Enodo Rights. ‘Practical Deﬁnitions of Cause, Contribute and Directly Linked to Inform Business Respect for Human Rights’ (2017) Available at <https://www. business-humanrights.org/sites/default/ﬁles/documents/Debevoise-Enodo-Practical-Meaning-of- Involvement-Draft-2017-02-09.pdf>. 142 ibid 7–8. A. Divergence between International Standards and the UNGP Ruggie took issue with the notion of ‘signiﬁcant’ or ‘substantial’ contribution to harm, discussed in Section III.A, noting the risk of employing language inconsistent with the UNGP, potentially undermining its spirit.140 Ruggie’s criticism is based on a discussion paper prepared by the law ﬁrm Debevoise & Plimpton on the three categories for attribution.141 In addition to using the term ‘substantial contribution’, Ruggie also criticised the idea that the concept of direct link and the presence of a business relation should be qualiﬁed by the presence of ‘mutual commercial beneﬁts’.142 The discussion paper equated direct link to the presence of a beneﬁt to the company, a concept extraneous to the UNGP that limits the scope of attribution. While the notion of signiﬁcant contribution was left in the ﬁnal draft of the Guidance, no reference to the concept of ‘mutual commercial beneﬁt’ can be found. Ruggie also publicly commented on the Thun Group paper discussed in Section III.B and reiterated his concern with the introduction of language deviating from the UNGP. In a letter, Ruggie noted the misconstruction of UNGP 13 by collapsing ‘cause’ and ‘contribution’ into a single category, and the introduction of the term of ‘proximity to harm’.143 Following Ruggie’s intervention and NGOs pressure, the Thun Group slightly revised its position, conceding that banks could contribute to human rights impact ‘under p 143 JG Ruggie, ‘Comments on Thun Group of Banks Discussion Paper on the Implications of UN Guiding Principles 13 & 17 in a Corporate and Investment Banking Context’ (2017) Available at <https://www.business-humanrights.org/sites/default/ﬁles/documents/Thun%20Final.pdf>. https://doi.org/10.1017/S0020589320000469 Published online by Cambridge University Press 157 Polycentricity and Polyphony in International Law exceptional circumstances’.144 However, the paper failed to address the substance of Ruggie’s criticism. In 2016 Ruggie also developed recommendations for FIFA to embed human rights in its global operations.145 Having received express requests on three occasions from the ﬁnancial sector, the UN Oﬃce of the High Commissioner for Human Rights (OHCHR) has also oﬀered (formally non-binding) interpretative clariﬁcations concerning the UNGP and HRDD. 144 Thun Group of Banks, ‘Paper on the Implication of UN Guiding Principles 13b and 17 in a Corporate and Investment Banking Context’ (2017) at 1. <https://www.business-humanrights.org/ sites/default/ﬁles/documents/2017_12_Thun%20Group%20of%20Banks_Paper_UNGP%2013b %20and%2017.pdf>. 145 JG Ruggie, ‘‘‘For the Game. For the World.’’ FIFA and Human Rights’ Corporate Responsibility Initiative Report No 68 (Harvard Kennedy School, MA 2016). 146 One is a response to a request made by the NGOs SOMO and OECD Watch: <http://www. ohchr.org/Documents/Issues/Business/LetterSOMO.pdf>. The other is a response to a request by the OECD Working Group on Responsible Business Conduct: <https://www.ohchr.org/ Documents/Issues/Business/LetterOECD.pdf>. 147 See the response to the OECD Working Group on Responsible Business Conduct, at 5. 148 Available at <https://www.ohchr.org/Documents/Issues/Business/InterpretationGuiding Principles.pdf>. 149 See the response to BankTrack, at 10. 150 United Nations Human Rights Oﬃce of the High Commissioner, ‘The Corporate Responsibility to Respect Human Rights: An Interpretative Guide’ (2012) HR/PUB/12/02. 144 Thun Group of Banks, ‘Paper on the Implication of UN Guiding Principles 13b and 17 in a Corporate and Investment Banking Context’ (2017) at 1. <https://www.business-humanrights.org/ sites/default/ﬁles/documents/2017_12_Thun%20Group%20of%20Banks_Paper_UNGP%2013b %20and%2017.pdf>. 145 JG Ruggie, ‘‘‘For the Game. For the World.’’ FIFA and Human Rights’ Corporate Responsibility Initiative Report No 68 (Harvard Kennedy School, MA 2016). https://doi.org/10.1017/S0020589320000469 Published online by Cambridge University Press A. Divergence between International Standards and the UNGP Two requests made in 2013 covered the issue of minority shareholding, and speciﬁcally whether it could constitute a business relationship capable of giving rise to a direct link between a human rights impact and ﬁnancial institutions.146 Having responded positively to this question, the two 2013 documents also accepted that project ﬁnancing could amount to a direct link, something which is not supported by the text of the UNGP, and rejected the frequently employed terminology of ‘indirect impact’ or ‘indirect link’ (ie impacts caused by the client and not by the bank). In particular, it stressed that ﬁnancing oﬀered to a ﬁrm for a speciﬁc project would directly link a ﬁnancial institution to all adverse human rights impacts generated by that project.147 Finally, in 2017, the OHCHR was approached by the NGO BankTrack for additional interpretative guidance following the Thun Group’s paper.148 In its response, the OHCHR reiterated the distinction between contribution and direct link and noted that proximity was not a factor determining the boundaries of human rights responsibilities. It also noted that disputes about the presence of ‘contribution’ rather than ‘direct link’ could be settled through stakeholder dialogue or grievances processes,149 thereby identifying possible means of solving interpretative tensions. It should also be mentioned that the OHCHR published early interpretative guidance on the UNGP in 2012150 which, however, left several questions open, in particular on the concepts of attribution and leverage. The UN Working Group on Business and Human Rights was established in the immediate aftermath of the UNGP, with its mandate extended in 2014 and 2017. Although not actively involved in interpreting UNGP, it has come to https://doi.org/10.1017/S0020589320000469 Published online by Cambridge University Press 158 International and Comparative Law Quarterly resemble an orchestrator that convenes and facilitates the work of other actors.151 Over the years, the UN Working Group has engaged with and supported a number of non-UN initiatives with the goal of achieving increased consistency, coordination and deﬁnitional clarity in the interpretations and understanding of the UNGP, while promoting dissemination and uptake.152 A key focus is to identify good practices while ensuring that the UNGP remain the authoritative reference point for business and human rights, and that various interpretative instruments do not undermine the integrity of the framework.153 A fourth actor playing a critical role in steering the interpretation of the UNGP, and in particular the concept of leverage, has been the Shift Project. 151 KW Abbott and D Snidal, ‘Strengthening International Regulation through Transnational New Governance: Overcoming the Orchestration Deﬁcit’ (2009) 42 VandJTransnatlL 551. 152 151 KW Abbott and D Snidal, ‘Strengthening International Regulation through Transnational New Governance: Overcoming the Orchestration Deﬁcit’ (2009) 42 VandJTransnatlL 551. 152 MK Addo, ‘The Reality of the United Nations Guiding Principles on Business and Human Rights’ (2014) 14 HRLRev 133. 153 UN Working Group on the Issue of Human Rights and Transnational Corporations and Other Business Enterprises, Report of the Working Group on the Issue of Human Rights and Transnational Corporations and Other Business Enterprises, Human Rights Council, UN Doc A/HRC/20/29 (10 April 2012) 11–12. 154 <https://www.shiftproject.org>. 155 Shift Project, ‘Embedding Respect for Human Rights within a Company’s Operation’ Shift Workshop Report No 1 (June 2012) available at <https://www.shiftproject.org/resources/ publications/embedding-respect-human-rights>. 156 Shift Project, ‘Respecting Human Rights through Global Supply Chains’ Shift Workshop Report No 2 (October 2012) available at <https://www.shiftproject.org/resources/publications/ respecting-human-rights-global-supply-chains>. 157 Shift Project, ‘Using Leverage in Business Relationships to Reduce Human Rights Risks’ (November 2013) available at <https://www.shiftproject.org/resources/publications/leverage- business-relationships-reduce-human-rights-risk/#>. 158 Shift Project, ‘Human Rights Due Diligence in High-Risk Circumstances: Practical Strategies for Business’ (March 2015) available at <https://www.shiftproject.org/resources/publications/ human-rights-due-diligence-high-risk-circumstances/>. https://doi.org/10.1017/S0020589320000469 Published online by Cambridge University Press g ( ) 152 MK Addo, ‘The Reality of the United Nations Guiding Principles on Business and Human Rights’ (2014) 14 HRLRev 133. 153 156 Shift Project, ‘Respecting Human Rights through Global Supply Chains’ Shift Workshop Report No 2 (October 2012) available at <https://www.shiftproject.org/resources/publications/ respecting-human-rights-global-supply-chains>. 157 159 Over time, additional clariﬁcations emerged. For example, the OECD Guidances all contain examples of leverage in speciﬁc circumstances. 160 Shift Project (n 157) 6. 161 Shift Project, ‘Business and Human Rights Impact: Identifying and Prioritising Human Rights Risks’ Workshop Report (15–16 January 2014) Social and Economic Council (SER) The Hague. Available at <https://www.shiftproject.org/resources/publications/business-human-rights- impacts-identifying-prioritizing-risks/>. 162 European Commission, ‘ICT Sector Guide on Implementing the UN Guiding Principles on Business and Human Rights’ (2014). Available at <https://publications.europa.eu/en/publication- detail/-/publication/ab151420-d60a-40a7-b264-adce304e138b>. 163 ILO-IOE, ‘Child labour guidance tool for business. How to do business with respect for children’s right to be free from child labour. ILO International Programme on the Elimination of Child Labour; International Organisation of Employers (Geneva: ILO 2015). Available at <https://www.unglobalcompact.org/library/3881>. 164 Ruggie (n 17). A. Divergence between International Standards and the UNGP Shift describes itself as the leading centre of expertise on the UNGP. Established by Ruggie in 2011, Shift’s experts work in close contact with businesses, governments, civil society and international organisations to turn the UNGP into practice through education and training, advocacy and facilitation.154 Shift was among the ﬁrst organisations to cooperate closely with business and has provided some pioneering guidance on the UNGP. Over time it has compiled a considerable number of documents, mostly in the form of guidelines to help business operationalise components of HRDD. A series of workshops with companies have contributed to clarify a number of practical elements of HRDD, including the embedding of human rights in corporate operations,155 the concept of ‘direct link’ and value chain due diligence,156 the use of leverage,157 and due diligence in high-risk circumstances.158 Even though its guidance documents can hardly be considered as soft law, the impact of Shift’s work should not be underestimated. In particular, its work on leverage represents the ﬁrst clariﬁcation oﬀered on this critical concept based on 156 Shift Project, ‘Respecting Human Rights through Global Supply Chains’ Shift Workshop Report No 2 (October 2012) available at <https://www.shiftproject.org/resources/publications/ respecting-human-rights-global-supply-chains>. 157 157 Shift Project, ‘Using Leverage in Business Relationships to Reduce Human Rights Risks’ (November 2013) available at <https://www.shiftproject.org/resources/publications/leverage- business-relationships-reduce-human-rights-risk/#>. p g 158 Shift Project, ‘Human Rights Due Diligence in High-Risk Circumstances: Practical Strategies for Business’ (March 2015) available at <https://www.shiftproject.org/resources/publications/ human-rights-due-diligence-high-risk-circumstances/>. https://doi.org/10.1017/S0020589320000469 Published online by Cambridge University Press 159 Polycentricity and Polyphony in International Law corporate experience.159 The Guidance identiﬁes ﬁve categories of leverage (traditional commercial leverage, business leverage, leverage with business partners, leverage through bilateral engagement, and through multi- stakeholder collaboration), with concrete examples and suggestions on how to identify opportunities and strategies.160 By cooperating with other actors, Shift has been able to spread this understanding of leverage to public bodies and international organisations. Guidance published with the Dutch Social and Economic Council,161 the European Commission,162 as well as by the International Labour Organisation and the International Organisation of Employers163 all reﬂect the approaches identiﬁed in Shift’s workshops. https://doi.org/10.1017/S0020589320000469 Published online by Cambridge University Press p 163 ILO-IOE, ‘Child labour guidance tool for business. How to do business with respect for children’s right to be free from child labour. ILO International Programme on the Elimination of Child Labour; International Organisation of Employers (Geneva: ILO 2015). Available at <https://www.unglobalcompact.org/library/3881>. 164 Ruggie (n 17). IV. THE THREAT OF POLYPHONY AND POSSIBLE SOLUTIONS The analysis above highlights considerable challenges in achieving consistent and fully aligned interpretation in an open polycentric regime where many actors can, and are in fact invited to, oﬀer interpretation of key elements. The underlying idea behind the UNGP was to support and steer a process of bottom- up emergence and convergence between various transnational regulatory regimes aiming at establishing human rights responsibility, bypassing States (and their disagreements). One of the purposes of the UNGP was to encourage rule emergence by and through interpretative intermediaries, ie to stimulate a global conversation on the principle of business respect for human rights. The UNGP have largely been successful on this front. Disseminating and embedding social norms is an indispensable method for inducing changes in conduct.164 The UNGP polycentric approach presents, however, its own risks, and these become clear where principles need to be reﬁned and clariﬁed through interpretation. An open polycentric system—characterised by the lack of barriers to expansive interpretations nor boundaries nor bodies that can oﬃcially reject ‘deviant’ interpretation—opens up the challenge of misaligned interpretations, and even interpretations which are contrary to the constitutive principles. Diﬀerent intermediaries, while contributing to the assertion and institutionalisation of the general idea of corporate https://doi.org/10.1017/S0020589320000469 Published online by Cambridge University Press 160 International and Comparative Law Quarterly responsibility to respect human rights, also hinder the institutionalisation of some of the equally important details. This may occur because of intermediaries operating at diﬀerent moments in time, on the basis of diﬀerent organisational goals, or the use of diﬀerent processes. More problematically, it also occurs because polycentricity—in the absence of clear interpretative responsibilities—enables powerful economic interests to undertake the fundamentally public functions of interpreting international soft law. Certain intermediaries arguably emerged in order to provide instrumental rule interpretation congenial to corporations, as it happens in the case of the Thun Group of Banks. Alignment of the intermediaries’ goals with those of the principal regime is crucial in determining how they will fulﬁl their intermediary function. International organisations whose mandate is close to the UNGP goals generate fewer discrepancies. ‘Obstructionists’ that intervene arguably to frustrate the goals of the regime generate substantial deviation.165 In this ‘polyphonic’ scenario, where no organisation or actor can claim interpretative monopoly over the language in the UNGP, rule creators must attempt to retain or regain control over the interpretation of the rules in question,166 and steer intermediates towards consistency. 165 L Brès, S Mena and ML Salles-Djelic, ‘Exploring the Formal and Informal Roles of Regulatory Intermediaries in Transnational Multistakeholder Regulation’ (2019) 13 RegGov 133. 166 P Goodrich, Reading the Law: A Critical Introduction to Legal Method and Techniques (Basil Blackwell 1984) 123. 167 ‘… the Guiding Principles on Business and Human Rights that I developed …’, emphasis added, Ruggie (n 5) 5. 168 Deva (n 67) 85–6. 169 R Hahn and C Weidtmann, ‘Transnational Governance, Deliberative Democracy, and the Legitimacy of ISO 26000: Analyzing the Case of a Global Multistakeholder Process’ (2016) 55 Business & Society 90. See also Aseeva (n 138). 170 N De Silva, ‘Intermediaries Complexity in Regulatory Governance: The International Criminal Court’s Use of NGOs in Regulating International Crimes’ (2017) 670 AnnalsAmAcadPol&SocSci 170. IV. THE THREAT OF POLYPHONY AND POSSIBLE SOLUTIONS As suggested earlier, diﬀerent actors operate on the basis of distinct organisational goals, and draft their standards involving speciﬁc groups and on the basis of their own procedures. In this light, a certain degree of variation would be diﬃcult to avoid. Inclusive participation is not the rule, as the limited stakeholder participation in ISO 26000 drafting demonstrates,169 It is diﬃcult to pinpoint the underlying reasons for deviations and discrepancies between the approaches of international organisations. As suggested earlier, diﬀerent actors operate on the basis of distinct organisational goals, and draft their standards involving speciﬁc groups and on the basis of their own procedures. In this light, a certain degree of variation would be diﬃcult to avoid. Inclusive participation is not the rule, as the limited stakeholder participation in ISO 26000 drafting demonstrates,169 and as evidenced by the Thun Group approach, which has involved only business actors. A decentralised process of interpretation (and implementation) cannot ensure that the inclusive process that led to the adoption of the UNGP can be successfully replicated. Quite the opposite, inclusiveness may be more of an obstacle to the formation of detailed rules than it is to the adoption of broad principles, or may contribute to the emergence of particular interpretations. Further research is needed, but this may occur because of internal disagreement or because, when operating by consensus, the temptation may be either to avoid the more controversial issues, or to settle for the less ambitious positions advanced by of businesses. and as evidenced by the Thun Group approach, which has involved only business actors. A decentralised process of interpretation (and implementation) cannot ensure that the inclusive process that led to the adoption of the UNGP can be successfully replicated. Quite the opposite, inclusiveness may be more of an obstacle to the formation of detailed rules than it is to the adoption of broad principles, or may contribute to the emergence of particular interpretations. Further research is needed, but this may occur because of internal disagreement or because, when operating by consensus, the temptation may be either to avoid the more controversial issues, or to settle for the less ambitious positions advanced by of businesses. The analysis illustrates that polycentric regimes relying on the interpretative work of multiple intermediaries and the lack of an authority to challenges ‘deviant’ interpretation are exposed to the risk of a lack of consistency when it comes to detail. IV. THE THREAT OF POLYPHONY AND POSSIBLE SOLUTIONS The lack of a body with a clear coordinating role has meant that various actors have attempted to steer intermediaries. Their degree of success varies, highlighting the diﬃculty of steering other regulatory actors towards a desired outcome. Interpretation provided by these ‘steering actors’ does not adhere to the traditional methods of interpreting the law, such as linguistic, systemic, and teleological interpretative techniques. This is quite striking considering that, in the end, the UNGP constitute international law, however soft. Apart from the uncontroversial role of the OHCHR in providing interpretative guidance, albeit non-binding, Ruggie’s interventions are arguably made possible because of the moral authority he continues, and decides, to exercise as ‘creator’ of the UNGP. That Ruggie sees himself as a main architect behind the UNGP is clear from his writings,167 and from his limited receptivity to input concerning possible changes to the core concepts of the framework.168 The centralising eﬀect of Ruggie’s interventions and his position as the ‘main creator’ are diﬃcult to reconcile with the UNGP’ focus on polycentricity, pluralism and consultation. The additional reliance on Shift, established by Ruggie as a centre of expertise on the UNGP, is a creative attempt to insulate the code from other inﬂuences and to develop it in accordance with its founding principles through multi-stakeholder workshops https://doi.org/10.1017/S0020589320000469 Published online by Cambridge University Press 161 Polycentricity and Polyphony in International Law and consensus. In spite of this unconventional approach, the interpretative work of Shift has been widely accepted and is now reﬂected in guidance documents produced by public authorities and international organisations. Attempts at steering are, then, grounded on rationales as diverse as the formal authority of the OHCHR, the moral authority of Ruggie and, in line with bottom-up processes underlying the emergence of the UNGP, the involvement of corporations and other stakeholders. and consensus. In spite of this unconventional approach, the interpretative work of Shift has been widely accepted and is now reﬂected in guidance documents produced by public authorities and international organisations. Attempts at steering are, then, grounded on rationales as diverse as the formal authority of the OHCHR, the moral authority of Ruggie and, in line with bottom-up processes underlying the emergence of the UNGP, the involvement of corporations and other stakeholders. It is diﬃcult to pinpoint the underlying reasons for deviations and discrepancies between the approaches of international organisations. IV. THE THREAT OF POLYPHONY AND POSSIBLE SOLUTIONS Empirically, considerable divergence is possible, as ISO 26000 demonstrates. While polycentricity has been instrumental in establishing business responsibility to respect human rights, deviations in interpretation and polyphony could undermine rule acceptance. In particular, they may hinder the establishment and institutionalisation of the responsibility itself and a consistent message about the boundaries of such responsibility, as corporations shop around for interpretations that better suit their interests. Where intermediaries have divergent goals, regulators should intervene in their activities and centralise their functions.170 In the case of the UNGP an organisation responsible for interpretative work could be designated, leaving https://doi.org/10.1017/S0020589320000469 Published online by Cambridge University Press 162 International and Comparative Law Quarterly implementation and operationalisation of management processes to other actors with no ‘barriers to entry’. Currently, the OHCHR provides non-binding interpretations, but has done so only in the few cases in which it was requested to intervene. A possible solution would be to have national courts or OECD National Contact Points taking up interpreting functions.171 Even if national courts themselves produce diﬀering interpretations, the transnational nature of much business activity means that corporations tend to adopt the approach found in the most restrictive jurisdiction in which they work, and this can have an ‘aligning’ eﬀect. Courts decisions could also inﬂuence guidance issued by organisations such as the OECD. It would however be preferable if centralised interpretive functions were exercised by international courts and tribunals or international organisations. The possible adoption of a binding treaty on business and human rights may return some interpretive functions to bodies traditionally entrusted with that role. A Zero Draft submitted in 2017 to the Open-ended Intergovernmental Working Group on Transnational Corporations and Other Business Enterprises with Respect to Human Rights attracted considerable criticism.172 The goals of a possible Treaty are to strengthen respect of human rights in business activities, prevent human rights violations and ensure access to remedies. The Revised Draft submitted in July 2019 makes the connection with the UNGP more explicit and includes an obligation for States to introduce mandatory HRDD legislation.173 The Draft is better aligned to both the spirit and the language of the UNGP than was the Zero Draft.174 However, the text refers to ‘abuses that may arise from their own business activities, or from their contractual relationships’. It must be noted that direct linkage encompasses situations connected to business relationships, which is a broader concept than merely contractual relationships. 171 HH Koh, ‘Transnational Public Law Litigation’ (1991) 100 YaleLJ 2347. 172 p 174 Holding, with respect to the categories of attribution, that corporations should exercise due diligence with respect to their own activities, those of entities under direct and indirect control, and those directly linked to its operations, products or services. Open-ended Intergovernmental Working Group on Transnational Corporations and Other Business Enterprises with Respect to Human Rights. Zero Draft, art 9. Available at <https://www.business-humanrights.org/sites/default/ﬁles/ documents/DraftLBI.pdf>. 171 HH Koh, ‘Transnational Public Law Litigation’ (1991) 100 YaleLJ 2347. 172 D Cassel, ‘The Third Session of the UN Intergovernmental Working Group on a Business and Human Rights Treaty’ (2018) 3 BHRJ 277. 173 Open-ended Intergovernmental Working Group on Transnational Corporations and Other Business Enterprises with Respect to Human Rights. Revised Draft, art 5. Available at <https:// www.ohchr.org/Documents/HRBodies/HRCouncil/WGTransCorp/OEIGWG_RevisedDraft_LBI. pdf>. 174 Holding, with respect to the categories of attribution, that corporations should exercise due diligence with respect to their own activities, those of entities under direct and indirect control, and those directly linked to its operations, products or services. Open-ended Intergovernmental Working Group on Transnational Corporations and Other Business Enterprises with Respect to Human Rights. Zero Draft, art 9. Available at <https://www.business-humanrights.org/sites/default/ﬁles/ documents/DraftLBI.pdf>. 172 D Cassel, ‘The Third Session of the UN Intergovernmental Working Group on a Business and Human Rights Treaty’ (2018) 3 BHRJ 277. https://doi.org/10.1017/S0020589320000469 Published online by Cambridge University Press g y ( ) 173 Open-ended Intergovernmental Working Group on Transnational Corporations and Other Business Enterprises with Respect to Human Rights. Revised Draft, art 5. Available at <https:// www.ohchr.org/Documents/HRBodies/HRCouncil/WGTransCorp/OEIGWG_RevisedDraft_LBI. pdf>. 174 , g ( ) 172 D Cassel, ‘The Third Session of the UN Intergovernmental Working Group on a Business and Human Rights Treaty’ (2018) 3 BHRJ 277. 173 Open-ended Intergovernmental Working Group on Transnational Corporations and Other Business Enterprises with Respect to Human Rights. Revised Draft, art 5. Available at <https:// www.ohchr.org/Documents/HRBodies/HRCouncil/WGTransCorp/OEIGWG_RevisedDraft_LBI. pdf>. 174 Holding, with respect to the categories of attribution, that corporations should exercise due diligence with respect to their own activities, those of entities under direct and indirect control, and 175 Open-ended Intergovernmental Working Group on Transnational Corporations and Other Business Enterprises with Respect to Human Rights. Revised Draft, art 7. 176 Open-ended Intergovernmental Working Group on Transnational Corporations and Other Business Enterprises with Respect to Human Rights. Draft Optional Protocol. Available at <https:// www.ohchr.org/Documents/HRBodies/HRCouncil/WGTransCorp/Session4/ZeroDraftOPLegally. pdf>. 177 Open-ended Intergovernmental Working Group on Transnational Corporations and Other Business Enterprises with Respect to Human Rights. Revised Draft, art 16.2. IV. THE THREAT OF POLYPHONY AND POSSIBLE SOLUTIONS On a more positive note, the Revised Draft expressly enumerates the components of HRDD as speciﬁed in the UNGP. The Revised Draft oﬀers various venues for interpretation. First, it speciﬁes States where alleged violations have occurred, victims are domiciled, or natural https://doi.org/10.1017/S0020589320000469 Published online by Cambridge University Press 163 Polycentricity and Polyphony in International Law sons involved are domiciled, all have jurisdiction over claims.175 or legal persons involved are domiciled, all have jurisdiction over claims.175 This approach increases the possibility of national litigation concerning the meaning and boundaries of HRDD, which could foster consistency and uniformity. Another option for decentralised interpretation would be the Draft Optional Protocol,176 establishing a National Implementation Mechanism responsible for the promotion and implementation of the Treaty. Among other objectives, the national body would be entrusted with review functions and of making recommendations concerning corporate operationalisation of HRDD practices, including that of single corporations. Remarkably, and most promisingly, centralised interpretation is provided for in the Revised Draft itself. Parties to the Treaty may agree to submit to the International Court of Justice (ICJ) disputes concerning the interpretation of the Treaty which cannot be solved by non-judicial means.177 Although in its current form the provision does not grant compulsory jurisdiction to the ICJ, it would make possible the unequivocal interpretation of the categories of attribution and other matters left open by the UNGP Framework. A possible Binding Treaty should, however, go much further establish a body which has the authority to provide binding interpretations of the UNGP. It could have the power to respond to requests for interpretation, or even do so proprio motu should it consider that guidance issued by intermediaries, or even national courts, was not in accordance with the UNGP. This would not undermine the progress made through the UNGP, the acceptance of HRDD and its interpretation in various fora. Decentralised interpretation, in line with polycentricity, would be retained. It could, however, oﬀer a much-needed institutional complement to the UNGP polycentric approach that is indispensable both for cementing the responsibility to respect human rights, as well as providing guidance to businesses in the operationalisation of their responsibilities. V. CONCLUSION Polycentric structures help ensure both norm diﬀusion and implementation by decentralised networks. However, in the ﬁeld of business and human rights an appraisal of the regulatory governance of the UNGP reveals challenges in interpretation and ensuring consistency concerning approaches to attribution. Remarkably, these challenges derive from the very governance arrangements chosen as an alternative to traditional legal instruments to establish and reﬁne https://doi.org/10.1017/S0020589320000469 Published online by Cambridge University Press 164 International and Comparative Law Quarterly corporate responsibility to respect human rights. While polycentricity has been instrumental to the diﬀusion and acceptance of the corporate responsibility to respect, it also risks degenerating into polyphony given the lack of centralised interpretative authority. It is therefore suggested that the informal mechanisms currently used for guiding the interpretation of key elements of the UNGP are supported by more formal institutions which are able to provide ﬁnal and authoritative interpretations when necessary. This would act as a guard against interpretations being inappropriately inﬂuenced by the economic interests of those currently engaging in interpretive activities. Drawing on the literature on regulation, this article has argued that it is crucial that detailed interpretive guidance is provided to multinationals, and that uncontested and clear Principles are needed in order to establish the corporate responsibility to respect. The polycentric structure of the UNGP, almost ten years since its establishment, has been successful in aﬃrming and diﬀusing the corporate responsibility to respect. We are now entering a stage where due diligence is about to become ‘mainstream’, with a growing number of companies actively supporting the introduction of mandatory HRDD legislation, and where precise and consistent guidance is needed. Corporations must receive unequivocal guidance about the boundaries of their responsibility to respect. The threats posed by HRDD’s indeterminate framework and obligations should not be underestimated, and are similar to the wild proliferation of ‘sustainability’ claims in the past decades.178 Similarly to the many private standards addressing sustainability in production practices that appeared in recent times, it is possible to make claims concerning the proper undertaking of due diligence by corporations, that may in fact be disconnected from actual positive impact. 178 A Marx and J Wouters, ‘Competition and Cooperation in the Market of Voluntary Sustainability Standards’ in Delimatsis (n 93) 215. 179 E Partiti, ‘Orchestration as a Form of Public Action: The EU Engagement with Voluntary Sustainability Standards’ 25 ELJ 115–16. V. CONCLUSION There is thus a risk that due diligence could similarly result in extensive ‘greenwashing’ and ‘bluewashing’ of corporate activities, as well as forum shopping for less ambitious HRDD standards and guidelines that better suit the businesses in question.179 Centralising interpretation is necessary in order to address and avoid conﬂicting approaches to operationalising HRDD and, ultimately, for embedding respect for human rights in corporate activities. It is consistent with the legal pluralism of the business and human rights ﬁeld and is an essential and indispensable component of eﬀective polycentric regimes. Adding this missing element to the informal structure of the UNGP would allow public authorities to eﬀectively intervene in conﬂicts between public and private orders through the provision of ‘formal’ interpretation, and to manage tensions between, and align, the various conﬂicting instruments. https://doi.org/10.1017/S0020589320000469 Published online by Cambridge University Press
https://openalex.org/W2890211832	https://bmcbioinformatics.biomedcentral.com/track/pdf/10.1186/s12859-018-2349-1	English	null	GeFaST: An improved method for OTU assignment by generalising Swarm’s fastidious clustering approach	BMC bioinformatics	2,018	cc-by	9,713	Mü and Nebel BMC Bioinformatics (2018) 19:321 https://doi.org/10.1186/s12859-018-2349-1 Mü and Nebel BMC Bioinformatics (2018) 19:321 https://doi.org/10.1186/s12859-018-2349-1 Abstract Background: Massive genomic data sets from high-throughput sequencing allow for new insights into complex biological systems such as microbial communities. Analyses of their diversity and structure are typically preceded by clustering millions of 16S rRNA gene sequences into OTUs. Swarm introduced a new clustering strategy which addresses important conceptual and performance issues of the popular de novo clustering approach. However, some parts of the new strategy, e.g. the fastidious option for increased clustering quality, come with their own restrictions. Results: In this paper, we present the new exact, alignment-based de novo clustering tool GeFaST, which implements a generalisation of Swarm’s fastidious clustering. Our tool extends the fastidious option to arbitrary clustering thresholds and allows to adjust its greediness. GeFaST was evaluated on mock-community and natural data and achieved higher clustering quality and performance for small to medium clustering thresholds compared to Swarm and other de novo tools. Clustering with GeFaST was between 6 and 197 times as fast as with Swarm, while the latter required up to 38% less memory for non-fastidious clustering but at least three times as much memory for fastidious clustering. Conclusions: GeFaST extends the scope of Swarm’s clustering strategy by generalising its fastidious option, thereby allowing for gains in clustering quality, and by increasing its performance (especially in the fastidious case). Our evaluations showed that GeFaST has the potential to leverage the use of the (fastidious) clustering strategy for higher thresholds and on larger data sets. Keywords: Sequence clustering, Operational taxonomic units, Microbial community analysis data sets, the amplicons are commonly grouped into oper- ational taxonomic units (OTUs). Over the years, diverse methods for OTU clustering have been developed, which can employ alignment-based or alignment-free [2] sim- ilarity measures and compute these exactly or approxi- mately. In addition, methods differ in how they deter- mine the clusters: (i) comparing sequences to a reference database and grouping those sequences which are similar to the same reference sequence (closed-reference cluster- ing), (ii) clustering sequences based on their distances among each other (de novo clustering), and (iii) a combina- tion of both using de novo clustering for those sequences that could not be assigned through closed-reference clus- tering (open-reference clustering). GeFaST: An improved method for OTU assignment by generalising Swarm’s fastidious clustering approach Robert Müller1,2* and Markus E. Nebel1,2,3 Correspondence: romueller@techfak.uni-bielefeld.de 1International Research Training Group “Computational Methods for the Analysis of the Diversity and Dynamics of Genomes”, Bielefeld University, Bielefeld, Germany 2Faculty of Technology, Bielefeld University, Bielefeld, Germany Full list of author information is available at the end of the article Background The advent of high-throughput sequencing (HTS) tech- nologies revolutionised the research in the life sciences and the resulting massive genomic data sets provide the basis for new insights into the diversity and dynamics of biological systems. For example, contemporary studies of the diversity and structure of microbial communities often involve sequencing millions of 16S rRNA gene sequences due to, e.g., its ubiquitous nature [1]. In order to facili- tate downstream analyses of the resulting huge amplicon Correspondence: romueller@techfak.uni-bielefeld.de 1International Research Training Group “Computational Methods for the Analysis of the Diversity and Dynamics of Genomes”, Bielefeld University, Bielefeld, Germany As pointed out by Westcott and Schloss [3], all three approaches have their strengths and weaknesses, but de novo clustering has become a favourite one – especially 2Faculty of Technology, Bielefeld University, Bielefeld, Germany Full list of author information is available at the end of the article 2Faculty of Technology, Bielefeld University, Bielefeld, Germany Full list of author information is available at the end of the article g y © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Swarm clustering Swarm [8] has been devised as an exact, two-phased, agglomerative de novo clustering algorithm that over- comes above problems by iteratively extending a cluster using a local clustering threshold t and starting from the most abundant amplicons. Here, a cluster (or OTU) can be viewed as an edge-weighted, rooted, acyclic and undirected graph G = (V, E, s, w) where V is the set of vertices (amplicons), E is the set of edges (links between amplicons), s is the root (seed amplicon), and w is the weight function assigning to each edge the distance between the incident amplicons. Using a scoring func- tion δ, Swarm considers the distance dδ between two amplicons as the number of differences in an optimal alignment based on the given δ. Then, the set of part- ners of an amplicon a in an amplicon pool A respective to a distance function d and a threshold t is defined as Pd(a, A, t) = {b ∈A \| d(a, b) ≤t}, with Swarm using d = dδ. C<b = C ∈C a∈C.V a.abundance < b C≥b = C ∈C a∈C.V a.abundance ≥b C<b = C ∈C a∈C.V a.abundance < b C≥b = C ∈C a∈C.V a.abundance ≥b C<b = C ∈C a∈C.V a.abundance < b C≥b = C ∈C a∈C.V a.abundance ≥b Fastidious clustering grafts light OTUs onto heavier ones by postulating the existence of a (virtual) linking amplicon (Fig. 1b). If such a virtual amplicon bridges the gap of size at most tf = 2 (with tf being the fastidious threshold) between the OTUs, then all amplicons of the light OTU (but not the virtual amplicon itself) are added to the heavy one. y In general, Swarm identifies the partners of an amplicon by iterating over the remaining amplicons in the pool and computing pairwise optimal alignments to determine the number of differences. In order to avoid a large number of unnecessary alignment computations, two amplicons have to pass a filtering step first, which compares their k- mer compositions to obtain an estimate of their similarity [9]. Furthermore, Swarm speeds up the alignment compu- tations by parallelisation through SIMD instructions. For t = 1, current versions of Swarm employ a dedicated algo- rithm which scales linearly with the number of amplicons. ∧a.abundance ≥b.abundance}. Moreover, Swarm offers a so-called fastidious clustering option for t = 1 from version 2.1.0 onwards in order to reduce the effect of under-grouping. To this end, Swarm distinguishes between light and heavy OTUs using a user- definable threshold b on their total abundance (with the sum of the abundances of the comprised amplicons being considered as the weight of an OTU). For a collection of OTUs C and threshold b, the light and heavy OTUs (C<b and C≥b, respectively) are defined as follows: Swarm clustering The partners of an amplicon are found by generating the microvariants of the current amplicon (i.e. all amplicons with an edit distance of 1 to it) and searching these in a hash table of the amplicons in the pool. Microvariants are also used in the fastidious clustering step, which is imple- mented with the help of a Bloom filter [10], a probabilistic dictionary, in which the microvariants of all amplicons of light OTUs are stored. Subsequently, the microvariants of the amplicons of heavy OTUs are cross-checked against the dictionary in order to identify the fastidious links. Its iterative clustering method for a pool A of amplicons works as follows (Fig. 1a): The most abundant amplicon in the pool is removed from it and serves as the seed s of a new OTU. Next, all amplicons in Pdδ(s, A, t) are transferred from A to the OTU (forming the first genera- tion of subseeds). For each such subseed s′, we determine Pdδ s′, A, t in order to find the second generation of sub- seeds. This process is iterated until no more amplicons can be added to the OTU, which is then closed. Start- ing with the most abundant amplicon in the remaining pool as the seed of the next OTU, the overall procedure is repeated until the pool is empty. In order to avoid over-grouping through long chains of consecutive links between amplicons (a common problem of single-linkage clustering), Swarm also implements an optional breaking mechanism to turn different centres of abundance into separate OTUs. Originally, breaking was realised in a separate phase using a parameterised script. In brief, it examined the abundances along such amplicon chains linking centres of abundance (usually star-shaped subgraphs with an abundant amplicon in its centre, which is surrounded by less abundant amplicons) and decided on breaking or not based on the ratio between the minimum and maximum observed abundance. More current ver- sions of Swarm use a non-parameterised breaking mech- anism, which is directly included in the growth phase described above and allows only monotonically decreas- ing abundances along consecutive links (outwards from the seed). Partners of an amplicon are then defined as Page 2 of 14 Page 2 of 14 Page 2 of 14 Mü and Nebel BMC Bioinformatics (2018) 19:321 P′ d(a, A, t) = {b ∈A \| d(a, b) ≤t because it does not depend on (the existence of) a refer- ence database. However, traditional de novo methods (e.g. [4–6]) are criticised for their sensitivity to the input order of the amplicons and their dependence on an arbitrary fixed global clustering threshold [7]. Pass-Join also proved that their approach is both correct and com- plete, i.e. it finds all pairs of similar sequences and only those. inverted indices (chosen based on the length of the cur- rently considered S) with a selection of substrings from S. Pass-Join finds similar sequences in a set of sequences using the pigeonhole principle and the inverted indices as described in Algorithm 1. Li et al. also propose some sophisticated methods for the substring selection (Algorithm 1, line 6), reducing the number of feasible substrings to only a few per seg- ment. Their most advanced method, multimatch-aware substring selection, makes use of the length and position of the segment as well as of the length difference of S and the indexed sequences in question and prunes the substring set by some clever considerations on where further match- ing substrings have to exist to satisfy the edit-distance threshold. The filtering step is based on a pigeonhole principle. For a given edit-distance threshold t, consider two sequences R and S where R is divided into t + 1 (disjoint) segments. Then, S has to contain a substring matching a segment of R if the edit distance de between R and S is at most t. The segments for this method are chosen using an even-partitioning scheme, limiting the maximum length difference of segments of R to 1. Furthermore, Li et al. suggest to reduce the complex- ity of the verification step (Algorithm 1, line 13) by computing only the bounded edit distance. They also improve on the traditional method [12] by, e.g., con- sidering the length difference of S and C as well as adding an early-termination check. In the present study, we lift some of the restrictions of Swarm by introduc- ing our exact, alignment-based de novo clustering tool GeFaST (Generalised Fastidious Swarming Tool), which in particular generalises the fastidious clustering option and makes it more broadly applicable. We assess the extended functionality in comparison with Swarm and other de novo tools by evaluating the clustering quality and performance on mock-community and natural data sets. In order to apply the pigeonhole principle efficiently, inverted indices mapping segments onto sequence iden- tifiers are built. Pass-Join As described in the previous section, determining the partners of the current subseed is a crucial step in the clustering strategy of Swarm. While the employed k-mer filter helps to avoid many unnecessary alignment compu- tations, iterating over the remaining pool for each subseed is still time-consuming. Similarly, setting up the Bloom Page 3 of 14 Mü and Nebel BMC Bioinformatics (2018) 19:321 a b Fig. 1 Schematic view of Swarm’s clustering strategy. a Starting from a seed, amplicons are added iteratively using a small local threshold t until the OTU reaches its natural limit when no more amplicons can be connected to it. b By postulating the existence of virtual linking amplicons, light OTUs are grafted onto heavy ones during the fastidious clustering step. Adapted from [8, Figure 1] a b Fig. 1 Schematic view of Swarm’s clustering strategy. a Starting from a seed, amplicons are added iteratively using a small local threshold t until the OTU reaches its natural limit when no more amplicons can be connected to it. b By postulating the existence of virtual linking amplicons, light OTUs are grafted onto heavy ones during the fastidious clustering step. Adapted from [8, Figure 1] filter and cross-checking microvariants for fastidious clus- tering can be expensive in terms of runtime and memory consumption. Both tasks come down to identifying sim- ilar sequences, which can be efficiently accomplished by adapting the segment filter introduced by Li et al. in Pass- Join [11], a tool originally proposed for computing string similarity joins on two sets of strings using the edit dis- tance. It follows a filter-and-verify approach to determine pairs of similar sequences efficiently, avoiding large pro- portions of unnecessary sequence comparisons. Li et al. also proved that their approach is both correct and com- plete, i.e. it finds all pairs of similar sequences and only those. filter and cross-checking microvariants for fastidious clus- tering can be expensive in terms of runtime and memory consumption. Both tasks come down to identifying sim- ilar sequences, which can be efficiently accomplished by adapting the segment filter introduced by Li et al. in Pass- Join [11], a tool originally proposed for computing string similarity joins on two sets of strings using the edit dis- tance. It follows a filter-and-verify approach to determine pairs of similar sequences efficiently, avoiding large pro- portions of unnecessary sequence comparisons. Li et al. Pass-Join Hence, for each sequence length l and segment index (i ∈[ 1 : t + 1]), the corresponding inverted index Il,i establishes the relation between observed seg- ments and all sequences of length l containing them as their i-th segment. For a given set of sequences S, we can then find (poten- tially) similar sequences by querying a subset of the Page 4 of 14 Mü and Nebel BMC Bioinformatics (2018) 19:321 Algorithm 1 Segment filter of Pass-Join Input: S = set of sequences, t = edit-distance threshold Output: A = {{R, S} \| R, S ∈S ∧de(R, S) ≤t} 1: Sort S first by string length and second lexicographi- cally; 2: for S ∈S do 3: for \|S\| −t ≤l ≤\|S\| do 4: Initialise candidate set C := ∅; 5: for 1 ≤i ≤t + 1 do 6: Select substrings W of S for lookup in Il,i; 7: for w ∈W do 8: Add Il,i(w) to C; 9: end for 10: end for 11: end for 12: for C ∈C do 13: Verify sequence pair (S, C); 14: if de(S, C) ≤t then 15: Add {S, C} to A; 16: end if 17: end for 18: Partition S and add its segments to I\|S\|,i, 1 ≤i ≤ t + 1; 19: end for Algorithm 1 Segment filter of Pass-Join Input: S = set of sequences, t = edit-distance threshold Output: A = {{R, S} \| R, S ∈S ∧de(R, S) ≤t} 1: Sort S first by string length and second lexicographi- cally; 2: for S ∈S do 3: for \|S\| −t ≤l ≤\|S\| do 4: Initialise candidate set C := ∅; 5: for 1 ≤i ≤t + 1 do 6: Select substrings W of S for lookup in Il,i; 7: for w ∈W do 8: Add Il,i(w) to C; 9: end for 10: end for 11: end for 12: for C ∈C do 13: Verify sequence pair (S, C); 14: if de(S, C) ≤t then 15: Add {S, C} to A; 16: end if 17: end for 18: Partition S and add its segments to I\|S\|,i, 1 ≤i ≤ t + 1; 19: end for Algorithm 1 Segment filter of Pass-Join Implementation details. Implementation details. Since GeFaST differs from the original versions of Swarm and Pass-Join’s segment filter, we subsequently describe the key aspects of our implementation. Pass-Join 1: Sort S first by string length and second lexicographi- cally; Segment filter. In order to enhance the segment filter, GeFaST deviates from its original version introduced by Li et al. in some respects. First, it applies a generalised pigeonhole principle [13], dividing amplicon sequences into t + k, k ≥1, segments of which at least k have to be matched. Second, GeFaST implements a bidirectional seg- ment filter [14] adding a pipelined second filtering step in order to increase the filtering capacity. Unlike in Pass-Join, all inverted indices (per pool) are constructed at once, because the amplicons are processed in an order based on their abundance (and not their length). Non-fastidious clustering. This first and mandatory clustering step explores the amplicon space in order to find the initial OTUs. For each amplicon pool, we start by building the inverted indices of the segment filter using all amplicons of this pool in order to facilitate the efficient computation of the amplicon partners. Subsequently, we determine the OTUs according to the iterative strategy described in “Swarm clustering” section. Algorithm 2 pro- vides a pseudocode description of how the amplicon space is explored in GeFaST. The optional breaking mechanism in our tool is identical to the non-parameterised one used in newer Swarm versions. The resulting OTUs are then handed over to the fastidious clustering step or directly to the output phase. Implementation GeFaST generalises the clustering strategy of Swarm and combines it with a refined version of the segment filter introduced in Pass-Join in order to find the pairs of simi- lar amplicons more efficiently during the computation of the OTUs. Our tool mimics the key features of Swarm and offers a similar command-line interface. Fastidious clustering. The second but optional cluster- ing step tries to refine the initial OTUs as also outlined in “Swarm clustering” section. GeFaST generalises the fastid- ious clustering in two ways. First, it is no longer restricted to input threshold t = 1. This is achieved by employing a second segment filter, for which we index only the ampli- cons from light OTUs and search grafting partners for the ones from heavy OTUs among them. In order to preserve the idea of a virtual linking amplicon, the segment filter is used with a fastidious threshold tf = 2 ∗t (as the default setting). Second, we capitalise on the flexibility of the seg- ment filter by making tf freely adjustable and independent of t. This allows for more or less conservative fastidious clustering as needed. The overall workflow of GeFaST (Fig. 2) consists of three main phases: preprocessing, swarm (or OTU) clus- tering and generating the outputs. The preprocessing allows to filter the input amplicons by length and alpha- bet. It also splits the overall set of amplicons into pools based on the clustering threshold t such that amplicons from different pools cannot be similar. As a result, each amplicon pool can then be handled separately in the clus- tering phase whose details are described below. Finally, the requested outputs are generated from the obtained OTUs, with GeFaST offering the same five output types as Swarm. Currently, the memory consumption of our tool is in O(T) words where T is the total length of all amplicons. GeFaST’s runtime complexity is dominated by the veri- fications (Algorithm 1, line 13) having an overall worst- case complexity in O(N2 · L · t), with N and L being the number of amplicons and their maximum length, respectively. Subsequently, we provide a more formal description of fastidious clustering in GeFaST. A grafting link can only be established between an amplicon from a light OTU and another one from a heavy OTU. Implementation Let Lb(C) = otu∈C<b otu.V, Hb(C) = otu∈C≥b otu.V Lb(C) = otu∈C<b otu.V, Hb(C) = otu∈C≥b otu.V Page 5 of 14 Mü and Nebel BMC Bioinformatics (2018) 19:321 Fig. 2 Workflow of GeFaST. The amplicons from one or more input files are preprocessed and grouped into pools. Within each pool, OTUs are formed by finding similar sequences using a segment filter. Optionally (denoted by dashed frames), OTUs can be refined by fastidious clustering. Finally, the different kinds of output are generated from the OTUs of all amplicon pools Fig. 2 Workflow of GeFaST. The amplicons from one or more input files are preprocessed and grouped into pools. Within each pool, OTUs are formed by finding similar sequences using a segment filter. Optionally (denoted by dashed frames), OTUs can be refined by fastidious clustering. Finally, the different kinds of output are generated from the OTUs of all amplicon pools be the collections of amplicons from all light and heavy OTUs, respectively. The set of potential grafting links is then defined as hi.abundance > hj.abundance hi.abundance > hj.abundance j ∨(hi.abundance = hj.abundance ∧li.abundance > lj.abundance). ∨(hi.abundance = hj.abundance Finally, the valid grafting links, which are used in the fastidious clustering step (Algorithm 3), are defined as Results In order to evaluate the performance of our tool as well as the clustering quality of the new fastidious clustering options, we conducted several comparative analyses on the following mock-community and natural data sets: • even: The even mock-community data set from the original Swarm paper [7]. Genome isolates of the V4 region of the 16S rRNA gene from 49 bacterial and 10 archaeal species were dereplicated to 143,162 unique amplicons of average length 271.2 bp (from 1,577,469 raw reads). More information on the composition of the mock community is available in Additional file 1: Section 1. Notes: Notes: (a) next_seed(A) obtains the amplicon with the highest abundance in A. (b) The amplicons in the subseed queue Q are sorted by generation and (within each generation) in descending order by abundance. (c) Similar to Swarm, ties between amplicons are broken through the lexicographical order of their identifiers in both cases. • uneven: The uneven mock-community data set from the original Swarm paper [7]. Genome isolates of the same origin as those for even were dereplicated to 55,621 unique amplicons of average length 263.6 bp (from 637,871 raw reads). In order to obtain a more realistic community structure (including a few abundant and many rare organisms), the genome isolates were distributed according to a log-normal distribution whose parameters were fitted from a soil microbial community. refer as scoring-function mode) as well, because it provides a lower bound for the number of differences in an opti- mal alignment. Moreover and in contrast to Swarm, the user can choose whether to run GeFaST in edit-distance or scoring-function mode. • eldermet: Natural data set obtained from the faecal microbiota of 170 human subjects as part of the ELDERMET project [16]. The 16S rRNA gene V4 region reads of all subjects were pooled and dereplicated to 4,183,843 unique amplicons of average length 250.8 bp (from 8,989,448 raw reads). Verification In order to verify whether two amplicons are similar or not, we use the length-aware verification method [11, Sec. 5.1.]. This dynamic-programming algo- rithm improves on a method developed by Ukkonen [12] to determine the bounded edit distance, reducing the number of diagonals to compute, and performs an early termination when it is guaranteed that the amplicons can- not be similar. Ld(C, t, b) ={(h, l) \| h ∈Hb(C) ∧l ∈Lb(C) ∧d(h, l) ≤t}. Ld(C, t, b) ={(h, l) \| h ∈Hb(C) ∧l ∈Lb(C) ∧d(h, l) ≤t}. Ld(C, t, b) ={(h, l) \| h ∈Hb(C) ∧l ∈Lb(C) ∧d(h, l) ≤t}. Vd(C, t, b) = {(hi, li) ∈Ld(C, t, b) \| ¬∃ hj, lj ∈Ld (C, t, b) . j < i ∧otu (li) = otu lj ¬∃ hj, lj ∈Ld (C, t, b) . j < i ∧otu (li) = otu lj For an amplicon l ∈Lb(C), there can be multiple poten- tial grafting partners h ∈Hb(C), but only the one with the highest abundance is actually considered during the grafting process. Furthermore, a light OTU is grafted at most once, even if there are potential grafting links to sev- eral heavy OTUs. Hereinafter, we assume that Ld(C, t, b) is sorted such that for all (hi, li) and (hj, lj) with i < j the following holds where otu(a) denotes the OTU containing amplicon a. where otu(a) denotes the OTU containing amplicon a. Edit-distance mode. The segment filter was originally developed just for the edit distance de, while Swarm uses dδ - based on some (user-specified) affine scoring func- tion δ - as the distance between two amplicons. Ld(C, t, b) ={(h, l) \| h ∈Hb(C) ∧l ∈Lb(C) ∧d(h, l) ≤t}. However, we can use the segment filter in this case (to which we will Page 6 of 14 Mü and Nebel BMC Bioinformatics (2018) 19:321 Algorithm 2 Non-fastidious clustering in GeFaST Input: A = amplicon pool, t = clustering threshold, d = distance function Output: C = collection of OTUs 1: C := ∅; 2: while A ̸= ∅do 3: ampl := next_seed(A); 4: otu := ({ampl}, ∅, ampl, ∅); 5: A := A \ {ampl}; 6: Q := {ampl}; // queue of subseeds to be processed 7: while Q ̸= ∅do 8: ampl := next_subseed(Q); 9: Q := Q \ {ampl}; 10: for p ∈P′ d(ampl, A, t) do 11: Add amplicon p and edge {ampl, p} with 12: weight d(ampl, p) to otu; 13: end for 14: Q := Q ∪P′ d(ampl, A, t); 15: A := A \ P′ d(ampl, A, t); 16: end while 17: C := C ∪{otu}; 18: end while Algorithm 2 Non-fastidious clustering in GeFaST Algorithm 3 Fastidious clustering in GeFaST Input: C = OTUs from non-fastidious clustering, tf = fastidious clustering threshold, b = abundance thresh- old, d = distance function Output: C = collection of refined OTUs 1: Determine Vd(C, tf , b); 2: for (h, l) ∈Vd(C, tf , b) do 3: otu(h).V = otu(h).V ∪otu(l).V; 4: otu(h).E = otu(h).E ∪otu(l).E ∪{{h, l}}; 5: otu(h).w = otu(h).w∪otu(l).w∪{{h, l} →d(h, l)}; 6: C := C \ otu(l); 7: end for Algorithm 3 Fastidious clustering in GeFaST Input: C = OTUs from non-fastidious clustering, tf = fastidious clustering threshold, b = abundance thresh- old, d = distance function Output: C = collection of refined OTUs 1: Determine Vd(C, tf , b); 2: for (h, l) ∈Vd(C, tf , b) do 3: otu(h).V = otu(h).V ∪otu(l).V; 4: otu(h).E = otu(h).E ∪otu(l).E ∪{{h, l}}; 5: otu(h).w = otu(h).w∪otu(l).w∪{{h, l} →d(h, l)}; 6: C := C \ otu(l); 7: end for Results In order to attain similar benefits in the scoring-function mode, we transfer the ideas of length- aware verification to Gotoh’s algorithm [15] for affine scoring functions. The dereplication of above data sets was performed using Swarm (v2.1.13) and, in addition, all reads that contained at least one ambiguous base (IUPAC code n resp. N) were removed. In our evaluations, we compared the de novo clustering tools GeFaST (v1.0.0), Swarm (v1.2.3 and v2.1.13), USEARCH ([4], v10.0.240_i86linux32), VSEARCH ([17], v2.7.1), CD-HIT ([6], v4.6.8), DNACLUST ([5], release 3) and Sumaclust ([18], v1.0.31). USE- ARCH (cluster_fast, cluster_smallmem) and Page 7 of 14 Mü and Nebel BMC Bioinformatics (2018) 19:321 VSEARCH (cluster_fast, cluster_smallmem, cluster_size) were included with different options and sorting criteria (abundance, length). differences between GeFaST’s edit-distance and scoring- function mode on both data sets were minute. However, Swarm (v.1.2.3) exceeded Swarm (v2) and GeFaST for t ≥7 (uneven) resp. t ≥4 (even). Due to fastidious cluster- ing, the recall rose (decreasingly) and the precision tended to decline (increasingly) for growing t on both data sets. As a consequence, the adjusted Rand index decreased again for t ≥5 (t ≥4) when activating fastidious cluster- ing with tf = t+1 (tf = 2∗t). For all t ≥2, we observed on both data sets that the increase (decrease) in recall (preci- sion) due to using tf = 2 ∗t was larger than the one due to tf = t + 1 (for t = 1, tf was obviously the same in both cases). Evaluation of clustering quality We assessed the clustering quality using ground truths and three metrics analogous to Mahé et al. [7]: the recall, measuring the proportion of amplicons from the same species that are grouped in the same OTU, the precision, quantifying the extent to which amplicons in an OTU are also from the same species, and - summarising both - the adjusted Rand index [19, 20], measuring the agreement between the OTUs and the taxonomic assignment and correcting for chance. We also tested the statistical significance of the differ- ences in clustering quality between the evaluated tools (see Additional files 1: Section 4, 2 and 3). The results of the performed paired t-tests (with a significance level of 0.05) hinted at statistically significant differences between the different modes and fastidious options of GeFaST as well as between GeFaST and other tools. The magnitude of the differences compared to the metric values was, how- ever, very small in the majority of the cases (often even below 1%). Clustering mock-community data First, we examined uneven and even with all the above tools using threshold t from 1 to 10 (resp. 0.99 to 0.90). Moreover, Swarm (v2.1.13) was executed with fastidious clustering for t = 1, while GeFaST was also run with an activated fastidious option for all thresholds t, once per fastidious threshold tf ∈{t + 1, 2 ∗t}. The 16S refer- ence data set for this analysis had been hand-picked from the Greengenes database [21] by the authors of Swarm based on the list of organisms in the mock communities as pointed out by Mahé et al. (pers. comm., 2017). To ensure reproducibility, the reference data set is accessible online and a link to it is included in Additional file 1: Section 1. For both mock communities, the ground truth was estab- lished by matching the sequences against above reference data set (through VSEARCH with a minimum sequence identity of 97% and the usearch_global option) and picking the closest hit. 83.2% of the sequences in uneven and 68.2% of the ones in even matched against the refer- ence. The results of analogous analyses based on ground truths derived with a minimum sequence identity of 95 resp. 99% are shown in Additional file 1: Section 2. Clustering natural data Second, we performed a quality analysis on the eldermet data set at the genus level (Fig. 4) using GeFaST (as the representative of the iterative approach) as well as USEARCH, VSEARCH, CD-HIT, DNACLUST and Sumaclust (all representing the classic de novo approach). Swarm and some options of GeFaST, USEARCH and VSEARCH were not included for performance reasons or based on the results on the mock-community data. In contrast to the mock-community analyses, we had to preprocess the natural data in order to derive a feasible ground truth. In brief, we started by match- ing the sequences from eldermet against the SILVA database ([22], release 128) with a minimum sequence identity of 95%. Among the sequences having a match in SILVA, we kept only those that could be assigned a complete unambiguous taxonomic classification up to the genus level. The reduced eldermet data set then contained 1,315,605 unique amplicons with an average length of 244.1 bp. We conducted this analysis at the genus level because the species information in the ref- erence databases is very incomplete and together with a minimum sequence identity of 97% less than 10% of the eldermet sequences would have passed the preprocessing. More details on the reduction steps are provided in Additional file 1: Section 3. For the actual evaluation, we generated five random subsamples of the The clustering quality behaved similarly on both mock- community data sets (Fig. 3). In general, the recall improved up to a threshold around t = 6, after which it levelled of or decreased slightly. The precision declined with increasing t for all tools but they differed notably in the extent of this decline. Only GeFaST, Swarm and one option of USEARCH could avoid larger drops for thresh- olds close to 10. With some exceptions, e.g. USEARCH (cluster_fast plus length sorting) on even, the over- all clustering quality (adjusted Rand index) peaked for medium to small thresholds. The overall clustering qual- ity of many tools dropped off at one or even both ends of the threshold range (e.g. DNACLUST). In constrast, GeFaST and Swarm remained relatively stable over all examined thresholds. Hence, they achieved a higher or similar clus- tering quality for the majority of thresholds (especially on uneven). Non-fastidious clustering with GeFaST and Swarm was almost identical in terms of clustering quality. Also, the Mü and Nebel BMC Bioinformatics (2018) 19:321 Page 8 of 14 Fig. Performance evaluation We compared the runtime and memory consumption of GeFaST (in scoring-function mode) and Swarm (v2.1.13) on eldermet in two ways. First, we used the full data set, but varied the threshold t from 1 to 10. Both tools were run without fastidious clustering for all thresh- olds. Again, the fastidious option was activated for all t (with tf set to t + 1 resp. 2 ∗t) for GeFaST and when possible (i.e. for t = 1) for Swarm. Second, we exam- ined different data set sizes while keeping threshold t constant. For that purpose, we randomly subsampled eldermet at various levels ranging from 5% to 100% (5% steps, three subsamples per level). Each of the 60 subsamples was then clustered with both tools for t ∈ {1, 2} (the fastidious option was activated when possible as above). The recall again rose with increasing threshold, but - in contrast to the previous evaluation - it started very low for all tools and achieved only a maximum recall of 0.68 through GeFaST for t = 9. The recall of the other tools usually stayed below the one of GeFaST and hardly sur- passed the level of 0.6 for t = 10. The precision, in turn, behaved almost as for the mock communities. Starting from high values around 0.97, it decreased gradually for all tools. The precision values of the tools spread out more notably beyond t = 7, with GeFaST showing the largest drop this time. Similar to the recall, GeFaST achieved the highest overall clustering quality with a maximum adjusted Rand index of 0.59 and usually outperformed the other tools. In contrast to the mock-community analysis, fastidious clustering did not have a notable impact on the overall clustering quality throughout this evaluation. In addition, we compared the performance of iterative swarm clustering and classic de novo clustering with a global threshold. To this end, we evaluated the runtime and memory consumption of GeFaST (scoring-function mode), USEARCH, VSEARCH, CD-HIT, DNACLUST and Sumaclust on the reduced eldermet data set described in the previous section. As before, threshold t ranged from 1 to 10 and the fastidious option of GeFaST was activated for all t (with tf set to t + 1 resp. 2 ∗t). We again tested the statistical significance of the qual- ity differences (see Additional files 1: Section 4 and 4). Clustering natural data 3 Comparison of clustering quality on uneven (top) and even (bottom) mock-community data set for ten different thresholds. Precision and recall (summarised in the adjusted Rand index) use the amplicons’ taxonomic assignments as the ground truth options, but attains double-digit percentages for those involving the other tools. reduced eldermet data set (each covering 80% of it). Subsequently, we computed the ground truth for each subsample and clustered each of them with the tools stated above for t from 1 to 10 (resp. 0.99 to 0.90). Performance evaluation As for the mock-community data, the paired t-tests (with a significance level of 0.05) showed a large proportion of statistically significant differences. The magnitude of the differences compared to the metric values was, as before, very small for comparisons between different GeFaST Mü and Nebel BMC Bioinformatics (2018) 19:321 Page 9 of 14 Fig. 4 Comparison of clustering quality on the reduced eldermet data set for ten different thresholds. The average values are determined from five random subsamples (each comprising 80% of the reduced data set). The standard deviation is indicated by the error bars Fig. 4 Comparison of clustering quality on the reduced eldermet data set for ten different thresholds. The average values are determined from five random subsamples (each comprising 80% of the reduced data set). The standard deviation is indicated by the error bars Fig. 4 Comparison of clustering quality on the reduced eldermet data set for ten different thresholds. The average values are determined from five random subsamples (each comprising 80% of the reduced data set). The standard deviation is indicated by the error bars Our analyses were performed in an LXC container under Debian GNU/Linux 8.7 (jessie) on an Intel Xeon E5-2687W v4 (3.00GHz) system with 256 GB of RAM. We measured the runtime and memory consumption of a program execution via the (external) command /usr/bin/time with the resource specifiers e and M, respectively. More precisely, specifier e returns the elapsed wall clock time, while M provides the maximum resident set size. all these finished computations were still faster than Swarm for t = 2. Additionally, GeFaST completed the computations for t ≤4 (non-fastidious and fastidious with tf = t + 1) resp. t ≤2 (fastidious with tf = 2 ∗t) in less or approximately the same time as Swarm for t = 1 with fastidious clustering. While there was a drastic difference between the runtime of Swarm for t = 1 and t = 2, the runtime of GeFaST grew more gradually as t increased. The memory consumption of non-fastidious clustering with GeFaST was continuously higher than the one of Swarm, while it was notably lower for fastidious cluster- ing. Furthermore, there was a huge difference w.r.t. the amount of additional memory used for the latter. Fas- tidious clustering increased the memory consumption of Swarm more than fivefold, whereas GeFaST’s memory footprint grew by less than 5%. Iterative clustering for different thresholds. Performance evaluation The development of the runtime and memory consump- tion dependent on clustering threshold t is depicted in Fig. 5. Within the time limit of 36 h, Swarm completed the computations only for t ≤2. In contrast, GeFaST computed all clusterings in time except for t = 9 and t = 10 with fastidious clustering using tf = 2 ∗t and Mü and Nebel BMC Bioinformatics (2018) 19:321 Page 10 of 14 Fig. 5 Comparison of runtime and memory consumption on eldermet for 1 ≤t ≤10 with and without fastidious clustering. The runtime was capped at 36 h Fig. 5 Comparison of runtime and memory consumption on eldermet for 1 ≤t ≤10 with and without fastidious clustering. The runtime was capped at 36 h Iterative clustering for different data set sizes. Figure 6 shows how runtime and memory consumption developed with increasing data set size. GeFaST was consistently faster than Swarm for both thresholds and all fastidious options. For example, GeFaST performed non-fastidious clustering almost seven times as fast as Swarm for t = 1. While the runtime of both tools behaved linearly in the data set size for t = 1, they showed a non-linear behaviour for t = 2, with Swarm displaying a much steeper incline. Hence, Swarm could only process subsets not larger than 50% of eldermet within the time limit of 10 h for t = 2. Moreover, even fastidious clustering with GeFaST was continuously faster than non-fastidious clustering with Swarm. GeFaST also increased the runtime considerably less than Swarm across the different subset sizes. On the permutations of the full eldermet data set (i.e. the 100% subsets), for instance, the average runtime of GeFaST rose from 93 s to 585 s by activating fastidious clustering with tf = 2, while it increased from 632 s to 6682 s for Swarm. Furthermore, the variation in the runtime on subsets of the same size tended to be stronger for Swarm. clustering with GeFaST, in turn, required only between 22 and 32% of the memory occupied by Swarm. On top of that, the additional memory consumption due to fastidi- ous clustering was much smaller when using GeFaST (less than 6% more memory compared to increasing fivefold or more using Swarm). In contrast to the runtime, there was no noticeable variation in the memory consumption of both tools on subsets of the same size irrespective of threshold and fastidious option. Performance evaluation Iterative versus classic de novo clustering. For an increasing clustering threshold t, GeFaST exhibited a con- trary behaviour - especially w.r.t. the runtime - compared to the other tools in this evaluation (Fig. 7). While the run- time of GeFaST increased for larger thresholds, it tended to decrease for the other tools. As a consequence, GeFaST clustered the data notably faster or similarly fast for t ≤4 but was also considerably slower for thresholds towards t = 10. The largest gains in runtime of the non-iterative tools occurred before t = 5, after which some of them (e.g. CD-HIT and DNACLUST) got slightly slower again. Among the non-iterative clustering approaches DNACLUST was the fastest and also the only one that showed low runtimes for thresholds down to t = 1. With respect to the memory consumption, the picture is more complex. Non-fastidious clustering with Swarm was consistently more memory-efficient, but the advan- tage seemed to grow smaller with increasing threshold. While the average memory advantage on subsets of up to 50% of eldermet was approximately 34% for t = 1, it was only slightly more than 26% for t = 2. Fastidious The memory consumption behaved similarly but the differences were not as distinct as for the runtime. Some non-iterative tools (e.g. USEARCH) required less memory Mü and Nebel BMC Bioinformatics (2018) 19:321 Page 11 of 14 Fig. 6 Comparison of runtime and memory consumption on differently sized subsets of eldermet for t = 1 and t = 2. The average values are determined from three random subsamples of the respective size, while the standard deviation is indicated by the error bars. The runtime was capped at 10 h Fig. 6 Comparison of runtime and memory consumption on differently sized subsets of eldermet for t = 1 and t = 2. The average values are determined from three random subsamples of the respective size, while the standard deviation is indicated by the error bars. The runtime was capped at 10 h Sumaclust, which required more than three times as much memory than the others throughout the comparison. Sumaclust, which required more than three times as much memory than the others throughout the comparison. for higher thresholds, while the memory usage of oth- ers such as DNACLUST was basically independent of t. Performance evaluation The memory consumption of GeFaST was very similar to the one of the other tools for small thresholds but, in contrast to them, increased slightly for higher thresh- olds. However, the outlier in terms of memory usage was for higher thresholds, while the memory usage of oth- ers such as DNACLUST was basically independent of t. The memory consumption of GeFaST was very similar to the one of the other tools for small thresholds but, in contrast to them, increased slightly for higher thresh- olds. However, the outlier in terms of memory usage was Discussion While the two mock-community data sets span a number of phyla, these often comprise more than one class and even several species per class to make the data more representative and to not rule out effects such as single-linkage chaining from the outset. On the mock- community data, GeFaST showed a smaller dependence on the clustering threshold and was often similarly good or even slightly better than the classic de novo tools. On the natural data, in contrast, the threshold had a large impact on all tools, but the clustering quality was notably higher for GeFaST. Moreover, the edit-distance and scoring-function mode (using the default parameters borrowed from Swarm) did only differ slightly, hinting at the possibility to use the potentially faster edit-distance mode without impairing the quality. Future work is going to address GeFaST’s runtime and memory consumption as well as the achievable clus- tering quality. On the one hand, we will work on the performance of the segment filter for higher thresh- olds and explore the benefits of parallelising (parts of) GeFaST’s workflow. On the other hand, we plan to intro- duce memory-saving succinct data structures [25] for the key data structures of GeFaST in order to investi- gate their applicability to sequence clustering in terms of runtime. In our analyses, fastidious clustering improved the qual- ity only on the mock-community data sets. The reasons for the lack of effect on natural data require further anal- ysis but potential factors are a relatively low number of light OTUs and the restriction to the genus level. Based on our evaluation, benefits in clustering quality from fastidious clustering might be expected for thresholds up to 5. Beyond that the clustering became too greedy and aggregated supposedly different species, thus impair- ing the clustering quality. Many of the differences were found to be statistically significant but their relative size were often small as well. Hence, their practical significance is yet to be examined through, e.g., more biologically motivated metrics such as diversity measures or heritability [24]. With respect to the clustering quality, we will exam- ine the effects of over- and under-grouping more closely. This will involve the exploration of alternative breaking mechanisms and the analysis of how strongly fastidious clustering affects clusters obtained by them, e.g. the one used in older versions of Swarm which produced clusters of higher quality for high thresholds during our analy- ses. Discussion GeFaST adds to the list of de novo clustering tools by extending the iterative approach of Swarm. Therefore, we Fig. 7 Comparison of runtime and memory consumption on the reduced eldermet data set for ten different thresholds. The average values are determined from five random subsamples (each comprising 80% of the reduced data set). The standard deviation is indicated by the error bars Fig. 7 Comparison of runtime and memory consumption on the reduced eldermet data set for ten different thresholds. The average values are determined from five random subsamples (each comprising 80% of the reduced data set). The standard deviation is indicated by the error bars Page 12 of 14 Page 12 of 14 Mü and Nebel BMC Bioinformatics (2018) 19:321 compared GeFaST to Swarm and other current de novo tools for a range of clustering thresholds and on dif- ferent taxonomy levels and kinds of data. Our analyses showed that there are notable differences between the classic approach using a global threshold and the iterative one of Swarm and GeFaST. in these increases. On the one hand, the number of sub- strings per sequence to check during the filtering grows polynomially in the threshold. On the other hand, higher thresholds increase the number of inverted indices to be held in memory. On top of that, there were also distinct differences between GeFaST and Swarm. As described above, increas- ing threshold t led to a relatively gradual growth in run- time for GeFaST, whereas there was a much larger change between t = 1 and t ̸= 1 for Swarm, which applies a dedicated algorithm in the former case. Compared to Swarm, the runtime of GeFaST benefits from the efficient determination of potential amplicon partners due to the segment filter and their fast verification through bounded computations with early termination. Furthermore, the effect of fastidious clustering on runtime and memory consumption was largely different between the two tools. This asymmetry stems from the use of a more memory- intensive Bloom filter and the lengthy cross-checking of microvariants in Swarm compared to another seg- ment filter in GeFaST in order to facilitate the fastidious clustering step. We evaluated the clustering quality on natural and mock-community data, with the latter being acknowl- edged as a trade-off between simulated and natural data by being of biological origin but also of known composi- tion [23]. Author details 1 1International Research Training Group “Computational Methods for the Analysis of the Diversity and Dynamics of Genomes”, Bielefeld University, Bielefeld, Germany . 2Faculty of Technology, Bielefeld University, Bielefeld, Germany . 3IMADA, Southern Denmark University, Odense, Denmark . Received: 13 December 2017 Accepted: 29 August 2018 Received: 13 December 2017 Accepted: 29 August 2018 Funding g This work is funded by the International DFG Research Training Group GRK 1906/1. We also acknowledge the support of the publication fee by Deutsche Forschungsgemeinschaft and the Open Access Publication Funds of Bielefeld University. Availability of data and material GeFaST and the workflow to reproduce the analysis (including all scripts) freely available at https://github.com/romueller/gefast and https://github. com/romueller/gefast-paper-analysis, respectively. The data and results of the analyses are available from Bielefeld University (http://doi.org/10.4119/unibi/ 2918928). Conclusions We thank the anonymous reviewers for their helpful remarks and suggestions on a previous version of this article. We introduced GeFaST, an exact, alignment-based de novo clustering tool which generalises the fastidious clus- tering approach of Swarm to arbitrary thresholds in order to reduce under-grouping in a broader range of settings. Comparisons with Swarm and other current de novo clustering tools on mock-community and natural data showed a competitive or even better clustering quality with GeFaST in a variety of settings. Some results also indicated at improvements due to fastidious clustering for small and medium thresholds up to 5 that might be beneficial to downstream analyses. In addition, our tool outperformed Swarm in terms of runtime throughout our analyses and was also faster than the other tools for thresholds up to 4. Depending on the clustering threshold and the fastidious option, GeFaST was between 6 and 197 times as fast as Swarm. However, Swarm used up to 38% less memory for non-fastidious clustering, but required at least three times as much memory as GeFaST for fastid- ious clustering. Furthermore, our tool scaled better with increasing data set size (especially for t > 1) at the cost of a moderately increased memory footprint. It could also complete computations for higher thresholds and / or with fastidious clustering faster than Swarm with less demanding parameters. Discussion Further evaluations of fastidious clustering will also address the effect of its parameters, i.e. the fastidious clustering threshold tf and the abundance boundary b between light and heavy OTUs. The subsequent evalua- tions will include further comparisons with Swarm and other tools on mock and natural data sets (down to the species level, if possible) as well as the use of a more extensive set of metrics. As pointed out by Westcott and Schloss [3], this is sensible in order to obtain a more objective quality assessment since there is a wide range of approaches all having their assets and drawbacks. In order to explore the limits of GeFaST, we also repeated the mock-community analysis for ground truths based on different sequence similarities. The effect of changing the ground truth was similar for all tools in our evaluation, suggesting that GeFaST is equally well-suited for OTU analyses at different levels of granularity. Our evaluations also showed large differences between the tools in terms of their performance. Most of the clas- sic de novo tools tended to need less time and memory for larger clustering thresholds, most likely due to the decreasing number of clusters they had to build and main- tain. On the contrary, GeFaST’s runtime and, to a lesser extent, its memory consumption increased with a growing threshold. The employed segment filter was a major factor Moreover, we will continue to investigate the character- istics and limits of GeFaST, e.g. whether there is a relation between the clustering threshold and the expected ampli- con length in terms of the clustering quality or whether Page 13 of 14 Mü and Nebel BMC Bioinformatics (2018) 19:321 there is a minimum sequence length for the iterative approach to work properly. chemistry; OTU: Operational taxonomic unit; rRNA: Ribosomal ribonucleic acid; SIMD: Single instruction, multiple data chemistry; OTU: Operational taxonomic unit; rRNA: Ribosomal ribonucleic acid; SIMD: Single instruction, multiple data Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. References d 1. Janda JM, Abbott SL. 16S rRNA Gene Sequencing for Bacterial Identification in the Diagnostic Laboratory: Pluses, Perils, and Pitfalls. J Clin Microbiol. 2007;45(9):2761–4. https://doi.org/10.1128/JCM.01228-07. Additional file 2. Bonham-Carter O, Steele J, Bastola D. Alignment-free genetic sequence comparisons: a review of recent approaches by word analysis. Brief Bioinform. 2014;15(6):890–905. https://doi.org/10.1093/bib/bbt052. Additional file 1: Supplement containing information on the mock-community data, the analyses and additional results. (PDF 336 KB) Additional file 2: Tabular data showing results on the statistical significance of differences in clustering quality on the uneven data set. (CSV 220 KB) Additional file 3: Tabular data showing results on the statistical significance of differences in clustering quality on the even data set. (CSV 219 KB) Additional file 4: Tabular data showing results on the statistical significance of differences in clustering quality on the eldermet data set. (CSV 86 KB) 3. Westcott SL, Schloss PD. De novo clustering methods outperform reference-based methods for assigning 16S rRNA gene sequences to operational taxonomic units. PeerJ. 2015;3:1487. https://doi.org/10.7717/ peerj.1487. 4. Edgar RC. Search and clustering orders of magnitude faster than BLAST. Bioinformatics. 2010;26(19):2460. https://doi.org/10.1093/bioinformatics/ btq461. 5. Ghodsi M, Liu B, Pop M. DNACLUST: accurate and efficient clustering of phylogenetic marker genes. BMC Bioinformatics. 2011;12(1):271. https:// doi.org/10.1186/1471-2105-12-271. 6. Fu L, Niu B, Zhu Z, Wu S, Li W. CD-HIT: accelerated for clustering the next-generation sequencing data. Bioinformatics. 2012;28(23):3150. https://doi.org/10.1093/bioinformatics/bts565. Authors’ contributions RM designed, implemented and evaluated the software. MN supervised all aspects of the GeFaST project. RM wrote the paper with contributions from MN. All authors read and approved the final manuscript. Ethics approval and consent to participate Mü and Nebel BMC Bioinformatics (2018) 19:321 8. Mahé F, Rognes T, Quince C, de Vargas C, Dunthorn M. Swarm v2: highly-scalable and high-resolution amplicon clustering. PeerJ. 2015;3: 593. https://doi.org/10.7717/peerj.1420. 8. Mahé F, Rognes T, Quince C, de Vargas C, Dunthorn M. Swarm v2: highly-scalable and high-resolution amplicon clustering. PeerJ. 2015;3: 593. https://doi.org/10.7717/peerj.1420. 9. Ukkonen E. Approximate string-matching with q-grams and maximal matches. Theoretical Computer Science. 1992;92(1):191–211. https://doi. org/10.1016/0304-3975(92)90143-4. 10. Bloom BH. Space/time trade-offs in hash coding with allowable errors. Commun ACM. 1970;13(7):422–6. https://doi.org/10.1145/362686.362692 10. Bloom BH. Space/time trade-offs in hash coding with allowable errors. Commun ACM. 1970;13(7):422–6. https://doi.org/10.1145/362686.362692. 11. Li G, Deng D, Feng J. A Partition-Based Method for String Similarity Joins with Edit-Distance Constraints. ACM Trans Database Syst. 2013;38(2): 9:1–9:33. https://doi.org/10.1145/2487259.2487261. 11. Li G, Deng D, Feng J. A Partition-Based Method for String Similarity Joins with Edit-Distance Constraints. ACM Trans Database Syst. 2013;38(2): 9:1–9:33. https://doi.org/10.1145/2487259.2487261. 12. Ukkonen E. Algorithms for approximate string matching. Information and Control. 1985;64(1):100–18. https://doi.org/10.1016/S0019- 9958(85)80046-2. 13. Lin C, Yu H, Weng W, He X. Large-Scale Similarity Join with Edit-Distance Constraints. In: Bhowmick SS, Dyreson CE, Jensen CS, Lee ML, Muliantara A, Thalheim B, editors. Database Systems for Advanced Applications. DASFAA 2014. Lecture Notes in Computer Science vol. 8422. Cham: Springer International Publishing; 2014. p. 328–42. https://doi.org/ 10.1007/978-3-319-05813-9_22. 14. Huang Y, Niu B, Song C. Web-Age Information Management: 16th International Conference. WAIM 2015. Lecture Notes in Computer Science vol. 9098. In: Dong XL, Yu X, Li J, Sun Y, editors.; 2015. p. 400–12. https://doi.org/10.1007/978-3-319-21042-1_32. 400–12. https://doi.org/10.1007/978-3-319-21042-1_32. 15. Gotoh O. An improved algorithm for matching biological sequences. J Mol Biol. 1982;162(3):705–8. https://doi.org/10.1016/0022- 2836(82)90398-9. 16. Claesson MJ, Cusack S, O’Sullivan O, Greene-Diniz R, de Weerd H, Flannery E, Marchesi JR, Falush D, Dinan T, Fitzgerald G, Stanton C, van Sinderen D, O’Connor M, Harnedy N, O’Connor K, Henry C, O’Mahony D, Fitzgerald AP, Shanahan F, Twomey C, Hill C, Ross RP, O’Toole PW. Composition, variability, and temporal stability of the intestinal microbiota of the elderly. Proc Natl Acad Sci. 2011;108(Supplement 1):4586–91. https://doi.org/10.1073/pnas. 1000097107. 17. Rognes T, Flouri T, Nichols B, Quince C, Mahé F. VSEARCH: a versatile open source tool for metagenomics. PeerJ. 2016;4:2584. https://doi.org/ 10.7717/peerj.2584. 18. Mercier C, Boyer F, Bonin A, Coissac É. SUMATRA and SUMACLUST: fast and exact comparison and clustering of sequences. Programs Abstr SeqBio Workshop. 2013;14:27–28. 19. Rand WM. Objective Criteria for the Evaluation of Clustering Methods. J Am Stat Assoc. 1971;66(336):846–50. https://doi.org/10.2307/2284239. 20. Hubert L, Arabie P. Abbreviations 7. Mahé F, Rognes T, Quince C, de Vargas C, Dunthorn M. Swarm: robust and fast clustering method for amplicon-based studies. PeerJ. 2014;2:593. https://doi.org/10.7717/peerj.593. bp: Base pair; GeFaST: Generalised fastidious swarming tool; HTS: High-throughput sequencing; IUPAC: International union of pure and applied Page 14 of 14 Mü and Nebel BMC Bioinformatics (2018) 19:321 25. Jacobson GJ. Succinct static data structures. Pittsburgh, PA, USA: PhD thesis, School of Computer Science; 1988. Mü and Nebel BMC Bioinformatics (2018) 19:321 Comparing partitions. J Classif. 1985;2(1):193–218. https://doi.org/10.1007/BF01908075. 21. DeSantis T. Z, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K, Huber T, Dalevi D, Hu P, Andersen GL. Greengenes, a Chimera-Checked 16S rRNA Gene Database and Workbench Compatible with ARB. Appl Environ Microbiol. 2006;72(7):5069–72. https://doi.org/10.1128/AEM.03006-05. 22. Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, Peplies J, Glöckner FO. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 2013;41(D1): 590–6. https://doi.org/10.1093/nar/gks1219. 23. Bokulich NA, Rideout JR, Mercurio WG, Shiffer A, Wolfe B, Maurice CF, Dutton RJ, Turnbaugh PJ, Knight R, Caporaso JG. mockrobiota: a Public Resource for Microbiome Bioinformatics Benchmarking. mSystems. 2016;1(5):. https://doi.org/10.1128/mSystems.00062-16. 24. Jackson MA, Bell JT, Spector TD, Steves CJ. A heritability-based comparison of methods used to cluster 16s rRNA gene sequences into operational taxonomic units. PeerJ. 2016;4:2341. https://doi.org/10.7717/ peerj.2341. 25. Jacobson GJ. Succinct static data structures. Pittsburgh, PA, USA: PhD thesis, School of Computer Science; 1988.
https://openalex.org/W4385561607	https://ojs.academicon.pl/np/article/download/4737/5005	Polish	null	Dominik Zamiatała, Zakony męskie w polityce władz komunistycznych w latach 1945-1989, T. 1 - Problematyka organizacyjno-personalna, Kielce 2009, ss. 720	Nasza Przeszłość	2,010	cc-by-sa	3,663	„N asza P rz e sz ło ść ” t. 113: 2 0 1 0 , s. 3 6 5 -3 7 3 . J A N U S Z Z B U D N IE W E K O SP P E Dotyka kwestii trudnych, przemilczanych, ale naszpikowanych skar gami agentów i prostodusznych duchownych zakonnych i diecezjal nych. Wynika z nich, że zakonnicy bywali nierzadko żywymi tarczami w rękach ordynariuszy i prymasa Wyszyńskiego, który aczkolwiek sam był symbolem męczeństwa, nie doznał jednak większych udręk nad tych, którzy byli i są strukturach Kościoła i państwa ostatnią kategorią duchowieństwa, nierzadko jakby wyjęci spod parasola Wspólnoty Ko ścielnej, zauważani o ile byli przydatni w ogólnej statystyce represji i walce o prawa duchownych i ogółu wierzących (np. s. 216, 242). Susuła zamieszczony w „Tygodniku Powszechnym”. On jako asystent kościelny począł nam dowodzić o nieortodoksyjności maryjnej linii Prymasa, wobec czego artykułu nie przyjął, bo jak uzasadniał - „inną linią w tym wzglądzie ma nasz tygodnik”. Nie da się zaprzeczyć, że w skargach prowincjałów i ludzi obserwu jących sytuację w Polsce były powody do narzekania na antyzakonną linię episkopatu polskiego. Mówiono to, co potwierdził ks. Zamiatała ze skarg, że „zakony w Polsce są degradowane, gdyż nie posiadają swego przedstawiciela na konferencjach episkopatu (wyróżnienie J. Z), za konnicy są pomijani w nominacjach na biskupów, a jedynie są zatrud niani na podrządnych stanowiskach w Kościele” (s. 326). Problem ten dał się zaobserwować w głosowaniu polskich hierarchów na Soborze Watykańskim II, gdzie zaledwie dwóch spośród wszystkich (bp Kazi mierz Kowalski i bodajże Edmund Nowicki) głosowali za zachowaniem egzempcji zakonów. W świetle narosłych problemów, wymienionych w pracy, episkopat traktował zakony jako element, któremu można było wiele zakazy wać, by wspomnieć zakaz kontaktów z Urzędem Bezpieczeństwa, prowadzenia ksiąg inwentarzowych, wizytacji domów, seminariów itp. Prowincjał franciszkanów, o. Celestyn Niezgoda, w swoim utyskiwa niu na panujące wobec zakonów stosunki miał stwierdzić, że „żądanie władz państwowych jest jasne i nie budzi zastrzeżeń, lecz jako prowin cjał podlega episkopatowi i musi wykonywać jego polecenia, gdyż w przeciwnym wypadku Prymas zmyje mu głową... słowem zakony «są bite z obu stron» " (s. 217). Prymas Wyszyński w istocie zastrzegł sobie niewątpliwie wiele, m.in. prawo kontroli wyjeżdżających za granice, nawet wyższych przełożonych, czego autor wprawdzie nie zauważył, niemniej był to jeden z podstawowych warunków, bez spełnienia którego - nie mówiąc o kancelaryjnej opłacie - i mając na względzie tajnych współpracowników - wyjazd był niemożliwy. Natomiast wzywał niekarnych, którzy łamali jego instrukcje lub ustalenia epi skopatu (s. 223). Autor przytoczył kilka odważnych i znamiennych wypowiedzi z tajnych rozmów, a także o otwartym konflikcie zakonów z episkopatem w 1964 roku (s. 216-217). Wspomniał bodaj wzorcową opozycję prowincjała dominikanów o. J A N U S Z Z B U D N IE W E K O SP P E J A N U S Z Z B U D N IE W E K O SP P E Rec.: Dominik Zamiatała, Zakony m ęskie w polityce władz ko m unistycznych w> latach 1945-1989. T. 1 - Problem atyka organi- zacyjno-personalna, Kielce 2009, ss. 720. Obrodziło w ostatnich kilku latach studiami nad polityką władz PRL w sprawach różnorakich represji wobec Kościoła, w tym szczególnie wobec zakonów. Po interesującym studium s. E. Kaczmarek pod wy zywającym tytułem: Dlaczego przeszkadzały? (Warszawa 2007) i zna komitym studium habilitacyjnym s. dr Agaty Mirek: Siostry zakonne w obozach pracy w latach 1954-1956, (Lublin 2008) - w Wydawnic twie Kieleckim „Jedność” ukazała się kolejna potężna praca na temat polityki represyjnej władz komunistycznych w latach 1945-1989, liczą ca 718 stron. Jej autorem jest ks. dr Dominik Zamiatała, klaretyn, ad iunkt w Instytucie Politologii Uniwersytetu Kardynała Stefana Wyszyń skiego w Warszawie. Autor dał się już poznać w kręgu historyków naj nowszej historii Kościoła z cennej monografii Caritas. Działalność i likwidacja organizacji 1945-1950, Lublin 2000, ss. 332 i kilkudziesię ciu artykułów poświęconych torpedowaniu działalności Kościoła przez wyszukane represje, lub po prostu likwidacje jego instytucji. Niemal we wszystkich dotyka ściśle zamkniętego okresu państwa polskiego pod sztuczną nazwą PRL, a zatem istotnych odniesień do życia Kościoła, jego działalności duszpasterskiej i charytatywnej i co istotne - wielo aspektowych restrykcji władz partyjnych wobec instytucji kościelnych, inwigilacji duchowieństwa, represji wobec zakonów i ich działalności edukacyjnej, wychowawczej i charytatywnej, rozsadzania jedności ko ścielnej przez płatnych agentów, szeroko rozbudowanej akcji propa gandy w prasie, restrykcji wobec wierzących i praktykujących na wszystkich szczeblach życia codziennego itp. Studium pt. Zakony męskie w polityce władz komunistycznych w la tach 1945-1989, Problematyka organizacyjno-personalna, jest pozy cją niezwykłą pod wieloma względami. Opracowana na postawie akt zdeponowanych w 13 archiwach państwowych i kościelnych liczących kilka tysięcy fascykułów, m.in.: z Instytutu Pamięci Narodowej, Biura J A N U S Z Z B U D N IE W E K O S P P E R ec.: D. Z A M IA T A Ł A , Z A K O N Y M Ę S K IE W P O L IT Y C E ... 367 366 Politycznego PZPR, kilku ministerstw, Urzędu d/s. Wyznań, akt Epi skopatu, kilku zakonów i ogromny zestaw źródeł drukowanych oraz stosownej literatury. Dotyka wspólnot zakonnych, które zbierały pierw sze i najcięższe uderzenia reżimu komunistycznego w struktury Kościo ła. J A N U S Z Z B U D N IE W E K O SP P E Dyskretny unik w kwestiach politycznie drażliwych, to bezsprzecznie ważki atut jego studium, ale też i pewien niedosyt, że na tle polityki kościelnej nie podjął dyskusyjnego stanowiska państwa, obliczonego najwyraźniej na trwałe niegdyś przymierze ze wschodnim sojusznikiem strzegącym swojego wasala murem berliń skim i silnym kordonem czerwonej armii. zakonników. Toteż nie ulega kwestii, że na ich pomoc integracyjną wła dze PRL liczyły, oficjalnie je wspierały w nadziei stabilizacji i poloniza- cji, ale z drugiej strony niszczyły funkcjonujące domy i ośrodki działal ności edukacyjnej i charytatywnej, w których pozostawali jeszcze auto chtoni. Autor dotknął tego tematu jakby mimochodem (ponieważ wy kraczał on poza jego zainteresowania), ale nie zauważył choćby kilkoma zdaniami o eksmisji zakonnic niemieckich, które przeżyły exodus do obozów pracy w głąb Polski z powodu, co tu dużo mówić - klasycznego nacjonalistycznego szowinizmu1. Los ten dotknął przy okazji kilku sióstr Polek, które zdążyły już dołączyć do swojego zgromadzenia, aczkolwiek w większości niemieckiego. Czy ten problem znalazł swoje odbicie w stosunku do zakonników niemieckiego pochodzenia, odpowiedzi nie znalazłem. Wiadomo natomiast choćby z badań Albina Patoły1 2 3, że ide ologicznie sklecone władze PRL obeszły się z duchowieństwem nie mieckim bezwzględnie, czego do dzisiaj nie mogą wybaczyć bratnie wspólnoty niemieckiego pochodzenia. Niestety to nie pierwszy kłopot, za który płacimy wymówkami w kontaktach z Niemcami. Wracając jednak do kwestii obsady Ziem Odzyskanych polskim duchowieństwem zakonnym, dotykamy znamiennej taktyki władz, któ ra z jednej strony tolerowała zakonników w pracy kościelnej, lecz z drugiej wykorzystywała, choćby pojedyncze osobistości zakonne, dla „nowego ładu”. Nieufność repatriantów do wojskowych, żydowskich czy rosyjskich komisarzy, była zrozumiała, otwarta natomiast do altru- istycznie nastawionych zakonników i wypędzonych ze wschodnich rubieży duszpasterzy. To był niezwykły atut, który tolerowali świadomi swej bezradności komisarze, a z drugiej sukces, który utrwalał nowy porządek kościelny „na ziemiach po wiekach odzyskanych”. W działalności władz PRL było wiele irracjonalnych potknięć z powodu obłędnej polityki związania się sojuszem ze Związkiem Radzieckim, zamiast konstruktywnej współpracy na rzecz pojednania z pokonanym narodem i poszukiwania statusu integracyjnego w gra nicach orzeczonych Traktatem Jałtańskim. To był błąd zaślepionych kabotynów zaprzedanych Rosji począwszy od prezydenta B. Bieruta po sekretarzy partii: W. Gomułki, E. Gierka, W. Jaruzelskiego i pierwsze go wolnego, ale naiwnego premiera, za którego upadł mur berliński, lecz poprzez grubą kreskę snuł on naiwną wizję braterskiej zgody w pustych słowach i liturgicznym uścisku. J A N U S Z Z B U D N IE W E K O SP P E Krzysztofa Kasznicy, który stał w wyraźnym sporze do linii politycznej Prymasa (s. 286) i jeśli wolno przypuszczać, to od niego zaczęła się kontrowersyjna linia liberalizmu kościelnego, czy też populizmu polskich dominikanów - jakże nie bezpiecznie ustawionych nawet wobec niezbywalnych wartości Ko ścioła Powszechnego. Piszę to z goryczą i niemałym żalem, bowiem sam doświadczyłem smutnej z nim rozmowy, gdy w roku 1960 zanio słem wespół z moim współbratem napisaną replikę na artykuł Jacka Nie wdając się w ten nieprzyjemny i nietykalny z gruntu problem stosunków wewnątrz Kościoła w Polsce, ks. Zamiatała pokazał, jaką rolę odegrali zakonnicy, gdy dzięki nim - niemal natychmiast po za kończeniu wojny powstały podstawowe obszary społecznej działalno ści życia religijnego, głównie szkoły, domy opieki, potężna gałąź cha rytatywna, a nawet misyjna w kraju i poza jego granicami, a na sa mym końcu praca duszpasterska w kilkuset parafiach - nierzadko jako wymuszona na rzecz integracji ludności z rubieży wschodnich na Ziemiach Odzyskanych, - później co prawda odbierana przez ordyna riuszy, gdy umocniły się szeregi duchowieństwa diecezjalnego. Zagadnienie duszpasterstwa na Ziemiach Odzyskanych, choć znalazło się w pracy dopiero w VI rozdziale, stanowi złożony problem począw szy od kwestionowania przez państwo podejmowanych wysiłków za konników w kościołach i dobrach swego zakonu, głównie niemieckiej proweniencji. Represje wobec pozostałych w nich autochtonów, a także duchowieństwa diecezjalnego, jeśli przeżyli represje wojsk sowieckich, oraz bezpardonowe ich deportacje z terenów anektowanych do Polski, a tu z kolei - księży zza Buga do znoszenia moralnych udręk w sprawie prowadzenia ksiąg inwentarzowych, czy negowanie im praw do ubez pieczeń zdrowotnych, nie były zachętą do pracy w niepewnym terenie. Tragiczne w skutkach niedopełnienie prawa własności Ziem Odzyska nych po zakończeniu wojny, a także po rozpadzie komunizmu - stało się zadaniem, które podjęli ci, którzy gdyby nie kierowali się duchem wiary i troską duszpasterską wśród wypędzonych rodaków ze Wschodu, nie byłoby realne i skuteczne bez zaangażowania kilkuset bezinteresownych Rec.: D. Z A M I A T A Ł A , Z A K O N Y M Ę S K IE W P O L IT Y C E ... 369 J A N U S Z Z B U D N I E W E K O S P P E 368 Politycznych wątków ks. Zamiatała w zasadzie unika, choć nie obce mu były zapewne z opowiadań trudnego exodusu za chlebem własnej rodziny. 1 A. M i r e k, Siostry zakonne w obozach pracy w PRL w latach 1954-1956, Lublin 2009. 2 A. P a t o ł a, Życie i działalność ks. Kazimierza Borcza (¡913-1973), Częstochowa 1990, mps. 3 Zob. „Rzeczpospolita” 17 V I 2008. J A N U S Z Z B U D N IE W E K O SP P E sie ostatni akt infiltracji w życie Kościoła - przy pozyskaniu co najmniej najważniejszej osoby z watykańskiej dyplomacji, przysłanej do Polski i krajów bloku sowieckiego, by ugruntować sojusz z reżimem bloku wschodniego. Autor nie dotknął tego tematu, ponieważ go nie intereso wał, albo nic natrafił na jasne dowody. Znajdują się one m.in. w siatce szpiegowskiej zastawionej na paulinów pod koniec lat siedemdziesią tych, o czym wypadnie napisać kiedyś odrębne studium, ponieważ śla dowe akta sprawy na szczęście zachowały się. Dotyka ich w swoich wspomnieniach Dominik Morawski na przykładzie „fałszywek” pod kopujących autorytet prymasa Wyszyńskiego4. Z osobistej obserwacji chciałbym zrobić zastrzeżenia co do opinii Autora, że polski eksperyment tolerowania zakonów nie miał tutaj swojego represyjnego odpowiednika w innych państwach bloku so wieckiego. Przede wszystkim w innych znalazły się one realiach ani żeli u naszych południowych sąsiadów. Co więcej, tuż po „wyzwole niu” zakony były konieczną - choć niechcianą - podporą w kształto waniu ładu duchowego i politycznego po zniszczeniach wojennych, a także integracji ludności wypędzonych ze wschodnich rubieży Rzecz pospolitej. Tymczasem, gdy umocnił się aparat komunistycznego bez prawia, społeczeństwo poznawało jego obłudne machinacje, rosło zubożenie, zaciskała się obroża wolnego słowa - wtedy na generalną z zakonami rozprawę było już za późno. Toteż osiedlonym od dawna zakonnikom nie zabraniano pełnienia funkcji plebańskich, nie ograni czano głoszenia misji czy rekolekcji, pozwolono kilku wspólnotom prowadzić szkoły (pijarzy, niepokalanki, urszulanki), kierować do mami dla starców i kalek, natomiast usuwano ich z czynnego duszpa sterstwa w szkołach jako katechetów lub kapelanów w szpitalach i wojsku. Zakony przeżyły w tym układzie najgorszy okres do śmierci Stalina i polskiego Października, bowiem późniejsze represje komuni stów, posługując się parafrazą kard. J. Glempa, przypominały już przysłowiowe szczekanie kundelków, z którymi coraz raźniej poczy nały sobie zakony, wspierając na różny sposób ruchy wolnościowe aż do oskarżenia komunizmu o zbrodnie i ludobójstwo. Największy werbunek tajnych współpracowników spośród zakon ników i duchowieństwa w ogóle - nie wyłączając absolutnie bisku pów - przybrał na sile w okresie „sekretarzowania partii” za E. Gier ka. Sięgnięto wówczas m.in. do infiltracji młodej gwardii przyszłych duchownych, mianowicie do kleryków w seminariach. Okolicznością sprzyjającą tej akcji stały się pobory do wojska z bardziej opozycyj nych seminariów, gdzie według instrukcji zdeterminowanego do szpiku kości komunisty, Wojciecha Jaruzelskiego5 polegała ona na stosowa niu swoistego prania mózgów i szantażowania na rzecz zniechęcenia do życia zakonnego i stanu duchownego. J A N U S Z Z B U D N IE W E K O SP P E Jak ona przebiega, kto z niej korzysta, jakie budzą się resentymenty naszych sąsiadów ze Wschodu i Zachodu odczuwamy niczym oskubane gęsi po stracie rubieży wschod nich, a niepewni, czy na „ziemiach przydzielonych Polsce” wrócą daw ni właściciele, czy też znajdą tu swoją drugą ojczyznę powszechnie nielubiani Żydzi'. A zatem problem bytowania i sensu pracy, tworzenia własnych struktur domowych powtarza się nie tylko na faktycznych wypędzonych zza Buga, Niemna i Willi, lecz także na ich potomkach. Lecz oto na tej płaszczyźnie pojawił się kolejny destrukcyjny ele ment walki władzy państwowej z hierarchią Kościoła, lub też z su werennymi wspólnotami zakonnymi. Stanowisko kard. A. Hlonda w kwestii obsadzania polskimi duchownymi stolic biskupich na Zie miach Odzyskanych nie budziło sprzeciwu władz PRL. Zakonnicy cieszyli się tutaj swoimi uprawnieniami, stąd rysowała się nadzieja postawienia ich ponad jurysdykcyjnymi ordynariuszami, a w wypadku niesubordynacji, traktowano ich jako swoistych rewizjonistów. Aparat władzy świadomy był, że walka z zakonnikami w zasadzie nie zdawa ła egzaminu, ponieważ ich status nie należał do uprzywilejowanych w Kościele. Zakonnicy niczego nie tracili, nawet gdy spotykało ich prześladowanie, więzienie a nawet kary pozbawienia życia. Tak było w ciągu wielu wieków historii. Zdeterminowani ślubami posłuszeń stwa i ubóstwa na służbę wśród represji i oczywistej kary, po prostu dyspozycyjni w misji Kościoła! Toteż najlepszym rozwiązaniem by wały w ostatnich dwóch stuleciach kasaty domów, więzienia, eksmisje poza granice kraju nieposłusznych indywiduów, wyróżnianie lojal nych wysokimi stanowiskami w ramach struktur diecezjalnych, na gradzanie stosownymi gratyfikacjami itp. Każdy z tych modeli miał swoje odbicie w latach zaciskania obroży komunistycznej wobec za konów i w różnym wydaniu można je tutaj przywoływać. 370 J A N U S Z Z B U D N IE W E K O S P P E R ec.: D . Z A M IA T A Ł A , Z A K O N Y M Ę S K IE W P O L IT Y C E ... 371 W polityce państwa wobec Kościoła zakonnicy byli postrzegani jako emisariusze Watykanu, element wrogi systemowi komunistycz nemu i narzędzie swoistej „demagogii” wobec wierzących katolików, a nawet obojętnych religijnie tzw. „patriotów”. Ponieważ na domiar wszystkiego podrywali totalitarne stanowisko do wszechwładzy w pań stwie i stanowionego przez siebie prawa, nie mieściły się w realiach marksistowskiego systemu. 4 D. M o r a w s k i , Pomost na Wschód, Londyn - Lublin 1991, s. 44 i n. 5 Zob. A. L c s i ń s k i, Rola kadry wojskowej wobec alumnów - żołnierzy w świetle „Wytycznych gen. Wojciecha Jaruzelskiego”, [w:] Kompanie kleryckie w Wojsku Polskim 1959-1980, Szczecin 2009, s. 53-83. , 5 Zob. A. L c s i ń s k i, Rola kadry wojskowej wobec alumnów - żołnierzy w świetle „Wytycznych gen. Wojciecha Jaruzelskiego”, [w:] Kompanie kleryckie w Wojsku Polskim 1959-1980, Szczecin 2009, s. 53-83. 4 D. M o r a w s k i , Pomost na Wschód, Londyn - Lublin 1991, s. 44 i n. J A N U S Z Z B U D N IE W E K O SP P E Toteż właśnie wokół tego seminarium narosło w gronie paulińskiej wspólnoty tak wiele domy słów i protestów, że wiązaliśmy je z naciskiem komunistów, jako karę za uwolnienie wędrującego obrazu Matki Bożej w roku 1972, lub po prostu jako wyraz poparcia władzy generalnej dla zjednoczenia semi nariów, do czego po cichu zmierzały od dawna władze oświatowe. , g p y Reasumując ocenę książki ks. dra hab. Dominika Zamiatały, wypa da stwierdzić, że zawiera ona tak wielką ilość cennego materiału fak tograficznego, że będąc świadkiem opisanych w niej wydarzeń, wi działem w nich siebie śledzącego tamtą rzeczywistość, a kolekcjonując wycinki prasowe i notując własne opinie, jakbym już wtedy postrzegał kto knuł intrygi, jakie partie, tzw. katolickie dojdą do głosu i jakie żniwo zbierać będziemy po śmierci Prymasa Tysiąclecia i polskiego Papieża, którego liberałowie uważają za swego patrona. Osobista kon frontacja setek akt na zlecenie władz mojej wspólnoty, przybliżyła mi obraz mechanizmów UB i SB, z którymi spotkałem się w kwestii starań o paszport na Węgry w 1968 roku. Dzisiaj mogę wyznać - na szczę ście bezskutecznie. Ale iluż właśnie w osławionym czasie „gierkow skiej odwilży” uległo presji wojażowania po świecie za cenę współ pracy, szpiegostwa na przełożonych, na prymasa Wyszyńskiego i na jego program obrony praw Kościoła, czy determinację odnowy spo łecznej poprzez kult maryjny. Bilans zachowań duchowieństwa wypa da mimo wszystko pozytywny. Potwierdzają to badania ks. Zamiatały. Toteż książkę jego uważam za swego rodzaju katechizm, jak postę pować, by nie dać się złamać, umieć zrezygnować z zaszczytów, nie podać ręki dawcy złudnej sławy, pieniądza czy butelki „Brandy”. Au tor monografii zebrał fakty, dal do nich stosowny komentarz; - ocenę moralną zaprezentuje, jak sądzę w zapowiedzianym drugim tomie, poświęconym działalności duszpasterskiej zakonów. Będziemy czekać na to dzieło z niecierpliwością i czytać z tą samą pasją jak obecne. y p p j ąg ą j Zakony polskie w okresie PRL przeżyły trzy etapy upokorzeń re strykcji i złośliwych zawirowań. Omawia je ks. Zamiatała w szczegó łach. Przypomnijmy, że pierwszy etap po zakończeniu wojny to czas aresztowań, pokazowych sądów i tortur więziennych. Za sekretarzowa nia partii W. Gomułki nie stosowano już aresztowań, nakładano kary pieniężne za nielojalność władzy, za krytykę ustroju w kazaniach i kon taktach z ludźmi za żelazną kurtyną. Agitacja antyzakonna i antyko ścielna w mediach stała się przysłowiowa papką polskiego odbiorcy środków masowego przekazu, a kierujący nią filozofowie i socjologo wie (m.in. L. Kołakowski, E. Janusz ZBUDNIEWEK OSPPE - prof. zw., dr hab., kierownik Katedry Historii - Kościoła Średniowiecznego w Uniwersytecie Kardynała Stefana Wyszyńskiego, redaktor nacz. „Studia Claromontana”. J A N U S Z Z B U D N IE W E K O SP P E Akcje infiltracji wewnątrz wspólnot seminaryjnych znalazły swoje odbicie niemal we wszystkich seminariach, w których zwerbowano kilku chwiejnych lub naiwnych. Nie inaczej też było w kwestii agitacji nas rzecz rezygnacji ze studiów seminaryjnych. Ten ostatni element ideologicznej propagandy przy niósł skutek raczej mizerny, by nie powiedzieć odwrotny. Niejeden płatny nauczyciel nie radził sobie z teoriami marksistowskimi, inni nie mogli wynaleźć sposobów dręczenia alumnów, nawet gdy sprowadza no im zalotne dla uciech dziewczyny. Ostatecznie skutek był ten, że zarówno inicjator „Wytycznych” represji jak też generalicja, stwarza jąc im szanse dostania się na studia uniwersyteckie, czy podjęcie in tratnej pracy, tracili nadzieję, by można z nich uczynić ateistów, lub namówić do powrotu w szeregi wolnych obywateli. Analiza powyższych problemów w świetle zebranej przez ks. Zamia- tałę dokumentacji nie jest pierwszą na temat mechanizmów władzy komunistycznej wobec zakonów. Podejmowali ją w różnych opracowa niach historycy, opublikowano także szereg materiałów represyjnych i operacyjnych. Tym razem jednak przeprowadzenie pełnej analizy aktów prawnych i przywołanie faktów pokazuje wyrafinowany kierunek dzia łalności zmierzający do zneutralizowania roli zakonów, wykazania ich bezużyteczności, a jeśli w ostatnim okresie chcieli wykorzystać ich jako przysłowiowy klin w walce z kierunkiem ideologicznym kard. Wyszyń skiego, to bynajmniej nic z sympatii do zakonów. Był to w pewnym sen Nie był to jedyny sposób walki z istnieniem i działalnością zakonów. Głośnym echem odbiły się kasaty małych seminariów i restrykcyjne wilcze listy, które blokowały kontynuowanie nauki w państwowych J A N U S Z Z B U D N IE W E K O S P P E R cc.: D . Z A M IA T A Ł A , Z A K O N Y M Ę S K IE W P O L IT Y C E ... 372 373 gimnazjach z wyjątkiem techników. Należę do pokolenia tych „wyrzut ków”, którzy nie mogli być przyjęci do żadnego gimnazjum pomimo protekcji trzech bliskich krewnych, którzy pełnili stanowiska dyrekto rów takich szkół. Pozostał tylko jeden wariant, niestrzeżony restryk cjami w kursach i szkołach korespondencyjnych. A potem znów nauka w tajnych gimnazjach zakonu łącznie z egzaminami maturalnymi, które w latach odwilży usiłowano bezskutecznie nostryfikować. W tej sytu acji biegaliśmy po całej Polsce, by przejść od nowa wszystkie etapy szkoły ponadpodstawowej i osiągnąć świadectwo dojrzałości. to decyzja wyjątkowo spolegliwego rektora o. Kazimierza Szafrańca, który przyjął dwóch delegatów na rozmowę przy kawie, nie podejmu jąc żadnej wizytacji lokalu seminaryjnego. J A N U S Z Z B U D N IE W E K O SP P E Ciupak) byli czołowymi nośnikami walki i modelowej współpracy. To wówczas podjęto naciski na weryfikację nauczania w wyższych seminariach poprzez wizytacje kuratorów Mini sterstwa Oświaty, domagano się list alumnów i wykładowców, zakresu wykładów itp. W trzecim okresie, zwanym gierkowskim, podjęto kolej ną intrygancką, czy wręcz diabelską akcję, którą poznajemy na przykła dach totalnej szpiegomanii i donosicielstwa, jakich nie stosowano w tak szerokim zakresie w poprzednich represjach. To wówczas nasiliły się agitacje wobec działalności kard. Wyszyńskiego, przeciw któremu mo bilizowano obóz liberalnych katolików, nie wykluczając oczywiście tzw. posoborowych zakonników i księży z dwóch conajmniej opinio twórczych ośrodków cieszących poczytnymi pismami. Czyta się o nich z setnych rozmów ubeków z tajnymi współpracownikami, w których niemal z reguły sprawy stosunków z Prymasem i z wrogo nastawionymi do rządu biskupami były priorytetem werbunku i gratyfikacji. Głębszej analizy wymaga sprawa narodzin Papieskiej Akademii Teologicznej w Krakowie, która stała się ambicją kard. K. Wojtyły. Liczył on na jej wsparcie ze strony zakonów i zjednoczenia w niej kilku seminariów, czego nie poparły bodajże żadne władze seminaryj ne poza paulinami. Opinii tej przeczy żyjący generał o. Jerzy Tomziń- ski, wspominając, że zawieszenie własnego seminarium podyktowane było m.in. pomniejszoną liczbą powołań, a w kwestii wizytacji, była
https://openalex.org/W2789128215	https://www.e3s-conferences.org/10.1051/e3sconf/20183102011/pdf	English	null	Technical Analysis Feasibility Study on Smart Microgrid System in Sekolah Tinggi Teknik PLN	E3S web of conferences	2,018	cc-by	2,155	2 Technical Review The conditions of electricity in Indonesia is still inadequate, which the ratio of electrification is still low at 87% as of October 2015 (PT. PLN Persero). This shows that people access the energy is still limited. And also with the development of energy infrastructure in Energy for rural / remote and island - the outermost islands in general do not get adequate energy access. Dependence on electricity industry is more limited. Utilization of new and renewable energy and implementation of energy conservation are also not set optimal [1]. Research and development of renewable energy especially for Solar Power Generation based on smart- grid technology has been listed on RIP STT PLN 2013 - 2018. One of strategies to fulfill the electric energy needs is to utilize the potential of new and renewable energy (EBT) at the local site, In order to reduce transmission and distribution costs. Potential of Renewable Energy (ET) in local area is used to meet the needs of electrical energy in the area [4]. This will lead high concentration of renewable energy in into the grid. High concentrations of renewable energy sources can cause problems in stability, reliability and power quality on the main grid [5]. Smart microgrid is the key of the future grids where high concentration of renewable energy does not affect or disrupt with the quality of the grid. As the one of the way fulfil local needs for electricity, and in accordance with the policies of Indonesian government, in the past few years has been started the development of power systems of new and renewable energy (EBT) [2]. Due to the nature intermittent characteristic of new and renewable energy, we need a system to resolve the issue. Smart microgrid system or smart grid is one technology that can effectively and efficiently address the integration of new and renewable Energy in the electrical system for a large scale. A smart microgrid system can be interpreted as an electrical system consists of several Distributed Generations (DG) where the source is usually from EBT, such as photovoltaic panels, wind turbines, microturbons, with the addition of a storage system, load control, and an energy regulation system (Energy Management System - EMS). EMS allows smart microgrid systems to make its own free adjustment regardless grid positive in standalone mode (islanding). Technical Analysis Feasibility Study on Smart Microgrid System in Sekolah Tinggi Teknik PLN Heri Suyanto Lecturer, College of Engineering – PLN (Foundation for Education & Welfare PT. PLN (Persero). PLN tower. Jl. Outer West Lingkar, Duri Kosambi, Cengkareng, Indonesia 11750 Abstract. Nowadays application of new and renewable energy as main resource of power plant has greatly increased. High penetration of renewable energy into the grid will influence the quality and reliability of the electricity system, due to the intermittent characteristic of new and renewable energy resources. Smart grid or microgrid technology has the ability to deal with this intermittent characteristic especially if these renewable energy resources integrated to grid in large scale, so it can improve the reliability and efficiency of the grid. We plan to implement smart microgrid system at Sekolah Tinggi Teknik PLN as a pilot project. Before the pilot project start, the feasibility study must be conducted. In this feasibility study, the renewable energy resources and load characteristic at the site will be measured. Then the technical aspect of this feasibility study will be analyzed. This paper explains that analysis of ths feasibility study. © 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/). * Corresponding author: heri.suyanto@yahoo.com © 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/). https://doi.org/10.1051/e3sconf/20183102011 https://doi.org/10.1051/e3sconf/20183102011 E3S Web of Conferences 31, 02011 (2018) ICENIS 2017 * Corresponding author: heri.suyanto@yahoo.com 3 Research Methods Beban Gedung Lab Bengkel Elektro 5 - 10 kW The expected outcome of this research program is the synergies which mutually beneficial and supportive synergies between university (STT PLN) and industrial partners to produce hybrid power plant products designed and manufactured in a micro-distributed (micro-grid structure), and Supported by high technology, so it can be operated in local/ remote island to fulfil needs of housing and also support the operation of NKRI territorial integrity [9]. Box Panel Gasifer PLTSampah 14 kVA Methodology and research stages to be used follow: Methodology and research stages to be used follow: 250 Meter a. Measure New and Renewable Energy potential, the wind and solar energy at Sekolah Tinggi Teknik PLN. b. Analysis of irradiation data on selected area (local / remote island), refers to data from BMKG and Lapan and other agencies. c. Measure the load characteristics at the smart micro grid site. Beban Gedung Lab Bengkel Mesin 5 - 15 kW Beban Gedung Lab Bengkel Mesin 5 - 15 kW g d. Conducted an analysis of data on potential and characteristics of the load. Fig. 1. Blueprint STT PLN Smart microgrid System. e. Conduct the analysis of feasibility for smart micro grid system as the technical specifications of solar cells, and determining the electrical power control system based on "power electronics" based on the study of literature [10]. e. Conduct the analysis of feasibility for smart micro grid system as the technical specifications of solar cells, and determining the electrical power control system based on "power electronics" based on the study of literature [10]. This Installation that has been installed with a capacity of 14.5 kW with microgrid smart system On Grid and Off Grid. Fig. 2. Smart grid system design of Microgrid system for off grid at STT PLN 2 Technical Review This microgrid smart system can operate in two modes, On-Grid / Grid- connected and Off-Grid / Standalone / Islanding. Off- Grid / Standalone / Islanding operation mode is when circuit breaker isolates the system from the main grid (PLN), so power generator equipment, storage, load regulator, power quality controller and other system Since 2015 STT PLN has been conducting some research of smart microgrid, with stage: study potentials stats with (2015) and design of the smart microgrid laboratorium scale model (2016) [3], As the development of smart microgrid model and for application in the field, so as a first step of the implementation to start to establish the pilot project of smart microgrid system with in at the laboratory scale in STT PLN start in 2017 E3S Web of Conferences 31, 02011 (2018) E3S Web of Conferences 31, 02011 (2018) ICENIS 2017 https://doi.org/10.1051/e3sconf/20183102011 KWH METER EXIM 16500 VA JARINGAN PLN 3 Fasa Box Panel AC PLTS 14,5 kWp Beban Gedung Lab PLTS 4 kW Beban Gedung Lab Bengkel Mesin 5 - 15 kW 250 Meter 25 Meter AC Beban Gedung Lab Bengkel Elektro 5 - 10 kW Gasifer PLTSampah 14 kVA Box Sinkron Genset PLTSampah Box Panel Gasifer PLTSa AC Box Panel Genset Digester PLTSa Digester PLTSampah 2 kVA 200 meter 200 meter Fig. 1. Blueprint STT PLN Smart microgrid System. operation regulator are applied only to smart microgrid system [6]. In Off-Grid mode the load is supplied applied by DG, Diesel Generator (it backup) and Battery [7]. In an on-grid / grid connected mode, the microgrid smart system is also a controllable load, or the power generated from the grid is a backup / supplemental energy source. In On-Grid / Grid Connected mode this load is supplied by DG, grid (as backup) and Battery [8]. JARINGAN PLN 3 Fasa KWH METER EXIM 16500 VA 4 Results And Discussion Sekolah Tinggi Teknik PLN is located at Lingkar Barat, Duri Kosambi, Cengkareng, Jakarta Barat, Jakarta 11750. Applied load in the Campus main building of 100KW, and the load in the Laboratory of STT PLN is 15 KW. After the discussions among colleagues and professors of Sekolah Tinggi Teknik PLN and by direct observation at the location it was determined that the project smart microgrid system will be built right at Laboratory at Research and Training Center For Renewable Energy Power System. Presence of solar Laboratory will save the electricity bill from PT. PLN Blueprint New and Renewable Energy STT PLN as follows according to Figure 1 among others as follows: 1. Solar Power Plant (PLTS) capacity 14.5 kWp Fig. 2. Smart grid system design of Microgrid system for off grid at STT PLN 2. Bayu / wind power plant (PLTB) capacity of 20 kW 3. Biomass Power Plant 16 kVA consisting of: Our Solar Power Plants Capacity of 17 kW / peak has already Integrated KWH Meter Exim Export and Import and has installed with advanced technology monitoring system. This design is made as a Research by knowing new and renewable energy technologies especially Solar Power Generation and as a saver of 3. Biomass Power Plant 16 kVA consisting of: - Waste Power Plants T gasifer 14 kVA - Waste Power Plants digester of 2 kVA 4. With KWH Export and Import KWH meter system (Exim) from PT. PLN (Persero) and Battery - Waste Power Plants digester of 2 kVA 4. With KWH Export and Import KWH meter system (Exim) from PT. PLN (Persero) and Battery 2 2 https://doi.org/10.1051/e3sconf/20183102011 E3S Web of Conferences 31, 02011 (2018) ICENIS 2017 E3S Web of Conferences 31, 02011 (2018) electricity usage from PT. PLN (Persero) and the electricity cost down. Figure of generated electricity from PLTS monitoring system. Fig.5. Monthly Electricity Curve of PLTS Fig. 3. Smart System Design of Microgrid system on grid at STT PLN Fig.5. Monthly Electricity Curve of PLTS Fig. 3. Smart System Design of Microgrid system on grid at STT PLN 5 Conclusion  From the analysis conducted on the technical aspects of the feasibility study of the application of Smart Microgrid System Project in Sekolah Tinggi Teknik PLN is feasible with high potential availability and installation area of equipment as we expected. Fig. 4. Daily electricity curve of PLTS Fig. 4. Daily electricity curve of PLTS  Microgrid Smart System Project in Sekolah Tinggi Teknik PLN Research and Training Center Laboratory for Renewable Energy Power system STT PLN as New and Renewable Energy Technology Research and as a saver of electricity usage from PT. PLN.  Blueprint of smart microgrid system for New and Renewable Energy STT PLN consists of PLTS capacity 14.5 kWp, PLTB capacity 20 kW, PLTbiomass with 14 kVA Gasifer PLTS garbage and 2 kVA of waste PLTS Digester) and Battery. 6. Smart Houses interacting with Smart Grids to achieve next generation efficiency and sustainability, Dr. Anke Weidlich, SAP Research 11.02.2009 7. IEC Smart Grid Standardization Roadmap, Prepared by SMB Smart Grid Strategic Group (SG3) June 2010; Edition 1.0 8. R. Ramakumar, “Integrated Renewable Energy System – Micro Grid (IRES-MG) For Sustainable Development”, ICSUNIDO, Trieste, Italy, April (2012) 9. “The Smart Grid: Coming Of Age”, Electric Power Research Institute Journal, EPRI,Inc. (2009). 10. T.Nakata, K. Kubo and A. Lamont, “Design For Renewable Energy System With Application To Rural Area In Japan”, white paper. References Fig. 4. Daily electricity curve of PLTS Fig. 4. Daily electricity curve of PLTS 1. CERTS Microgrid Symposium, Northern Power Systems 1. CERTS Microgrid Symposium, Northern Power Systems It shows the maximum peak at 11:00 to 14:00 hours to reach 10 kW so that one can reach 70 kW in the position of maximum results Daily and Daily Average reaches 40 kW. y 2. Clarke,S., “Electricity Generation Using Small Wind Turbines At Your Home Or Farm”, Fact Sheet, September (2003) y 2. Clarke,S., “Electricity Generation Using Small Wind Turbines At Your Home Or Farm”, Fact Sheet, September (2003) 3. Jay Stuller, “An Electric Revolution”, Galvin Electricity Initiative From the curve above, electricity generated from Solar Power Generation can reach 1.4 MWh.and Biomass Power Plant is in the design stage For Bayu / Wind Power Generation and Biomass Power Plant is still under costumer. 4. J. B. (Secretary of U. S. D. of C. P. D.G. (Director of N. I. of S. and Technology), NIST Framework and Roadmap for Smart Grid Interoperability Standards , NISTSpecial Publication 1108R2 NIST Framework and Roadmap for Smart Grid Interoperability Standards, NIST Speci. Energy Independence and Security Act of 2007, (2012). This Microgrid Project Smart will be applied as Research and Learning on New and Renewable Energy at the Research and Training Center Laboratory for Renewable Energy Power System STT PLN. y ( ) 5. Kurt Yeager, “The Microgrid Revolution”, Oktober (2010) y ( ) 5. Kurt Yeager, “The Microgrid Revolution”, Oktober (2010) 3 3 E3S Web of Conferences 31, 02011 (2018) ICENIS 2017 https://doi.org/10.1051/e3sconf/20183102011 Development”, ICSUNIDO, Trieste, Italy, April (2012) 9. “The Smart Grid: Coming Of Age”, Electric Power Research Institute Journal, EPRI,Inc. (2009). 7. IEC Smart Grid Standardization Roadmap, Prepared by SMB Smart Grid Strategic Group (SG3) June 2010; Edition 1.0 10. T.Nakata, K. Kubo and A. Lamont, “Design For Renewable Energy System With Application To Rural Area In Japan”, white paper. 8. R. Ramakumar, “Integrated Renewable Energy System – Micro Grid (IRES-MG) For Sustainable 4 4
https://openalex.org/W2994763760	https://link.springer.com/content/pdf/10.1007/s12541-019-00261-3.pdf	English	null	External Kinematic Calibration of Hybrid Kinematics Machine Utilizing Lower-DOF Planar Parallel Kinematics Mechanisms	International journal of precision engineering and manufacturing/International Journal of the Korean Society of Precision Engineering	2,019	cc-by	16,816	1 Introduction For a precision manipulation, an accurate pose of the mov- ing platform is the aim to be achieved during its controlled motion. Since the control is commonly performed in the joint space, forward kinematics of the mechanism is required to transform the active joint positions to the pose of the mov- ing platform. As the trajectory is commonly planned in the task space, inverse kinematics is needed to transform the planned trajectory to the joint space in which the control is applied. Consequently, the accuracy of the kinematics affects positioning accuracy. A kinematic calibration is commonly conducted to estimate the kinematic parameters accurately and subsequently, a compensation is performed by correct- ing the software, which is, in this case, the kinematics of the machine. For a precision manipulation, an accurate pose of the mov- ing platform is the aim to be achieved during its controlled motion. Since the control is commonly performed in the joint space, forward kinematics of the mechanism is required to transform the active joint positions to the pose of the mov- ing platform. As the trajectory is commonly planned in the task space, inverse kinematics is needed to transform the planned trajectory to the joint space in which the control is applied. Consequently, the accuracy of the kinematics affects positioning accuracy. A kinematic calibration is commonly conducted to estimate the kinematic parameters accurately and subsequently, a compensation is performed by correct- ing the software, which is, in this case, the kinematics of the machine. The parallel kinematics mechanisms (PKMs) which consist of a base, some legs, and a moving platform in a closed- chain configuration have been proposed and developed to achieve more accuracy in the precision manipulation. Fur- thermore, hybrid kinematics machines (HKMs) have been introduced and developed to integrate the advantages of both the serial kinematics mechanism (SKM) and PKM. Any of the three schemes can compose an HKM: (1) serially con- necting two or more PKMs, (2) serially connecting a PKM with an SKM, or (3) serially combining two or more PKMs, or a PKM with an SKM, through a rigid connection. In the third scheme, the rigid connection is considered a type of serial connection. In some applications such as machining, a PKM is typically used together with an SKM or another PKM and therefore becomes an HKM. Online ISSN 2005-4602 Print ISSN 2234-7593 Online ISSN 2005-4602 Print ISSN 2234-7593 International Journal of Precision Engineering and Manufacturing (2020) 21:995–1015 https://doi.org/10.1007/s12541-019-00261-3 International Journal of Precision Engineering and Manufacturing (2020) 21:995–1015 https://doi.org/10.1007/s12541-019-00261-3 REGULAR PAPER Abstract This paper presents the implementation of nonlinear least squares and iterative linear least squares algorithms for exter- nal kinematic calibration of a hybrid kinematics machine composed of two 3PRR planar parallel kinematics mechanisms by utilizing a laser tracker. First the hand-eye and robot-world transformations were obtained by a separable closed-form solution and refined by the nonlinear least squares. Subsequently, the geometric parameters of the machine’s mechanisms were estimated using the two algorithms. Due to the rank deficiency, we implemented the nonlinear least squares algorithm through a subset selection approach in which we performed the estimation in two steps. We iterated the closed-form solution of the linear least squares until the solution converges to the actual values. We have shown that the nonlinear least squares algorithm successfully refined the hand-eye and robot-world transformations and outperformed the iterative linear squares algorithm in the estimation of the geometric parameters of the mechanisms. Keywords Calibration · Planar parallel mechanism · Hybrid kinematics · Least squares External Kinematic Calibration of Hybrid Kinematics Machine Utilizing Lower‑DOF Planar Parallel Kinematics Mechanisms Abdur Rosyid1 · Bashar El‑Khasawneh1 · Anas Alazzam1 Received: 7 February 2019 / Revised: 24 June 2019 / Accepted: 22 August 2019 / Published online: 13 December 2019 © The Author(s) 2019 * Bashar El‑Khasawneh bashar.khasawneh@ku.ac.ae 1 Mechanical Engineering Department, Khalifa University of Science and Technology, Abu Dhabi, United Arab Emirates * Bashar El‑Khasawneh bashar.khasawneh@ku.ac.ae 1 Mechanical Engineering Department, Khalifa University of Science and Technology, Abu Dhabi, United Arab Emirates 1 Introduction Moreover, more attention has been given to the use of lower degree-of-free- dom (DOF) PKMs due to less complexity in their modeling. In general, the kinematic calibration can be either an external calibration, a constrained calibration, or a self-cal- ibration [1]. The external and constrained calibration can be conducted offline, and the number of calibration poses is usually limited. Here the number and the choice of the cali- bration poses become an issue. The number of the calibra- tion poses should be adequate, whereas the selection of the calibration poses is shown to be vital as it affects the quality of the calibration. According to Bai and Theo [2], the opti- mal number of calibration poses is 10 or more. Moreover, Vol.:(0123456789) 1 3 International Journal of Precision Engineering and Manufacturing (2020) 21:995–1015 996 a full pose measurement typically requires less number of calibration poses to get an accurate estimation [1]. mentioned earlier that a minimum of ten calibration poses should be used while a full pose measurement can reduce the required number of calibration poses. Accordingly, a full pose measurement of twelve unique poses is taken in this work. The unrepeated coordinate values for each DOF of the poses provide the uniqueness of the poses. The geometric parameters of the PKMs, which include the length of all the legs and the moving platform as well as the position of the joints connecting the legs with the moving platform, and the actual relative position between the two conjugated PKMs are the parameters to be calibrated. The model function to be used in the calibration is the forward kinematics of the machine’s mechanisms. Finally, the accuracy improvement was evaluated after performing the compensation. In a nutshell, the kinematic calibration is performed by firstly determining the parameters to be calibrated, followed by modeling, measurement (data collection), identification (estimation), and compensation (correction) [3, 4]. Various algorithms in the identification stage are used to minimize the residual error. To mention some of them in the kine- matic calibration of PKMs and HKMs, Vischer and Clavel [5] used the Levenberg–Marquardt algorithm for kinematic calibration of a Delta robot. Yang et al. [6] used a linear least squares algorithm for kinematic calibration of a spa- tial 3-DOF PKM. Patel and Ehmann [7] as well as Iurascu and Park [8] applied the total least squares algorithm for kinematic calibration of PKMs. 1 Introduction Yu [9] used nonlinear least squares for kinematic calibration of a hexapod. Wang et al. [10] used minimal linear combinations of error parameters for kinematic calibration of a spatial 3-DOF PKM in a five- axis HKM milling machine. Wang et al. [11] applied the Markov Chain Monte Carlo method for kinematic calibra- tion of an HKM. Liu et al. [12] proposed the use of the genetic algorithm for kinematic calibration of a Stewart plat- form while Fan et al. [13] proposed it for an HKM polishing machine. p g p This paper is organized into several sections. Section 1 gives an introduction to the paper and describes its novelty. Section 2 provides the kinematics of the individual mecha- nism and subsequently presents the solution of its forward kinematics by using both Euler angles and quaternions rep- resentations. The quaternions are used to overcome the for- mulation singularity. Section 3 presents the hand-eye and robot-world calibration which should be performed before the calibration of the mechanism geometric parameters. Sec- tion 4 presents the kinematics of the complete mechanism with the external measurement device in the setup. Sec- tion 5 presents the use of nonlinear least squares to refine the hand-eye and robot-world calibration and to estimate the mechanism geometric parameters. Section 6 presents the use of iterative linear least squares to estimate the mechanism geometric parameters. In Sect. 7, the estimates of the mecha- nism geometric parameters obtained by both the nonlinear least squares and the iterative linear least squares are used for compensation. The accuracy of the position tracking cor- responding to the estimates obtained by the two algorithms is compared. Finally, Sect. 8 concludes the paper. While researchers [14, 15] investigate the kinematic calibration of five-axis SKMs, this paper discusses the kin- ematic calibration of a five-axis HKM. This paper uses an external kinematic calibration of an HKM machine tool utilizing two different identification algorithms, namely a nonlinear least squares algorithm and an iterative linear least squares algorithm. In particular, a subset selection approach is proposed to overcome the rank deficiency in the nonlinear least squares algorithm. Furthermore, an iteration is intro- duced to be used in the linear least squares to achieve a solution convergence. This scheme is the first novelty of this paper. The implementation of the two algorithms as well as a comparison of the estimation accuracy provided by both algorithms is presented. 1 Introduction This comparative study is another contribution of this paper. The HKM machine tool was built by conjugating two lower-DOF, planar PKMs having a simi- lar topology but different geometric parameters. The planar state of the PKMs as will be shown results in a configuration degeneracy which indicates unidentified parameters in the off-plane direction of the mechanism. A physical adjustment should be conducted to impose a certain value, typically zero for convenience, of the unidentified parameters. The imple- mentation of the mentioned algorithms to this new topology of HKM composed of planar PKMs is another novelty of this paper. 1 3 2.1 Kinematics Equations 2 The prototype of the hybrid kinematics machine point in the moving platform can also be used as the moving platform is rigid. The selection of point (x, y) is mainly for more convenience. Besides, the orientation of the platform is defined as the angle made by the platform with respect to the fixed base. Upon the dimensional optimization of the mecha- nism by considering its workspace, stiffness magnitude, and stiffness uniformity, the mechanism is simplified to a topology with two coincident upper joints as shown in Fig. 1b. As a result, xp2 = xp3 = xp. The optimization procedure is discussed in [17]. Figure 2 shows the hybrid scheme of the machine. The conjugation of the two planar PKMs in perpendicular direc- tions easily creates the required spatial workspace and provides six DOFs. Fig. 1 Schematic of the a general and b optimized mechanism topol- ogy (with the Z-axis perpendicular to the paper through the origin) The tool spindle is oriented horizontally to avoid redun- dancy between the rotation of the spindle and the rotation of the upper mechanism. This results in the proposed five-axis machine configuration. The five-axis machine has translational DOFs in x, y, and z directions as well as rotational DOFs about the Z and Y axes. Moreover, there is a redundancy between the two mechanisms in the X translational motion. This redun- dancy is advantageous as it provides an additional workspace in the x-direction. However, this also results in more com- plexity in determining which mechanism to move when a translation in the x-direction is required. In this case, one can pre-determine which mechanism is fixed while another mecha- nism is moved. A more systematic approach is a determination using an optimization scheme based on a defined objective to be optimized, such as energy consumption (control effort), accuracy, or stiffness. general motion consisting of translation in X-axis, trans- lation in Y-axis, and rotation about the Z-axis, where the three axes are orthogonal and follow the right-hand rule. It consists of a fixed base, three legs, a moving platform, and several joints. The joints used in an order starting from the fixed base are prismatic (P) joints, revolute (R) joints which connect the sliders with the legs, and other revolute (R) joints which connect the legs with the moving platform. Therefore, the mechanism is called the 3PRR PKM. 2.1 Kinematics Equations The HKM calibrated in this work is used for five-axis machining and composed by the third scheme of hybridiza- tion. A PKM which serves as the workpiece platform (lower platform) is conjugated with another PKM as the spindle platform (upper platform). Both the lower and upper plat- forms use planar PKMs because they are easier to design than spatial PKMs and more geometrical constraints on the DOFs will minimize the error due to joints’ errors. The lower and upper platforms use an identical PKM topology called the 3PRR, planar PKM as depicted in Fig. 1a and optimized to a simpler topology as shown in Fig. 1b. This PKM has three DOFs which adequately enable required Laser tracker, which is capable of providing a full pose measurement, was used to conduct the kinematic calibration. The laser tracker offers a high accuracy pose measurement with easy use, although it is relatively expensive [16]. It was 1 3 1 3 International Journal of Precision Engineering and Manufacturing (2020) 21:995–1015 997 point in the moving platform can also be used as the moving platform is rigid. The selection of point (x, y) is mainly for more convenience. Besides, the orientation of the platform is defined as the angle made by the platform with respect to the fixed base. Upon the dimensional optimization of the mecha- nism by considering its workspace, stiffness magnitude, and stiffness uniformity, the mechanism is simplified to a topology with two coincident upper joints as shown in Fig. 1b. As a result, xp2 = xp3 = xp. The optimization procedure is discussed in [17]. Figure 2 shows the hybrid scheme of the machine. The conjugation of the two planar PKMs in perpendicular direc- tions easily creates the required spatial workspace and provides six DOFs Fig. 1 Schematic of the a general and b optimized mechanism topol- ogy (with the Z-axis perpendicular to the paper through the origin) Fig. 2 The prototype of the hybrid kinematics machine point in the moving platform can also be used as the moving platform is rigid. The selection of point (x, y) is mainly for more convenience. Besides, the orientation of the platform is Fig. 2 The prototype of the hybrid kinematics machine Fig. 1 Schematic of the a general and b optimized mechanism topol- ogy (with the Z-axis perpendicular to the paper through the origin) Fig. 2 The prototype of the hybrid kinematics machine Fig. 2.1 Kinematics Equations The prismatic joints are actuated, have translation along X-axis, and are implemented by using sliders moving along the base through guideway. The use of the sliders creates more workspace and enables the moving platform to move upward and downward as well as to tilt. The actuation is provided by linear motors which give more accuracy compared with pneumatic/hydraulic pistons and lead screws. f The kinematics equations of the proposed parallel mecha- nism can be obtained easily by the algebraic approach. Accord- ing to the geometry of the mechanism shown in Fig. 1, the following geometric relations are found for the legs: In this proposed mechanism, three legs are required to con- strain the mechanism fully. Furthermore, two neighboring legs are maintained in a crossing configuration to avoid singularity as well as maximize the useful workspace and the stiffness of the mechanism. Point (x, y) in the moving platform is used to evaluate the general motion of the moving platform. Any other (1) L2 1 = (x + xp2 cos 휃−x1)2 + (y + xp2 sin 휃)2 (1) L2 1 = (x + xp2 cos 휃−x1)2 + (y + xp2 sin 휃)2 1 3 1 3 1 3 International Journal of Precision Engineering and Manufacturing (2020) 21:995–1015 International Journal of Precision Engineering and Manufacturing (2020) 21:995–1015 998 In this paper, only the forward kinematics of the mechanism (2) L2 2 = (x −x2)2 + (y)2 (3) L2 3 = (x + xp3 cos 휃−x3)2 + (y + xp3 sin 휃)2 (9) x2 −(2x2)x + y2 = L2 2 −x2 2 = c2 (10) x2 −(2x3 + 2xp3 cos 휃)x + y2 + (2xp3 sin 휃)y = 2x3xp3 cos 휃+ L2 3 −x2 3 −x2 p3 = c5 (2) (9) x2 −(2x2)x + y2 = L2 2 −x2 2 = c2 L2 2 = (x −x2)2 + (y)2 (9) anism (3) (10) x2 −(2x3 + 2xp3 cos 휃)x + y2 + (2xp3 sin 휃)y = 2x3xp3 cos 휃+ L2 3 −x2 3 −x2 p3 = c5 (3) (10) In this paper, only the forward kinematics of the mechanism is discussed as it is required in the kinematic calibration. In this paper, only the forward kinematics of the mechanism is discussed as it is required in the kinematic calibration. 2.2 Forward Kinematics with Known θ (13) x = ( f −ec b d −ea b ) (14) y = c −ax b The complete forward kinematics of the mechanism at hand which includes the determination of the angle θ can be seen in [17]. In this paper, only the determination of x and y given the angle θ is presented for the kinematic calibration pur- pose. In the case of a known θ, the value of x and y can be obtained through a more straightforward derivation. Let us rearrange the kinematics Eqs. (1), (2), and (3) in the follow- ing set of equations: (13) x = ( f −ec b d −ea b ) (14) y = c −ax b (13) (14) 2.1 Kinematics Equations Furthermore subtracting (9) from = 2x3xp3 cos 휃+ L3 −x3 −xp Furthermore, subtracting (9) from (8) and (10) yields a more compact set of equations as follows: The kinematics of the mechanism has the following constraints: (11) (−2x1)x + (2xp2 cos 휃)x + (2x2)x + (2xp2 sin 휃)y = ax + by = c4 −c2 = c (4) 0 ≤L1 ≤ymax 0 ≤L2 ≤ymax 0 ≤L3 ≤ymax 0 ≤xp2 ≤xp3 ≤xmax L1 + L2 −xp2 ≤xmax L1 + L3 −xp2 + xp3 ≤xmax (4) 0 ≤L1 ≤ymax 0 ≤L2 ≤ymax 0 ≤L3 ≤ymax 0 ≤xp2 ≤xp3 ≤xmax L1 + L2 −xp2 ≤xmax L1 + L3 −xp2 + xp3 ≤xmax (11) (12) x2 −(2x2)x + y2 = L2 2 −x2 2 = c2 x2 −(2x2)x + y2 = L2 2 −x2 2 = c2 (12) (4) where a = −2x1 + 2x2 + 2xp2 cos 휃 b = 2xp2 sin 휃 c = c4 −c2 d = −2x3 + 2x2 + 2xp3 cos 휃 e = 2xp3 sin 휃 f = c5 −c2 a = −2x1 + 2x2 + 2xp2 cos 휃 where ymax and xmax are the vertical and horizontal limits of the machine volume having the numerical values of 1200 mm and 1700 mm, respectively. Those constraints indicate that the mechanism should always be inside the specified machine volume, the sliders should not interfere with each other, and two adjacent legs must always be cross- ing each other. Equations (11) and (12) have to be solved simultaneously as x and y exist in both of the equations. The solution is given by: 2.3 Forward Kinematics Using Quaternion 3 The position vectors defined in the mechanism Using Euler angles, (15) can be expanded to the following: Using Euler angles, (15) can be expanded to the following: x = x2 + L2 cos 훼2 Table 1 The link lengths corresponding to values of θ near zero when solving for x (16) y = L2 sin 훼2 x = x1 + L1 cos 훼1 −xp2 cos 휃 y = L1 sin 훼1 −xp2 sin 휃 x = x3 + L3 cos 훼3 −xp3 cos 휃 y = L3 sin 훼3 −xp3 sin 휃 (16) From (16), we know that we have six equations with nine variables, i.e. x, y, θ, α1, α2, α3, x1, x2, and x3. However, three variables among the nine variables are known. In other words, we have six equations with six unknowns. A quaternion can be written as: A quaternion can be written as: (17) q = q0 + q1̂i + q2̂j + q3̂k = [ q0 q1 q2 q3 ]T (17) where i, j, and k are the unit vectors in x, y, and z directions, respectively. A rotation in a three-dimensional space can be repre- sented by a rotation matrix R in terms of quaternions as follows: Fig. 3 The position vectors defined in the mechanism (18) R = ⎡ ⎢ ⎢⎣ 1 −2q2 2 + q2 3 2q1q2 + q0q3 2q0q2 −q1q3 2q1q2 + q0q3 1 −2q2 1 + q2 3 2q2q3 −q0q1 2q1q3 −q0q2 2q0q1 −q2q3 1 −2q2 1 + q2 2 ⎤ ⎥ ⎥⎦ to different values of θ near zero, given the expected actual values of L1= 600 mm, L2 = 600 mm, and L3 = 750 mm. On the other hand, division by zero in (14) is very sensi- tive. Even a minimal value of θ replacing an exact zero θ leads to a significant error in the solution, i.e. the value of y. Therefore, (14) cannot be used to solve for y in the case of zero tilting angle of the moving platform. In such a case, either one of the following two ways can be used. First, using an iterative technique by spanning over possible values of y and checking with the inverse kinematics. 2.3 Forward Kinematics Using Quaternion For any set of x, y, and θ, it should be observed if it retrieves a known set of x1, x2, and x3. However, the accuracy of this technique is affected by the interval of y used in the iteration. Besides, this technique is computationally intensive. Second, solving the forward kinematics by using quaternion. The reason for using the quaternion is because a formulation singularity causes this problem due to the use of Euler angle θ. There- fore, avoiding using the Euler angle by using the quaternion will avoid the formulation singularity.i (18) ⎦ The rotation of the legs and the moving platform of the mechanism, which is about the Z-axis, can be written in the quaternion notation in terms of the Euler angles α1, α2, α3, and θ as follows: (19) q 훼i = ⎡ ⎢ ⎢ ⎢⎣ q0(훼i) q1(훼i) q2(훼i) q3(훼i) ⎤ ⎥ ⎥ ⎥⎦ = ⎡ ⎢ ⎢ ⎢⎣ cos 훼i∕2 0 0 sin 훼i∕2 ⎤ ⎥ ⎥ ⎥⎦ ; i = 1, 2, 3 (19) q 훼i = ⎡ ⎢ ⎢ ⎢⎣ q0(훼i) q1(훼i) q2(훼i) q3(훼i) ⎤ ⎥ ⎥ ⎥⎦ = ⎡ ⎢ ⎢ ⎢⎣ cos 훼i∕2 0 0 sin 훼i∕2 ⎤ ⎥ ⎥ ⎥⎦ ; i = 1, 2, 3 (20) q(휃) = ⎡ ⎢ ⎢ ⎢⎣ q0(휃) q1(휃) q2(휃) q3(휃) ⎤ ⎥ ⎥ ⎥⎦ = ⎡ ⎢ ⎢ ⎢⎣ cos (휃∕2) 0 0 sin (휃∕2) ⎤ ⎥ ⎥ ⎥⎦ (19) (20) q(휃) = ⎡ ⎢ ⎢ ⎢⎣ q0(휃) q1(휃) q2(휃) q3(휃) ⎤ ⎥ ⎥ ⎥⎦ = ⎡ ⎢ ⎢ ⎢⎣ cos (휃∕2) 0 0 sin (휃∕2) ⎤ ⎥ ⎥ ⎥⎦ (20) Recall the mechanism at hand defined by some position vectors as shown in Fig. 3. 2.3 Forward Kinematics Using Quaternion (5) x2 + y2 + 2xxp2 cos 휃+ 2yxp2 sin 휃 −2x1x −2x1xp2 cos 휃= L2 1 −x2 1 −x2 p2 = c1 (6) x2 + y2 −2x2x = L2 2 −x2 2 = c2 (7) x2 + y2 + 2xxp3 cos 휃+ 2yxp3 sin 휃 −2x3x −2x3xp3 cos 휃= L2 3 −x2 3 −x2 p3 = c3 (5) x2 + y2 + 2xxp2 cos 휃+ 2yxp2 sin 휃 −2x1x −2x1xp2 cos 휃= L2 1 −x2 1 −x2 p2 = c1 (5) Observing the solution of the forward kinematics given in (13) and (14), we realize that division by zero occurs when θ = 0 and therefore b = 0. In this situation, x still can be solved by using (13) by converting zero θ to a minimal value near (above or under) zero. The effect of the closeness of θ to zero can be evaluated by observing the value of the solution, i.e. x and y, as well as the corresponding links’ dimension if we apply the solution. The latter shows a more obvious effect. After scanning some values of θ close to zero, it is shown that θ should not be larger than 1 × 10−5. This will give an accurate solution to four decimal places. Using a closer value of θ to zero increases further the accuracy. Table 1 shows the values of the link lengths corresponding (6) x2 + y2 −2x2x = L2 2 −x2 2 = c2 (6) (7) x2 + y2 + 2xxp3 cos 휃+ 2yxp3 sin 휃 −2x3x −2x3xp3 cos 휃= L2 3 −x2 3 −x2 p3 = c3 (7) Collecting x and y in (5)–(7) in the left-hand sides yields: Collecting x and y in (5)–(7) in the left-hand sides yields: (8) x2 −(2x1 + 2xp2 cos 휃)x + y2 + (2xp2 sin 휃)y = 2x1xp2 cos 휃+ L2 1 −x2 1 −x2 p2 = c4 (8) 1 3 International Journal of Precision Engineering and Manufacturing (2020) 21:995–1015 999 Table 1 The link lengths corresponding to values of θ near zero when solving for x θ = 1 × 10−3 θ = 1 × 10−4 θ = 1 × 10−5 L1 (mm) 600.0025 600.0003 600.0000 L2 (mm) 600.0000 600.0000 600.0000 L3 (mm) 750.0040 750.0004 750.0000 Fig. 2.3 Forward Kinematics Using Quaternion The geometric relations in the mechanism can also be written using a vectorial approach as follows: Substituting any of (19)–(20) into (18) as they have a similar form to get the corresponding rotation matrix, the rotation about the Z-axis can be written in a rotation matrix in terms of the quaternions as follows: (15) rp = X2 + R2S2 rp = X1 + R1S1 −R4Sp2 rp = X3 + R3S3 −R4Sp3 (21) R = ⎡ ⎢ ⎢⎣ 1 −2q2 3 −2q0q3 0 2q0q3 1 −2q2 3 0 0 0 1 ⎤ ⎥ ⎥⎦ rp = X2 + R2S2 rp = X1 + R1S1 −R4Sp2 rp = X3 + R3S3 −R4Sp3 (21) R = ⎡ ⎢ ⎢⎣ 1 −2q2 3 −2q0q3 0 2q0q3 1 −2q2 3 0 0 0 1 ⎤ ⎥ ⎥⎦ (21) (15) International Journal of Precision Engineering and Manufacturing (2020) 21:995–1015 1000 Now we can express the geometric relations (16) by utilizing the rotation matrices in terms of the quaternions (21): Now we can express the geometric relations (16) by utilizing the rotation matrices in terms of the quaternions (21): Now we can get y in terms of the knowns by substituting (34) into (23): Now we can express the geometric relations (16) by utilizing the rotation matrices in terms of the quaternions (21): Now we can get y in terms of the knowns by substituting (34) into (23): Now we can express the geometric relations (16) by utilizing the rotation matrices in terms of the quaternions (21): Now we can get y in terms of the knowns by substituting (34) into (23): (22) x = x2 + L2(1 −2q3(훼2)2) (23) y = 2L2q0(훼2)q3(훼2) (24) x = x1 + L1(1 −2q3(훼1)2) −xp2(1 −2q3(휃)2) It is worth mentioning that (35) applies for all values of θ, not only for θ equal or close to zero. Therefore, it is more practical to use (35) instead of (14) to compute y for all cases. (35) y = √( L2 2 −(x −x2 )2) (22) x = x2 + L2(1 −2q3(훼2)2) (23) y = 2L2q0(훼2)q3(훼2) (24) x = x1 + L1(1 −2q3(훼1)2) −xp2(1 −2q3(휃)2) (25) y = 2L1q0(훼1)q3(훼1) −2xp2q0(휃)q3(휃) (26) x = x3 + L3(1 −2q3(훼3)2) −xp3(1 −2q3(휃)2) (27) y = 2L3q0(훼3)q3(훼3) −2xp3q0(휃)q3(휃) (35) (23) 3 Hand‑Eye and Robot‑World Calibration The kinematic calibration presented in this paper utilized a Leica absolute laser tracker in combination with a T-Mac TMC30-F reflector to measure the platform poses. The laser tracker head is automatically controlled to track the pose of the reflector rigidly attached on the platform. Therefore, the laser tracker measures the pose of the reflector instead of the platform. The use of a similar laser tracker for the external kinematic calibration of a serial kinematics mechanism can be found in [18]. The laser tracker in combination with the reflector used in this work is capable of measuring all rigid body DOFs which include both position and orientation, i.e. three translational DOFs and three rotational DOFs. Figure 4 shows the local frame of the reflector. As shown in the figure, the origin of the reflector frame takes place at the center of the reflector prism whereas the three axes of the reflector frame are aligned with the geometry of the reflec- tor. The accuracy of the laser tracker is ± 15 μm for the position and 0.01 degree for the rotation.l Furthermore, we can write the unit norm constraints of the quaternions as follows: (28) q0(훼i)2 + q1(훼i)2 + q2(훼i)2 + q3(훼i)2 = 1; i = 1, 2, 3 (29) q0(휃)2 + q1(휃)2 + q2(휃)2 + q3(휃)2 = 1 (28) 휃)2 + q1(휃)2 + q2(휃)2 + q3(휃)2 = 1 (29) Since in this case: (30) q1(훼1) = q2(훼1) = q1(훼2) = q2(훼2) = q1(훼3) = q2(훼3) = q1(휃) = q2(휃) = 0 (30) then we can rewrite (28)–(29) as follows: (31) q0(훼i)2 + q3(훼i)2 = 1; i = 1, 2, 3 (32) q0(휃)2 + q3(휃)2 = 1 q0(훼i)2 + q3(훼i)2 = 1; i = 1, 2, 3 (31) Since the laser tracker is an external device, the reflec- tor pose is provided with respect to the measurement frame fixed to the laser tracker frame. Figure 5 depicts the trans- formations among the machine base frame FB, the platform frame FP, the measurement frame FM, and the reflector frame (32) To this point, we can see that we have ten equations with thirteen variables, i.e. x, y, q0(α1), q3(α1), q0(α2), q3(α2), q0(α3), q3(α3), q0(θ), q3(θ), x1, x2, and x3. However, three variables among the 13 variables are known. In other words, we have ten equations with ten unknowns. For ease, x can be computed using (13). T2X1 = X2T1 (36) For a pair of mechanism poses as illustrated in Fig. 6, we have the following: For a pair of mechanism poses as illustrated in Fig. 6, we have the following: (37a) T21X1 = X2T11 (37b) T22X1 = X2T12 T21X1 = X2T11 (37a) (37b) It can be observed from Fig. 6 that X1 and X2 are constant, whereas T1 and T2 change with the mechanism pose. It can be observed from Fig. 6 that X1 and X2 are constant, whereas T1 and T2 change with the mechanism pose. Upon manipulating (37a) and (37b), we have: Upon manipulating (37a) and (37b), we have: where (38) AX1 = X1B (38) AX1 = X1B AX1 = X1B (38) where where (39) A = T−1 22 T21 = [ RA tA 0 1 ] (40) B = T−1 12 T11 = [ RB tB 0 1 ] y g To solve for X1 and X2, at least three different poses of the mechanism should be measured and T1 should be available. In this case, T1 is obtained based on the nominal values of the mechanism geometry. Hence, the estimation of X1 and X2 assumes that the nominal values of the mechanism geometry are good enough. These nominal values are to be calibrated later based on the obtained X1 and X2. The estimation of X1 and X2 can be classified into two broad categories: separable (sequential) and simultaneous techniques. The former tech- niques include [19–23] whereas the latter techniques include [24–31]. In the former techniques, X1 is first obtained and subsequently, X2 is computed based on the obtained X1. This approach is more straightforward but gives a less precise solution. On the other hand, the latter techniques estimate both X1 and X2 simultaneously. In this work, the former approach is applied only to obtain the initial values for the next algorithm proposed to refine the solution. (39) A = T−1 22 T21 = [ RA tA 0 1 ] (39) (40) B = T−1 12 T11 = [ RB tB 0 1 ] (40) Notice that A and B are homogeneous transformation matrices. RA and RB are rotation matrices whereas tA and tB are position vectors. Equation (38), commonly known as AX = XB problem, is the hand-eye calibration problem. A separable closed-form hand-eye calibration following [19] is employed here. 3 Hand‑Eye and Robot‑World Calibration When θ is zero, we can replace it with a small value near zero to accurately compute x. Once θ and x are obtained, we can use any pair of (22)–(23), (24)–(25), or (26) – (27). Using (22)–(23) is easier since we do not involve q3(θ) for which we need to convert θ to get q3(θ). From (22), we have: To this point, we can see that we have ten equations with thirteen variables, i.e. x, y, q0(α1), q3(α1), q0(α2), q3(α2), q0(α3), q3(α3), q0(θ), q3(θ), x1, x2, and x3. However, three variables among the 13 variables are known. In other words, we have ten equations with ten unknowns. Fig. 4 The T-Mac TMC30-F reflector and its local frame For ease, x can be computed using (13). When θ is zero, we can replace it with a small value near zero to accurately compute x. Once θ and x are obtained, we can use any pair of (22)–(23), (24)–(25), or (26) – (27). Using (22)–(23) is easier since we do not involve q3(θ) for which we need to convert θ to get q3(θ). From (22), we have: (33) q3(훼2) = √( 1 2 −x −x2 2L2 ) (33) Substituting (33) into (31), we obtain the following: (34) q0(훼2) = √( 1 2 + x −x2 2L2 ) (34) Fig. 4 The T-Mac TMC30-F reflector and its local frame International Journal of Precision Engineering and Manufacturing (2020) 21:995–1015 1001 Fig. 5 Transformations T1, T2, X1, and X2 Fig. 6 Transformations in a pair of mechanism poses Fig. 6 Transformations in a pair of mechanism poses Fig. 6 Transformations in a pair of mechanism poses Fig. 6 Transformations in a pair of mechanism poses Fig. 5 Transformations T1, T2, X1, and X2 Fr. T1 is the transformation from the machine base frame to the platform. This is given by the forward kinematics of the mechanism. T2 is the transformation from the measure- ment frame to the reflector frame. X1 is the transformation from the reflector frame to the platform, whereas X2 is the transformation from the measurement frame to the machine base frame. The platform or end-effector is commonly called a hand whereas the reflector is called an eye. A calibration to estimate the transformation X1 is accordingly called the hand-eye calibration. 3 Hand‑Eye and Robot‑World Calibration The measurement frame is also com- monly called the world frame and consequently a calibration to estimate the transformation X2 is usually called the robot- world calibration. Since the laser tracker at hand is capable of providing the six-DOF pose of the reflector, T2 is wholly known. Unfortunately, X1 and X2 are unknown. As a result, they should be estimated before calibrating the mechanism.f (36) T2X1 = X2T1 T2X1 = X2T1 T2X1 = X2T1 To this point, we solve for the rotation matrix of the transformation X1.i (41) (42) ̄PB = 2 sin 𝛼(B) 2 ⎡ ⎢ ⎢⎣ n(B) 1 n(B) 2 n(B) 3 ⎤ ⎥ ⎥⎦ ; 0 ≤𝛼(B) ≤𝜋 Step 10: Define a linear system involving the translation vector of the transformation X1, namely tX1, as follows: (42) (50) GtX1 = (RB −I)tX1 = d = RX1tA −tB (50) Step 6 Define υ and S as follows: Solve the linear system (50) by using linear least squares: (43) 𝜐= ⎡ ⎢ ⎢⎣ 𝜐x 𝜐y 𝜐z ⎤ ⎥ ⎥⎦ = ̄PA + ̄PB (44) S = skew(휐) = ⎡ ⎢ ⎢⎣ 0 −휐z 휐y 휐z 0 −휐x −휐y 휐x 0 ⎤ ⎥ ⎥⎦ (43) 𝜐= ⎡ ⎢ ⎢⎣ 𝜐x 𝜐y 𝜐z ⎤ ⎥ ⎥⎦ = ̄PA + ̄PB (44) S = skew(휐) = ⎡ ⎢ ⎢⎣ 0 −휐z 휐y 휐z 0 −휐x −휐y 휐x 0 ⎤ ⎥ ⎥⎦ Solve the linear system (50) by using linear lea Equations (50) and (51) hold in general for th sional Euclidean space. Since the mechanism planar, the dimensions of (50) and (51) should to a two-dimensional Euclidean space. Having t nism on the XY plane, the elements correspon tX1 = (GTG)−1GTd (43) (43) (43) (51) tX1 = (GTG)−1GTd (51) tX1 = (GTG)−1GTd (51) Equations (50) and (51) hold in general for three-dimen- sional Euclidean space. Since the mechanism at hand is planar, the dimensions of (50) and (51) should be reduced to a two-dimensional Euclidean space. Having the mecha- nism on the XY plane, the elements corresponding to the Z-axis in (50) and (51) should be suppressed. If it is not sup- pressed and accordingly x, y, and z elements of tX1 are all to be solved, the system will be rank deficient. This indicates a configuration degeneracy. Physically, this means that there is no reference in the z-direction to which the z element of tX1 should be defined. As a result, an infinite number of pos- sible values of z exist. (44) S = skew(휐) = ⎡ ⎢ ⎢⎣ 0 −휐z 휐y 휐z 0 −휐x −휐y 휐x 0 ⎤ ⎥ ⎥⎦ (44) Step 7 We have the following linear system: Step 7 We have the following linear system: (45) S ̄̄PX1 = ̄PA −̄PB S ̄̄PX1 = ̄PA −̄PB (45) With only a pair of poses, this linear system is singular. T2X1 = X2T1 Instead of describing the hand-eye calibration procedure in a derivation format, the proce- dure is described briefly here in a sequential manner as the following: Step 1 Find the transformation T1 of two different poses. This is obtained by the forward kinematics of the mecha- nism using the nominal values of the mechanism geom- etry, namely L1, L2, L3, xp2, and xp3. i Let T1, T2, X1, and X2 be homogeneous transformation matrices which contain both rotation and translation com- ponents. The estimation of X1 can be derived based on the following relation among the transformation matrices: 1 3 1002 International Journal of Precision Engineering and Manufacturing (2020) 21:995–1015 Step 8 Compute ̄PX1 as follows: Step 2 Find the transformation T2 of the two poses. This is given directly by the measurement of the full pose of the reflector by using the laser tracker. In this work, the measurement of the reflector orientation is provided in quaternions. Step 8 Compute ̄PX1 as follows: (48) ̄PX1 = 2 ̄̄PX1 √ 1 + ‖‖‖ ̄̄PX1 ‖‖‖ 2 (48) Step 3 Compute the matrices A and B for the two poses by using (39) and (40). Step 9 Convert ̄PX1 to the rotation matrix of the transfor- mation X1, namely RX1: Step 4 Convert the rotation matrices RA and RB into an axis-angle representation defined by a rotation angle α and a rotation axis (n1, n2, n3).i (49) RX1 = ⎛ ⎜ ⎜ ⎜⎝ 1 − ̄PX1 2 2 ⎞ ⎟ ⎟ ⎟⎠ I + 1 2 ̄PX1 ̄PT X1 + 4 − ̄PX1 2 skew̄PX1 (49) Step 5 Define ̄PA and ̄PB as follows: Step 5 Define ̄PA and ̄PB as follows: (41) ̄PA = 2 sin 𝛼(A) 2 ⎡ ⎢ ⎢⎣ n(A) 1 n(A) 2 n(A) 3 ⎤ ⎥ ⎥⎦ ; 0 ≤𝛼(A) ≤𝜋 where I is an identity matrix whereas skew ( ̄PX1 ) is a skew-symmetric matrix defined in a similar fashion to (44). Equation (53) also implies that the zero coordinate of the l to represent this kinematics. Figure 7 shows all the homoge- neous transformation matrices in the combined mechanism. For convenience, a similar matrix notation is used for the upper mechanism. The asterisk superscripts indicate trans- formation matrices belonging to the upper mechanism.l to represent this kinematics. Figure 7 shows all the homoge- neous transformation matrices in the combined mechanism. Z-axis is aligned with the reflector. For convenience, a similar matrix notation is used for the upper mechanism. The asterisk superscripts indicate trans- formation matrices belonging to the upper mechanism.l l After the hand-eye transformation X1 has been estimated, the robot-world transformation X2 can be easily estimated by rearranging (36): The transformation among the laser tracker, the reflec- tor, and the lower mechanism has been described earlier in (36). Similarly, the homogeneous transformation of the laser tracker, the reflector, and the upper mechanism can be written as: (54) X2 = T2X1T−1 1 X2 = T2X1T−1 1 (54) Using n different mechanism poses in the measurement, we accordingly have n different T1 and T2. As a result, there will be n different X2. To obtain a single X2, which is expected to be constant, we average the elements of T1 and T2 in an element-wise manner to get an averaged, constant X2. (55) T∗ 2X∗ 1 = X∗ 2T∗ 1 T∗ 2X∗ 1 = X∗ 2T∗ 1 (55) Alternatively, the following homogeneous transforma- tion can also be used: It is worth mentioning that the selected poses should be completely different from each other in all coordinates in order to get the best estimates. In this case, the x, y, and θ values should be completely different among the poses. In this work, twelve experimental measurement data was care- fully taken in which the x, y, and θ values of the poses are entirely different. (56) T∗ 2X∗ 1 = X2X0T∗ 1 (56) where X0 denotes the homogeneous transformation from the machine base frame to the base frame of the upper mecha- nism. Using (56), we have: (57) T∗ 2 = X2X0T∗ 1 (X∗ 1 )−1 (58) T∗ 1 = (X2X0 )−1T∗ 2X∗ 1 (57) T2X1 = X2T1 Therefore, at least two pairs of poses, i.e. three different poses, are required. Perform Step 1 to Step 7 for all n poses and accordingly stack (45) from all pairs of poses to compose the following overdetermined system: Equations (49) and (51) completely define the transforma- tion matrix X1: (52) X1 = [ RX1 tX1 0 1 ] (46) ⌢ S ̄̄PX1 = ⎡ ⎢ ⎢ ⎢⎣ S1 S2 ⋮ Sn ⎤ ⎥ ⎥ ⎥⎦ ̄̄PX1 = b = ⎡ ⎢ ⎢ ⎢⎣ ̄PA −̄PB 1 ̄PA −̄PB 2 ⋮ ̄PA −̄PB n ⎤ ⎥ ⎥ ⎥⎦ (52) (46) where, in the case of planar mechanism working on the XY plane: (53) tX1 = ⎡ ⎢ ⎢⎣ tX1(x) tX1(y) 0 ⎤ ⎥ ⎥⎦ Solve for ̄̄PX1 by using linear least squares: Solve for ̄̄PX1 by using linear least squares: (53) (47) ̄̄PX1 = ( ⌢ S T ⌢ S )−1 ⌢ S T b (53) tX1 = ⎢ ⎢⎣ tX1(y) 0 ⎥ ⎥⎦ (47) ̄̄PX1 = ( ⌢ S T ⌢ S )−1 ⌢ S T b tX1 = ⎢ ⎢⎣ tX1(y) 0 ⎥ ⎥⎦ ̄̄PX1 = ( ⌢ S T ⌢ S )−1 ⌢ S T b (47) 1 3 1003 International Journal of Precision Engineering and Manufacturing (2020) 21:995–1015 Equation (53) also implies that the zero coordinate of the Z-axis is aligned with the reflector. 4 Kinematics of the Combined Mechanism with the External Measurement Device (58) Another way to express the kinematics involving the laser tracker and the reflector is by using vector notation. Figure 8 shows the position vectors in the combined mech- anism. The superscript indicates the frame in which a posi- tion vector r is expressed, whereas the subscript indicates a point defined by the position vector. The subscripts and superscripts M, B, O, P, and r respectively represent the measurement frame, machine base frame, base frame of The pose measurement is always provided with respect to the measurement frame. Since the kinematic calibration uses an external pose-measurement device, an extended kinematic model should be established to include the hand- eye and robot-world transformations as described earlier. A homogeneous transformation is very convenient to be used Fig. 7 Homogeneous transformations in the combined mechanism with a laser tracker and reflectors, namely T1, T2, X1, X2 (for the lower mechanism), T∗ 1 , T∗ 2 , X∗ 1, X∗ 2 (for the upper mechanism), and X0 which connects both mechanisms Fig. 8 Position vectors in the combined mechanism with a laser tracker and reflectors, where the subscripts indicate the points of interest whereas the superscripts indicate the reference frames Fig. 7 Homogeneous transformations in the combined mechanism with a laser tracker and reflectors, namely T1, T2, X1, X2 (for the lower mechanism), T∗ 1 , T∗ 2 , X∗ 1, X∗ 2 (for the upper mechanism), and X0 which connects both mechanisms Fig. 8 Position vectors in the combined mechanism with a laser tracker and reflectors, where the subscripts indicate the points of interest whereas the superscripts indicate the reference frames 1 3 1 3 1004 International Journal of Precision Engineering and Manufacturing (2020) 21:995–1015 (60) RO P,U = Rz (휃U ) the upper mechanism, platform frame, and reflector frame. For example, a notation rM B denotes the position vector of the machine base frame origin B with respect to the meas- urement frame M. RO P,U = Rz (휃U ) (60) where Rz is an elementary rotation matrix about Z-axis whereas θL and θU are the rotation angles of the lower and upper mechanisms, respectively.l The additional subscripts L and U indicate the lower and upper mechanism, respectively. The transformation between frames may involve a rotation matrix R which represents the rotational transformation between two frames. 4 Kinematics of the Combined Mechanism with the External Measurement Device Similarly, a notation RM B , as an example, denotes the rotation matrix from the machine base frame B to the measurement frame M. The reflectors are attached to the platforms, as shown in Fig. 6, such that: (61) RP,L r,L = I (61) RP,L r,L = I (62) RP,U r,U = Rx ( −휋 2 ) RP,L r,L = I (61) (62) RP,U r,U = Rx ( −휋 2 ) (62) For more clarity, mainly when dealing with rotational transformation, Fig. 9 shows only the orientation of all the frames. The drawn axes indicate the positive directions of the axes. The orientation of the axes shown in this figure follows the right-hand rule in which the cross product between the X and Y axes gives the direction of the Z-axis. For more con- venience, the base frame of the upper mechanism is oriented such that its X and Y axes create the planar workspace of the upper mechanism. Accordingly, the positive Z-axis is pointing downward. Using such an orientation, the inverse and forward kinematics of both the lower and upper mechanisms can be written on the XY plane. where I and Rx are an identity matrix and an elementary rota- tion matrix about the X-axis, respectively. This means that the lower reflector frame is aligned with the lower platform frame whereas the upper reflector frame is also aligned but rotated about the X-axis by –π/2 rad. This arrangement can be made with the aid of measurement devices such as digital calipers, digital depth gage, and precision square.l The position of the reflector mounted on the lower platform can be expressed in the measurement frame as follows: (63) rM r,L = rM B + RM B rB r,L = rM B + RM B ( rB P,L + RB P,LrP,L r,L ) (63) Since the mechanisms are planar, by referring to Fig. 6, their rotation about their base frames can be represented by an elementary rotation matrix about the Z-axis, i.e.: where RM B denotes the rotation matrix from the machine base frame to the measurement frame whereas RB r,L denotes the rotation matrix from the reflector frame to the machine base frame.l (59) RB P,L = Rz (휃L ) RB P,L = Rz (휃L ) RB P,L = Rz (휃L ) (59) Since the reflector frame is aligned with the platform frame, i.e. 4 Kinematics of the Combined Mechanism with the External Measurement Device the orientation transformation between them is represented by an identity rotation matrix RP,L r,L , the rotation of the reflector frame in the measurement frame is given by: Fig. 9 Orientation of the frames (drawn axes indicate positive direc- tions) namely the measurement frame M, the lower machine base frame B, the upper machine base frame O, the lower platform frame P,L, the upper platform frame P,U, the lower reflector frame r,L, and the upper reflector frame r,U (64) RM r,L = RM P,L = RM B RB P,LRP,L r,L = RM B RB P,L (64) As a result, we have: As a result, we have: (65) RB P,L = (RM B )−1RM r,L (65) Substituting (65) into (63), we obtain: (66) rM r,L = rM B + RM B ( rB P,L + (RM B )−1RM r,LrP,L r,L ) (66) (67) rM r,L = rM B + RM B rB P,L + RM r,LrP,L r,L (67) Both the position of the reflector rM r,L and the orientation of the reflector RM r,L , expressed in the measurement frame, are measured by using the laser tracker. The rotation matrix RM B and the position vector rM B can be estimated by using the robot-world calibration, whereas the position vector rP,L r,L can be estimated by using the hand-eye calibration. Fig. 4 Kinematics of the Combined Mechanism with the External Measurement Device 9 Orientation of the frames (drawn axes indicate positive direc- tions) namely the measurement frame M, the lower machine base frame B, the upper machine base frame O, the lower platform frame P,L, the upper platform frame P,U, the lower reflector frame r,L, and the upper reflector frame r,U 1005 and Manufacturing (2020) 21:995–1015 1005 International Journal of Precision Engineering and Manufacturing (2020) 21:995–1015 The position of the platform end-effector in the base frame rB P,L is given by the forward kinematics, i.e.: frame of the upper mechanism rO P,U is given by the forward kinematics, i.e.: (68) rB P,L = ⎡ ⎢ ⎢⎣ xL yL zL ⎤ ⎥ ⎥⎦ = ⎡ ⎢ ⎢⎣ f1(x1, x2, x3, PL) f2(x1, x2, x3, PL) 0 ⎤ ⎥ ⎥⎦ (75) rO P,U = ⎡ ⎢ ⎢⎣ xU yU zU ⎤ ⎥ ⎥⎦ = ⎡ ⎢ ⎢⎣ f1(x4, x5, x6, PU) f2(x4, x5, x6, PU) 0 ⎤ ⎥ ⎥⎦ (75) rO P,U = ⎡ ⎢ ⎢⎣ xU yU zU ⎤ ⎥ ⎥⎦ = ⎡ ⎢ ⎢⎣ f1(x4, x5, x6, PU) f2(x4, x5, x6, PU) 0 ⎤ ⎥ ⎥⎦ (68) (75) where f1 and f2 are the forward kinematics equations and PL contains all the geometric parameters of the lower mechanism. where f1 and f2 are the forward kinematics equations and PU contains all the geometric parameters of the upper mechanism. For convenience, the Z-axis of the machine base frame is assumed to be aligned with the Z-axis of the reflector frame. 4 Kinematics of the Combined Mechanism with the External Measurement Device In other words, the z coordinate of the reflector frame origin with respect to the machine base frame is zero: The transformation from the machine base frame B to the base frame of the upper mechanism O can be obtained from the following relations: (76) RM O = RM B RB O (76) RM O = RM B RB O (77) rM O = rM B + RM B rB O RM O = RM B RB O (76) (69) rP,L r,L = ⎡ ⎢ ⎢⎣ xr,L yr,L zr,L ⎤ ⎥ ⎥⎦ = ⎡ ⎢ ⎢⎣ xr,L yr,L 0 ⎤ ⎥ ⎥⎦ (77) rM O = rM B + RM B rB O (69) (77) From (76) and (77), we obtain: From (76) and (77), we obtain: Similarly, the position of the reflector mounted on the upper platform can be expressed in the measurement frame as follows: (78) RB O = (RM B )−1RM O (79) rB O = (RM B )−1(rM O −rM B ) (78) RB O = (RM B )−1RM O RB O = (RM B )−1RM O (78) (79) rB O = (RM B )−1(rM O −rM B ) (70) rM r,U = rM O + RM O rO r,U = rM O + RM O ( rO P,U + RO P,UrP,U r,U ) (79) rB O = (RM B )−1(rM O −rM B ) (70) rM r,U = rM O + RM O rO r,U = rM O + RM O ( rO P,U + RO P,UrP,U r,U ) (70) (79) rB O = (RM B )−1(rM O −rM B ) (70) + RM O ( rO P,U + RO P,UrP,U r,U ) (79) rB O = (RM B )−1(rM O −rM B ) (70) (79) Figure 10 shows the laser tracker fixed sitting beside the prototype machine. Figure 11 depicts the reflector mounted on the lower and upper platforms. Since only one reflector is available, the reflector was first mounted on the lower platform and subsequently mounted on the upper platform, or vice versa. In each setup, the estimation of the hand-eye transformation, robot-world transformation, and the geometric parameters of the lower mechanism was conducted. 4 Kinematics of the Combined Mechanism with the External Measurement Device As mentioned earlier, the reflector should The rotation of the reflector frame in the measurement frame, which is measured by using the laser tracker, can be written as: (71) RM r,U = RM O RO P,URP,U r,U (71) Solving for RO P,U from (71) yields: Solving for RO P,U from (71) yields: Solving for RO P,U from (71) yields: (72) RO P,U = (RM O )−1RM r,U ( RP,U r,U )−1 (72) RO P,U = (RM O )−1RM r,U ( RP,U r,U )−1 (72) Because we have (62), then: Because we have (62), then: Because we have (62), then: Fig. 10 The laser tracker with a reflector installed on the machine (73) RO P,U = (RM O )−1RM r,U ( Rx ( −휋 2 ))−1 (73) Substituting (73) into (70), we obtain: Substituting (73) into (70), we obtain: Substituting (73) into (70), we obtain: (74) rM r,U = rM O + RM O rO r,U = rM O + RM O ( rO P,U + ((RM O )−1RM r,U ( Rx ( −휋 2 ))−1) rP,U r,U ) (74) ) In fact, both the position of the reflector rM r,U and the ori- entation of the reflector RM r,U , expressed in the measurement frame, are measured by using the laser tracker. The rota- tion matrix RM O and the position vector rM O can be estimated by using the robot-world calibration, whereas the position vector rP,U r,U can be estimated by using the hand-eye calibra- tion. The position of the platform end-effector in the base Fig. 10 The laser tracker with a reflector installed on the machine Fig. 10 The laser tracker with a reflector installed on the machine 1 3 1 3 International Journal of Precision Engineering and Manufacturing (2020) 21:995–1015 1006 Fig. 11 The reflector mounted a on the upper platform and b on the lower platform (80) ̃Y = Y(P) + v ̃Y = Y(P) + v (80) The noise υ is typically Gaussian with zero mean and a standard deviation is given by the uncertainty of the measure- ment device. 4 Kinematics of the Combined Mechanism with the External Measurement Device The model function Y for the lower mechanism is given by (67), i.e.: (81) Y = rM r,L = rM B + RM B rB P,L + RM r,LrP,L r,L (81) whereas the model function Y for the upper mechanism is given by (74), i.e.: (82) Y = rM r,U = rM O + RM O rO r,U = rM O + RM O ( rO P,U + ((RM O )−1RM r,U ( Rx ( −휋 2 ))−1) rP,U r,U ) (82) The residual error is given by: The residual error is given by: (83) e = ΔY = ̃Y −̂Y( ̂P) (83) Notice that the tilde (~) mark on a parameter indicates a measured parameter whereas a hat mark indicates an esti- mated parameter. Given m measurements, the measured function values ̃Y , the estimated function values ̂Y , and accordingly the residual errors e should be stacked in a sin- gle vector. Fig. 11 The reflector mounted a on the upper platform and b on the lower platform For the calibration of the lower mechanism, the residual error is defined as: (84) e = ̃Y −̂Y( ̂P) = ̃rM r,L −̂rM r,L be installed to satisfy (61) and (62). To verify this align- ment, one should observe the estimated rotation matrix of the reflector frame with respect to the platform frame, namely RX1,L = RP,L r,L and RX1,U = RP,U r,U . After the estimation was conducted for both the mechanisms, the transforma- tion from the machine base frame B to the base frame of the upper mechanism O can be estimated. be installed to satisfy (61) and (62). To verify this align- ment, one should observe the estimated rotation matrix of the reflector frame with respect to the platform frame, namely RX1,L = RP,L r,L and RX1,U = RP,U r,U . After the estimation was conducted for both the mechanisms, the transforma- tion from the machine base frame B to the base frame of the upper mechanism O can be estimated. (84) The measured position vector ̃rM r,L is provided by the laser tracker, whereas the estimated position vector ̂rM r,L is evalu- ated by using (67) or (81). 4 Kinematics of the Combined Mechanism with the External Measurement Device Similarly, the residual error for the calibration of the upper platform is defined as: (85) e = ̃Y −̂Y( ̂P) = ̃rM r,U −̂rM r,U (85) While the laser tracker provides the measured position vec- tor ̃rM r,U , the estimated position vector ̂rM r,U is evaluated by using (74) or (82). 1 3 J = HT k WHk (92) Stage 3 Estimation of the position of the base frame of the upper mechanism,rB O. Stage 3 Estimation of the position of the base frame of the upper mechanism,rB O. The matrix J should have a full rank in order to give a trusted solution. This full rank indicates that all the parameters are independent and therefore fully identified. In other words, a rank deficiency by n indicates that n parameters are dependent and therefore unidentified. The determination of the unidentified parameters can be done mathematically or through knowledge on the parameter dependency of the physical system. In a system with a rank deficiency, the dependent parameters should be elimi- nated until a full-rank system is obtained. In such a case, the nominal parameter values can be used. The dependent parameters can be estimated in the next step employing the estimates of the independent parameters having been obtained previously. This is commonly called the subset Stage 4 Estimation of the geometric parameters of the upper mechanism. Similar to the case of the lower plat- form, this consists of two steps. The iterations in the GLSDC keeps running until the norm of ∆F is less than a specified threshold. This is the stopping criteria of the GLSDC algorithms. In this work, the norm (∆F) of 1.0 × 10−10 is used. 5.1 Nonlinear least squares algorithm Besides the position vectors ̃rM r,L and ̃rM r,U , the other param- eters given by measurements are the orientation matrices RM r,L and RM r,U , as well as the active joint positions which contrib- ute to the forward kinematics solution of both mechanisms.i In this section, Gaussian least squares differential correction (GLSDC) algorithm [32–34] (also called the Gauss–Newton nonlinear least squares) is applied for the refinement of the hand-eye calibration as well as the calibration of the mecha- nism geometric parameters. Using this algorithm, the nonlin- earity of the system is taken into consideration. The estimation is conducted to find the estimates of the parameters ̂P which minimizes the following cost function: (86) F = ‖e‖2 = 1 2eTWe (86) Let P and Y be the parameters to be estimated and the model function, respectively, then the measured function values are given with a noise υ as follows: and accordingly minimizes the residual error in (83)–(85). The cost function F in (86) indicates the norm or the square of the residual error. The matrix W denotes the weight matrix 1 3 1 3 1007 International Journal of Precision Engineering and Manufacturing (2020) 21:995–1015 which is, in this work, chosen to be an identity matrix. Using GLSDC, the parameter estimates are computed iteratively as follows: Fig. 12 Flowchart of the subset selection in the implementation of GLSDC (87) ̂Xk+1 = ̂Xk + Δx where (87) ̂Xk+1 = ̂Xk + Δx ̂Xk+1 = ̂Xk + Δx (87) where where (88) Δx = (HT k WHk )−1HT k Wek (88) and the subscript k indicates the k-th iteration.i A modification to (88) called the Levenberg–Marquardt algorithm (also called the damped least squares) had been proposed to speed up the convergence in the case of initial values far from the “true” values. Since the initial val- ues assigned in this work are considered close enough to the “true” values, the GLSDC is sufficient to give a fast convergence.f The Jacobian matrix H is obtained by differentiating the model function with respect to the estimated parameters: Fig. 12 Flowchart of the subset selection in the implementation of GLSDC (89) H = 휕Y 휕P = [ Hx Hy Hz ]T = [ 휕Yx 휕P 휕Yy 휕P 휕Yz 휕P ]T (89) selection which results in a sequential estimation, i.e. the estimation is conducted in several steps. 5.1 Nonlinear least squares algorithm This approach assumes that the nominal parameter values should be good enough. Figure 12 shows how the subset selection is implemented in the GLSDC algorithm. As shown in the flowchart, if J does not have a full rank N then only M parameters are firstly estimated where M < N is the rank of J. This, as will be discussed soon, includes the following sequential estimation stages: where Yx, Yy, and Yz denote the x, y, and z components of the model function Y. Given m measurements, the Jacobian matrix H should be stacked as follows: (90) Hx = ⎡ ⎢ ⎢ ⎢⎣ 휕Yx1 휕P ⋮ 휕Yxm 휕P ⎤ ⎥ ⎥ ⎥⎦ ; Hy = ⎡ ⎢ ⎢ ⎢⎣ 휕Yy1 휕P ⋮ 휕Yym 휕P ⎤ ⎥ ⎥ ⎥⎦ ; Hz = ⎡ ⎢ ⎢ ⎢⎣ 휕Yz1 휕P ⋮ 휕Yzm 휕P ⎤ ⎥ ⎥ ⎥⎦ (91) H3mx5 = [ Hx Hy Hz ]T (90) Hx = ⎡ ⎢ ⎢ ⎢⎣ 휕Yx1 휕P ⋮ 휕Yxm 휕P ⎤ ⎥ ⎥ ⎥⎦ ; Hy = ⎡ ⎢ ⎢ ⎢⎣ 휕Yy1 휕P ⋮ 휕Yym 휕P ⎤ ⎥ ⎥ ⎥⎦ ; Hz = ⎡ ⎢ ⎢ ⎢⎣ 휕Yz1 휕P ⋮ 휕Yzm 휕P ⎤ ⎥ ⎥ ⎥⎦ (90) (91) H3mx5 = [ Hx Hy Hz ]T Stage 1 Estimation of the hand-eye and robot-world trans- formation parameters to refine the closed-form solution obtained previously. (91) The squared system Jacobian matrix in each iteration is the inverted part in (88), i.e.: Stage 2 Estimation of the geometric parameters of the lower mechanism. This consists of two steps. Stage 2 Estimation of the geometric parameters of the lower mechanism. This consists of two steps. (92) J = HT k WHk J = HT k WHk 5.2 Refinement of the Hand‑Eye and Robot‑World Calibration As the rotation matrix RM B is defined in terms of quaternions, namely q0, q1, q2, and q3, the position vector rM B is defined by the components xB, yB, and zB, and the position vector rP,L r,L is defined by the components xr and yr while zr= 0, the parameters to be estimated are: Tables 2 and 3 show both the estimates obtained using the closed-form solution as described in Sect. 1 and those obtained through the refinement using the GLSDC, for the lower and upper mechanisms, respectively. (93) P = [ q0 q1 q2 q3 xB yB zB xr yr ]T (93) Similarly for the upper mechanism, since the orientation of the reflector frame is aligned with that of the platform frame with a rotation of –π/2 about X-axis, the remaining parameters which should be estimated to establish the hand- eye and robot-world transformations are the position and orientation of the machine base frame with respect to the measurement frame, namely rM O and RM O , as well as the posi- tion of the reflector with respect to the lower platform frame, namely rP,U r,U . As the rotation matrix RM O is defined in terms of quaternions, namely q0, q1, q2, and q3, the position vec- tor rM O is defined by the components xO, yO, and zO, and the position vector rP,U r,U is defined by the components xr and zr while yr= 0, the parameters to be estimated are: As a partial benchmark, the available nominal value of yr is 185 mm. Since the reflector is mounted well on the lower platform, the deviation from the nominal value should not be too large. The estimated value of yr = 186.087565 mm is considered acceptable. This indicates that the refinement works well. After the hand-eye and robot-world transformations for both the lower and upper mechanisms have been performed, the transformation from the machine base frame B to the base frame of the upper mechanism O can be easily com- puted by using (78) and (79). 5.2 Refinement of the Hand‑Eye and Robot‑World Calibration After the procedure described in Sect. 1 has been per- formed to obtain the rough estimates of the hand-eye and 1 3 1 3 International Journal of Precision Engineering and Manufacturing (2020) 21:995–1015 1008 Table 3 The estimated hand-eye and robot-world transformation parameters for the upper mechanism Parameter Closed-form solution GLSDC solution q0 1.041334 − 0.004111 q1 0.147820 1.000361 q2 − 0.130918 0.002880 q3 0.033675 − 0.003159 xB (mm) − 710.871725 − 869.180182 yB (mm) 1788.690134 2800.708342 zB (mm) − 526.167776 − 269.454137 xr (mm) − 99.726060 − 109.462822 yr (mm) 229.062687 175.912422 Table 3 The estimated hand-eye and robot-world transformation parameters for the upper mechanism robot-world transformations, a refinement is conducted by using the obtained estimates as initial values in an itera- tive estimation using GLSDC. For the lower mechanism, since the orientation of the reflector frame is aligned with that of the platform frame, the remaining parameters which should be estimated to establish the hand-eye and robot- world transformations are the position and orientation of the machine base frame with respect to the measurement frame, namely rM B and RM B , as well as the position of the reflector with respect to the lower platform frame, namely rP,L r,L . As the rotation matrix RM B is defined in terms of quaternions, namely q0, q1, q2, and q3, the position vector rM B is defined by the components xB, yB, and zB, and the position vector rP,L r,L is defined by the components xr and yr while zr= 0, the parameters to be estimated are: robot-world transformations, a refinement is conducted by using the obtained estimates as initial values in an itera- tive estimation using GLSDC. For the lower mechanism, since the orientation of the reflector frame is aligned with that of the platform frame, the remaining parameters which should be estimated to establish the hand-eye and robot- world transformations are the position and orientation of the machine base frame with respect to the measurement frame, namely rM B and RM B , as well as the position of the reflector with respect to the lower platform frame, namely rP,L r,L . 5.2 Refinement of the Hand‑Eye and Robot‑World Calibration The obtained rotation matrix is: (94) P = [ q0 q1 q2 q3 xO yO zO xr zr ]T (95) RB O = ⎡ ⎢ ⎢⎣ 1.001862 −0.001390 −0.001831 −0.001834 −0.002087 −1.001861 0.001386 1.001862 −0.002090 ⎤ ⎥ ⎥⎦ (94) (95) The evaluation of the squared system Jacobian matrix J for both the lower and upper mechanisms shows that its rank is 9 (full rank) and therefore all the parameters in (93) and (94) can be estimated in a single step for each mechanism. whereas the position vector from B to O is: whereas the position vector from B to O is: (96) rB O = ⎡ ⎢ ⎢⎣ xB O yB O zB O ⎤ ⎥ ⎥⎦ = ⎡ ⎢ ⎢⎣ −2.084870 898.462981 −637.302275 ⎤ ⎥ ⎥⎦ (96) Table 2 The estimated hand-eye and robot-world transformation parameters for the lower mechanism Parameter Closed-form solution GLSDC solution q0 0.698220 0.704535 q1 0.710261 0.708877 q2 − 0.001228 0.004105 q3 0.003017 0.000943 xB (mm) − 520.639720 − 866.594463 yB (mm) 2175.139684 2169.653698 zB (mm) − 861.763839 − 1170.828004 xr (mm) 141.733177 143.192701 yr (mm) 182.660182 186.087565 Table 2 The estimated hand-eye and robot-world transformation parameters for the lower mechanism This shows that the origin of the X-axis of the base frame of the upper mechanism O is shifted by − 2.084870 mm from that of the machine base frame B. In the design, xB O is expected to be zero. However, the estimated value shows that it is not zero. This occurs because there may be an error in the manufacturing of the machine base structure. International Journal of Precision Engineering and Manufacturing (2020) 21:995–1015 1009 Table 4 The estimates of geometric parameters of the lower mecha- nism (xp2 = xp3 = xp) Parameter Estimate Nominal value Error L1 (mm) 486.000000 486.000000 0.000000 L2 (mm) 500.985068 500.000000 0.985068 L3 (mm) 506.857577 507.000000 − 0.142423 xp (mm) 285.866049 285.000000 0.866049 This is because the nominal value of L1 is used in the first step and subsequently it is estimated after the other parame- ters have been estimated. In other words, the nominal values of L1 are retrieved in this two-step estimation. 6 Estimation of the Geometric Parameters Using Iterative Linear Least Squares In this section, a linear error model is used for the kinematic calibration. Accordingly, the closed-form linear least squares estimation can be employed. From the kinematic relations given in (1)–(3), a small perturbation can be introduced to all the parameters which include the geometric parameters P, the active joint positions q, and the platform pose X. This small perturbation which represents a small error is a first-order approximation. Following (1)–(3), the perturbed kinematics can be written by differentiating the kinematics with respect to all parameters: Table 5 The estimates of geometric parameters of the upper mecha- nism (xp2 = xp3 = xp) Table 5 The estimates of geometric parameters of the upper mecha- nism (xp2 = xp3 = xp) Parameter Estimate Nominal value Error L1 (mm) 600.000000 600 0.000000 L2 (mm) 475.001025 475 0.001025 L3 (mm) 604.000966 604 0.000966 xp (mm) 280.001333 280 0.001333 (98) L1훿l1 = (x + xp2 cos(휃) −x1) (훿x + 훿xp2 cos(휃) −xp2 sin(휃)훿휃−훿x1) + (y + xp2 sin(휃))(훿y + 훿xp2 sin(휃) + xp2 cos(휃)훿휃) 5.3 Estimation of the Mechanism Geometric Parameters (98) For each mechanism, there are five geometric parameters to be estimated, namely l1, l2, l3, xp2 and xp3. In the case of xp2= xp3= xp, which is the case of the prototype machine, there are four geometric parameters to be estimated, i.e. l1, l2, l3,and xp. Therefore, the parameters to be estimated are: (99) L2훿l2 = (x −x2)(훿x −훿x2) + y훿y (99) ( L3훿l3 = (x + xp3 cos(휃) −x3) (훿x + 훿xp3 cos(휃) −xp3 sin(휃)훿휃−훿x3) + (y + xp3 sin(휃))(훿y + 훿xp3 sin(휃) + xp3 cos(휃)훿휃) (97) P = [ L1 L2 L3 xp ]T (97) (100) (100) The observation shows that the squared system Jacobian matrix J gives only a rank of 4 in the case of five parameters to be estimated, i.e. when xp2≠ xp3, and a rank of 3 in the case of four parameters to be estimated, i.e. when xp2= xp3= xp. This is because one of all the geometric parameters to be estimated is dependent on the other four parameters. In other words, all the geometric parameters but one are known then the one parameter remaining can be calculated by using the closed-form geometric relation of the mechanism. In a matrix form, (98)–(100) can be written compactly as: In a matrix form, (98)–(100) can be written compactly as: (101) Jx훿X + Jq훿q = Jp훿P Jx훿X + Jq훿q = Jp훿P (101) (101) where where (102) Jx = ⎡ ⎢ ⎢⎣ ̂X1 y + xp2 sin(𝜃) ̂X1xp2(cos(𝜃) −sin(𝜃)) (x −x2) y 0 ̂X3 y + xp3 sin(𝜃) ̂X3xp3(cos(𝜃) −sin(𝜃)) ⎤ ⎥ ⎥⎦ (102) Another approach is to estimate all the geometric param- eters in two steps. In the first step, L2, L3, xp2, and xp3 are estimated. In this step, the nominal value of L1 is used in the estimation iterations. In the second step, the remaining parameter, i.e. L1, is estimated. Although the nominal value of L1 is used in the first step, L1 is subsequently estimated in the second step. Using twelve unique calibration poses for each of the lower and upper mechanisms, the estimates of the geometric parameters of the lower and upper mechanisms along with their nominal values are shown in Tables 4 and 5. The errors are defined as the difference between the esti- mates and the nominal values. It can be observed in Tables 4 and 5 that the estimate of L1 is similar to its nominal value. 5.3 Estimation of the Mechanism Geometric Parameters (103) Jq = ⎡ ⎢ ⎢⎣ −̂X1 0 0 0 x2 −x 0 0 0 −̂X3 ⎤ ⎥ ⎥⎦ (104) Jp = ⎡ ⎢ ⎢⎣ L1 0 0 −̂X1(cos 𝜃+ sin 𝜃) 0 0 L2 0 0 0 0 0 L3 0 −̂X3(cos 𝜃+ sin 𝜃) ⎤ ⎥ ⎥⎦ 1 3 International Journal of Precision Engineering and Manufacturing (2020) 21:995–1015 1010 i.e.: 105) 106) 107) 108) 109) 110) Step 2 Measure the poses, Xmeasured. Step 3 Calculate δX = Xmeasured − X. Step 4 Perform the inverse kinematics to obtain the active joint positions q = f(X,P). Step 5 Measure the active joint positions, qmeasured. Step 6 Calculate δq = qmeasured – q. Step 7 Calculate A(X, 훿X, q, 훿q, P) = Jx훿X + Jq훿q [Eq. (110)]. Step 8 Compose the vector B(X,q,P). Step 9 Repeat Step 1 to Step 8 for m measurements. Compose ̄A and ̄B as in (113). Step 10 Check the rank of ̄BT ̄B. Step 11 If ̄BT ̄B has full rank, then compute the linear least squares solution (115). Otherwise, eliminate the dependent parameters either mathematically, such as through SVD, or based on knowledge on the physical system. (105) ̂X1 = x + xp2 cos(𝜃) −x1 (106) ̂X3 = x + xp3 cos(𝜃) −x3 (107) 훿X = [ 훿x 훿y 훿휃]T (108) 훿q = [ 훿x1 훿x2 훿x3 ]T (109) 훿P = [ 훿L1 훿L2 훿L3 훿xp2 훿xp3 ]T L ll h i h l f h d id b ll d A i (105) ̂X1 = x + xp2 cos(𝜃) −x1 (106) ̂X3 = x + xp3 cos(𝜃) −x3 (107) 훿X = [ 훿x 훿y 훿휃]T (108) 훿q = [ 훿x1 훿x2 훿x3 ]T (109) 훿P = [ 훿L1 훿L2 훿L3 훿xp2 훿xp3 ]T (105) (109) Let all the terms in the left-hand side be called A, i.e.: A = Jx훿X + Jq훿q (110) A = Jx훿X + Jq훿q and B = Jp , then we can rewrite (101) as: and B = Jp , then we can rewrite (101) as: (111) A = B훿P (111) In real practice, the measured poses Xmeasured is often given by an external measurement device such as a camera or a laser tracker as in this work. As a result, Xmeasured is given with respect to a measurement frame which is not aligned with the machine base frame. Step 1 Assign the poses to be visited, X. 5.3 Estimation of the Mechanism Geometric Parameters Accordingly, the assigned poses X should be defined with respect to the measurement frame. The values of X with respect to the measurement frame can be derived from (67) or (81) for the lower mechanism and from (74) or (82) for the upper mechanism. Having m measurements, we can stack (110) into the following overdetermined system: where (112) ̄A = ̄B𝛿P (112) ̄A = ̄B𝛿P where (113) ̄A = ⎡ ⎢ ⎢ ⎢⎣ A1 A2 ⋮ Am ⎤ ⎥ ⎥ ⎥⎦ ; ̄B = ⎡ ⎢ ⎢ ⎢⎣ B1 B2 ⋮ Bm ⎤ ⎥ ⎥ ⎥⎦ (113) From (67) or (81), we obtain: From (67) or (81), we obtain: The estimation of 훿P is aimed at minimizing the fol- lowing cost function: (117) XL = rB P,L = (RM B )−1( rM r,L −rM B −RM r,LrP,L r,L ) (117) In a similar fashion, from (74) or (81) we have: (114) F = ‖‖ ̄B𝛿p −̄A‖‖ 2 = 1 2 ( ̄B𝛿p −̄A)2 (114) (118) XU = rO P,U = (RM O )−1 ( rM r,U −rM O −RM r,U ( Rx ( −휋 2 ))−1 rP,U r,U ) As the problem is an overdetermined system, a closed- form solution for δp is given by the following linear least squares: (118) ) The sequential procedure to implement the linear least squares estimation based on the linear model when an exter- nal pose measurement device is used can be summarized as follows: (115) 𝛿P = (̄BT ̄B)̄BT ̄A (115) Since (115) denotes the errors in the geometric param- eters, the estimated geometric parameters are given by: Step 1 Estimate the hand-eye and robot-world transfor- mations. (116) ̂P = P + 𝛿P (116) ̂P = P + 𝛿P Step 2 Assign X with respect to the machine base frame FB for the lower mechanism and with respect to the base frame of the upper mechanism FO for the upper mecha- nism. where ̂P and P denote the estimated and nominal values of the geometric parameters, respectively. Assuming that the measurement is given with respect to the machine base frame, the following is the sequential procedure to implement the linear least squares estimation based on the abovementioned linear error model: Step 3 Measure the reflector position with respect to the measurement frame, namely rM r,L for the lower mechanism and rM r,U for the upper mechanism. 5.3 Estimation of the Mechanism Geometric Parameters r,U Step 4 Calculate Xmeasured by utilizing (117) for the lower mechanism and (118) for the upper mechanism. Step 1 Assign the poses to be visited, X. 1 3 International Journal of Precision Engineering and Manufacturing (2020) 21:995–1015 1011 Step 5 Perform Step 3 to Step 11 as described for the pose measurement in the machine base frame. Step 5 Perform Step 3 to Step 11 as described for the pose measurement in the machine base frame. Table 6 Estimated geometric parameters of the lower mechanism obtained by using iterative linear least squares Parameter Estimated value Nominal value Error 훿P L1 (mm) 485.994173 486 − 0.005827 L2 (mm) 500.009894 500 0.009894 L3 (mm) 507.009070 507 0.009070 xp (mm) 285.000000 285 0.000000 Table 6 Estimated geometric parameters of the lower mechanism obtained by using iterative linear least squares Using the same measurement data as used in the GLSDC, the resulting overdetermined linear system (112) with all the geometric parameters to be estimated has a full-ranked ̄BT ̄B . Therefore, the estimation can be performed straight- forwardly in a single step. However, the estimates given by a single run of the algorithm are not satisfying. They are inconsistent and not guaranteed to be the optimum solution. Some coordinates may have an error less than that of the uncalibrated one, but some other coordinates may have an even more significant error than that of the uncalibrated one. This implies that the estimation algorithm fails to minimize the cost function, i.e. the residual errors. least squares is depicted in Fig. 13. It can be seen in the flow- chart that the estimated parameter values are updated and subsequently supplied to the new linear system which will be solved iteratively until the stopping criteria as discussed is achieved. Furthermore, the norm of the residual is evaluated between any two consecutive iterations. If the norm of the residual is increasing, the new parameter values should be subtracted by the parameter errors. Otherwise, they should be summed. Using twelve unique calibration poses for each of the lower and upper mechanisms, the iterative linear least squares (124) provides the estimated geometric parameters of the lower and upper mechanisms as shown in Tables 6 and 7, respectively. In a similar manner with the two-step estima- tion using the nonlinear least squares, the errors are defined as the difference between the estimated and nominal values. 5.3 Estimation of the Mechanism Geometric Parameters To overcome this problem, the linear least squares algorithm as described above needs to be iterated until a minimum norm of the residual errors ε is achieved. Since this iterative linear least squares algorithm requires higher computational cost compared to the iterative nonlinear least squares, one can also stop the algorithm after a certain amount of time or a certain number of iterations and evaluate the obtained estimates. The flowchart of the iterative linear Fig. 13 Flowchart of the iterative linear least squares f To this point, it still cannot be judged whether the esti- mates of the geometric parameters obtained by using the nonlinear least squares or those obtained by the iterative linear squares are more accurate as the true values of the geometric parameters are unknown. To evaluate and com- pare the accuracy of the estimates obtained by using both the algorithms, the pose errors will be evaluated in the next section upon the compensation of the kinematic parameters. 7 Compensation After the estimation of the geometric parameters has been done, compensation should be conducted to improve the accuracy of the machine. This is performed by replacing the nominal geometric parameter values in the kinematics by the estimated ones. Since two estimation techniques have been implemented, the estimates from both the techniques 1 3 Table 7 Estimated geometric parameters of the upper mechanism obtained by using iterative linear least squares Parameter Estimated value Nominal value Error 훿P L1 (mm) 599.995369 600 − 0.004631 L2 (mm) 474.992761 475 − 0.007239 L3 (mm) 603.988235 604 − 0.011765 xp (mm) 280.000000 280 0.000000 Table 7 Estimated geometric parameters of the upper mechanism obtained by using iterative linear least squares Fig. 13 Flowchart of the iterative linear least squares International Journal of Precision Engineering and Manufacturing (2020) 21:995–1015 1012 Fig. 14 Pose errors of a x coordinates, b y coordinates, and c angles θ of the lower mechanism at twelve data points (poses) before calibra- tion and after calibration using NLS and iterative LLS Fig. 15 Pose errors of a x coordinates, b y coordinates, and c angles θ of the lower mechanism at twelve data points (poses) after calibra- tion using NLS and iterative LLS Fig. 15 Pose errors of a x coordinates, b y coordinates, and c angles θ of the lower mechanism at twelve data points (poses) after calibra- tion using NLS and iterative LLS Fig. 14 Pose errors of a x coordinates, b y coordinates, and c angles θ of the lower mechanism at twelve data points (poses) before calibra- tion and after calibration using NLS and iterative LLS degree. The calibration using GLSDC provides an average accuracy of less than 0.004 mm for the position and less than 0.0002 degrees for the orientation, whereas the iterative linear least squares algorithm provides an average accuracy of less than 0.01 mm for the position and a similar aver- age accuracy in the orientation to the GLSDC. In general, both the calibration algorithms can improve the position accuracy by around 0.2 mm. To illustrate more clearly, the plots of the pose errors of the lower mechanism correspond- ing to only the calibrated parameters at the twelve different poses are shown in Fig. 15. The plots show clearly that the GLSDC performs better in the position accuracy than the iterative linear least squares. However, both algorithms do not perform differently in the orientation accuracy. 1 3 7 Compensation The circle is centered at (400, 300) mm and has a radius of 50 mm. As shown in the figure, the contour corresponding to the nominal parameters was at a glance very close to that corresponding to the calibrated (estimated) parameters. In order to compare the accuracy, contour errors which are defined as the difference between the nominal coordinates and the calibrated coordinates were plotted as shown in Fig. 17. Since the planar contour has two coordi- nates, i.e. x and y, the contour errors should be evaluated for each coordinate. The figures depict the contour errors along postures the error is zero while at other postures the error starts increasing. 8 Conclusion It is shown that the nonlinear least squares algorithm suc- cessfully refined the hand-eye and robot-world transfor- mation previously obtained by using a simple separable technique. The evaluation of the contouring error also shows that the nonlinear least squares algorithm estimates more accurately the geometric parameters of the machine’s Fig. 16 Test contour performed by the lower mechanism (blue: using nominal values, red: using calibrated values). (Color figure online) Fig. 17 Contour errors of a the absicca (x) coordinates and b ordinate (y) coordinates of the test contour Fig. 16 Test contour performed by the lower mechanism (blue: using nominal values, red: using calibrated values). (Color figure online) Fig. 16 Test contour performed by the lower mechanism (blue: using nominal values, red: using calibrated values). (Color figure online) The improved accuracy mentioned above is position and orientation accuracy in the case of position tracking. To evaluate the effect of calibration in the accuracy of contour tracking, a test contour was executed using both the nominal and estimated parameters. Since the estimated parameters obtained by using the nonlinear least squares are more accu- rate than those obtained by the iterative linear least squares, they are used in the test contour. In this work, a full circle was selected as the test contour. Figure 16 shows the contour performed by the lower mechanism and therefore lying on the XY plane. The circle is centered at (400, 300) mm and has a radius of 50 mm. As shown in the figure, the contour corresponding to the nominal parameters was at a glance very close to that corresponding to the calibrated (estimated) parameters. 7 Compensation In order to compare the accuracy, contour errors which are defined as the difference between the nominal coordinates and the calibrated coordinates were plotted as shown in Fig. 17. Since the planar contour has two coordi- nates, i.e. x and y, the contour errors should be evaluated for each coordinate. The figures depict the contour errors along the whole trajectory of the contour. It is shown in the figures that the contour corresponding to the calibrated parameters is more accurate by around 0.2 mm than that corresponding to the nominal (uncalibrated) parameters. This is consist- ent with the position accuracy of around 0.2 mm provided by both the calibration algorithms. The remaining position accuracy is basically due to the posture and/or dynamics of the mechanism which is a task for the control system to overcome. Along the trajectory of the circle, the mechanism posture keeps changing. As can be seen in Fig. 17, at some Fig. 17 Contour errors of a the absicca (x) coordinates and b ordinate (y) coordinates of the test contour postures the error is zero while at other postures the error starts increasing. 7 Compensation It can be seen in these plots that the iterative nonlinear least squares algorithm outperforms the iterative linear least squares in the pose error suppression. In addition to that, the former algorithm also outperforms the latter in the computational cost as the former converges faster than the latter. were used for the compensation. To evaluate the accuracy of the mechanism pose, pose errors are defined for all coor- dinates of the pose, i.e. x, y, and θ at the twelve different poses. Figure 14 shows a comparison between the pose errors of the uncalibrated and calibrated lower mechanism at the twelve different poses. For conciseness, the pose errors of the uncalibrated and calibrated upper mechanism are not shown in this paper as their behavior is similar to those of the lower mechanism. The pose errors are presented for each coordinate of the pose, i.e. x, y, and θ. It can be seen that the both the iterative nonlinear least squares (NLS, i.e. GLSDC) and the iterative linear least squares (LLS) algorithms suc- cessfully suppress the pose errors to values very close to zero. Some of the plots in this figure cannot show clearly the difference between the pose errors corresponding to both the algorithms as the error curves look coincident although they have a small difference. It is shown that the average position accuracy before the calibration is around 0.9 mm whereas the average orientation accuracy is less than 0.2 International Journal of Precision Engineering and Manufacturing (2020) 21:995–1015 1013 Fig. 17 Contour errors of a the absicca (x) coordinates and b ordinate (y) coordinates of the test contour The improved accuracy mentioned above is position and Fig. 16 Test contour performed by the lower mechanism (blue: using nominal values, red: using calibrated values). (Color figure online) The improved accuracy mentioned above is position and orientation accuracy in the case of position tracking. To evaluate the effect of calibration in the accuracy of contour tracking, a test contour was executed using both the nominal and estimated parameters. Since the estimated parameters obtained by using the nonlinear least squares are more accu- rate than those obtained by the iterative linear least squares, they are used in the test contour. In this work, a full circle was selected as the test contour. Figure 16 shows the contour performed by the lower mechanism and therefore lying on the XY plane. 8 Conclusion It is shown that the nonlinear least squares algorithm suc- cessfully refined the hand-eye and robot-world transfor- mation previously obtained by using a simple separable technique. The evaluation of the contouring error also shows that the nonlinear least squares algorithm estimates more accurately the geometric parameters of the machine’s mechanisms than the iterative linear least squares algo- rithm. In return to this higher accuracy, the nonlinear least squares algorithm should be performed in two steps by employing the subset selection approach in which m parameters should be firstly fixed at their nominal val- ues while the rest of the parameters are estimated (where N is the total number of parameters to be estimated, r is the rank of the system Jacobian matrix, and m = N − r). Subsequently, the previously fixed parameter(s) should be estimated in the next estimation step. This implies that 1 3 International Journal of Precision Engineering and Manufacturing (2020) 21:995–1015 1014 the fixed parameter(s) in the first step should have a con- siderably accurate value. Among all the estimated param- eters, the parameter(s) which is believed to have the most accurate value(s) can be chosen as the fixed parameter(s). Such dependency to the nominal values of the parameters does not only occur in this situation; it also occurs in the hand-eye and robot-world calibration which assumes some degree of accuracy of the nominal values of the mecha- nism geometric parameters. On the other hand, the closed- form solution of the linear least squares should be iterated until it converges to the actual values. Otherwise, it may give estimates of the parameters that are worse than the nominal values due to divergence from the actual values. Finally, using the compensated kinematics, it is shown that both the position tracking error and the contour tracking error were reduced. 10. Wang, L.-P., et al. (2011). Kinematic calibration of the 3-DOF parallel module of a 5-axis hybrid milling machine. Robotica, 29, 535–546. 10. Wang, L.-P., et al. (2011). Kinematic calibration of the 3-DOF parallel module of a 5-axis hybrid milling machine. Robotica, 29, 535–546. 11. Wang, Y., Wu, H., & Handroos, H. (2011). Markov Chain Monte Carlo (MCMC) methods for parameter estimation of a novel hybrid redundant robot. Fusion Engineering and Design, 88, 1863–1867. 12. Liu, Y., et al. (2007). Calibration of a Steward Parallel robot using genetic algorithm. In IEEE International Conference on Mechatronics and Automation. Harbin, China. 8 Conclusion 13. Fan, C., et al. (2015). Calibration of a parallel mechanism in a serial-parallel polishing machine tool based on genetic algorithm. International Journal of Advanced Manufacturing Technology, 81, 27–37. 14. Lee, K.-I., Lee, J.-C., & Yang, S.-H. (2018). Optimal on-machine measurement of position-independent geometric errors for rotary axes in five-axis machines with a universal head. International Journal of Precision Engineering and Manufacturing, 19(4), 545–551.i 15. Lee, D.-M., et al. (2011). Identification and measurement of geo- metric errors for a five-axis machine tool with a tilting head using a double ball-bar. International Journal of Precision Engineering and Manufacturing, 12(2), 337–343. Acknowledgements This research was partially supported by the Khalifa University Internal Research Fund. 16. Chen-Gang, et al. (2014). Review on kinematics calibration tech- nology of serial robots. International Journal of Precision Engi- neering and Manufacturing, 15(8), 1759–1774. Open Access This article is distributed under the terms of the Crea- tive Commons Attribution 4.0 International License (http://creativeco mmons.org/licenses/by/4.0/), which permits unrestricted use, distribu- tion, 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. 17. Rosyid, A., El-Khasawneh, B. S., & Alazzam, A. (2018). Genetic and hybrid algorithms for optimization of non-singular 3PRR planar parallel kinematics mechanism for machining application. Robotica, 36, 1–26. 18. Chen, D., et al. (2018). A Compensation method for enhancing aviation drilling robot accuracy based on Co-Kriging. Interna- tional Journal of Precision Engineering and Manufacturing, 19(8), 1133–1142. References In IEEE/RSJ International Conference on Intelligent Robots and Systems (IROS) 2017. Vancouver, BC, Canada. 30. Zhao, Z. (2018). Simultaneous robot-world and hand-eye calibra- tion by the alternative linear programming. Pattern Recognition Letters. 31. Zhuang, H., Roth, Z. S., & Sudhakar, R. (1994). Simultaneous robot-world and tool-flange calibration by solving homogeneous transformation equations of the form AX = YB. IEEE Transac- tions on Robotics and Automation, 10(4), 549–554. Anas Alazzam is an Associate Professor in the Mechanical Engineering Department at Khalifa University, Abu Dhabi, UAE. Prior to joining Khalifa University in 2012, he was part of the research and development team at the Canadian Space Agency (CSA), Saint Hubert, Canada. He completed his PhD in the area of Micro-Electro Mechanical Systems (MEMS) in 2010 from Concordia University, Montreal, Canada. He has research background in microflu- idics, Dielectrophoresis, and healthcare applications of microdevices. He contributed to advance- ment of knowledge in the field of separation using micro-fluidic means. Dr. Alazzam has published over 80 peer reviewed papers in prestigious journals and conferences and a member of several academic associa- tions. His research interests include microsystems, cells separation, micro- and macro- fabrication, and bio-applications of microfluidic systems. 32. Gauss, K. F. (1963). Theory of the motion of the heavenly bodies moving about the sun in conic sections. A translation of Theoria Motus. New York, NY: Dover Publications. 33. Saaty, T. L. (1981). Modern nonlinear equations. NY: Dover Publications. 34. Crassidis, J. L., & Junkins, J. L. (2012). Optimal estimation of dynamic systems (2nd ed.). Boca Raton: CRC Press. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Abdur Rosyid received his Ph.D. in Engineering from Khalifa University of Science and Tech- nology, Abu Dhabi, UAE. He is currently working as a post-doc- toral research fellow at the same university. His research interests include parallel and hybrid kin- ematics mechanisms (kinemat- ics, dynamics, gravity compen- sation, control, design optimization, and parameter estimation) as well as robotics for manufacturing. Bashar El‑Khasawneh is currently the chair of the graduate pro- gram at the Mechanical Engi- neering Department and an asso- ciate professor of mechanical engineering at Khalifa Univer- sity. He served as the Associate Chair for the department from 2013-2016. Before that he acted as an academic advisor for the British University in Dubai to develop an accredited industrial engineering program. References 1 3 1 3 1 3 International Journal of Precision Engineering and Manufacturing (2020) 21:995–1015 1015 27. Li, H., et al. (2016). Simultaneous hand-eye and robot-world cali- bration by solving the AX = YB problem without correspondence. IEEE Robotics and Automation Letters, 1(1), 145–152. 27. Li, H., et al. (2016). Simultaneous hand-eye and robot-world cali- bration by solving the AX = YB problem without correspondence. IEEE Robotics and Automation Letters, 1(1), 145–152. (JUST), within that time he served as a chairman for the department for two years. Between 1998-2002 he worked for Caterpillar Incorpo- rated in the technical center and for KLA-Tencor with the technology group both in USA. He worked extensively on technical and managerial assistance for many industries. He founded a design and development pre-incubator at the JUST University to train and qualify students to become entrepreneurs. He obtained his PhD in Mechanical Engineering from University of Illinois at Urbana-Champaign, USA (1997). He is a member of the Advisory Board of the Innovation Lab at the Dubai Health Authority (DHA) and a member of the editorial board of IJAER. His current research is focused on developing new concepts of advanced machining centers based on parallel kinematics mechanism and machining in harsh environments by adapting to the environment/ geometry and be able to perform machining/welding tasks. (JUST), within that time he served as a chairman for the department for two years. Between 1998-2002 he worked for Caterpillar Incorpo- rated in the technical center and for KLA-Tencor with the technology group both in USA. He worked extensively on technical and managerial assistance for many industries. He founded a design and development pre-incubator at the JUST University to train and qualify students to become entrepreneurs. He obtained his PhD in Mechanical Engineering from University of Illinois at Urbana-Champaign, USA (1997). He is a member of the Advisory Board of the Innovation Lab at the Dubai Health Authority (DHA) and a member of the editorial board of IJAER. His current research is focused on developing new concepts of advanced machining centers based on parallel kinematics mechanism and machining in harsh environments by adapting to the environment/ geometry and be able to perform machining/welding tasks. 28. Liu, Y., et al. (2017). Simultaneous calibration of hand-eye rela- tionship. Robot-World Relationship and Robot Geometric Param- eters with Stereo Vision, 710, 462–475. 29. Zhi X, Schwertfeger S (2017) Simultaneous hand-eye calibra- tion and reconstruction. References 19. Tsai, R. Y., & Lenz, R. K. (1989). A new technique for fully autonomous and efficient 3D robotics hand-eye calibration. IEEE Transactions on Robotics and Automation, 5(3), 345–358. 1. Merlet, J.-P. (2006). Parallel robots (2nd ed.). Dordrecht: Springer. 20. Daniilidis, K. (1999). Hand-eye calibration using dual quater- nions. The International Journal of Robotics Research, 18(3), 286–298. 2. Bai, S., & Theo, M. Y. (2003). Kinematic calibration and pose measurement of a medical parallel manipulator by optical position sensors. Journal of Robotic Systems, 20(4), 201–209. 21. Strobl, K.H. & Hirzinger, G. (2006). Optimal hand-eye calibra- tion. In International Conference on Intelligent Robots and Sys- tems 2006. Beijing, China. 3. Altuzarra, O., et al. (2009). Parallel kinematics for machine tools, in machine tool for high performance machining. London: Springer. 22. Shah, M. (2013). Solving the robot-world/hand-eye calibration problem using the Kronecker product. Journal of Mechanisms and Robotics, 5(3), 031007. 4. Roth, Z. S., Mooring, B., & Ravani, B. (1987). An overview of robot calibration. IEEE Journal of Robotics and Automation, 3(5), 377–385. 23. Heller, J., Henrion, D, & Pajdla, T. (2014). Hand-eye and robot- world calibration by global polynomial optimization. In IEEE International Conference on Robotics and Automation (ICRA) 2014. Hong Kong, China 5. Vischer, P., & Clavel, R. (1998). Kinematic calibration of the par- allel Delta robot. Robotica, 16, 207–218. 6. Yang, Z., Cheon, S.-U., & Yang, J. (2013). A kinematic calibration method of a 3-DOF secondary mirror of the giant Magellan. Jour- nal of Mechanical Sciences and Technology, 27(12), 3779–3786. 24. Dornaika, F., & Horaud, R. (1998). Simultaneous robot-world and hand-eye calibration. IEEE Transactions on Robotics and Automa- tion, 14(4), 617–622. 7. Patel, A. J., & Ehmann, K. F. (2002). Calibration of a hexapod machine tool using a redundant leg. International Journal of Machine Tools and Manufacture, 40, 489–512. 25. Li, A., Wang, L., & Wu, D. (2010). Simultaneous robot-world and hand-eye calibration using dual-quaternions and Kronecker product. International Journal of the Physical Sciences, 5(10), 1530–1536.l 8. Iurascu, C., & Park, F. C. (2003). Geometric Algorithms for Kin- ematic Calibration of Robots Containing Closed Loops. Journal of Mechanical Design, 125(1), 23–32. 26. Ernst, F., et al. (2012). Non-orthogonal tool/flange and robot/ world calibration. International Journal of Medical Robotics, 8(4), 407–420. 9. Yu, D. (2011). Kinematic calibration of a parallel robot using coordinate measuring machine. International Journal of the Phys- ical Sciences, 6(21), 4999–5004. References Between 2008 and 2010 he was the Man- ager of the Applied Research Programs at King Abdullah II Design and Development Bureau (KADDB), Jordan. Between 2002 to 2008 he held an assistant professor position in the Industrial Engineer- ing Department, at Jordan University of Science and Technology 1 3 1 3
https://openalex.org/W2989835856	https://zenodo.org/records/4514176/files/23-1699.pdf	English	null	A design of wideband high-power 3-dB quadrature coupler using defected ground structure for status data transmitting system	Bulletin of Electrical Engineering and Informatics	2,020	cc-by	4,924	Bulletin of Electrical Engineering and Informatics Vol. 9, No. 1, February2020, pp. 198~204 ISSN: 2302-9285, DOI: 10.11591/eei.v9i1.1699 Bulletin of Electrical Engineering and Informatics Vol. 9, No. 1, February2020, pp. 198~204 ISSN: 2302-9285, DOI: 10.11591/eei.v9i1.1699  198  198 198 Corresponding Author: The Anh Nguyen Dinh, Vietnam National Space Center, VNSC Building (A6), No. 18-Hoang Quoc Viet Street-Hanoi–Vietnam. Email: ndtanh@vnsc.org.vn The Anh Nguyen Dinh, Vietnam National Space Center, VNSC Building (A6), No. 18-Hoang Quoc Viet Street-Hanoi–Vietnam. Email: ndtanh@vnsc.org.vn A design of wideband high-power 3-dB quadrature coup using defected ground structure for status data transmitting system The Anh Nguyen Dinh1, Long Hoang Duc2, Duong Bach Gia3, Dragos Dancila4 1Vietnam National Space Center, Vietnam Academy of Science and Technology 2,4Department of Engineering Sciences, Solid State Electronics, Uppsala University, Sweden 3VNU University of Engineering and Technology, Hanoi, Vietnam Article Info ABSTRACT Article history: Received Aug 6, 2019 Revised Nov 26, 2019 Accepted Dec 12, 2019 The paper presents a wideband 3-dB quadrature coupler designed for operation at 2 GHz. The presented coupler is based on a broadside-coupled suspended structure in combination with a proposed defected ground structure (DGS) allowing for high power, wide-band and improved harmonic suppression performance. The experimental results show 0.2 dB of insertion loss, return loss of better than 18 dB and isolation of better than 25 dB in the frequency range from 1.74 to 2.67 GHz. The proposed coupler is able to be integrated in the status data transmitting system, which is suitable for vessel monitoring. The fundamental characteristics of the implemented coupler have been measured and verified. Keywords: 3-dB quadrature coupler Defected ground structure Divider RF combiner Status data transmitting system Vessel monitoring system This is an open access article under the CC BY-SA license. Corresponding Author: The Anh Nguyen Dinh, Vietnam National Space Center, VNSC Building (A6), No. 18-Hoang Quoc Viet Street-Hanoi–Vietnam. Email: ndtanh@vnsc.org.vn Keywords: 3-dB quadrature coupler Defected ground structure Divider RF combiner Status data transmitting system Vessel monitoring system Journal homepage: http://beei.org ABSTRACT The paper presents a wideband 3-dB quadrature coupler designed for operation at 2 GHz. The presented coupler is based on a broadside-coupled suspended structure in combination with a proposed defected ground structure (DGS) allowing for high power, wide-band and improved harmonic suppression performance. The experimental results show 0.2 dB of insertion loss, return loss of better than 18 dB and isolation of better than 25 dB in the frequency range from 1.74 to 2.67 GHz. The proposed coupler is able to be integrated in the status data transmitting system, which is suitable for vessel monitoring. The fundamental characteristics of the implemented coupler have been measured and verified. 1. INTRODUCTION High power, wide bandwidth, low-loss combiners or dividers with an average power handling capability of hundreds of Watts are used generally for combining high power amplifiers. The functionality of the coupler is to combine two input signals which have equivalent power levels and with a 90 degree relative phase difference. It can also separate an incoming signal into two output signals with similar amplitudes and a phase difference of 90 degrees. p p g Various methods are proposed to improve the performance of couplers [1-9]. Most of them are applied to low power systems. Typically, a 3-dB quadrature coupler which is suitable for printed circuit board implementation presented in [1]. It shows that the coupler can reduce the drawbacks of conventional thin-film microstrip line Lange coupler with the advantages of coplanar waveguide coupled line structures. Another 3-dB quadrature coupler using broadside-coupled coplanar waveguides [2] illustrates that the coupler with a broadside-coupled structure can easily be designed on a single-layer substrate printed circuit board, without using multi-layer substrates. However, such low-power couplers cannot be used directly for high power applications, because of the field breakdown effect [10] and thermals issues with the coupler. Defected Ground Structures (DGS) have been proposed for microwave applications such as filters [11-15], amplifiers [16], antennas [17-18], and wireless power transfers [19]. In this paper, we propose a new coupler design for high power applications, adopting a broadside-coupled suspended Journal homepage: http://beei.org 199 ISSN: 2302-9285  Bulletin of Electr Eng & Inf structure (BSS) and a defected ground structure (DGS) to improve bandwidth, reduce insertion loss and offer a truly high power solution. The proposed coupler is able to be integrated in the status data transmitting system, which is suitable for vessel monitoring [20-21]. The paper is organized as follow. Section 2 introduces the design considerations of the proposed coupler. The experimental results are provided in section 3 and discussed in the last section. structure (BSS) and a defected ground structure (DGS) to improve bandwidth, reduce insertion loss and offer a truly high power solution. The proposed coupler is able to be integrated in the status data transmitting system, which is suitable for vessel monitoring [20-21]. The paper is organized as follow. Section 2 introduces the design considerations of the proposed coupler. The experimental results are provided in section 3 and discussed in the last section. 2. THEORY AND DESIGN The presented power combiner is based on a broadside-coupled air suspended strip-line (BSS), realizing a tightly coupled structure. A 3-D drawing of the combiner is shown in Figure 1. The BSS structure is characterized by two transmission lines in which Z0,e and Z0,o are respectively the even and odd characteristic impedances. Expressions for the mode characteristic impedances, coupling ratio (C) and characteristic impedance (Z0) are given as following [22-23]: 𝑍0,𝑒= 𝑍0√ 1+𝐶 1−𝐶 (1) 𝑍0,𝑜= 𝑍0√ 1−𝐶 1+𝐶 (2) 𝑍0 = √𝑍0,𝑒× 𝑍0,𝑜 (3) 𝑍0,𝑒= 𝑍0√ 1+𝐶 1−𝐶 𝑍0,𝑜= 𝑍0√ 1−𝐶 1+𝐶 𝑍0 = √𝑍0,𝑒× 𝑍0,𝑜 𝑍0 = √𝑍0,𝑒× 𝑍0,𝑜 (3) The dimensions of the structure are determined for a given dielectric substrate and substra te thicknes following [22, 24]: dimensions of the structure are determined for a given dielectric substrate and substra te thickness wing [22, 24]: 𝑊 𝑏= 1 𝜋[𝑙𝑛 1+𝑀 1−𝑀− 𝑆 𝑏𝑙𝑛 1+𝑀 𝑘 1−𝑀 𝑘 ] (4) 𝑆 𝑏= 0.0017𝑍𝑜√𝜖𝑟(1−𝐶 1+𝐶) 1/2 𝑙𝑛 1+𝑘 1−𝑘 (5) re 𝑊 𝑏= 1 𝜋[𝑙𝑛 1+𝑀 1−𝑀− 𝑆 𝑏𝑙𝑛 1+𝑀 𝑘 1−𝑀 𝑘 ] (4) 𝑊 𝑏= 1 𝜋[𝑙𝑛 1+𝑀 1−𝑀− 𝑆 𝑏𝑙𝑛 1+𝑀 𝑘 1−𝑀 𝑘 ] (4) 𝑆 𝑏= 0.0017𝑍𝑜√𝜖𝑟(1−𝐶 1+𝐶) 1/2 𝑙𝑛 1+𝑘 1−𝑘 (5) (5) Where, 𝑀= [(𝑘 𝑏 𝑆−1) ∕(1 𝑘 𝑏 𝑆−1)] 1/2 𝑁= 60𝜋 𝑍0√𝜖𝑟(1−𝐶 1+𝐶) 1/2 𝑀= [(𝑘 𝑏 𝑆−1) ∕(1 𝑘 𝑏 𝑆−1)] 1/2 𝑁= 60𝜋 𝑍0√𝜖𝑟(1−𝐶 1+𝐶) 1/2 (a) (b) Figure 1.(a) The 3D drawing of the presented quadrature coupler at 2 GHz, (b) The proposed DGS structure introduces the tree-shaped ground slot patterning in the 3D model (a) (b) (b) (a) Figure 1.(a) The 3D drawing of the presented quadrature coupler at 2 GHz, (b) The proposed DGS structure introduces the tree-shaped ground slot patterning in the 3D model A design of wideband high-power 3-dB quadrature coupler using defected ground… (The Anh Nguyen Dinh) ISSN:2302-9285 ISSN:2302-9285 200 for 𝑁≤1 𝑘= [1 −(0.5𝑒𝑥𝑝(𝜋𝑁) −1 0.5𝑒𝑥𝑝(𝜋𝑁) + 1) 4 ] 1/2 𝑘= [1 −(0.5𝑒𝑥𝑝(𝜋𝑁) −1 0.5𝑒𝑥𝑝(𝜋𝑁) + 1) 4 ] 1/2 𝑘= (0.5𝑒𝑥𝑝(𝜋𝑁) −1 ( ) ) 2 for 𝑁≥1 for 𝑁≥1 for 𝑁≥1 𝑘= (0.5𝑒𝑥𝑝(𝜋𝑁) −1 0.5𝑒𝑥𝑝(𝜋𝑁) + 1) 2 𝑘= (0.5𝑒𝑥𝑝(𝜋𝑁) −1 0.5𝑒𝑥𝑝(𝜋𝑁) + 1) 2 For a given coupling ratio with air as dielectric substrate (𝜖𝑟=1), the ratios 𝑆𝑏 ⁄ and 𝑊𝑏 ⁄ can be computed following the formulas (4) and (5) in which S, b, and W are defined in Figure 2. 2. THEORY AND DESIGN For the 3-dB coupler design, the initial dimensions are obtained as follows: W=6.9 mm, b=6.35 mm, S=1.4 mm, t=0 mm. Due to Ohmic loss characteristics at a high-power level, the thickness of strip conductors is increased to 1.5 mm, thus allowing an improved average power handling capability. Though, the high thickness of the strip conductors takes adverse effect on the accuracy of the formulas (4) and (5). The thickness correction is numerically calculated by using the computer-aided design software CST. The numerical results of the structural dimensions of the coupler are given as follows: W=5.4mm, b=10.8mm, S=0.8mm forthe 1.5 mm of strip conductor thickness. The proposed tree-shaped defected ground structure (DGS) in association with a bevel at the corner edge of each strip conductors allows improving the bandwidth as well as the harmonic rejection as shown in Figure 1(a) and Figure 1(b). A trade-off between bandwidth and average power handling capability must be taken into consideration using beveling techniques. This reduces the high strength of the electric field at the beveled edges as shown in Figure 3. The tree-shaped structure is symmetrically placed at the top and bottom of the ground plane as shown in Figure 1(b). Figure 2. Variations of S/b and W/b as a function of coupling ratio with air substrate (left). Geometry of broadside-coupled suspended strip-line (right) in which S is the space between strip conductors, t is the thickness of conductors, W is the width of strip conductors, and b is the ground spacing Figure 2. Variations of S/b and W/b as a function of coupling ratio with air substrate (left). Figure 2. Variations of S/b and W/b as a function of coupling ratio with air substrate (left). eometry of broadside-coupled suspended strip-line (right) in which S is the space between strip con Figure 2. Variations of S/b and W/b as a function of coupling ratio with air substrate (left). Geometry of broadside-coupled suspended strip-line (right) in which S is the space between strip conductors, t is the thickness of conductors, W is the width of strip conductors, and b is the ground spacing Figure 3. The electric field lines pattern (cross-view). The simulation is performed using a signal of 1 W fed into the output Figure 3. The electric field lines pattern (cross-view). The simulation is performed using a signal of 1 W fed into the output Bulletin of Electr Eng & Inf, Vol. Bulletin of Electr Eng & Inf Typical defected ground structures Case DGS structure Bandwidth (MHz) Case DGS structure Bandwidth (MHz) 1 715 5 875 2 867 6 720 3 890 7 843 4 834 8 930 The optimization of the DGS structure is implemented following a set of requirements, i.e. bandwidth, S parameters, power handling capability (RF breakdown), etc. Table 1 illustrates some typical Defected Ground Structures with corresponding simulated bandwidths. In our design, the tree-shaped structure is optimally designed with 5 branches using the CST simulation software. For the presented coupler design, the final simulation parameters are: l1=4.35 mm; l2=4.5 mm; the bevel length l3=7.39 mm, for the initial design the bevel length is 0 mm; the length of the strip conductor is a quarter-wavelength at 2 GHz; l4=42 mm; l5=30.5 mm; l6=4 mm, in which l2 and l6 are step matching sections at ports; the spacing grounds b=10.8 mm; the spacing between strip-lines S=0.8 mm; d1=2mm; d2=7.5mm; d3=12.5mm; d4=7.17mm; d5=7.17mm; d6=7.17 mm; d7=14.5 mm; d8=9.5 mm; n1=2 mm; n2=4.5 mm. The strip-line and the housing are made of copper. The overall dimension of the coupler is 30 mm x 65 mm x 38 mm (WxLxh). The electric field pattern of the combiner is described in Figure 3 for 1 W output power. For 1 kW signal, this translates into 0.126 MV/m of the maximum E-field, which is lower than the threshold breakdown electric field of the dry air [10]. The simulated results are demonstrated in Figure 4 for both cases with and without the DGS structure. It is clear that using DGS in the coupler design has remarkable advantages in term of bandwidth and return loss improvement. Table1. Typical defected ground structures Case DGS structure Bandwidth (MHz) Case DGS structure Bandwidth (MHz) 1 715 5 875 2 867 6 720 3 890 7 843 4 834 8 930 Table1. Typical defected ground structures The optimization of the DGS structure is implemented following a set of requirements, i.e. bandwidth, S parameters, power handling capability (RF breakdown), etc. Table 1 illustrates some typical Defected Ground Structures with corresponding simulated bandwidths. In our design, the tree-shaped structure is optimally designed with 5 branches using the CST simulation software. Bulletin of Electr Eng & Inf For the presented coupler design, the final simulation parameters are: l1=4.35 mm; l2=4.5 mm; the bevel length l3=7.39 mm, for the initial design the bevel length is 0 mm; the length of the strip conductor is a quarter-wavelength at 2 GHz; l4=42 mm; l5=30.5 mm; l6=4 mm, in which l2 and l6 are step matching sections at ports; the spacing grounds b=10.8 mm; the spacing between strip-lines S=0.8 mm; d1=2mm; d2=7.5mm; d3=12.5mm; d4=7.17mm; d5=7.17mm; d6=7.17 mm; d7=14.5 mm; d8=9.5 mm; n1=2 mm; n2=4.5 mm. The strip-line and the housing are made of copper. The overall dimension of the coupler is 30 mm x 65 mm x 38 mm (WxLxh). The electric field pattern of the combiner is described in Figure 3 for 1 W output power. For 1 kW signal, this translates into 0.126 MV/m of the maximum E-field, which is lower than the threshold breakdown electric field of the dry air [10]. The simulated results are demonstrated in Figure 4 for both cases with and without the DGS structure. It is clear that using DGS in the coupler design has remarkable advantages in term of bandwidth and return loss improvement. Figure 4. The simulated results of the coupler as function of frequency with and without DGS Figure 4. The simulated results of the coupler as function of frequency with and without DGS Bulletin of Electr Eng & Inf 201  201 ISSN: 2302-9285  The design of a conventional hybrid coupler based on the calculated dimensions is adapted considering the bevel technique, as to extend the bandwidth, which is normally from 400 MHz to 600 MHz for other combiners. We propose further optimization of the design using the new DGS structure, without any changes in the coupling structure realized previously. The design of a conventional hybrid coupler based on the calculated dimensions is adapted considering the bevel technique, as to extend the bandwidth, which is normally from 400 MHz to 600 MHz for other combiners. We propose further optimization of the design using the new DGS structure, without any changes in the coupling structure realized previously. Table1. Typical defected ground structures Case DGS structure Bandwidth (MHz) Case DGS structure Bandwidth (MHz) 1 715 5 875 2 867 6 720 3 890 7 843 4 834 8 930 The optimization of the DGS structure is implemented following a set of requirements, i.e. bandwidth, S parameters, power handling capability (RF breakdown), etc. Table 1 illustrates some typical Defected Ground Structures with corresponding simulated bandwidths. In our design, the tree-shaped structure is optimally designed with 5 branches using the CST simulation software. For the presented coupler design, the final simulation parameters are: l1=4.35 mm; l2=4.5 mm; the bevel length l3=7.39 mm, for the initial design the bevel length is 0 mm; the length of the strip conductor is a quarter-wavelength at 2 GHz; l4=42 mm; l5=30.5 mm; l6=4 mm, in which l2 and l6 are step matching sections at ports; the spacing grounds b=10.8 mm; the spacing between strip-lines S=0.8 mm; d1=2mm; d2=7.5mm; d3=12.5mm; d4=7.17mm; d5=7.17mm; d6=7.17 mm; d7=14.5 mm; d8=9.5 mm; n1=2 mm; n2=4.5 mm. The strip-line and the housing are made of copper. The overall dimension of the coupler is 30 mm x 65 mm x 38 mm (WxLxh). The electric field pattern of the combiner is described in Figure 3 for 1 W output power. For 1 kW signal, this translates into 0.126 MV/m of the maximum E-field, which is lower than the threshold breakdown electric field of the dry air [10]. The simulated results are demonstrated in Figure 4 for both cases with and without the DGS structure. It is clear that using DGS in the coupler design has remarkable advantages in term of bandwidth and return loss improvement. Table1. 2. THEORY AND DESIGN 9, No. 1, February2020 : 198 – 204 Bulletin of Electr Eng & Inf Bulletin of Electr Eng & Inf 3. RESULTS AND DISCUSSION The fabricated coupler is designed at the center operating frequency of 2 GHz as shown in Figure 5. We use the PNA-N5221A vector network analyzer (VNA) to measure the S-parameters of the combiner. The simulated and measured results are in good agreement a s shown in Figure 6. The bandwidth is measured up to 930 MHz. The insertion loss is measured on the order of −3.02±0.2 dB. The maximum return loss A design of wideband high-power 3-dB quadrature coupler using defected ground… (The Anh Ng   202 202 202 ISSN:2302-9285 at port 1 and 3 are measured approximately 40 dB, while on the order of 30 dB at port 2 and 4 due to a sligh t mismatch between the conductor of the coaxial connector and the transmission lines. It results from the manual assembly. Figure 5. The proposed DGS-coupler is shown Figure 6. A comparison of the simulated and measured results of the coupler is shown The phase shift between ports is illustrated in Figure 7. The phase shift between two Output ports of coupler is 90±1 degree. There is a high agreement between the simulation result and the measurement result. Our results are compared with the recent published work in Table 2. It can be seen that the power handling capacibility can reach to 1 kW by using copper busbar for transmission line. The bandwidth and return loss can be improved by using DGS. Thus, our coupler can be used in the status data transmitting system as presented in [20-21]. Figure 6. A comparison of the simulated and measured results of the coupler is shown Figure 5. The proposed DGS-coupler is shown Figure 6. A comparison of the simulated and measured results of the coupler is shown Figure 5. The proposed DGS-coupler is shown The phase shift between ports is illustrated in Figure 7. The phase shift between two Output ports of coupler is 90±1 degree. There is a high agreement between the simulation result and the measurement result. Our results are compared with the recent published work in Table 2. It can be seen that the power handling capacibility can reach to 1 kW by using copper busbar for transmission line. The bandwidth and return loss can be improved by using DGS. Thus, our coupler can be used in the status data transmitting system as presented in [20-21]. Figure 7. Phase shift among ports Figure 7. REFERENCES [1] J. Chiu, C. Lin and Y. Wang, “A 3-dB quadrature coupler suitable for PCB circuit design,” in IEEE Transactions on Microwave Theory and Techniques, vol. 54, no. 9, pp. 3521-3525, Sept. 2006. [1] J. Chiu, C. Lin and Y. Wang, “A 3-dB quadrature coupler suitable for PCB circuit design,” in IEEE Transactions on Microwave Theory and Techniques, vol. 54, no. 9, pp. 3521-3525, Sept. 2006. y q pp p [2] C. Chang, J. Chiu, H. Chiu and Y. Wang, “A 3-dB quadrature coupler using broadside-coupled coplanar waveguides,” inIEEE Microwave and Wireless Components Letters, vol. 18, no. 3, pp. 191-193, March 2008. [3] Jikwon Kim, and Jong-GwanYook, “A miniaturized 3 dB 90degree hybrid coupler using coupled-line section with spurious rejection,” in IEEE Microwave and Wireless Components Letters, vol.24, no.11, pp. 766-768, November 2014. [4] S. Jung, R. Negra and F. M. Ghannouchi, “A design methodology for miniaturized 3-dB branch-line hybrid couplers using distributed capacitors printed in the inner area,” in IEEE Transactions on Microwave Theory and Techniques, vol. 56, no. 12, pp. 2950-2953, Dec. 2008 [5] K. M. Cheng and S. Yeung, “A novel rat-race coupler with tunable power dividing ratio, ideal port isolation, and return loss performance,” inIEEE Transactions on Microwave Theory and Techniques, vol. 61, no. 1, pp. 55-60, Jan. 2013. [6] S. I. Shams and A. A. Kishk, “Design of 3-dB hybrid coupler based on RGW technology,” inIEEE Transactions on Microwave Theory and Techniques, vol. 65, no. 10, pp. 3849-3855, Oct. 2017. [7] S. Liao and J. Peng, “Compact planar microstrip branch-line couplers using the quasi-lumped elements approach with nonsymmetrical and symmetrical T-shaped structure,” inIEEE Transactions on Microwave Theory and Techniques, vol. 54, no. 9, pp. 3508-3514, Sept. 2006. [8] Kai-Yu Tsai, Hao-Shun Yang, Jau-Horng Chen, and Yi-Jan Emery Chen, “A miniaturized 3 dB brand-line hybrid coupled with harmonics suppression,” in IEEE Microwave and Wireless Components Letters, vol. 21, no. 10, pp. 537-539, October 2011. pp [9] H. Yoon and B. Min, “Two section wideband 90° hybrid coupler using parallel-coupled three-line,” in IEEE Microwave and Wireless Components Letters, vol. 27, no. 6, pp. 548-550, June 2017. [10] L. Gould and L. W. Roberts et al., “Breakdown of air at microwave frequencies,”J. Appl. Phys., vol. 27, no. 10, pp. 1162-1170, 1956. [11] A. Abdel-Rahman, A. K. Verma, A. Boutejdar and A. S. 3. RESULTS AND DISCUSSION Phase shift among ports Bulletin of Electr Eng & Inf, Vol. 9, No. 1, February2020 : 198 – 204 Table 2. The comparison between our research and previous published researchs References Frequency (MHz) Bandwidth (MHz) Power Handling Capability (W) Insertion Loss (dB) Isolation Loss (dB) Return Loss (dB) Manufacture Material [1] 1800÷2800 1000 - 3÷0.1 Better than 20 Better than 20 Printed Circuit Board (PCB) [2] 2100÷2700 600 - 3.2÷0.1 Better than 19 Better than 19 PCB [25] 1770÷2200 570 - 3÷0.5 Better than 20 Better than 20 PCB [26] 2000÷2800 800 - 3÷1 Betterthan 14 Better than 15 PCB [This work] 1740÷2670 930 Up to 1 kW 3.02÷0.2 Better than 25 Better than 18 Copper BusBar Table 2. The comparison between our research and previous published researchs References Frequency Bandwidth Power Handling Insertion Isolation Loss Return Loss Bulletin of Electr Eng & Inf, Vol. 9, No. 1, February2020 : 198 – 204 Bulletin of Electr Eng & Inf, Vol. 9, No. 1, February2020 : 198 – 204 203  Bulletin of Electr Eng & Inf ISSN: 2302-9285 4. CONCLUSION We analyzed and successfully designed a 3-dB coupler using a newly proposed DGS structure. Our coupler has a bandwidth of 930 MHz. The insertion loss, return loss, a nd isolation loss of this coupler are 3.02÷0.2, 18 dB and 25 dB respectively. The power handling capability of the design can further be improved by choosing the higher thickness of the strip-conductors and the proper connectors, i.e. 7/16 type. The design methodology can be applied to any frequency range of interest up to 3 GHz, and it could be adapted for other combining structures such as e.g. the Wilkinson and Gysel types, as to expand their nominal bandwidths. The results prove that our coupler can be used in the status data transmitting system. ACKNOWLEDGEMENTS This research is granted by Vietnam Space Science and Technology Program through the national projects titled "Research, Design, Integrate, Launch and Operate a Nano Satellite-VT-CN.02/17-20". This research is granted by Vietnam Space Science and Technology Program through the national projects titled "Research, Design, Integrate, Launch and Operate a Nano Satellite-VT-CN.02/17-20". design of wideband high-power 3-dB quadrature coupler using defected ground… (The Anh Nguyen Dinh REFERENCES Bhartia, “The design of broadside-coupled stripline circuits,” inIEEE Transactions on Microwa Theory and Techniques, vol. 29, no. 2, pp. 165-168, Feb. 1981. h [22] I. J. Bahl and P. Bhartia, “The design of broadside-coupled stripline circuits,” inIEE Theory and Techniques, vol. 29, no. 2, pp. 165-168, Feb. 1981. th y q pp [23] David M. Pozar, “Microwave Engineering 4th ed,” John Wiley & Sons, Inc., 2012. pp [23] David M. Pozar, “Microwave Engineering 4th ed,” John Wiley & Sons, Inc., 2012. [24] S. B. Cohn, “Thickness corrections for capacitive obstacles and strip conductors,” inIRE Transactions on Microwave Theory and Techniques, vol. 8, no. 6, pp. 638-644, November 1960. [25] L. Wang, G. Wang and J. Sidén, “High-performance tight coupling microstrip directional coupler with frag type compensated structure,” in IET Microwaves, Antennas & Propagation, vol. 11, no. 7, pp. 1057-1063, 201 [26] K Di d A Ki hk “Wid b d h b id l ith l t i ll it h bl h diff f 5] L. Wang, G. Wang and J. Sidén, “High-performance tight coupling microstrip directional coupler with fragmen type compensated structure ” in IET Microwaves Antennas & Propagation vol 11 no 7 pp 1057-1063 2017 yp p , , p g , , , pp , [26] K. Ding and A. Kishk, “Wideband hybrid coupler with electrically switchable phase-difference performance,” in IEEE Microwave and Wireless Components Letters vol 27 no 11 pp 992-994 Nov 2017 yp p pp [26] K. Ding and A. Kishk, “Wideband hybrid coupler with electrically switchable phase-difference performance,” in IEEE Microwave and Wireless Components Letters, vol. 27, no. 11, pp. 992-994, Nov. 2017. BIOGRAPHIES OF AUTHORS The Anh Nguyen Dinh received the B.S Degree and M.Sc Degree in Electronics and Telecommunications Technology from University of Engineering and Technology, Vietnam National University in 2009 and 2011, respectively. From 2012 to 2015, he was a researcher in Communications and Television Develoment., JSC. Since 2016, he has been a researcher in Vietnam Space Center, Vietnam Academy of Science and Technology. Now, he is a Ph.D student in Vietnam National University. His researches are in fields of microwave engineering, communications in satellites, ground station and radio systems. The Anh Nguyen Dinh received the B.S Degree and M.Sc Degree in Electronics and Telecommunications Technology from University of Engineering and Technology, Vietnam National University in 2009 and 2011, respectively. From 2012 to 2015, he was a researcher in Communications and Television Develoment., JSC. Since 2016, he has been a researcher in Vietnam Space Center, Vietnam Academy of Science and Technology. Now, he is a Ph.D student in Vietnam National University. His researches are in fields of microwave engineering, communications in satellites, ground station and radio systems. Long Hoang Duc was born in Hanoi, Vietnam, in 1987. He received the bachelor’s degree (Hons.) in electronics and telecommunications from the University of Engineering and Technology (UET), VNU, in 2009, and the M.Sc. degree from Poles UniversitairesFrancais Hanoi in 2012. He is currently pursuing the Ph.D. degree at FREIA laboratory, Uppsala University Sweden. His current research interests include the area of RF PA design. Duong Bach Gia Ph.D degree in wireless physics from University of HaNoi in 1988. From 1988 to 1990, he was a researcher assisstant in Leningrad University, Russia. From 1991 to 2005, he was a researcher in academy of air force. He has been a lecturer and head of electronics and telecommunications center, University of Engineering and Technology, Vietnam National University since 2006. He was promoted to Associate Professor in 2009 and to Professor in 2016. His research forcuses on RF analog singal processing, RF chip design, radar engineering and technology, automatic control. Email: duongbg@vnu.edu.vn. Dragos Dancila received the electrical engineering degree from the Universitcatholique de Louvain (UCL), Louvain-la-Neuve, Belgium, in 2006, and the Ph,D. degree in applied sciences from UCL in collaboration with IMEC, Leuven, Belgium, in 2011. REFERENCES Omar, “Compact stub type microstrip bandpass filter using defected ground plane,” inIEEE Microwave and Wireless Components Letters, vol. 14, no. 4, pp. 136-138, April 2004. p [12] D. Ahn, J. Park, C. Kim, J. Kim, Y. Qian and T. Itoh, “A design of the low-pass filter using the novel microstrip defected ground structure,” inIEEE Transactions on Microwave Theory and Techniques, vol. 49, no. 1, pp. 86-93, Jan. 2001. [13] M. K. Mandal and S. Sanyal, “A novel defected ground structure for planar circuits,” in IEEE Microwave and Wireless Components Letters, vol. 16, no. 2, pp. 93-95, Feb. 2006. [14] S. U. Redman, A. F. Sheta, and M. Alkanhal, “Compact bandstop filter using defected ground structure,”in Saudi Int. Electron. Commun. Photon. Conf. (SIECPC), pp. 1-4, Apr, 2011. [15] Jiayuan Lu, Jianpeng Wang and Hui Gu, “Design of compact balanced ultra-wideband bandpass filter with half mode dumbbell DGS,”Electronics Letters 28th, Vol. 52, No. 9, pp. 731-732, April 2016. [16] Jong-Sik Lim, Ho-Sup Kim, Jun-Seek Park, Dal Ahn and Sangwook Nam, “A power amplifier with efficiency improved using defected ground structure,” inIEEE Microwave and Wireless Components Letters, vol. 11, no. 4, pp. 170-172, April 2001. pp p [17] Y. J. Sung, M.Kim, and Y.S.Kim, “Harmonics reduction with defected ground structure for a microstrip patch antenna,” inIEEE Antennas and Wireless Propagation Letters, vol. 2, pp. 111-113, 2003. [18] Debatosh Guha, Chandrakanta Kumar, and Surendra Pal, “Improved cross-polarization characteristics of circular microstrip antenna employing arc-shaped Defected Ground Structure (DGS),” inIEEE Antennas and Wireless Propagation Letters, vol. 8, pp. 1367-1369, 2009. [19] S. Hekal, A. B. Abdel-Rahman, H. Jia, A. Allam, A. Barakat and R. K. Pokharel, “A novel technique for compact size wireless power transfer applications using defected ground structures,” in IEEE Transactions on Microwave Theory and Techniques, vol. 65, no. 2, pp. 591-599, Feb. 2017.  204 ISSN:2302-9285 ISSN:2302-9285 [20] The Anh Nguyen Dinh, Le Xuan Huy, Tuan Anh Vu, and Duong Bach Gia, “A status data transmitting system for vessel monitoring,”International Journal of Electronic and Computer Engineering (IJECE), vol. 8, no. 2, pp. 917-925, April 2018. pp , p [21] The Anh Nguyen Dinh, Minh Ngo Duc, Duong Bach Gia, “Design of an S-band vessel monitoring system using satellites,”International Journal of Applied Engineering Research, Vol. 13, No. 8, 2018, pp. 6063-6068. , f pp g g pp 2] I. J. Bahl and P. BIOGRAPHIES OF AUTHORS Currently, he is an Associate Lecturer with the Microwave Group at the Department of Engineering Sciences, Solid State Electronics, Uppsala University, Uppsala, Sweden, where he is also a Researcher with the FREIA Laboratory, and is involved in solid-state RF power amplifier development. His current research interests include adaptive and integrated antennas, RF-MEMS technology, and RF sensors for biomedical applications, such as skin cancer detection and glucose monitoring. Bulletin of Electr Eng & Inf, Vol. 9, No. 1, February2020 : 198 – 204
https://openalex.org/W2119259097	https://europepmc.org/articles/pmc3741130?pdf=render	English	null	Use of a Dual Reporter Plasmid to Demonstrate Bactofection with an Attenuated AroA- Derivative of Pasteurella multocida B:2	PloS one	2,013	cc-by	8,632	Use of a Dual Reporter Plasmid to Demonstrate Bactofection with an Attenuated AroA- Derivative of Pasteurella multocida B:2 Sarah Othman¤, Andrew J. Roe, Roger Parton, John G. Coote Sarah Othman¤, Andrew J. Roe, Roger Parton, John G. Coote Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom Sarah Othman*¤, Andrew J. Roe, Roger Parton, John G. Coote Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom nity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom Abstract ¤ Current address: Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Serdang, Selangor, Malaysia nt of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Serdang, Selangor, ¤ Current address: Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra M Malaysia ¤ Current address: Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Pu Malaysia antigen for another disease of the target animal, thus providing heterologous protection against both HS and a second disease. Bactofection, bacteria-mediated transfer of expressible DNA, is a promising approach to express plasmid-encoded heterologous proteins in different cell types, including phagocytic and non- phagocytic mammalian cells. Various studies have demonstrated bactofection with attenuated strains of invasive bacteria. For example, vaccination of mice with attenuated Salmonella typhimurium transformed with plasmid DNA encoding listeriolysin (a virulence factor of Listeria monocytogenes) induced specific antibody as well as T-cell responses to listeriolysin [8]. In a separate study, fluorescent dendritic cells (DC) were demonstrated after oral administration of S. typhimurium harboring plasmid DNA encoding green fluorescent protein (GFP). These data provided evidence that this delivery system could target relevant immune cells, leading to efficient induction of an immune response [9]. Mice vaccinated with an attenuated Shigella vaccine harbouring measles virus genes induced a vigorous measles virus antigen-specific response [10]. An attenuated L. monocytogenes strain has also been used for delivery of eukaryotic expression vectors to the cytoplasm of murine macrophage cell lines [11]. This technology has also been demonstrated with a plant pathogen, Agrobacterium tumefaciens, which contains a virulence plasmid that promotes transfer of an expression cassette from the plasmid into the plant. Although wounded plants are its natural targets, plasmid transfer by A. PLOS ONE \| www.plosone.org August 2013 \| Volume 8 \| Issue 8 \| e71524 Abstract A reporter plasmid pSRG has been developed which expresses red fluorescent protein (RFP) from a constitutive prokaryotic promoter within Pasteurella multocida B:2 and green fluorescent protein (GFP) from a constitutive eukaryotic promoter within mammalian cells. This construct has been used to determine the location and viability of the bacteria when moving from the extracellular environment into the intracellular compartment of mammalian cells. Invasion assays with embryonic bovine lung (EBL) cells and an attenuated AroA- derivative of Pasteurella multocida B:2 (strain JRMT12), harbouring the plasmid pSRG, showed that RFP-expressing bacteria could be detected intracellularly at 3 h post-invasion. At this stage, some EBL cells harbouring RFP-expressing bacteria were observed to express GFP simultaneously, indicating release of the plasmid into the intracellular environment. At 5 h post-invasion, more EBL cells were expressing GFP, while still harbouring RFP-expressing bacteria. Concurrently, some EBL cells were shown to express only GFP, indicating loss of viable bacteria within these cells. These experiments proved the functionality of the pSRG dual reporter system and the potential of P. multocida B:2 JRMT12 for bactofection and delivery of a DNA vaccine. Citation: Othman S, Roe AJ, Parton R, Coote JG (2013) Use of a Dual Reporter Plasmid to Demonstrate Bactofection with an Attenuated AroA- Derivative of Pasteurella multocida B:2. PLoS ONE 8(8): e71524. doi:10.1371/journal.pone.0071524 Editor: Bruce W. Banfield, Queen’s University, Canada Editor: Bruce W. Banfield, Queen’s University, Canada Received March 24, 2013; Accepted June 29, 2013; Published August 12, 2013 hman 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 Othman et al. This is an open-access article distributed under the terms of the Creative Commons Attribut unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: SO is funded by Universiti Putra Malaysia (http://www.upm.edu.my/) and Ministry of Higher Education, Malaysia (http://www.mohe.gov.my/portal/). 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. Introduction The numerous serotypes of Pasteurella multocida are associated with a variety of disease syndromes in a wide range of agricultural, domestic and feral animal species [1]. P. multocida serotypes B:2 and E:2 are associated with haemorrhagic septicaemia (HS) of cattle, water buffaloes and occasionally other species, resulting in major economic losses, mainly in South East Asia [2,3]. Several types of killed, whole-cell vaccines have been used for prevention and control of the disease including alum-precipitated vaccine and oil-adjuvant vaccine but, in general, immunity is short-lived. Countries where the disease is endemic resort to routine prophylactic vaccination. A live-attenuated vaccine for HS might be a better alternative vaccine as, by mimicking the ability of the pathogen to infect the host, but without causing disease, a more prolonged protective immune response may be induced. A live- attenuated vaccine developed by Tabatabaei et al. [4], by mutating a housekeeping gene aroA, proved to be highly attenuated yet protective in mice and in cattle [4–6]. In later work, Othman et al. [7] reported on the ability of the live vaccine strain to adhere to and to invade embryonic bovine lung (EBL) cells with an efficiency similar to the wild-type strain. The live vaccine strain was also able to survive intracellularly in the EBL cells for at least 7 hours. The long term aim of the present work is to exploit the capacity of the live P. multocida aroA vaccine strain to invade mammalian cells in order to deliver plasmid DNA encoding a protective August 2013 \| Volume 8 \| Issue 8 \| e71524 August 2013 \| Volume 8 \| Issue 8 \| e71524 1 PLOS ONE \| www.plosone.org Bactofection and P. multocida B:2 tumefaciens has also been demonstrated to yeast and fungi. In vitro, it can also target human cell lines such as HeLa cells [12]. These reports clearly demonstrate bacterial versatility as delivery vectors. As this approach has not previously been used with Pasteurella, we have developed a bactofection model system for use with this organism that employs a dual-expression plasmid for protein expression in both prokaryotic and eukaryotic cells in order to understand the fate of the plasmid DNA carrying antigenic genes after delivery by the live-vaccine strain in vivo. transformed cells were then spread onto selective plates containing appropriate antibiotics and the plates were incubated at 37uC to obtain transformant colonies. For E. coli, plasmid DNA was transformed into E. Preparation of Embryonic Bovine Lung (EBL) Cells Preparation of Embryonic Bovine Lung (EBL) Cells EBL cells (German Collection of Microorganisms and Cell Cultures, no. ACC192) were grown as a monolayer in Minimal Essential Medium (MEM 2279, Sigma) containing streptomycin (100 mg/ml), penicillin (100 U/ml) and 15% (v/v) foetal calf serum (FCS, Sigma) in a humidified atmosphere of 5% (v/v) CO2 and 95% (v/v) air at 37uC. Cells taken from liquid nitrogen stock were cultured for two days before seeding into a 24-well tissue- culture plate and incubated overnight. EBL Cell Viability Assessment The trypan blue exclusion method was used. A mixture containing 10 ml of trypan blue (0.4% w/v) (Sigma) and 10 ml of the cell suspension was placed in an improved Neubauer (1/400 mm260.1 mm depth) chamber and viewed with a light micro- scope and a 610 objective lens. Cells stained blue were counted as dead whereas clear cells were counted as viable. Bacterial Strains and Growth Conditions Bacterial strains used in this study were: P. multocida B:2 JRMT12, an aroA deletion mutant of P. multocida strain 85020 [4] and E. coli XL-1BLUE (Amersham Biosciences). P. multocida was grown on Brain Heart Infusion (BHI) agar or in BHI broth (Difco) media and E. coli was grown on Luria-Bertani (LB) agar or in LB broth (Difco) media at 37uC and shaken at 180 rpm. General Molecular Biology Techniques y Plasmid extractions were performed using Qiaprep spin columns (Qiagen). DNA concentrations were measured using a NanoDrop ND-1000 UV-visible spectrophotometer (NanoDrop Technologies). Unless otherwise stated, restriction enzymes were obtained from New England Biolabs and used according to the manufacturer’s protocol. PCR was performed according to standard procedures [13,14] using Taq DNA polymerase (NEB,UK). RED primers with internal PciI sites (bold fonts) (RED Forward: 59- CTACATGTCCGCGCCAACCG 239, RED Reverse: 59- GCACATGTTCTACTGGGAG -39) were used to amplify the PsodC-DsRed fragment from plasmid pMK- RED. A MegaBACE1000 (96 capillary) sequencer, which used Big Dye (Applied Biosystems) and ET-Dye Terminator (Amersham Bioscience) chemistries, was employed for DNA sequencing. The sequencing was done by The Sequencing Unit, University of Dundee, Scotland. Plasmids Plasmid pMK-Express [13] is a broad-host range plasmid that can propagate in HAP (Haemophilus, Actinobacillus & Pasteurella) bacteria. An A. pleuropneumoniae promoter, Psod-C, was incorpo- rated into plasmid pMK-Express upstream of the gfpmut3 gene coding for GFP. This constitutive promoter was able to express the GFP protein in Pasteurella [13]. Plasmid pDsRed-Monomer (Clontech) encodes red fluorescent protein (RFP) from the DsRed fragment. Plasmid pEGFP-N1TM (Clontech) encodes green fluorescent protein (GFP) from a constitutive eukaryotic promoter PCMVIE and is able to replicate in E. coli and eukaryotic cells. P. multocida Invasion of EBL Cells This was done as previously described (Othman et al., 2012) (N.B. ref [7]). Briefly, bacteria were harvested from 18-h cultures in BHI broth, washed twice in phosphate-buffered saline (PBS) and suspended in MEM without antibiotics. The bacterial suspension was adjusted to an OD600nm corresponding to approximately 16107 colony forming units (CFU)/ml to allow a final multiplicity of infection (MOI) of 100 bacteria/ mammalian cell. Previous work had shown this to be the optimum MOI in our system (7). The concentration of viable EBL cells in one well of a 24-well tissue culture plate was assessed by trypan blue and bacterial suspension was then added at the appropriate concentration to each well containing EBL monolayer. The plate was centrifuged at 12006g for 5 min in a Heraeus 3 S-R centrifuge to encourage close contact between the EBL and bacterial cells. After incubation in a CO2 incubator at 37uC for 2 h, the wells were washed gently with PBS to remove the unattached bacteria. The washing step was repeated twice. One millilitre of MEM with antibiotics [polymyxin (50 mg/ml) and gentamicin (50 mg/ml)] was added to each well and the plate was incubated for 1 h at 37uC in 5% (v/v) CO2 to kill any remaining extracellular bacteria. The wells were then washed twice with PBS to remove the antibiotics before further manipulation. Introduction coli XL- 1BLUE using the heat shock protocol specified by the manufac- turer (Amersham Biosciences). August 2013 \| Volume 8 \| Issue 8 \| e71524 Construction of ‘‘traffic-light’’ Plasmid, pSRG g p Fig. 1 shows the cloning strategy for the construction of pSRG. First, plasmid pMK-RED was generated. It was derived from plasmid pMK-Express [13] and contained the DsRed fragment from pDsRed-Monomer (Clontech). The gfpmut3 gene from pMK- Express was removed by digestion with BamHI and NotI and replaced by the DsRed fragment cut from pDsRed-monomer to produce pMK-RED (Fig. 1A). Plasmid pMK-RED was then used as a template for PCR with RED primers to amplify the PsodC- DsRed fragment with PciI sites at each end. This amplimer was cut with PciI and cloned into the unique PciI site of pEGFP-N1 (Fig. 1B, C). Ligation of this fragment into pEGFP-N1 was performed at a ratio of 1:2 (plasmid:fragment) to obtain plasmid pSRG (Fig. 1D). The ligation mixture was then transformed into E. coli XL-1BLUE. Plasmid pSRG was purified using Qiaprep Spin Miniprep Kit (Qiagen) according to manufacturer’s instruc- tion and confirmed by restriction enzymes digestion. After antibiotic treatment to eliminate extracellular and adherent bacteria, the monolayer was washed twice with PBS. CellMask plasma membrane stain (GE Healthcare) (5 mg/ml) (diluted according to manufacturer’s instructions) was added at 200 ml per well. The chamber-slide was then incubated for 7 min at 37uC in an atmosphere supplemented with 5% (v/v) CO2. The staining solution was removed from the well and the cells washed three times with PBS. Pre-warmed paraformaldehyde (PFA) 200 ml at 4% (v/v) was added to each well and slides incubated for 30 min at 37uC in an atmosphere supplemented with 5% (v/v) CO2. PFA was then removed and cells were washed three times with PBS. The wells were then removed from the chamber-slide and the slide was left to dry. y y g After that, pSRG was electroporated into P. multocida B:2 JRMT12 and positive transformants were selected on BHI agar supplemented with kanamycin (50 mg/ml). A strain harbouring pSRG was assessed for plasmid stability after repeated subculture. P. multocida B:2 JRMT12 pSRG was plated on BHI agar with and without kanamycin (50 mg/ml). After incubation for 24 h, selected colonies from both plates were streaked to fresh plates. After a further 24 h, a dozen colonies were picked from both plates and inoculated into BHI broth with or without kanamycin (50 mg/ml) respectively. After 24 h, plasmids were purified and analysed by restriction digestion with PciI for confirmation of their identity. Construction of ‘‘traffic-light’’ Plasmid, pSRG Strains were subcultured daily into fresh broth medium for 14 days and plasmids from each culture were purified and analysed every alternate day. After 14 days, P. multocida B:2 JRMT12 pSRG cultured without antibiotic was found to be still harbouring the plasmid in each of the 12 clones subcultured. Thus, pSRG was stably maintained in P. multocida B:2 JRMT12 in the absence of antibiotic. Fluorescence mounting medium (DAKO) was used to cover the fixation area on the chamber-slide. A cover slip was then gently applied on top of the medium to avoid air bubble formation. Any gap between the cover slip and the chamber-slide was then sealed- off using nail varnish to prevent air pockets from forming. The slide was left for 10 min to allow the mounting medium to set. It was then cleaned and labelled before storage in the dark. Fluorescent Staining of Bacteria Bacteria were grown in BHI broth with vigorous shaking at 37uC until an OD600nm of 0.3–0.5 was obtained. One millilitre of bacterial suspension was taken and centrifuged at 13 0006g for 1 minute. The pellet was resuspended in 1 ml of PBS and the OD600nm was measured. An aliquot of 200 ml was transferred from the PBS suspension into a flat-bottom 96-well plate. All samples were done in triplicate. Fluorescence intensity (FI) of RFP was then measured. A vial of Cye5Dye (Amersham Bioscience) was mixed with 100 ml of PBS according to the manufacturer’s instructions and the dye was then mixed with bacterial suspension at a ratio of 1:20 (25 ml of dye to 500 ml of bacterial suspension). The mixture was then incubated in the dark at room temperature for 1 h to allow staining of bacterial free-peptide groups on the outer membrane. After incubation, the suspension was pelleted at 13 0006g for 1 min and washed with 1 ml of PBS before being re-centrifuged. The washing step was repeated until the suspension was no longer coloured. Expression of RFP from pSRG in P. multocida B:2 JRMT12 Figure 2 (I) shows the fluorescence intensity (FI) in relative fluorescence units (RFU) of P. multocida B:2 JRMT12 pSRG plotted against bacterial density (OD600nm). Immediately after inoculation into BHI broth, at OD600nm 0.07, the strain was clearly expressing RFP (FI .2500 RFU). This fluorescence decreased initially but then increased with increasing bacterial density, to .3000 RFU when the OD600nm had reached 0.79. After that, the RFP expression was seen to decline. The results indicated that the RFP expression from this strain peaked in the late exponential phase of growth. For fixation, an aliquot of the suspension was mixed (1:1) with 4% (v/v) PFA. About 20 ml of the fixation mixture was spotted onto a clean spot-slide which was then left at room temperature to dry. Fluorescence mounting medium (DAKO) was used to cover the fixation area on the spot-slide and the slides prepared for storage as described. Fluorescence Microscopy (FM) Fluorescence imaging was done with a Zeiss AxioImager M1 upright fluorescence microscope (Carl Zeiss) connected to a Hamamatsu Orca 03 and QIClick digital charge-coupled device (CCD) camera. The system allows visualization of fluorescence at high spatial resolution (120061300 pixels) and high bit depth (12- bit gray scale). Acquisition, visualization, measurement and restoration of the images were then processed with Volocity software (Perkin Elmer). Results Fluorescent staining of EBL cells. EBL cells were treated according to the invasion assay above. The only difference for fluorescence microscopy (FM) slide preparation was that the EBL cells were seeded in a chamber-slide (NUNC) rather than a 24-well plate, so that cells could be fixed directly to the slide after removing the well from the chamber slide. Construction of ‘‘traffic-light’’ Plasmid, pSRG Assessment of RFP and GFP Expression in Bacteria An overnight culture of P. multocida grown in BHI broth was diluted 1 in 100 into 500 ml of pre-warmed BHI broth and grown at 37uC to an optical density at 600 nm (OD600nm) of 0.5–0.7 measured using a cell density meter (WPA CO8000, Biochrom Ltd.). The cells were harvested by centrifugation and then made electrocompetent by concentrating 100-fold and washing three times with ice-cold 10% (v/v) glycerol before suspension in ice- cold 10% (v/v) glycerol. Electrocompetent cells (50 ml) plus 10– 200 ng of PCR product, plasmid or ligation mixture were used. After mixing, electroporation was done with a Bio-Rad pulse controller (MicroPulser Electroporator, Bio-Rad) set at 2.5 kV (field strength 12.5 kV/cm2). Shocked cells were added to 1 ml of BHI and incubated at 37uC for 4 h. Aliquots of 50–100 ml of Assessment of RFP and GFP Expression in Bacteria This was monitored using a fluorimeter (FLUOstar OPTIMA, BMG Labtech). BHI broth (100 ml), supplemented with appro- priate antibiotics at a final concentration of 50 mg/ml, was inoculated with 1 ml of an overnight culture of P. multocida B:2 or E. coli XL-1BLUE and shaken at 180 rpm, 37uC. At hourly intervals, 1 ml samples of culture were taken and the OD600nm was measured. The suspension was then centrifuged at 13 000 x g for 1 min and the pellet resuspended in 1 ml of PBS. The OD600nm was measured again with PBS as standard and 200 ml aliquots placed in wells of a 96-well flat-bottom dish (Corning, USA) in triplicate. The fluorescence intensity (FI) at 550 nm for GFP and 590 nm for RFP was measured in the fluorimeter with a August 2013 \| Volume 8 \| Issue 8 \| e71524 2 PLOS ONE \| www.plosone.org Bactofection and P. multocida B:2 fluorescent light filter and excitation at 490 nm and 520 nm, respectively. All samples were compared with bacteria without plasmids to eliminate background fluorescence. Samples were taken at hourly intervals until the OD600nm reached stationary phase (,2.0). Control experiments showed negligible expression of GFP in P. multocida JRMT12 throughout the growth cycle, indicating that the PCMVIE promoter was not able to drive transcription of GFP in this prokaryotic background. Intracellular Trafficking of P. multocida B:2 JRMT12 pSRG within EBL Cells py In Figure 2 (II), a set of images was captured from the spot slide prepared with P. multocida B:2 JRMT12 pSRG taken at OD600nm 0.79. Images were taken of the same field using the 1006objective lens under three different light paths to ease visualization. Image A, taken under DIC (Differential Interference Contrast) (white) light, showed a heavy cluster of bacteria. When the same field of the slide was captured in B, where the image was taken under filtered fluorescent light (Excitation = 649 nm, Emis- sion = 670 nm) (blue) to visualize the counterstain Cy5 Dye, the bacteria were more defined. This image was taken at 0.6 mm splicing through the slide in order to view a single layer of the bacteria. The slide was set to be sectioned optically at 0.3 mm intervals from top to bottom; an image was captured at every 0.3 mm depth at a total section stacking of 3.0 mm. The Volocity software was then utilized to deconvolve images captured by reversing the optical distortion to produce a sharper and clearer image. This technique also eliminated background light diffrac- tion. Image C was taken under filtered fluorescent light (Exc. = 557 nm, Ems. = 585 nm) (red) to visualize bacteria ex- pressing RFP from plasmid pSRG and under filtered fluorescent light (Exc. = 649 nm, Ems. = 670 nm) (blue) to show the counter- stain in order to localize the RFP expression in the bacteria. The image showed purplish pink coccobacilli of P. multocida B:2 JRMT12 pSRG expressing RFP. Image D shows a 3-dimensional (3D) image of image C to generate a 3D model of the RFP- expressing bacteria. The vertical elongated shape of the bacteria is EBL cells and bacteria were prepared as above but with the modification that the bacterium, P. multocida B:2 JRMT12 pSRG, was grown to OD600nm 0.79 (at maximum RFP expression) before use in the invasion assay. Slides were prepared for FM and viewed under the x100 objective lens of the Zeiss AxioImager M1 microscope (Carl Zeiss). Five sets of images are displayed in Figure 3. Sets A and B were captured from slides prepared after the standard invasion time of 3 h, while sets C and D were captured from slides prepared at 5 h post-invasion. All images in a set were captured in the same field of the slides but under different light paths. Visualization of RFP Expression from P. multocida B:2 JRMT12 pSRG via Fluorescence Microscopy (FM) The bacteria were cultured as described before, and sampling was done between OD600nm 0.7 and 0.8 in order to capture the PLOS ONE \| www.plosone.org August 2013 \| Volume 8 \| Issue 8 \| e71524 3 Bactofection and P. multocida B:2 Figure 1. Construction of the ‘‘traffic light’’ reporter plasmid, pSRG. (A) Plasmid pMK-RED was constructed from plasmid pMK-Express [13] containing the DsRed.M1 gene from plasmid pDsRed-Monomer (Clontech) inserted between the BamHI and NotI sites in place of the gfpmut3 gene. (B) Eukaryotic expression plasmid pEGFP-N1TM (Clontech) was digested with PciI. (C) Amplification of the PsodC-DsRed fragment with RED primers containing PciI sites. (D) Plasmid pSRG with two expression systems, a red reporter system that functions in prokaryotic cells driven by the sodC promoter and a green reporter system that functions in eukaryotic cells driven by the PCMVIE promoter. doi:10.1371/journal.pone.0071524.g001 Figure 1. Construction of the ‘‘traffic light’’ reporter plasmid, pSRG. (A) Plasmid pMK-RED was constructed from plasmid pMK-Express [13] containing the DsRed.M1 gene from plasmid pDsRed-Monomer (Clontech) inserted between the BamHI and NotI sites in place of the gfpmut3 gene. (B) Eukaryotic expression plasmid pEGFP-N1TM (Clontech) was digested with PciI. (C) Amplification of the PsodC-DsRed fragment with RED primers containing PciI sites. (D) Plasmid pSRG with two expression systems, a red reporter system that functions in prokaryotic cells driven by the sodC promoter and a green reporter system that functions in eukaryotic cells driven by the PCMVIE promoter. doi:10.1371/journal.pone.0071524.g001 presumed to be an artefact of iterative restoration after image manipulation using Volocity software. bacteria during maximum RFP expression. After confirming RFP expression, in the fluorimeter, bacteria were prepared for microscopy. Intracellular Trafficking of P. multocida B:2 JRMT12 pSRG within EBL Cells (II) Visualization of P. multocida B:2 JRMT12 pSRG via fluorescence microscopy. Images of P. multocida B:2 JRMT12 pSRG were taken of the same field under three different light paths; A: DIC (white) light, B: filtered fluorescent light (Exc. = 649 nm, Ems. = 670 nm) (blue) to visualize the counterstain, Cy5 Dye (Amersham Bioscience), C: two filtered lights; filtered fluorescent light (Exc. = 557 nm, Ems. = 585 nm) (red) to visualize bacteria expressing RFP from plasmid pSRG and filtered fluorescent light (Exc. = 649 nm, Ems. = 670 nm) (blue) to visualize the counterstain, Cy5 Dye in order to show that RFP expression was within the bacteria, D: 3D imaging of the 2D image from C using Volocity software. doi:10.1371/journal.pone.0071524.g002 were found not to be expressing GFP and no RFP-expressing bacteria were found intracellularly. The remaining 31 (44%) EBL cells were found to harbour a number of RFP-expressing P. multocida B:2 JRMT12 pSRG intracellularly and, of these, 12 (17% of total) EBL cells were already expressing GFP. No EBL cells expressing GFP without harbouring RFP-expressing bacteria were found at this stage. As in set A, images in set B were also captured at 3 h post- invasion. In this set, again a single EBL cell is clearly defined under the DIC (white) light (i) and filtered fluorescent blue light demonstrated the EBL cell membrane in blue when stained by the plasma membrane stain CellMask (ii). Under red and blue filtered fluorescent light, RFP expression (red) was visualised as in image A (iii). By switching the filtered fluorescent light path into green and blue, the EBL cell was seen to express GFP within the cell cytoplasm (iv). 3D images (v & vi) were then generated from the 2D images using the Volocity software. The data could be interpreted to indicate that, in this EBL cell, free plasmid was available for expression of GFP in the cytoplasm of the cell. The presence of fewer RFP-expressing bacteria within this cell might mean that others had been lysed so that they were no longer able to express RFP but had released plasmid into the cytoplasm to generate GFP. The obvious presence of bacteria at the periphery of the EBL cell, expressing RFP, might indicate that these bacteria had not yet been taken up by the cell but had nevertheless resisted washing and antibiotic treatment. Intracellular Trafficking of P. multocida B:2 JRMT12 pSRG within EBL Cells In set A, the first image (i) shows a single EBL cell taken under DIC (white) light. Under blue- filtered fluorescent light, the plasma membrane was stained blue (ii). An overlay of blue- and red-filtered fluorescent light showed the cell membrane as blue and the internalized P. multocida B:2 JRMT12 pSRG as red (iii). The 3D image (iv) displayed was generated from the 2D image using Volocity software. However, when the fluorescent light path was changed to green to visualize GFP, no green fluorescence was found to be expressed by this EBL cell (not shown). Hence, at 3 h post-invasion, internalized bacteria were found in this EBL cell but no expression of GFP was detected. This indicated that the bacteria were still intact and had not released plasmid into the intracellular environment of the EBL cell. August 2013 \| Volume 8 \| Issue 8 \| e71524 4 PLOS ONE \| www.plosone.org Bactofection and P. multocida B:2 Figure 2. (I) Assessment of RFP expression by P. multocida B:2 JRMT12 pSRG during growth. P. multocida B:2 JRMT12 without plasmid acted as a control for background fluorescence. Fluorescence was plotted against OD600nm using Microsoft Excel software and data were corrected for background fluorescence. Experiments were performed in triplicate and the error bars indicate standard deviations of the means. (II) Visualization of P. multocida B:2 JRMT12 pSRG via fluorescence microscopy. Images of P. multocida B:2 JRMT12 pSRG were taken of the same field under three different light paths; A: DIC (white) light, B: filtered fluorescent light (Exc. = 649 nm, Ems. = 670 nm) (blue) to visualize the counterstain, Cy5 Dye (Amersham Bioscience), C: two filtered lights; filtered fluorescent light (Exc. = 557 nm, Ems. = 585 nm) (red) to visualize bacteria expressing RFP from plasmid pSRG and filtered fluorescent light (Exc. = 649 nm, Ems. = 670 nm) (blue) to visualize the counterstain, Cy5 Dye in order to show that RFP expression was within the bacteria, D: 3D imaging of the 2D image from C using Volocity software. doi:10.1371/journal.pone.0071524.g002 Bactofection and P. multocida B:2 Figure 2. (I) Assessment of RFP expression by P. multocida B:2 JRMT12 pSRG during growth. P. multocida B:2 JRMT12 without plasmid acted as a control for background fluorescence. Fluorescence was plotted against OD600nm using Microsoft Excel software and data were corrected for background fluorescence. Experiments were performed in triplicate and the error bars indicate standard deviations of the means. August 2013 \| Volume 8 \| Issue 8 \| e71524 Intracellular Trafficking of P. multocida B:2 JRMT12 pSRG within EBL Cells Each of the remaining 32 (53%) EBL cells were found to be expressing GFP and of these, 12 (20% of total) cells were found to be still harbouring some RFP-expressing P. multocida B:2 JRMT12 pSRG intracellularly whereas the others expressed only GFP. Images A (iii), B (iii) and C (iii) show the capacity of P. multocida B:2 JRMT12 pSRG, internalised within EBL cells, to express RFP from pSRG. RFP expression would have taken place within intact bacteria, driven by the Psod-C promoter. Images B (iv), C (iv) and D (iv) show the ability of EBL cells to express GFP from pSRG which must have been released from the internalized P. multocida B:2 JRMT12 pSRG in order for the eukaryotic promoter PCMV IE to drive expression of GFP within the EBL cell cytoplasm. The images also illustrate the variation in the timing of both RFP and GFP expression with one cell showing RFP expression as late as 5 h post-invasion (image C) and another cell expressing GFP as early as 3 h (images A and B). From observation of 70 EBL cells at 3 h post-invasion, three categories of EBL cells were found. Some were found without any bacteria internally (no RFP expression), some had internalized bacteria but the mammalian cells were not expressing GFP and some, the lowest percentage, harboured internalized viable P. multocida B:2 JRMT12 pSRG but at the same time expressed GFP. Observation of 60 cells at 5 h post-invasion, demonstrated three categories of EBL cells, some without any bacteria internally, some harbouring internalized viable P. Figure 3. Localization of prokaryotic and eukaryotic protein expression. The images show EBL cells after invasion with P. multocida B:2 JRMT12 pSRG. Images were captured at 3 h (A and B) and 5 h (C and D) post-invasion. In A, an EBL cell was viewed (i) under DIC (white) light, (ii) by counterstaining with CellMaskTM plasma membrane stain (GE Healthcare) (blue), (iii) merged 2D images of (ii) and image captured under filtered fluorescent light (Exc. = 557 nm, Ems. = 585 nm) (red) to visualize internalized RFP-expressing bacteria and (iv) a 3D image of (iii). In B and C, an EBL cell was viewed (i) under DIC (white) light, (ii) by counterstaining with CellMaskTM plasma membrane stain (GE Health- care) (blue), (iii) merged 2D images of (ii) and image captured under filtered fluorescent light (Exc. = 557 nm, Ems. Intracellular Trafficking of P. multocida B:2 JRMT12 pSRG within EBL Cells Images in sets C and D were captured at 5 h post-invasion. In set C, under DIC (white) light, a single EBL cell on a monolayer was captured (i). When viewed under filtered fluorescent blue light, the labelled EBL membrane boundaries were visible as a blue fluorescence (ii). When viewed with red and blue filtered fluorescent light paths, the image showed some internalized bacteria (red) within membrane boundaries (blue) (iii). Green and blue fluorescent light paths showed green fluorescence in the EBL cell cytoplasm within membrane boundaries (blue) (iv). Both 2D images were then processed with Volocity software to generate their respective 3D images. In these, localization of RFP (v) and GFP (vi) expression was clearly displayed within the labelled (blue) membrane boundaries. Seventy EBL cells were assessed for RFP or GFP expression from slides prepared at 3 h post-invasion. Of these cells, 39 (56%) August 2013 \| Volume 8 \| Issue 8 \| e71524 PLOS ONE \| www.plosone.org 5 Bactofection and P. multocida B:2 Figure 3. Localization of prokaryotic and eukaryotic protein expression. The images show EBL cells after invasion with P. multocida B:2 JRMT12 pSRG. Images were captured at 3 h (A and B) and 5 h (C and D) post-invasion. In A, an EBL cell was viewed (i) under DIC (white) light, (ii) by counterstaining with CellMaskTM plasma membrane stain (GE Healthcare) (blue), (iii) merged 2D images of (ii) and image captured under filtered fluorescent light (Exc. = 557 nm, Ems. = 585 nm) (red) to visualize internalized RFP-expressing bacteria and (iv) a 3D image of (iii). In B and C, an EBL cell was viewed (i) under DIC (white) light, (ii) by counterstaining with CellMaskTM plasma membrane stain (GE Health- care) (blue), (iii) merged 2D images of (ii) and image captured under filtered fluorescent light (Exc. = 557 nm, Ems. = 585 nm) (red), (iv) merged 2D images of (ii) and image captured under filtered fluorescent light (Exc. = 488 nm, Ems. = 507 nm) (green) to visualize expression of GFP by the mammalian cells, (v) a 3D image of (iii) and (vi) a 3D image of (iv). In D, an EBL cell was viewed (i) under DIC (white) light, (ii) by counterstaining with CellMaskTM plasma membrane stain (GE Health- care) (blue), (iii) merged 2D images of (ii) and image captured under filtered fluorescent light (Exc. = 488 nm, Ems. Intracellular Trafficking of P. multocida B:2 JRMT12 pSRG within EBL Cells = 507 nm) (green) and (iv) a 3D image of (iii). The merged 3D images were included to demonstrate the localization of either RFP or GFP expression. Fluorescence emitted by EBL cells infected with JRMT12 strain harbouring pMK-RED (E) with the same microscope settings, was used as a negative control. Each cell was viewed (i) under DIC (white) light, (ii) by counterstaining with CellMaskTM plasma membrane stain (GE Healthcare) (blue), (iii) merged 2D images of (ii) and image captured under filtered fluorescent light (Exc. = 557 nm, Ems. = 585 nm) (red) to visualize internalized RFP-expressing bacteria and (iv) merged 2D images of (ii) and image captured under filtered fluorescent light (Exc. = 488 nm, Ems. = 507 nm) (green). doi:10.1371/journal.pone.0071524.g003 Set D images, captured at 5 h post-invasion, show a single EBL cell taken under DIC (white) light (i) and the same cell viewed under filtered fluorescent blue light revealed the labelled cell membrane (ii). Switching to both green and blue filtered fluorescent light showed expression of GFP by the EBL cell, displayed as green fluorescence within the cell membrane (blue) (iii). The 3D image (iv) was then generated from the 2D image using Volocity software. When the cell was viewed under red and blue filtered fluorescent light paths, the image showed only the EBL cell with the membrane boundaries outlined in blue fluorescence (image not shown). These data indicated that, at this stage, internalized RFP-expressing bacteria had been lysed so that they were no longer able to express RFP but had released the plasmid into the EBL cytoplasm for GFP expression controlled by the eukaryotic promoter. As a negative control, EBL cells were infected with the JRMT12 strain harbouring pMK-RED (Figure 1), which expresses only RFP. This control was analysed using the same microscopy settings as for sets A–D. Panel E shows that internalised bacteria were expressing RFP while the infected EBL cells emitted no detectable green autofluorescence. This confirms the conclusion that fluorescence measured in the GFP channel as in panels B-D could only be attributed to expression from pSRG. Sixty EBL cells were assessed from slides prepared at 5 h post- invasion. Of these, 28 (47%) were found not to be expressing GFP and no RFP-expressing bacteria were found intracellularly. Intracellular Trafficking of P. multocida B:2 JRMT12 pSRG within EBL Cells Plasmid DNA then localises in the nucleus (blue circle) to allow expression of the GFP gene by the eukaryotic promoter, which is followed by GFP formation in the cytosol (green colouration in cytoplasm). Blue arrows point to microscopic images taken at different stages of the invasion assay; (A) P. multocida B:2 JRMT12 pSRG expressing RFP. Bacteria counterstained with Cy5 Dye (blue). (B) EBL cell at 3h post-invasion showing intracellular P. multocida B:2 JRMT12 pSRG expressing RFP. The EBL plasma membrane is counterstained blue. (C) Optical sectioning of the EBL cell in B at 0.3 mm intervals from top to bottom, confirmed the intracellular location of the RFP-expressing bacteria. (D) EBL cell in B at 3h post-invasion showing intracellular P. multocida B:2 JRMT12 pSRG expressing RFP. (E) The same EBL cell in D expressing GFP from intracellular plasmid DNA. (F) EBL cell expressing GFP at 5h post-invasion with P. multocida B:2 JRMT12 pSRG. Magnification x100; counterstaining with Cy5 Dye (blue) for bacteria and CellMask (blue) for EBL cells. doi:10.1371/journal.pone.0071524.g004 was to visualize trafficking of a potential delivery plasmid from P. multocida B:2 into EBL cells using a plasmid able to propagate and express a reporter protein in P. multocida and also to express a reporter protein in mammalian cells. As outlined in Figure I, the ‘‘traffic-light’’ plasmid termed pSRG was designed with a promoter, Psod-C, active in Pasteurella bacteria upstream of a DsRed.M1 gene coding for RFP, together with the virally-derived promoter, PCMVIE, for expression in eukaryotes, upstream of the EGFP gene coding for GFP. pSRG, at 5700 bp, was successfully propagated and maintained stably in the P. multocida B:2 strain JRMT12 for at least 14 days without the presence of selective antibiotic. Assessment of RFP expression through the bacterial growth cycle showed that JRMT12, harbouring pSRG, expressed RFP maximally during the late exponential phase. Fluorescence microscopy showed P. multocida B:2 JRMT12 pSRG expressing RFP from individual bacteria. multocida B:2 JRMT12 pSRG expressing RFP while at the same time expressing GFP, and some EBL cells without any RFP expression but which expressed GFP. The last category was not found at 3 h post-invasion and the second category of EBL cells found at 3 h post-invasion was not found at 5 h post-invasion. Taken together, these data present good evidence that P. multocida B:2 JRMT12 can carry plasmid pSRG into EBL cells and, while the bacteria remain viable, they will express RFP. Intracellular Trafficking of P. multocida B:2 JRMT12 pSRG within EBL Cells = 585 nm) (red), (iv) merged 2D images of (ii) and image captured under filtered fluorescent light (Exc. = 488 nm, Ems. = 507 nm) (green) to visualize expression of GFP by the mammalian cells, (v) a 3D image of (iii) and (vi) a 3D image of (iv). In D, an EBL cell was viewed (i) under DIC (white) light, (ii) by August 2013 \| Volume 8 \| Issue 8 \| e71524 PLOS ONE \| www.plosone.org 6 August 2013 \| Volume 8 \| Issue 8 \| e71524 Bactofection and P. multocida B:2 Figure 4. Bactofection pathway for DNA vaccine delivery. Yellow arrows indicate a schematic pathway for the bacterial plasmid delivery process in EBL cells. After internalization of the bacteria and phagolysosome fusion, the escape of the plasmid DNA (pSRG) from the vacuole into the cytosol after bacterial degradation results in the transfer of the pSRG to the intracellular environment of the EBL cell. Plasmid DNA then localises in the nucleus (blue circle) to allow expression of the GFP gene by the eukaryotic promoter, which is followed by GFP formation in the cytosol (green colouration in cytoplasm). Blue arrows point to microscopic images taken at different stages of the invasion assay; (A) P. multocida B:2 JRMT12 pSRG expressing RFP. Bacteria counterstained with Cy5 Dye (blue). (B) EBL cell at 3h post-invasion showing intracellular P. multocida B:2 JRMT12 pSRG expressing RFP. The EBL plasma membrane is counterstained blue. (C) Optical sectioning of the EBL cell in B at 0.3 mm intervals from top to bottom, confirmed the intracellular location of the RFP-expressing bacteria. (D) EBL cell in B at 3h post-invasion showing intracellular P. multocida B:2 JRMT12 pSRG expressing RFP. (E) The same EBL cell in D expressing GFP from intracellular plasmid DNA. (F) EBL cell expressing GFP at 5h post-invasion with P. multocida B:2 JRMT12 pSRG. Magnification x100; counterstaining with Cy5 Dye (blue) for bacteria and CellMask (blue) for EBL cells. doi:10.1371/journal.pone.0071524.g004 Figure 4. Bactofection pathway for DNA vaccine delivery. Yellow arrows indicate a schematic pathway for the bacterial plasmid delivery process in EBL cells. After internalization of the bacteria and phagolysosome fusion, the escape of the plasmid DNA (pSRG) from the vacuole into the cytosol after bacterial degradation results in the transfer of the pSRG to the intracellular environment of the EBL cell. Intracellular Trafficking of P. multocida B:2 JRMT12 pSRG within EBL Cells Loss of RFP expression at 5 h post-invasion plus expression of GFP would indicate release of pSRG into the cytoplasm from the non-viable bacteria, where the plasmid was able to express GFP from the PCMVIE promoter. Discussion multocida B:2. However, in future work, more evidence of DNA transfer could perhaps be obtained by demonstrating splicing of the plasmid- encoded eukaryotic mRNA or expression in the presence of antibiotics that block prokaryotic protein synthesis. p y p y Figure 4 is a schematic diagram to model the bactofection pathway of P. multocida B:2 JRMT12 pSRG after infection of EBL cells and also incorporates some of the FM images obtained. In Figure 4A, P. multocida B:2 JRMT12 pSRG is shown expressing RFP before invading the EBL cells. At 3 h post-invasion, bacterial RFP expression was seen intracellularly (Figures 4B & 4C). Some infected cells harbouring viable P. multocida B:2 JRMT12 pSRG expressing RFP also expressed GFP (Figures 4D & 4E). This suggested that there was death of the bacteria from an early stage of infection. At 5 h post-invasion, the majority of the GFP- expressing cells were Pasteurella-negative (Figure 4F). In conclusion, our ‘‘traffic-light’’ plasmid pSRG, encoding dual promoters downstream of respective fluorescent reporters, represents a convenient and useful tool for analysing the different steps of the P. multocida B:2 infectious process by FM. Together with the live- attenuated P. multocida B:2 strain JRMT12, it has the potential to act as a vaccine, not only against HS, but also to provide immunity to other diseases, by replacing the eukaryotic GFP reporter gene with genes for protective antigens from other disease-causing organisms. In this regard, it will be of considerable value in future work to evaluate the persistence of GFP expression in EBL cells and, more importantly, to assess the persistence of antigen expression in vivo. In addition, our understanding of Pasteurella infection could be broadened by live-cell imaging for better visualization during time-course experiments and by flow-cytom- etry to further evaluate how bacterial dosage relates to plasmid delivery and protein expression. p y The high level of bacterial invasion (10–47%) found in this work and a previous study (7) indicates the potential of the system as a delivery method for foreign antigens. However, differences in behaviour are observed among attenuated bacterial strains in the hostile environment inside mammalian cells. Bacteria such as S. flexneri, L. monocytogenes or E. coli, carrying the virulence plasmid of S. flexneri, invade their host cells and then escape from a vacuole into the cytosol. Discussion Bactofection (bacteria-mediated transfer of expressible DNA) can be applied for genetic vaccination, gene therapy and production of therapeutic proteins [15]. A development of the work described here would be to apply bactofection to genetic vaccination against cattle diseases. Hence, in this study, our aim An invasion assay with P. multocida B:2 JRMT12 pSRG was undertaken to demonstrate bacterial entry, plasmid release and reporter protein expression within EBL cells. At 3 h post-invasion, bacteria were seen intracellularly, along with RFP expression and August 2013 \| Volume 8 \| Issue 8 \| e71524 PLOS ONE \| www.plosone.org 7 Bactofection and P. multocida B:2 in some cells green fluorescence was localised only in the cytoplasm and not in the nucleus. FM images suggested that, at 3 h post-invasion, RFP-expressing bacteria were found intracellu- lary in EBL cells. Some of these cells were expressing GFP indicating that bacterial lysis had occurred in some cases and plasmid had been released into the cytoplasm. Images captured at 5 h post-invasion showed that some 20% of EBL cells still contained viable P. multocida B:2 JRMT12 pSRG as demonstrated by RFP expression. This confirmed findings of Othman et al [7], on the intracellular survival of the live-vaccine strain, that viable intracellular bacteria could be detected from 3–7 h post-invasion although the number decreased with time. FM images at 5 h post- invasion showed some EBL cells containing viable P. multocida B:2 JRMT12 pSRG were also expressing GFP, an indicator of bacterial lysis and the presence of pSRG in the EBL cell cytoplasm. The 3D images provided a better visualization of the model system to demonstrate localization of expression of each fluorescent protein. Other EBL cells captured at 5 h post-invasion were negative for RFP expression yet were expressing GFP. This suggested that all the internalised bacteria had been lysed and free plasmid DNA had been delivered outside of the phagocytic vacuoles. The 3D images showed localization of eukaryotic GFP expression in the cytosol of the EBL cell. cells and some internalised bacteria appeared to be ruptured and in the process of degradation (7). Thus, it can be assumed that the attenuated aroA mutant dies through metabolic attenuation and then releases pSRG which must transfer from the vacuoles into the cytoplasm and then be delivered to the nuclear compartment, by an unknown mechanism. The above results have indicated that it is possible to obtain efficient bactofection using P. Discussion Once in the cytosol they die, for example because they are auxotrophic, conditionally express autolysins or are killed by the addition of antibiotics to the cultures. This results in lysis of the bacteria and release of the expression plasmids. Some of these plasmids find their way into the nucleus where the open reading frames are expressed under the control of the eukaryotic transcription elements [16–19]. Bacteria such as S. typhimurium, S. typhi or E. coli expressing the invasion gene, inv, of Y. pseudotuberculosis, invade their host cells and remain in the vacuole. There they die, for example due to metabolic attenuation, and release their expression plasmids. By an unknown mechanism, these plasmids cross the vesicular membrane and reach the cell nucleus of the host cells where they are expressed [16,20,21]. Our previous work showed by transmission electron microscopy that invasive aroA P. multocida B:2 were confined to the vacuoles of EBL Author Contributions Conceived and designed the experiments: JGC RP. Performed the experiments: SO. Analyzed the data: SO AJR. Contributed reagents/ materials/analysis tools: AJR. Wrote the paper: SO JGC RP AJR. Conceived and designed the experiments: JGC RP. Performed the experiments: SO. Analyzed the data: SO AJR. Contributed reagents/ materials/analysis tools: AJR. Wrote the paper: SO JGC RP AJR. 5. Hodgson JC, Finucane A, Dagleish MP, Ataei S, Parton R, et al. (2005) Efficacy of vaccination of calves against hemorrhagic septicemia with a live aroA derivative of Pasteurella multocida B:2 by two different routes of administration. Infect Immun 73: 1475–1481. 1. De Alwis MCL (1992) Pasteurellosis in production animals: A Review. ACIAR Proceedings 43: 11–22. 3. De Alwis MC (1999) Haemorrhagic septicaemia-a general review. ACIAR Proceedings 57: 99–112. 6. Dagleish MP, Hodgson JC, Ataei S, Finucane A, Finlayson J, et al. (2007) Safety and protective efficacy of intramuscular vaccination with a live aroA derivative of Pasteurella multocida B:2 against experimental hemorrhagic septicemia in calves. Infect Immun 75: 5837–5844. 2. Carter GR, De Alwis MCL (1989) Haemorrhagic septicaemia. In: Adlam C, Rutter JM, editors. Pasteurella and pasteurellosis. London: Academic Press. 131–160. ( ) Proceedings 43: 11–22. 17. Anderson RJ, Pasetti MF, Sztein MB, Levine MM, Noriega FR (2000) DguaBA attenuated Shigella flexneri 2a strain CVD 1204 as a Shigella vaccine and as a live mucosal delivery system for fragment C of tetanus toxin. Vaccine 18: 2193– 2202. 18. Darji A, Zur Lage S, Garbe AL, Chakraborty T, Weiss S (2000) Oral delivery of DNA vaccines using attenuated Salmonella typhimurium as carrier. FEMS Immunol Med Microbiol 27: 341–349. 16. Grillot-Courvalin C, Goussard S, Huetz F, Ojcius DM, Courvalin P (1998) Functional gene transfer from intracellular bacteria to mammalian cells. Nat Biotechnol 16: 862–866. 21. Fennelly GJ, Khan SA, Abadi MA, Wild TF, Bloom BR (1999) Mucosal DNA vaccine immunization against measles with a highly attenuated Shigella flexneri vector. J Immunol 162: 1603–1610. y 20. Leung KY, Finlay BB (1991) Intracellular replication is essential for the virulence of Salmonella typhimurium. Proc Natl Acad Sci U S A 88: 11470–11474. 19. Hense M, Domann E, Krusch S, Wachholz P, Dittmar KE, et al. (2001) Eukaryotic expression plasmid transfer from the intracellular bacterium Listeria monocytogenes to host cells. Cell Microbiol 3: 599–609. Bactofection and P. multocida B:2 References 1. De Alwis MCL (1992) Pasteurellosis in production animals: A Review. ACIAR Proceedings 43: 11–22. 8. Darji A, Guzma CA, Gerstel B, Wachholz P, Timmis KN, et al. (1997) Oral somatic transgene vaccination using attenuated S. typhimurium. Cell 91: 765–775. 2. Carter GR, De Alwis MCL (1989) Haemorrhagic septicaemia. In: Adlam C, Rutter JM, editors. Pasteurella and pasteurellosis. London: Academic Press. 131–160. 9. Paglia P, Medina E, Arioli I, Guzman CA, Colombo MP (1998) Gene transfer in dendritic cells, induced by oral DNA vaccination with Salmonella typhimurium, results in protective immunity against a murine fibrosarcoma. Blood 92: 3172– 3176. 3. De Alwis MC (1999) Haemorrhagic septicaemia-a general review. ACIAR Proceedings 57: 99–112. 10. Fennelly GJ, Khan SA, Abadi MA, Wild TF, Bloom BR (1999) Mucosal DNA vaccine immunization against measles with a highly attenuated Shigella flexneri vector. J Immunol 162: 1603–1610. 4. Tabatabaei M, Liu Z, Finucane A, Parton R, Coote J (2002) Protective immunity conferred by attenuated aroA derivatives of Pasteurella multocida B:2 strains in a mouse model of hemorrhagic septicemia. Infect Immun 70: 3355– 3362. 11. Dietrich G, Bubert A, Gentschev I, Sokolovic Z, Simm A, et al. (1998) Delivery of antigen-encoding plasmid DNA into the cytosol of macrophages by attenuated suicide Listeria monocytogenes. Nat Biotechnol 16: 181–185. y g 12. Kunik T, Tzfira T, Kapulnik Y, Gafni Y, Dingwall C, et al. (2001) Genetic transformation of HeLa cells by Agrobacterium. Proc Natl Acad Sci U S A 98: 1871–1876. 6. Dagleish MP, Hodgson JC, Ataei S, Finucane A, Finlayson J, et al. (2007) Safety and protective efficacy of intramuscular vaccination with a live aroA derivative of Pasteurella multocida B:2 against experimental hemorrhagic septicemia in calves. Infect Immun 75: 5837–5844. 13. Bosse´ JT, Durham AL, Rycroft AN, Kroll JS, Langford PR (2009) New plasmid tools for genetic analysis of Actinobacillus pleuropneumoniae and other pasteurella- ceae. Appl Environ Microbiol 75: 6124–6131. 14. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning:A laboratory manual. 2nd ed. Cold Spring Harbor Laboratory Press. 7. Othman S, Parton R, Coote J (2012) Interaction between mammalian cells and Pasteurella multocida B:2. Adherence, invasion and intracellular survival. Microb Pathog 52: 353–358. 15. Liu MA (2011) DNA vaccines: an historical perspective and view to the future. Immunol Rev 239: 62–84. References August 2013 \| Volume 8 \| Issue 8 \| e71524 August 2013 \| Volume 8 \| Issue 8 \| e71524 PLOS ONE \| www.plosone.org 8 Bactofection and P. multocida B:2 19. Hense M, Domann E, Krusch S, Wachholz P, Dittmar KE, et al. (2001) Eukaryotic expression plasmid transfer from the intracellular bacterium Listeria monocytogenes to host cells. Cell Microbiol 3: 599–609. y g 20. Leung KY, Finlay BB (1991) Intracellular replication is essential for the virulence of Salmonella typhimurium. Proc Natl Acad Sci U S A 88: 11470–11474. 21. Fennelly GJ, Khan SA, Abadi MA, Wild TF, Bloom BR (1999) Mucosal DNA vaccine immunization against measles with a highly attenuated Shigella flexneri vector. J Immunol 162: 1603–1610. PLOS ONE \| www.plosone.org August 2013 \| Volume 8 \| Issue 8 \| e71524 PLOS ONE \| www.plosone.org 9 9
https://openalex.org/W4361940699	https://figshare.com/articles/journal_contribution/Supplementary_Data_from_Effects_of_Thymoquinone_in_the_Expression_of_Mucin_4_in_Pancreatic_Cancer_Cells_Implications_for_the_Development_of_Novel_Cancer_Therapies/22485036/1/files/39936588.pdf	English	null	Supplementary Data from Effects of Thymoquinone in the Expression of Mucin 4 in Pancreatic Cancer Cells: Implications for the Development of Novel Cancer Therapies	null	2,023	cc-by	464	Supplementary Figure Legends Supplementary Figure Legends Supplementary Figure 1: Effect of TQ in cell viability and MUC4 expression on CD18/HPAF cells. (A) Cytotoxicity of TQ in CD18/HPAF cells after being incubated with the drug for 24 hr. The data represents the mean of quadruplicate values ± SE. (B) Western blot analysis of MUC4 expression in CD18/HPAF cells after being incubated with different doses of TQ and different time intervals. Twenty µg of protein lysates were resolved on 2% SDS agarose gells. β-actin was used as the loading control. Supplementary Figure 2: Downregulation of MUC4 expression on CD18/HPAF cells by TQ. (A) Measurement of MUC4 transcripts in cells incubated with TQ by RT-PCR. The housekeeping gene β-actin was used as a control. (B) Western blot analysis of MUC4 expression after being incubated with TQ in the presence of the proteasomal inhibitor (PrI) MG132. Experimental samples included media only, PrI only, TQ only, and PrI with TQ. Twenty µg of protein lysates were resolved in 2% SDS agarose gels. β-actin was used as the loading control. (C) Western blot analysis of total STAT1 and its phosphorylated form (pSer-STAT1) of cells incubated with TQ. Forty µg of protein lysates were resolved in 10% SDS-PAGE gels. Supplementary Figure 3: Effect of TQ in the motility of CD18/HPAF cells and the expression of HER2 and FAK. (A) Optical microscopy images (4x) of the wound healing assay of CD18/HPAF cells after being incubated with different doses of TQ for 24 hr. Dashed yellow lines indicate the migration progress of the cells in the 24 hr period. (B) Western blot analysis of MUC4 and HER2 expression in CD18/HPAF cells after TQ treatment. Twenty µg of protein lysates were resolved in 2% SDS agarose gels. β-actin was used as the loading control. (C) Western blot analysis of focal adhesion kinase (FAK) and its phosphorylated form in CD18/HPAF cells after TQ treatment. Forty µg of protein lysates were resolved in 10% SDS- PAGE gels. Supplemental Table 1 pp Gene Fold change Function of protein coded Upregulated STC2 6.51 Overexpression result in growth restriction IL24 5.40 Induce apoptosis of cancer cells TRIB3 4.99 Regulates activation of MAP kinases ERRFl1 4.55 Induced during cell stress ; mediates cell signaling GADD45A 4.30 Respond to environmental stresses by mediating activation of the p38/JNK pathway CYP27B1 3.04 Catalyze reactions involved in drug metabolism RND3 2.76 Regulate the organization of the actin cytoskeleton Downregulated TRIM24 0.354 Mediates transcriptional control by interaction with nuclear receptors including the estrogen, retinoic acid, and vitamin D3 receptors S100A4 0.322 Function in motility, invasion, tubulin polymerization, and tumor metastasis MMP7 0.316 Involved in breakdown of extracellular matrix in metastasis; involved in wound healing RORA 0.286 Interact with the product of a tumor metastasis suppressor candidate gene MMP13 0.261 Role in tumoral process and metastasis
https://openalex.org/W4233660698	https://archimer.ifremer.fr/doc/00585/69695/67582.pdf	English	null	Increase of dissolved inorganic carbon and decrease of pH in near surface waters of the Mediterranean Sea during the past two decades	null	2,017	cc-by	9,008	Correspondence: Liliane Merlivat (merlivat@locean.upmc.fr) Received: 4 July 2017 – Discussion started: 6 July 2017 Received: 4 July 2017 – Discussion started: 6 July 2017 Revised: 3 September 2018 – Accepted: 4 September 2018 – Published: 21 September 2018 Revised: 3 September 2018 – Accepted: 4 September 2018 – Published: 21 September 2018 Abstract. Two 3-year time series of hourly measurements of the fugacity of CO2 (f CO2) in the upper 10 m of the sur- face layer of the northwestern Mediterranean Sea have been recorded by CARIOCA sensors almost two decades apart, in 1995–1997 and 2013–2015. By combining them with the alkalinity derived from measured temperature and salinity, we calculate changes in pH and dissolved inorganic carbon (DIC). DIC increased in surface seawater by ∼25 µmol kg−1 and f CO2 by 40 µatm, whereas seawater pH decreased by ∼0.04 (0.0022 yr−1). The DIC increase is about 15 % larger than expected from the equilibrium with atmospheric CO2. This could result from natural variability, e.g. the increase between the two periods in the frequency and intensity of winter convection events. Likewise, it could be the signa- ture of the contribution of the Atlantic Ocean as a source of anthropogenic carbon to the Mediterranean Sea through the Strait of Gibraltar. We then estimate that the part of DIC accumulated over the last 18 years represents ∼30 % of the total inventory of anthropogenic carbon in the Mediterranean Sea. have complex direct and indirect impacts on marine organ- isms and ecosystems (Gattuso and Hansson, 2011). Empiri- cal methods to estimate the anthropogenic CO2 penetration in the ocean since the industrial revolution have improved over the past few decades (Chen and Millero, 1979; Gruber et al., 1996; Sabine et al., 2008; Touratier and Goyet, 2004, 2009; Woosley et al., 2016). As the concentration of anthro- pogenic carbon, Cant, cannot be distinguished from the nat- ural background of DIC through total DIC measurements, these methods are based on the analysis of different chem- ical properties of the water column. Direct estimates of the anthropogenic CO2 absorption in the sea surface layers are difﬁcult due to the large natural variability driven by physi- cal and biological phenomena. Bates et al. (2014) have ex- tracted the trend from the large variability based on the anal- ysis of a long time series (monthly or seasonal sampling). For the global surface ocean, Lauvset et al. Correspondence: Liliane Merlivat (merlivat@locean.upmc.fr) (2015) have used the Surface Ocean CO2 Atlas (SOCAT) database (Bakker et al., 2014) combined with an interpolation method. Estimates of anthropogenic storage in the Mediterranean Sea differ by about a factor of 2 (Huertas et al., 2009; Touratier and Goyet, 2009). In addition to the anthropogenic signal, oceanic DIC can also be the signature of a strong inter-annual variability. In the North Atlantic, for instance, McKinley et al. (2011) have shown that the long-term trend emerges only after more than 25 years because of natural variability. Biogeosciences, 15, 5653–5662, 2018 https://doi.org/10.5194/bg-15-5653-2018 © Author(s) 2018. This work is distributed under the Creative Commons Attribution 4.0 License. Biogeosciences, 15, 5653–5662, 2018 https://doi.org/10.5194/bg-15-5653-2018 © Author(s) 2018. This work is distributed under the Creative Commons Attribution 4.0 License. 1 Introduction The concentration of atmospheric carbon dioxide (CO2) has been increasing rapidly over the 20th century. As a result, the concentration of dissolved inorganic carbon (DIC) in the near surface ocean increases, which drives a decrease in pH in order to maintain a chemical equilibrium. These changes A high-frequency sampling of the seawater carbon chem- istry at the air-water interface over extended periods of time is useful in assessing the trends and variability of DIC. In Published by Copernicus Publications on behalf of the European Geosciences Union. Increase of dissolved inorganic carbon and decrease in pH in near-surface waters in the Mediterranean Sea during the past two decades Liliane Merlivat1, Jacqueline Boutin1, David Antoine2,3, Laurence Beaumont4, Melek Golbol3, and Vincenzo Vellucci3 1Sorbonne Université-CNRS-IRD-MNHN, LOCEAN, 75005 Paris, France 2Remote Sensing and Satellite Research Group, School of Earth and Planetary Sciences, Curtin University, Perth, Australia 3Sorbonne Université-CNRS, Laboratoire d’Océanographie de Villefranche, LOV, 06230 Villefranche-sur-Mer, France 4Division Technique INSU-CNRS, 92195 Meudon CEDEX, France Liliane Merlivat1, Jacqueline Boutin1, David Antoine2,3, Laurence Beaumont4, Melek Golbol3, and Vincenzo Vellucci3 1Sorbonne Université-CNRS-IRD-MNHN, LOCEAN, 75005 Paris, France 2Remote Sensing and Satellite Research Group, School of Earth and Planetary Sciences, Curtin University, Perth, Australia 3Sorbonne Université-CNRS, Laboratoire d’Océanographie de Villefranche, LOV, 06230 Villefranche-sur-Mer, France 4Division Technique INSU-CNRS, 92195 Meudon CEDEX, France Correspondence: Liliane Merlivat (merlivat@locean.upmc.fr) 2.2 Analytical methods At DYFAMED, f CO2 measurements at 2 m were provided by an anchored ﬂoating buoy ﬁtted with a CARIOCA sen- sor. At BOUSSOLE, measurements were carried out from a mooring normally dedicated to radiometry and optical measurements where two CARIOCA sensors were attached. Both monitored f CO2 hourly at the 3 and 10 m depths (al- though only one of the two depths was equipped with a func- tional sensor at some periods); S and T were monitored at the same two depths using a Seabird SBE 37-SM MicroCat in- strument. The CARIOCA sensors were adapted to work un- der pressure in the water column. They were swapped about every 6 months, with serviced and calibrated instruments re- placing those which were previously deployed. The accuracy of CARIOCA f CO2 measurements by the spectrophotomet- ric method based on the optical absorbance of a solution thy- mol blue diluted in seawater is estimated at 2 µatm during both periods. Hood and Merlivat (2001) have reported agree- ment between the f CO2 measured by CARIOCA buoys, similar to the one deployed at DYFAMED, with ship-based measurements during a number of ﬁeld programs and with an accuracy of 2 µatm and a precision of 5 µatm. Figure 1. The area of the northwestern Mediterranean Sea show- ing the southern coast of France, the Island of Corsica, the main current branches (gray arrows), the location of the DYFAMED site (43◦25′ N, 7◦52′ E, red star) (http://doi.org/10.17882/43749, last access: 14 September 2018) and the BOUSSOLE buoy (43◦22′ N, 7◦54′ E, black star) in the Ligurian Sea. this paper we analyse two 3-year time series of hourly fugac- ity of CO2, (f CO2), measured with autonomous CARIOCA sensors (Copin-Montégut et al., 2004; Merlivat and Brault, 1995) in 1995–1997 and 2013–2015 at two nearby locations in the northwestern Mediterranean Sea (Fig. 1). Using mea- sured f CO2, temperature (T ) and salinity (S), we derive the other variables of the carbonate system (pH and DIC). The experimental setting is ﬁrst described, and the recent data obtained over the 2013–2015 period are presented. Com- bined with the 1995–1997 measurements previously pub- lished (Hood and Merlivat, 2001), we estimate the decrease in pH and the increase in DIC. 2.2 Analytical methods The results are discussed with respect to the contributions of the exchange with atmospheric CO2, the possible impact of vertical mixing and recent esti- mates of the transport of anthropogenic carbon from the At- lantic Ocean over a 18-year period. At Boussole, newly designed f CO2 sensors have been cal- ibrated using in situ seawater samples taken at 5 and 10 m depths during the monthly servicing cruises to the mooring. The total alkalinity (Alk) and DIC of the samples were de- termined by potentiometric titration using a closed cell ac- cording to the method developed by (Edmond, 1970). Certi- ﬁed reference materials (CRMs) supplied by A. G. Dickson (Scripps Institution of Oceanography, San Diego, USA) were used for calibration (Dickson et al., 2007). The accuracy is estimated at 3 µmol kg−1 for both DIC and Alk. f CO2 is calculated using the dissociation constants of Mehrbach re- ﬁtted by Dickson and Millero (Dickson and Millero, 1987; Mehrbach et al., 1973) and as recommended by Álvarez et al. (2014) for the Mediterranean Sea. Uncertainty in f CO2 derived from an individual sample is expected to be on the or- der of 5 µatm (Millero, 2007). About eight samples have been used to calibrate each CARIOCA sensor so that the uncer- tainty of the absolute calibration of each f CO2 CARIOCA sensor is estimated at 1.8 µatm. In addition, we observe that the standard deviation of the difference between the CARI- OCA f CO2 and f CO2 computed with the monthly discrete samples (Fig. 2b) is equal to 4.4 µatm, consistent with the ex- pected precision on CARIOCA f CO2 of 5 µatm. Alk and S of the 56 samples taken at BOUSSOLE are linearly corre- L. Merlivat et al.: Increase of dissolved inorganic carbon and decrease in pH 5654 Figure 1. The area of the northwestern Mediterranean Sea show- ing the southern coast of France, the Island of Corsica, the main current branches (gray arrows), the location of the DYFAMED site (43◦25′ N, 7◦52′ E, red star) (http://doi.org/10.17882/43749, last access: 14 September 2018) and the BOUSSOLE buoy (43◦22′ N, 7◦54′ E, black star) in the Ligurian Sea. the Ligurian Sea. The two sites surrounded by the perma- nent geostrophic Ligurian frontal jet ﬂow are protected from coastal inputs (Antoine et al., 2008; Heimbürger et al., 2013; Millot, 1999). Monthly cruises are carried out at the same location. L. Merlivat et al.: Increase of dissolved inorganic carbon and decrease in pH 5655 Figure 2. Inter-annual variability of CARIOCA data on the BOUSSOLE mooring; left column represents a function of time and right column a function of months for a given year (blue for 2013, green for 2014 and red for 2015). (a, d) T, (b, e) f CO2 and (c, f) f CO2@13 ◦C. In (a–c), the dotted lines indicate the period affected by stratiﬁcation and internal waves (26 July to 1 October 2014 and 8 July to 1 October 2015). In (b), the open circles correspond to f CO2 data derived from DIC and alkalinity measurements of samples taken at 5 and 10 m. In (e), the thin lines indicate f CO2atm. Note that the colour code on (d–f) is different from (a–c). Figure 2. Inter-annual variability of CARIOCA data on the BOUSSOLE mooring; left column represents a function of time and right column a function of months for a given year (blue for 2013, green for 2014 and red for 2015). (a, d) T, (b, e) f CO2 and (c, f) f CO2@13 ◦C. In (a–c), the dotted lines indicate the period affected by stratiﬁcation and internal waves (26 July to 1 October 2014 and 8 July to 1 October 2015). In (b), the open circles correspond to f CO2 data derived from DIC and alkalinity measurements of samples taken at 5 and 10 m. In (e), the thin lines indicate f CO2atm. Note that the colour code on (d–f) is different from (a–c). lated according the following relationship; winter vertical mixing with the deeper Levantine Interme- diate Water (LIW) marked by extremes in temperature and salinity (Copin-Montégut and Bégovic, 2002). Temperature provides the main control of the seasonality of f CO2, from 350 µatm to more than 550 µatm in summer 2013 (Fig. 2b). The fugacity of CO2 in seawater is a function of tempera- ture, DIC, alkalinity, salinity and dissolved nutrients. In the oligotrophic surface waters of the Mediterranean Sea, the ef- fect of nutrients may be neglected. Temperature and DIC have the strongest inﬂuences. By normalising f CO2 to a constant temperature, the temperature effect can be removed and changes in f CO2 resulting from changes in DIC can be more easily identiﬁed. Figure 2c shows the variability of f CO2 normalised to the constant temperature of 13 ◦C, (f CO2@13) using the equation of Takahashi et al. (1993). L. Merlivat et al.: Increase of dissolved inorganic carbon and decrease in pH The underlying processes that govern the seasonal variabil- ity of f CO2@13 are successive winter mixing, biological activity (organic matter formation and remineralisation) and Alk (µmolkg−1) = 87.647S −785.5. (1) (1) The standard deviation of the Alk data around the regression line is equal to 4.4 µmol kg−1 (r2 = 0.89). The standard deviation of the Alk data around the regression line is equal to 4.4 µmol kg−1 (r2 = 0.89). 3.1 The BOUSSOLE mooring (2013–2015) time series 3.1 The BOUSSOLE mooring (2013–2015) time series Temperature and f CO2 were measured from February 2013 to February 2016. All seasons were well represented, with missing data only in May–July 2013. For some periods, si- multaneous measurements were made at the 3 and 10 m depths (Fig. 2a–c). The range of temperature (Fig. 2a) extends from 13 ◦C in winter up to 27 ◦C in summer, followed by progressive cool- ing in fall. The coldest temperature, 13 ◦C, results from the 2.1 The BOUSSOLE and DYFAMED sites Data collection was carried out at the BOUSSOLE site (43◦22′ N, 7◦54′ E) in 2013–2015 (Antoine et al., 2006, 2008) and at the DYFAMED site (43◦25′ N, 7◦52′ E) in 1995–1997 (Marty et al., 2002). These sites are 3 nauti- cal miles apart, both located in the Ligurian Sea, one of the basins of the northwestern Mediterranean Sea (Fig. 1). The water depth is ∼2400 m. The prevailing ocean currents are usually weak (< 20 cm s−1), because these sites are in the central area of the cyclonic circulation that characterises www.biogeosciences.net/15/5653/2018/ Biogeosciences, 15, 5653–5662, 2018 3.2.1 Sea surface temperature changes Monthly mean values of temperature have been computed for the two 3-year periods, 1995–1997 and 2013–2015. In 1995– 1997, f CO2 and T at 2 m were measured with CARIOCA sensors installed on a buoy at DYFAMED (Hood and Mer- livat, 2001). The mean annual temperature of hourly CAR- IOCA data is equal to 18.21 ◦C. For 2013–2015, tempera- ture measurements made on the BOUSSOLE mooring at 3 and 10 m have been used. For the April to September time interval, there are only data at the 3 m depth. In addition, temperature data measured half hourly at 0.7 m at a nearby meteorological buoy (43◦23′ N, 7◦50′ E) (https://doi.org/10. 6096/hymex.azurbuoy.thermosalinograph.20100308, last ac- cess: 19 September 2018; Rolland and Bouin, 2010) have been used (Fig. 3d). Mean annual temperatures are equal to 18.29 and 17.97 ◦C, respectively, based on the meteorolog- ical buoy and the BOUSSOLE mooring data. The two sets of data differ essentially during July and August, with the temperatures at 3 m colder than those at 0.7 m, which indi- cates a thermal gradient between the two depths during sum- mer. Therefore, for 2013–2015, we select the mean annual value computed with the meteorological buoy, 18.29 ◦C, as a better representation of the sea surface. This value is close to 18.21 ◦C computed for 1995–1997. Thus, no signiﬁcant change in SST is found between the 2 decades, with a mean value equal to 18.25 ◦C. During summer 2014, large differences between measure- ments at 3 and 10 m were observed (Fig. 2a–c between dashed lines). A detailed analysis of the temporal variability during that period underscores the role of inertial waves at the frequency of 17.4 h that create the observed differences between the 2 depths of observations, with the deeper wa- ters being colder and enriched in f CO2@13. Temperature and f CO2@13 variability is dominated by inertial waves. In particular, from 15 to 26 August 2014, the difference in T between the two depths is as large as 7.6 (5.1 ◦C on average). f CO2 decreases on average by 32.7 µatm, corresponding to an increase in f CO2@13 equal to 42.8 µatm. f 2 q µ The 2013–2015 seasonal and inter-annual variability of T , f CO2 and f CO2@13 is illustrated in Fig. 2–f. The larger inter-annual changes in temperature (Fig. www.biogeosciences.net/15/5653/2018/ Biogeosciences, 15, 5653–5662, 2018 L. Merlivat et al.: Increase of dissolved inorganic carbon and decrease in pH 5656 3.2 Decadal changes in hydrography the deepening of mixed layer in fall (Bégovic and Copin- Montegut, 2002; Hood and Merlivat, 2001). Biological pro- cesses account for the decline in f CO2@13 observed from March–April to late summer; the ensuing increase in surface f CO2@13 is associated with the deepening of the mixed layer in the fall or convection in winter, as the vertical dis- tribution of f CO2@13 at DYFAMED shows a maximum in the 50–150 m layer. Where a large remineralisation of or- ganic matter occurs, the productive layer is mostly between 0 and 40 m (Copin-Montégut and Bégovic, 2002). The con- tribution of air–sea exchange is not signiﬁcant (Bégovic and Copin-Montegut, 2002). Over the period 2013–2015, the air– sea CO2 ﬂux from the atmosphere to the ocean surface is equal to −0.45 mol m−2 yr−1. the deepening of mixed layer in fall (Bégovic and Copin- Montegut, 2002; Hood and Merlivat, 2001). Biological pro- cesses account for the decline in f CO2@13 observed from March–April to late summer; the ensuing increase in surface f CO2@13 is associated with the deepening of the mixed layer in the fall or convection in winter, as the vertical dis- tribution of f CO2@13 at DYFAMED shows a maximum in the 50–150 m layer. Where a large remineralisation of or- ganic matter occurs, the productive layer is mostly between 0 and 40 m (Copin-Montégut and Bégovic, 2002). The con- tribution of air–sea exchange is not signiﬁcant (Bégovic and Copin-Montegut, 2002). Over the period 2013–2015, the air– sea CO2 ﬂux from the atmosphere to the ocean surface is equal to −0.45 mol m−2 yr−1. 3.2.1 Sea surface temperature changes 2d) are observed during summer, both at 3 and 10 m depth, while over Febru- ary and March, a constant value of 13 ◦C is observed as the result of vertical mixing with the LIW. A very large inter- annual variability of f CO2@13 is observed for T < 14 ◦C (Fig. 2f). This is associated with the winter mixing at the mooring site, which is highly variable from year to year. Win- ter mixed-layer depth (MLD) varies between 50 and 160 m, at the top of the LIW over the 2013–2015 period (Coppola et al., 2016). The variable depth of the winter vertical mix- ing causes the difference in f CO2@13, as f CO2 increases with depth (Copin-Montégut and Bégovic, 2002). The deep- ening of the MLD is driven by episodic and intense mixing processes characterised by a succession of events lasting sev- eral days, which are related to atmospheric forcing (Antoine et al., 2008) and lead to increase in f CO2@13. Figure 2e illustrates the solubility control of the variability of f CO2, as f CO2 increases when T increases. Another cause of the inter-annual variability of f CO2 for T ∼14 ◦C is the timing of the spring increase in biological activity, which differs by a month between years. For instance, the increase happened at the beginning of April in 2013, when T ∼15–16 ◦C and by mid March in 2014, when T ∼14 ◦C. Another cause is the deepening of the mixed layer due to the fall cooling, which varies by a month between years. 3.2.2 Sea surface salinity changes The mean value of salinity and the standard error of the mean computed from 56 samples taken at BOUSSOLE in 2013– 2015 are respectively 38.19 and 0.02. In 1998–1999, ship measurements of surface salinity were made during monthly cruises at the DYFAMED site (Copin-Montégut et al., 2004). The mean salinity and the standard error of the mean of this set of 19 data are respectively 38.21 and 0.03. Thus, there is no signiﬁcant salinity change between the two decades. 3.3 Decadal changes in f CO2@13 3.3.1 Time series of f CO2@13 in 1995–1997 and 2013–2015 3.3 Decadal changes in f CO2@13 3.3.1 Time series of f CO2@13 in 1995–1997 and 2013–2015 3.3.1 Time series of f CO2@13 in 1995–1997 and 2013–2015 3.3.1 Time series of f CO2@13 in 1995–1997 and 2013–2015 The two time series of high-frequency data were analysed in order to quantify the change in f CO2@13 2 decades apart at the sea surface. To account for the inter-annual seasonal variability as well as irregular sampling, we performed an analysis of the change in f CO2@13 as a function of SST (Fig. 3a and b). For the 2013–2015 data set, we excluded summer data measured at 10 m depth as they were not rep- resentative of the surface mixed layer due to a strong strati- ﬁcation. Much larger f CO2@13 values are observed at low temperatures than at high temperatures, the decrease being Biogeosciences, 15, 5653–5662, 2018 www.biogeosciences.net/15/5653/2018/ L. Merlivat et al.: Increase of dissolved inorganic carbon and decrease in pH 5657 L. Merlivat et al.: Increase of dissolved inorganic carbon and decrease in pH 5657 Figure 3. (a) f CO2@13 as a function of temperature for hourly data in 2013, 2014 and 2015.The yellow dots indicate mean f CO2@13. (b) is similar to (a) but represents all hourly data in 1995–1997 (black) and in 2013–2015 (red). (c) is similar to (b) but represents average values per 1 ◦C interval (standard deviation as dotted line). The difference between the two periods is also displayed by the dashed blue curve (scale on the right axis; the mean difference over all SST is represented by the horizontal blue line). (d) Mean monthly sea surface temperature for 1993–1995 (black curve for CARIOCA sensors), 2013–2015 (green for CARIOCA sensors) and 2013–2015 (red for meteorological buoy). Corresponding mean annual values are indicated by dotted lines. Figure 3. (a) f CO2@13 as a function of temperature for hourly data in 2013, 2014 and 2015.The yellow dots indicate mean f CO2@13. (b) is similar to (a) but represents all hourly data in 1995–1997 (black) and in 2013–2015 (red). (c) is similar to (b) but represents average values per 1 ◦C interval (standard deviation as dotted line). The difference between the two periods is also displayed by the dashed blue curve (scale on the right axis; the mean difference over all SST is represented by the horizontal blue line). (d) Mean monthly sea surface temperature for 1993–1995 (black curve for CARIOCA sensors), 2013–2015 (green for CARIOCA sensors) and 2013–2015 (red for meteorological buoy). Corresponding mean annual values are indicated by dotted lines. 3.3.1 Time series of f CO2@13 in 1995–1997 and 2013–2015 in f CO2@13 between the two periods, df CO2@13, is de- rived in each temperature step, seen in the difference between column 2 and 6 of Table 1. The variability of this differ- ence is estimated as the quadratic mean of the standard de- viation in each time series. Both values are reported in Ta- ble 1, column 9 and 10 and in Fig. 3c. The distribution of each df CO2@13 values around the mean over all SST of df CO2@13 seems random and indicates no trend of depen- dency with SST (Fig. 3c). This suggests that the processes that control the seasonal variation of f CO2@13 at the sea surface have not changed over the last two decades. similar and strongly nonlinear for the two studied periods. As described in Sect. 3.1, large values at low temperatures result from mixing with enriched deep waters during win- ter, and low values for 26–28 ◦C temperatures occur at the end of summer after the biological drawdown of carbon. An increase in f CO2@13 between the two periods is evident across the range of temperatures. 3.3.2 Trend analysis and statistics To quantify the change in f CO2@13 between the two data sets, we proceed as follows: data are binned by 1 ◦C tem- perature intervals, thereby removing any potential seasonal weighting, especially towards the 13–14 ◦C winter months temperature. The measurements made in this temperature in- terval represent about 25 % of the total number of data for both periods. For each of the fourteen 1 ◦C steps, the mean and standard deviation of hourly f CO2@13 measurements are reported in Table 1 and in Fig. 3c. The mean temperature within each of the 1◦steps differ for the two periods, as the distribution of individual measurements are not identical. We have estimated the uncertainties in the estimates of the difference df CO2@13 with two methods. Firstly, the arithmetic mean of df CO2@13 is equal to 33.17 µatm, with a standard deviation (SD) and standard error (SE), respec- tively equal to 6.29 µatm and 1.68 µatm. A 95 % conﬁdence interval is thereby achieved within 1.96 SE, i.e. 3.29 µatm. A second approach consists of computing a weighted aver- age of the mean of df CO2@13. In this case, mean weighted value of df CO2@13 over the whole range of tempera- tures is estimated, the weights being equal to the vari- ance of df CO2@13 in each temperature step. The value of df CO2@13 is equal to 32.70 µatm. The weighted SD, and the associated SE, of the 14 data points are respectively For both data sets, a monotonic relationship between f CO2@13 and T is observed with correlation coefﬁcients respectively equal to −0.861 and −0.857. The difference www.biogeosciences.net/15/5653/2018/ At this temperature, the change in f CO2 at the sea surface is 41.4 ± 4.1 µatm. Thus the contribution of the increase in atmospheric CO2 is responsible for 84% ± 5 % of the increase in f CO2 mea- sured in the surface waters. With the same salinity and al- kalinity as previously, the measurement of the corresponding change in surface DIC, assuming air–sea equilibrium, would be 20.8 ± 1.3 µmol kg−1 (Table 2). equal to 4.85 and 1.30 µatm. A 95 % conﬁdence interval is achieved within 2.54 µatm. The difference between the two mean df CO2@13 estimates is 0.47 µatm, well below SE. In the following, we have chosen the former method. 3.4 Changes of seawater carbonate chemistry in surface waters We estimated the DIC and pH changes related to the in- crease in f CO2@13 measured at the sea surface 18 years apart, assuming a mean salinity equal to 38.2, a mean al- kalinity equal to 2562.3 µmol kg−1 following Eq. (1) and a mean in situ temperature (T ) equal to 18.25 ◦C. The dissoci- ation constants of Mehrbach reﬁtted by Dickson and Millero (Dickson and Millero, 1987; Mehrbach et al., 1973) were used. pH is calculated on the seawater scale. The uncer- tainty of df CO2@13, 3.3 µatm, has been propagated to com- pute the combined uncertainty in dDIC and dpHSWS. The uncertainties in the equilibrium constants are neglected in this propagation of uncertainties. Likewise, an implicit as- sumption is that there is no systematic error on DIC and pHSWS derived from f CO2@13 between the two time pe- riods; in particular, mean temperature and salinity remain the same (Sect. 3.2). This is further discussed in Sect. 4.1. We compute an increase in DIC and dDIC equal to 25.2 ± 2.7 µmol kg−1 (1.40 ± 0.15 µmol kg−1 yr−1) and a decrease in pHSWS and dpHSWS equal to −0.0397 ± 0.0042 pHSWS (−0.0022 ± 0.0002 pHSWS yr−1) (Table 2). 4.1 Time change in surface alkalinity High-frequency measurements of f CO2 and temperature over two periods of 3 years that are 2 decades apart have allowed the computation of an increase in DIC equal to 25.1 ± 2.3 µmol kg−1 assuming no change in alkalinity. In the range of salinity of the BOUSSOLE samples, 37.9 to 38.5, the alkalinity values computed with Eq. (1) are larger than those predicted by the relationship established for the DYFAMED site, with a mean difference equal to 10 ± 2 µmol kg−1 (Copin-Montégut and Bégovic, 2002). In both cases alkalinity measurements were made with a po- tentiometric method using the certiﬁed reference material www.biogeosciences.net/15/5653/2018/ www.biogeosciences.net/15/5653/2018/ Biogeosciences, 15, 5653–5662, 2018 5658 L. Merlivat et al.: Increase of dissolved inorganic carbon and decrease in pH Table 1. Distribution of temperature, f CO2@13, and increase df CO2@13 data binned by 1 ◦C temperature interval for the two periods 1995–1997 and 2013–2015. L. Merlivat et al.: Increase of dissolved inorganic carbon and decrease in pH 5658 Table 1. Distribution of temperature, f CO2@13, and increase df CO2@13 data binned by 1 ◦C temperature interval for the two periods 1995–1997 and 2013–2015. Table 1. Distribution of temperature, f CO2@13, and increase df CO2@13 data binned by 1 ◦C temperature interval for the two per 1995–1997 and 2013–2015. Time interval 1995–1997 Time interval 2013–2015 Temporal change T 1 f CO2@13 N Standard T 1 f CO2@13 N Standard df CO2@13 Standard ◦C µatm deviation ◦C µatm deviation µatm deviation µatm µatm µatm 13.45 331.58 1212 28.09 13.55 363.14 6869 18.07 31.56 33.40 14.45 305.28 495 26.02 14.43 337.16 3270 16.65 31.87 30.89 15.37 281.54 447 9.62 15.57 321.10 3112 11.09 39.56 14.68 16.44 274.43 182 8.53 16.42 313.79 1818 11.09 39.36 13.99 17.58 275.54 190 7.04 17.56 306.83 1528 14.65 31.29 16.25 18.47 277.34 300 9.04 18.45 296.57 2621 10.95 19.23 14.20 19.62 265.43 342 15.58 19.41 291.84 1406 13.45 26.40 20.59 20.50 258.08 529 14.15 20.50 293.16 1135 18.21 35.08 23.06 21.56 271.15 239 12.98 21.54 297.96 1200 20.41 26.82 24.19 22.49 250.75 742 13.66 22.49 290.27 2385 18.57 39.52 23.05 23.57 252.22 320 13.00 23.47 296.92 747 21.77 44.70 25.36 24.41 245.85 506 7.08 24.40 280.44 959 14.82 34.59 16.43 25.50 250.06 215 10.77 25.53 284.05 456 14.81 33.99 18.31 26.42 256.29 279 6.24 26.29 286.71 249 11.23 30.42 12.85 Table 1. Distribution of temperature, f CO2@13, and increase df CO2@13 data binned by 1 ◦C temperature interval for the two periods 1995–1997 and 2013–2015. xCO2 concentrations measured at the Lampedusa Island station (Italy) (35◦31′ N, 12◦37′ E) (http://ds.data.jma.go.jp/ gmd/wdcgg/, last access: 14 September 2018) (see Eq. 3 in Hood and Merlivat, 2001). Considering a mean annual in situ temperature equal to 18.25 ◦C and an atmospheric pres- sure of 1 atm, we derived a mean atmospheric f CO2 equal to 355.3 ± 0.8 µatm for 1995–1997 and 389.6 ± 0.9 µatm for 2013–2015, which is an increase of 34.3 ± 2.3 µatm (95 % conﬁdence interval) (Table 2). 3.5 Changes in atmospheric and seawater f CO2 The increase in atmospheric f CO2 from 1995–1997 to 2013–2015 was computed from the monthly atmospheric www.biogeosciences.net/15/5653/2018/ Biogeosciences, 15, 5653–5662, 2018 L. Merlivat et al.: Increase of dissolved inorganic carbon and decrease in pH 5659 Table 2. Seasonally detrended long-term and annual trends of seawater carbonate chemistry and atmospheric composition. df CO2a df CO2a dDICa dpHSWSc df CO2 dDIC annual dpHSWSc @13 µatm @T µatm µmol kg−1 pH unit @T annual µmol kg1 yr1 annual pH µatm yr−1 unit yr1 Sea surface 33.2 ± 3.3 41.4 ± 4.1 25.2 ± 2.7 −0.0397 ± 0.0042 2.30 ± 0.23 1.40 ± 0.15 −0.0022 ± 0.0002 Atmosphere 34.3 ± 2.3 20.8 ± 1.3b 1.91 ± 0.13 1.15 ± 0.07 Lampedusa data df CO2@Tair/ 0.83 ± 0.10 0.83 ± 0.09 df CO2@Tsea T , mean annual temperature equal to 18.25 ◦C; a change from 1995–1997 to 2013–2015; b dDICant; c dpHSWS computed at T . L. Merlivat et al.: Increase of dissolved inorganic carbon and decrease in pH 5659 T , mean annual temperature equal to 18.25 ◦C; a change from 1995– close to 2000 m (Coppola et al., 2016; Pasqueron de Fom- mervault et al., 2015). These episodes of strong and deep ver- tical mixing must have entrained DIC-rich LIW in the surface waters. This entrainment could be causing an increase in DIC between the 1995–1997 and 2013–2015 periods. Monthly surface samples collected at the Dyfamed time series sta- tion between 1998 and 2013 indicate an increasing DIC trend of 1.35 µmol kg−1 yr−1. This value is uncertain (r2 = 0.05) because of the large seasonal variability displayed in the monthly samples (Gemayel et al., 2015). Nevertheless, this value is closer to the trend we calculated between the two periods, 1993–1995 and 2013–2015 (1.40 µmol kg−1 yr−1) than to the trend inferred from the atmospheric increase (1.15 µmol kg−1 yr−1). In the DYFAMED time series, we ﬁnd no evidence that the strong increase in MLD observed during winters 1999 and especially 2006 resulted in a further increase in DIC. supplied by A. G. Dickson for calibration. It is difﬁcult to identify the cause for a possible change in alkalinity be- tween the two periods that are 18 years apart, while no salinity change has been observed. At a coastal site 50 km away from DYFAMED, Kapsenberg et al. 3.5 Changes in atmospheric and seawater f CO2 (2017) have mea- sured an increase in alkalinity unrelated to salinity over the period from 2007 to 2015. They attribute it to changes in freshwater inputs from land. However, based on data from Coppola et al. (2016), alkalinity in the upper 50 m at DY- FAMED did not change signiﬁcantly from 2007 to 2014 (3.204 µmol kg−1, P = 0.0794, r2 = 0.08). Thus, we cannot conclude whether the difference observed at DYFAMED and BOUSSOLE between the two periods is real or an artifact of measurement techniques. As a sensitivity test, we com- pute the expected changes in DIC and pH from 1995–1997 to 2013–2015 for a mean alkalinity increase in 10 µmol kg−1. We obtain annual changes of dDIC = +0.46 µmol kg−1 yr−1 and dpH = −0.0001 pH unit yr−1, which are well below er- rors estimated in Sect. 3.4. Hence, such a change in alkalin- ity does not signiﬁcantly affect the increase in DIC and the decrease in pH shown in Table 2. The monthly cruises of the Dyfamed time-series study have also been analysed in order to investigate the hydro- logical changes and some biological consequences over the period 1995–2007 (Marty and Chiavérini, 2010). These au- thors show that extreme convective mixing events such as those recorded in 1999 and 2006 are responsible for large increases in nutrient content in surface layers and conclude that biological productivity is increasing, especially during the 2003–2006 period, which could lead to a larger consump- tion of carbon, i.e. a decrease in DIC. 4.2 Drivers of the temporal change in DIC in surface waters The increase in sea surface DIC from 1995–1997 to 2013– 2015 is 25.2±2.7 µmol kg−1 (Table 2), whereas the expected contribution due to ocean uptake of anthropogenic CO2 is 20.8 ± 1.3 µmol kg−1. In order to interpret the difference be- tween these two values, we examine potential changes that may result from the inter-annual variability in local physical and biological processes or anthropogenic carbon invasion from lateral advection of Atlantic waters. 4.2.2 Anthropogenic carbon exchange through the Strait of Gibraltar. The concentration of oceanic anthropogenic carbon (Cant) is not a directly measurable quantity. To estimate it, several em- pirical methods have been developed. Flecha et al. (2012) computed the anthropogenic carbon inventory in the Gulf of Cadiz. They used observations made during a cruise in October 2008 throughout the oceanic area covered by the Gulf of Cadiz and the Strait of Gibraltar to estimate Cant with three methods, which are 1C* (Gruber et al., 1996), TrOCA (Touratier and Goyet, 2004; Touratier et al., 2007) 4.2.1 Natural variability Time series of the mixed layer depth (MLD) show a strong variability in winter at inter-annual scale. During the two pe- riods, 1995–1997 and 2013–2015, the winter MLD never ex- ceeded 220 m, whereas values over 300 m were observed in 1999 and especially in February and March 2006, with values L. Merlivat et al.: Increase of dissolved inorganic carbon and decrease in pH and ϕC0 T (Vázquez-Rodríguez et al., 2009). In the three cases, their results indicate a net import of Cant from the At- lantic towards the Mediterranean through Gibraltar. creasing and decreasing annual trends of DIC and pH ex- tends from 0.93 ± 0.24 to 1.89 ± 0.45 µmol kg−1 yr−1 and −0.0014 ± 0.0005 to −0.0026 ± 0.0006, respectively. The Revelle factor of surfaces waters vary from 9–10 in low lat- itudes to 12–15 in the subpolar time series sites, with higher Revelle factor values reﬂecting a reduced capacity to absorb atmospheric CO2. The data show that the increase in DIC is not only controlled by the buffer capacity of the water but also by the compounding effects of changes in physical fac- tors like the strengthening of winter mixing or larger air–sea uptake (Olafson et al., 2010). Schneider et al. (2010), using the transit-time distribu- tion method applied to a data set from a Mediterranean cruise in 2001, estimated a net anthropogenic carbon ﬂux across the Strait of Gibraltar into the Mediterranean Sea of 3.5 Tg C yr−1. Over the whole period from 1850 to 2001, this contribution of Cant represents almost 10 % of the to- tal Cant inventory of the Mediterranean Sea. Accordingly, about 90 % must have been taken directly from an equilib- rium with atmospheric CO2. Based on a high-resolution re- gional model, Palmiéri et al. (2015) computed the anthro- pogenic carbon storage in the Mediterranean basin. They concluded that 75 % of the total storage of Cant in the whole basin comes from the atmosphere and 25 % from net trans- port from the Atlantic through the Strait of Gibraltar. The ﬁndings of these two studies support our estimated change in DIC of (17 ± 10) % in addition to the direct contribution of air–sea exchange suggesting that it could result from the anthropogenic carbon input from the Atlantic Ocean towards the Mediterranean basin. The increase in DIC computed at DYFAMED is in the upper range of values reported in the other time series. A low Revelle factor of close to 10 characterises the Mediter- ranean Sea because of its warm and high-alkalinity waters. 5 Conclusion High-frequency ocean f CO2 measurements made by CAR- IOCA sensors have been used to calculate trends in f CO2, DIC and pH over a period of two decades, notwithstanding the short-time and natural seasonal variability of these prop- erties at the sea surface. We have estimated a large change in sea-surface carbonate chemistry, an increase in DIC and a decrease in pH. The computed increase in DIC is larger than the change expected from a chemical equilibrium with atmospheric CO2.This could be the result of the strong inter- annual variability of the winter mixing observed between the two periods of 1993–1995 and 2013–2015. Likewise, our re- sults support modelling work and analysis of vertical pro- ﬁles measurements that suggest that the Atlantic Ocean con- tributes a substantial amount of anthropogenic carbon to the Mediterranean basin, (17±10) %, which lies between the es- timates of 10 % (Schneider et al., 2010) and 25 % (Palmiéri et al., 2015). L. Merlivat et al.: Increase of dissolved inorganic carbon and decrease in pH Moreover, as the result of a relatively short deep-water re- newal time estimated to be 20–40 years in the western basin (Schneider et al., 2010), the waters of the Mediterranean Sea have a relatively high capacity to absorb anthropogenic CO2 from the atmosphere and transport it to deep water. The calculated decrease in pH in the surface water at DY- FAMED and in the global ocean are quite similar, despite the higher alkalinity of the Mediterranean Sea. Thermodynamic equilibrium calculations have highlighted the alkalinity’s ef- fect on the anthropogenic acidiﬁcation of the Mediterranean Sea (Palmiéri et al., 2015). Their results show that, notwith- standing a higher total alkalinity, the average anthropogenic change in the surface pH of the Mediterranean Sea does not differ signiﬁcantly from that of the average of global oceans. Huertas et al. (2009) and Schneider et al. (2010) re- port DICant surface concentrations respectively equal to 65– 70 µmol kg−1 at the Strait of Gibraltar in the years 2005– 2007 and close to 65 µmol kg−1 in the western basin in 2001. We extrapolate these ﬁgures to the year 2014, assuming a mean increased rate of DIC equal to 1.4 µmol kg−1 yr−1 as previously computed (Table 2). Taking into account the in- crease in DICant equal to 25.2 µmol kg−1 between 1995– 1997 and 2013–2015, we estimate that the contribution of the change in DICant over the last 18 years represents ∼30 % of the total change since the beginning of the industrial period (t> ∼1800). www.biogeosciences.net/15/5653/2018/ www.biogeosciences.net/15/5653/2018/ Biogeosciences, 15, 5653–5662, 2018 5660 References Álvarez, M., Sanleón-Bartolomé, H., Tanhua, T., Mintrop, L., Luchetta, A., Cantoni, C., Schroeder, K., and Civitarese, G.: The CO2 system in the Mediterranean Sea: a basin wide perspective, Ocean Sci., 10, 69–92, https://doi.org/10.5194/os-10-69-2014, 2014. Bates, N., Astor, Y., Church, M., Currie, K., Dore, J., Gonaález- Dávila, M., Lorenzoni, L., Muller-Karger, F., Olafsson, J., and Santa-Casiano, M.: A Time-Series View of Changing Ocean Chemistry Due to Ocean Uptake of Anthropogenic CO2 and Ocean Acidiﬁcation, Oceanography, 27, 126–141, 2014. Antoine, D., Chami, M., Claustre, H., d’Ortenzio, F., Morel, A., Bécu, G., Gentili, B., Louis, F., Ras, J., Roussier, E., Scott, A.J., Tailliez, D., Hooker, S. B., Guevel, P., Desté, J.-F., Dempsey, C., and Adams, D.: BOUSSOLE: A joint CNRS-INSU, ESA, CNES, and NASA ocean color calibration and validation activ- ity, NASA Tech. Memo. 2006-214147, 2006. Bégovic, M. and Copin-Montegut, C.: Processes controlling annual variations in the partial pressure of f CO2 in surface waters of the central northwestern Mediterranean sea (Dyfamed site), Deep- Sea Res. Pt. II, 49, 2031–2047, 2002. Chen, G. T. and Millero, F. J.: Gradual increase of oceanic CO2, Nature, 277, 205–206, 1979. Antoine, D., d’Ortenzio, F., Hooker, S. B., Bécu, G., Gentili, B., Tailliez, D., and Scott, A. J.: Assessment of uncertainty in the ocean reﬂectance determined by three satellite ocean color sen- sors (MERIS, SeaWiFS and MODIS-A) at an offshore site in the Mediterranean Sea (BOUSSOLE project), J. Geophys. Res., 113, https://doi.org/10.1029/2007JC004472, 2008. Copin-Montegut, C. and Bégovic, M.: Distributions of carbonate properties and oxygen along the water column (0–2000 m) in the central part of the NW Mediterranean Sea (Dyfamed site): inﬂu- ence of winter vertical mixing on air–sea CO2 and O2 exchanges, Deep-Sea Res. Pt. II 49, 2049–2066, 2002. Copin-Montégut, C., Bégovic, M., and Merlivat, L.: Variability of the partial pressure of CO2 on diel to annual time scales in the Northwestern Mediterranean Sea, Mar. Chem., 85, 169–189, 2004. Bakker, D. C. E., Pfeil, B., Smith, K., Hankin, S., Olsen, A., Alin, S. R., Cosca, C., Harasawa, S., Kozyr, A., Nojiri, Y., O’Brien, K. M., Schuster, U., Telszewski, M., Tilbrook, B., Wada, C., Akl, J., Barbero, L., Bates, N. R., Boutin, J., Bozec, Y., Cai, W.-J., Castle, R. D., Chavez, F. P., Chen, L., Chierici, M., Cur- rie, K., de Baar, H. J. W., Evans, W., Feely, R. A., Fransson, A., Gao, Z., Hales, B., Hardman-Mountford, N. J., Hoppema, M., Huang, W.-J., Hunt, C. L. Merlivat et al.: Increase of dissolved inorganic carbon and decrease in pH Boussole data (2013–2015) are available in the SEANOE database (Merlivat and Boutin, 2018). R., Wanninkhof, R., and Watson, A. J.: An update to the Surface Ocean CO2 Atlas (SOCAT version 2), Earth Syst. Sci. Data, 6, R., Wanninkhof, R., and Watson, A. J.: An update to the Surface Ocean CO2 Atlas (SOCAT version 2), Earth Syst. Sci. Data, 6, 69–90, https://doi.org/10.5194/essd-6-69-2014, 2014. B kk D C E Pf il B L d C S M t l N O’B i K 69–90, https://doi.org/10.5194/essd-6-69-2014, 2014. Bakker, D. C. E., Pfeil, B., Landa, C. S., Metzl, N., O’Brien, K. M., Olsen, A., Smith, K., Cosca, C., Harasawa, S., Jones, S. D., Nakaoka, S.-I., Nojiri, Y., Schuster, U., Steinhoff, T., Sweeney, C., Takahashi, T., Tilbrook, B., Wada, C., Wanninkhof, R., Alin, S. R., Balestrini, C. F., Barbero, L., Bates, N. R., Bianchi, A. A., Bonou, F., Boutin, J., Bozec, Y., Burger, E. F., Cai, W.-J., Castle, R. D., Chen, L., Chierici, M., Currie, K., Evans, W., Feather- stone, C., Feely, R. A., Fransson, A., Goyet, C., Greenwood, N., Gregor, L., Hankin, S., Hardman-Mountford, N. J., Harlay, J., Hauck, J., Hoppema, M., Humphreys, M. P., Hunt, C. W., Huss, B., Ibánhez, J. S. P., Johannessen, T., Keeling, R., Kitidis, V., Körtzinger, A., Kozyr, A., Krasakopoulou, E., Kuwata, A., Land- schützer, P., Lauvset, S. K., Lefèvre, N., Lo Monaco, C., Manke, A., Mathis, J. T., Merlivat, L., Millero, F. J., Monteiro, P. M. S., Munro, D. R., Murata, A., Newberger, T., Omar, A. M., Ono, T., Paterson, K., Pearce, D., Pierrot, D., Robbins, L. L., Saito, S., Salisbury, J., Schlitzer, R., Schneider, B., Schweitzer, R., Sieger, R., Skjelvan, I., Sullivan, K. F., Sutherland, S. C., Sutton, A. J., Tadokoro, K., Telszewski, M., Tuma, M., van Heuven, S. M. A. C., Vandemark, D., Ward, B., Watson, A. J., and Xu, S.: A multi- decade record of high-quality f CO2 data in version 3 of the Sur- face Ocean CO2 Atlas (SOCAT), Earth Syst. Sci. Data, 8, 383– 413, https://doi.org/10.5194/essd-8-383-2016, 2016. A Ch h C i G ál 69 90, https://doi.org/10.5194/essd 6 69 2014, 2014. Bakker, D. C. E., Pfeil, B., Landa, C. S., Metzl, N., O’Brien, K. Competing interests. The authors declare that they have no conﬂict of interest. Competing interests. The authors declare that they have no conﬂict of interest. Acknowledgements. Seawater samples were analysed for DIC and Alk by the SNAPO-CO2 at LOCEAN in Paris. The CO2Sys toolbox of Pierrot et al. (2006) has been used for the calculations of DIC and pH. The adaptation of CARIOCA sensors to high pressure has been supported by the BIO-optics and CARbon EXperiment (BIOCAREX) project, funded by the Agence Nationale de la Recherche (ANR, Paris). We are grateful for the helpful comments from Gilles Reverdin and the reviewers of the manuscript. Many thanks to Laurent Coppola, who kindly provided additional MLD data at Dyfamed. A., Mathis, J. T., Merlivat, L., Millero, F. J., Monteiro, P. M. S., Munro, D. R., Murata, A., Newberger, T., Omar, A. M., Ono, T., Paterson, K., Pearce, D., Pierrot, D., Robbins, L. L., Saito, S., Salisbury, J., Schlitzer, R., Schneider, B., Schweitzer, R., Sieger, Edited by: Jack Middelburg Reviewed by: two anonymous referees Edited by: Jack Middelburg Reviewed by: two anonymous referees Tadokoro, K., Telszewski, M., Tuma, M., van Heuven, S. M. A. C., Vandemark, D., Ward, B., Watson, A. J., and Xu, S.: A multi- decade record of high-quality f CO2 data in version 3 of the Sur- 4.3 Long-term trends in surface DIC and pH The annual changes in DIC and pHSWS calculated between 1995–1997 and 2013–2015 are respectively equal to 1.40 ± 0.15 µmol kg−1 and −0.0022 ± 0.0002. At the DYFAMED site, at 10 m, Marcellin Yao et al. (2016) studied the time variability of pH over 1995–2011 based on measurements of T , S, Alk and DIC sampled approximately once a month. They computed a mean annual decrease in pH, −0.003 ± 0.001, which is not signiﬁcantly different from our estimate. For the global surface ocean, Lauvset et al. (2015) have re- ported a mean rate of decrease in pH, −0.0018 ± 0.0004 for 1991–2011. This value is also within the limits of uncertainty of the pH change computed in our study. Bates et al. (2014) examined changes in surface seawa- ter CO2-carbonate chemistry at the locations of seven ocean CO2 time series that have been gathering sustained observa- tions from 15 to 30 years with monthly or seasonal sampling. Six stations are located in the Atlantic and Paciﬁc oceans in a latitudinal band between 10 and 68◦N. The range of in- Data availability. The time series data from Dyfamed (1995–1997) are available in the SOCAT v3 database (Bakker et al., 2016). The www.biogeosciences.net/15/5653/2018/ Biogeosciences, 15, 5653–5662, 2018 5661 References W., Huss, B., Ichikawa, T., Johan- nessen, T., Jones, E. M., Jones, S. D., Jutterström, S., Kitidis, V., Körtzinger, A., Landschützer, P., Lauvset, S. K., Lefèvre, N., Manke, A. B., Mathis, J. T., Merlivat, L., Metzl, N., Murata, A., Newberger, T., Omar, A. M., Ono, T., Park, G.-H., Pater- son, K., Pierrot, D., Ríos, A. F., Sabine, C. L., Saito, S., Salis- bury, J., Sarma, V. V. S. S., Schlitzer, R., Sieger, R., Skjelvan, I., Steinhoff, T., Sullivan, K. F., Sun, H., Sutton, A. J., Suzuki, T., Sweeney, C., Takahashi, T., Tjiputra, J., Tsurushima, N., van Heuven, S. M. A. C., Vandemark, D., Vlahos, P., Wallace, D. W. Coppola, L., Diamond Riquier, E., and Carval, T.: Dyfamed obser- vatory data, SEANOE, https://doi.org/10.17882/43749, 2016. Dickson, A. G. and Millero, F. J.: A comparison of the equilibrium constants for the dissociation of carbonic acid in seawater media, Deep-Sea Res., 34, 1733–1743, 1987. Dickson, A. G., Sabine, C. L., and Christian, J. R. (Eds.): Guide to Best Practices for Ocean CO2 Measurements, PICES Special Publication 3, 191 pp., 2007. Edmond, J. M.: High precision determination of titration alkalinity and total carbon dioxide content of seawater by potentiometric titration, Deep-Sea Res., 17, 737–750, 1970. L. Merlivat et al.: Increase of dissolved inorganic carbon and decrease in pH 5662 Flecha, S., Pérez, F. F., Navarro, G., Ruiz, J., Olivé, I., Rodríguez- Gálvez, S., Costas, E., and Huertas, I. E.: Anthropogenic carbon inventory in the Gulf of Cádiz, J. Marine Syst., 92, 67–75, 2012. Merlivat, L. and Brault, P.: CARIOCA BUOY, Carbon Dioxide Monitor, Sea Technol., 23–30, 1995. Millero, F. J.: The marine inorganic carbon cycle, Chem. Rev., 107, 308–341, 2007. Gattuso, J.-P. and Hansson, L.: Ocean Acidiﬁcation, Oxford Uni- versity Press, Oxford, UK, 352 pp., 2011. Millot, C.: Circulation in the Western Mediterranean Sea, J. Marine Syst., 20, 423–442, 1999. Gemayel, E., Hassoun, A. E. R., Benallal, M. A., Goyet, C., Ri- varo, P., Abboud-Abi Saab, M., Krasakopoulou, E., Touratier, F., and Ziveri, P.: Climatological variations of total alka- linity and total dissolved inorganic carbon in the Mediter- ranean Sea surface waters, Earth Syst. Dynam., 6, 789–800, https://doi.org/10.5194/esd-6-789-2015, 2015. Olafsson, J., Olafsdottir, S. R., Benoit-Cattin, A., and Takahashi, T.: The Irminger Sea and the Iceland Sea time series measure- ments of sea water carbon and nutrient chemistry 1983–2008, Earth Syst. Sci. Data, 2, 99–104, https://doi.org/10.5194/essd-2- 99-2010, 2010. Gruber, N., Sarmiento, J. L., and Stocker, T. F.: An improved method for detecting anthropogenic CO2 in the oceans, Global Biogeochem. Cy., 10, 809–837, 1996. Palmiéri, J., Orr, J. C., Dutay, J.-C., Béranger, K., Schneider, A., Beuvier, J., and Somot, S.: Simulated anthropogenic CO2 storage and acidiﬁcation of the Mediterranean Sea, Biogeosciences, 12, 781–802, https://doi.org/10.5194/bg-12-781-2015, 2015. Heimbürger, L.-E., Lavigne, H., Migon, C., D’Ortenzio, F., Es- tournel, C., Coppola, L., and Miquel, J.-C.: Temporal variabil- ity of vertical export ﬂux at the DYFAMED time-series station (Northwestern Mediterranean Sea), Progr. Oceanogr., 119, 59– 67, 2013. Pasqueron de Fommervault, O., Migon, C., D’Ortenzio, F., Ribera d’Alcalà, M., and Coppola, L.: Temporal variability of nutri- ent concentrations in the northwestern Mediterranean sea (DY- FAMED time-series station), Deep-Sea Res. Pt. I, 100, 1–12, 2015. Hood, E. M. and Merlivat, L.: Annual and interannual variations of f CO2 in the northwestern Mediterranean Sea: Results from hourly measurements made by CARIOCA buoys, 1995–1997, J. Mar. Res., 59, 113–131, 2001. Pierrot, D. E. L. and Wallace, D. W. R.: MS Excel Program Devel- oped for CO2 System Calculations, ORNL/CDIAC-105a, Car- bon Dioxide Information Analysis Center, Oak Ridge National Laboratory, U.S. Department of Energy, Oak Ridge, Tennessee, https://doi.org/10.3334/CDIAC/otg.CO2SYS_XLS_CDIAC105a, 2006. Huertas, I. E., Ríos, A. F., García-Lafuente, J., Makaoui, A., Rodríguez-Gálvez, S., Sánchez-Román, A., Orbi, A., Ruíz, J., and Pérez, F. L. Merlivat et al.: Increase of dissolved inorganic carbon and decrease in pH F.: Anthropogenic and natural CO2 exchange through the Strait of Gibraltar, Biogeosciences, 6, 647–662, https://doi.org/10.5194/bg-6-647-2009, 2009. Rolland, J. and Bouin, M. N.: Thermosalinograph, azur buoy [data set], CMM/CNRM (Météo-France), https://doi.org/10.6096/hymex.azurbuoy.thermosalinograph.20100308 (last access: 19 September 2018), 2010. Kapsenberg, L., Alliouane, S., Gazeau, F., Mousseau, L., and Gat- tuso, J.-P.: Coastal ocean acidiﬁcation and increasing total al- kalinity in the northwestern Mediterranean Sea, Ocean Sci., 13, 411–426, https://doi.org/10.5194/os-13-411-2017, 2017. Sabine, C. L., Feely, R. A., Millero, F. J., Dickson, A. G., Langdon, C., Mecking, S., and Greeley, D.: Decadal changes in Paciﬁc carbon, J. Geophys. Res., 113, C07021, https://doi.org/10.1029/2007JC004577, 2008. Lauvset, S. K., Gruber, N., Landschützer, P., Olsen, A., and Tjiputra, J.: Trends and drivers in global surface ocean pH over the past 3 decades, Biogeosciences, 12, 1285–1298, https://doi.org/10.5194/bg-12-1285-2015, 2015. Schneider, A., Tanhua, T., Körtzinger, A., and Wallace, D. W. R.: High anthropogenic carbon content in the eastern Mediterranean, J. Geophys. Res., 115, C12050, https://doi.org/10.1029/2010JC006171, 2010. Marcellin Yao, K., Marcou, O., Goyet, C., Guglielmi, V., Touratier, F., and Savy, J.-P.: Time variability of the north-western Mediter- ranean Sea pH over 1995–2011, Mar. Environ. Res., 116, 51–60, 2016. Takahashi, T., Olafson, J., Goddard, J. G., Chipman, D. W., and Sutherland, G.: Seasonal variations of CO2 and nutrients in the high-latitude surface oceans:a comparative study, Global Bio- geochem. Cy., 7, 843–878, 1993. Marty, J. C. and Chiavérini, J.: Hydrological changes in the Ligurian Sea (NW Mediterranean, DYFAMED site) during 1995–2007 and biogeochemical consequences, Biogeosciences, 7, 2117– 2128, https://doi.org/10.5194/bg-7-2117-2010, 2010. Touratier, F. and Goyet, C.: Applying the new TrOCA approach to assess the distribution of anthropogenic CO2 in the Atlantic Ocean, J. Marine Syst., 46, 181–197, 2004. Marty, J. C., Chiaverini, J., Pizay, M., D., and Avril, B.: Seasonal and interannual dynamics of nutrients and phytoplankton pig- ments in the western Mediterranean Sea at the DYFAMED time- series station (1991–1999), Deep-Sea Res. Pt. II, 49, 1965–1985, 2002. Touratier, F. and Goyet, C.: Decadal evolution of anthropogenic CO2 in the northwestern Mediterranean Sea from the mid-1990s to the mid-2000s, Deep-Sea Res. Pt. I, 56, 1708–1716, 2009. Touratier, F., Azouzi, L., and Goyet, C.: CFC-11, 14C and 3H trac- ers as a means to assess anthropogenic CO2 concentrations in the ocean, Tellus B, 59, 318–325, 2007. McKinley, G. A., Fay, A. R., Takahashi, T., and Metzl, N.: Conver- gence of atmospheric and North Atlantic carbon dioxide trends on multidecadal timescales, Nat. Geosci., 4, 606–610, 2011. www.biogeosciences.net/15/5653/2018/ Biogeosciences, 15, 5653–5662, 2018 www.biogeosciences.net/15/5653/2018/ L. Merlivat et al.: Increase of dissolved inorganic carbon and decrease in pH Vázquez-Rodríguez, M., Padin, X. A., Ríos, A. F., Bellerby, R. G. J., and Pérez, F. F.: An upgraded carbon-based method to estimate the anthropogenic fraction of dissolved CO2 in the Atlantic Ocean, Biogeosciences Discuss., 6, 4527–4571, https://doi.org/10.5194/bgd-6-4527-2009, 2009. Mehrbach, C., Culberson, C. H., Hawley, J. E., and Pytkow- icx, R. M.: Measurement of the apparent dissociation constants of carbonic acid in seawater at atmospheric pressure, Limnol. Oceanogr., 18, 897–907, 1973. g g Woosley, R. J., Millero, F. J., and Wanninkhof, R.: Rapid anthro- pogenic changes in CO2 and pH in the Atlantic Ocean: 2003– 2014, Global Biogeochem. Cy., 30, 70–90, 2016. Merlivat, L. and Boutin, J.: Mediterranean Sea surface CO2 partial pressure and temperature data, SEANOE, https://doi.org/10.17882/56709, 2018. Biogeosciences, 15, 5653–5662, 2018 www.biogeosciences.net/15/5653/2018/
https://openalex.org/W4366782548	https://www.qeios.com/read/7GKVRB/pdf	English	null	Review of: "A Proposed Heuristic for Guessing Distributions"	null	2,023	cc-by	322	Qeios, CC-BY 4.0 · Review, April 23, 2023 Review of: "A Proposed Heuristic for Guessing Distributions" Robert Veszteg Potential competing interests: No potential competing interests to declare. Qeios ID: 7GKVRB · https://doi.org/10.32388/7GKVRB Potential competing interests: No potential competing interests to declare. The proposed method for making a guess tries to find a possible probability distribution by matching the observed median to the theoretical median of all probability distributions under consideration. In the two examples described in the paper, the “layman” decision-maker is looking at a very specific family of distributions. In example 1, they are all symmetric. In example 2, it is the family of Pareto distributions. So who exactly chooses that family of distributions for the layman who then needs to match medians? In other words, what exactly do wee need to assume about the decision-maker's statsitical sophistication for this model to work? Later, for example 1, the authors claim that the chosen distribution is the best, becasue it seems to be in line with the observed income distribution from the United States. The trouble is that showing a single example where the method works is not a conclusive evidence. Using the data from the the very same link (3) that the authors are using, I have confirmed that for the real/observed US income distribution in 2021, distribution B performs better than disbribution A (the multinomial probabilities are 0.0974 vs 0.0799). But for 1971 that would be the exact opposite: distribution A fits better than B (0.1242 vs 0.0584). There is nothing in the proposed model that would help the decision-maker the correct guess for different problems, different years, different countries, etc. Relying on a simple criterion, e.g. the median, will not make wonders. The article in its second version is still very much misleading as it hides important assumptions. I suggest the authors to make major changes and take a much more rigorous approach. Qeios ID: 7GKVRB · https://doi.org/10.32388/7GKVRB 1/1
https://openalex.org/W2951108589	https://bmcbioinformatics.biomedcentral.com/track/pdf/10.1186/s12859-018-2203-5	English	null	Combining RNA-seq data and homology-based gene prediction for plants, animals and fungi	bioRxiv (Cold Spring Harbor Laboratory)	2,017	cc-by	9,569	© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Combining RNA-seq data and homology-based gene prediction for plants, animals and fungi Jens Keilwagen1, Frank Hartung1, Michael Paulini2, Sven O. Twardziok3 and Jan Grau4 ns Keilwagen1, Frank Hartung1, Michael Paulini2, Sven O. Twardziok3 and Jan Grau4 Abstract Background: Genome annotation is of key importance in many research questions. The identification of protein-coding genes is often based on transcriptome sequencing data, ab-initio or homology-based prediction. Recently, it was demonstrated that intron position conservation improves homology-based gene prediction, and that experimental data improves ab-initio gene prediction. Results: Here, we present an extension of the gene prediction program GeMoMa that utilizes amino acid sequence conservation, intron position conservation and optionally RNA-seq data for homology-based gene prediction. We show on published benchmark data for plants, animals and fungi that GeMoMa performs better than the gene prediction programs BRAKER1, MAKER2, and CodingQuarry, and purely RNA-seq-based pipelines for transcript identification. In addition, we demonstrate that using multiple reference organisms may help to further improve the performance of GeMoMa. Finally, we apply GeMoMa to four nematode species and to the recently published barley reference genome indicating that current annotations of protein-coding genes may be refined using GeMoMa predictions. Conclusions: GeMoMa might be of great utility for annotating newly sequenced genomes but also for finding homologs of a specific gene or gene family. GeMoMa has been published under GNU GPL3 and is freely available at http://www.jstacs.de/index.php/GeMoMa. Keywords: Homology-based gene prediction, RNA-seq, Genome annotation Keilwagen et al. BMC Bioinformatics (2018) 19:189 https://doi.org/10.1186/s12859-018-2203-5 Keilwagen et al. BMC Bioinformatics (2018) 19:189 https://doi.org/10.1186/s12859-018-2203-5 Background Genome annotation pipelines utilize three main sources of information, namely evidence from wet-lab transcrip- tome studies [1, 2], ab-initio gene prediction based on general features of (protein-coding) genes [3, 4], and homology-based gene prediction relying on gene models of (closely) related, well-annotated species [5–7]. The annotation of protein-coding genes is of critical importance for many fields of biological research includ- ing, for instance, comparative genomics, functional pro- teomics, gene targeting, genome editing, phylogenetics, transcriptomics, and phylostratigraphy. The process of annotating protein-coding genes to an existing genome (assembly) can be described as specifying the exact genomic location of genes comprising all (partially) cod- ing exons. A difficulty in gene annotation is distinc- tion between protein-coding genes, transposons and pseudogenes. Experimental data allow for inferring coverage of gene predictions and splice sites bordering their exons, which may assist computational ab-initio or homology-based approaches. Due to the progress in the field of next gener- ation sequencing, RNA-seq has revolutionized transcrip- tomics [8]. Today, RNA-seq data is available for a wide range of organisms, tissues and environmental conditions, and can be utilized for genome annotation pipelines. Correspondence: jens.keilwagen@julius-kuehn.de 1Institute for Biosafety in Plant Biotechnology, Julius Kühn-Institut (JKI) - Federal Research Centre for Cultivated Plants, D-06484, Quedlinburg, Germany Full list of author information is available at the end of the article Correspondence: jens.keilwagen@julius-kuehn.de 1Institute for Biosafety in Plant Biotechnology, Julius Kühn-Institut (JKI) - Federal Research Centre for Cultivated Plants, D-06484, Quedlinburg, Germany Full list of author information is available at the end of the article In recent years, several programs have been developed that combine multiple sources allowing for a more accu- rate prediction of protein-coding genes [9–11]. MAKER2 © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Page 2 of 12 Keilwagen et al. BMC Bioinformatics (2018) 19:189 mapped RNA-seq data. Introns passing this filter define donor and acceptor splice sites, which are treated inde- pendently within GeMoMa. If splice sites with experimen- tal evidence have been detected in a genomic region with a good match to an exon of a reference transcript, these are collected for further use. If no splice sites with experimen- tal evidence have been detected in a genomic region with a good match to an exon of a reference transcript, GeMoMa resorts to conserved dinucleotides allowing to identify gene models that are not covered by RNA-seq data due to, e.g., very specifically or lowly expressed transcripts. Com- bining two potential exons, all in-frame combinations using the collected donor and acceptor splice sites are tested and scored according to the reference transcript. The best combination is used for the prediction. is a pipeline that integrates support of different resources including ab-initio gene predictors and RNA-seq data [9]. CodingQuarry is a pipeline for RNA-Seq assembly- supported training and gene prediction, which is only recommended for application to fungi [10]. Recently, [11] published BRAKER1 a pipeline for unsupervised RNA- seq-based genome annotation that combines the advan- tages of GeneMark-ET [12] and AUGUSTUS [4]. Here, we present an extension of GeMoMa [7] that uti- lizes RNA-seq data in addition to amino acid sequence and intron position conservation. We investigate the per- formance of GeMoMa on publicly available benchmark data [11] and compare it with state-of-the-art competitors [9–11]. Subsequently, we demonstrate how combining homology- based predictions based on gene models from multiple reference organisms can be used to improve the perfor- mance of GeMoMa. Finally, we apply GeMoMa to four nematode species provided by Wormbase [13] and to the recently published barley reference genome [14], where GeMoMa predictions will be included into future versions of the corresponding genome annotations. Based on this experimental evidence, the improved version of GeMoMa provides several new properties reported for gene predictions. The most prominent fea- tures are transcript intron evidence (tie) and transcript percentage coverage (tpc). The tie of a transcript varies between 0 and 1, and corresponds to the fraction of introns (i.e., splice sites of two neighboring exons) that are supported by split reads in the mapped RNA-seq data. In case of transcripts comprising a single coding exon, NA is reported. The tpc of a transcript also varies between 0 and 1, and corresponds to the fraction of (cod- ing) bases of a predicted transcript that are also covered by mapped reads in the RNA-seq data. Further properties reported by GeMoMa are i) tae and tde, the percentages of acceptor and donor sites, respectively, with RNA-seq evidence, ii) minCov and avgCov, the minimum and aver- age coverage, respectively, of the predicted transcript, and iii) minSplitReads, the minimum number of split reads supporting any of the predicted introns of a transcript. Optionally, GeMoMa reports pAA and iAA, the percent- age of positive-scoring and identical amino acids in a pairwise alignment, if the reference protein is provided as input. Methods In this section, we describe recent extensions of GeMoMa to make use of evidence from RNA-seq data, the RNA-seq pipelines used and the data considered in the benchmark and application studies. GeMoMa using RNA-seq Blue items represent input data sets, green boxes represent GeMoMa modules, while grey boxes represent external modules. The GeMoMa Annotation Filter allows to combine predictions from different reference species and produces the final output. RNA-seq data is optional of GeMoMa now allows for joining and reducing such pre- dictions using various filters. Filtering criteria comprise the relative GeMoMa score of a predicted transcript, fil- tering for complete predictions (starting with start codon and ending with stop codon), and filtering for evidence from multiple reference organisms. In addition, GAF also joins duplicate predictions that originate from different reference transcripts. genes in the cluster looking for discarded predictions that do not overlap with any selected prediction, which are recovered. In the benchmark studies comparing GeMoMa with state-of-the-art competitors, we directly use the GAF results without any further filters on attributes reported by the GeMoMa pipeline. In addition to the modules for annotating a genome (assembly) described above, we also provide two addi- tional modules in GeMoMa for analyzing and comparing to prediction to a reference annotation. The module Com- pareTranscripts determines that CDS of the reference annotation with the largest overlap with the prediction utilizing the F1 measure as objective function [7]. The module AnnotationEvidence computes tie and tpc of all CDSs of a given annotation. Hence, these two modules can be used to determine, whether a prediction is known, partially known or new and whether the overlapping annotation has good RNA-seq support. Initially, GAF filters predictions based on their rela- tive GeMoMa score, i.e., the GeMoMa score divided by the length of the predicted protein. This filter removes spurious predictions. Subsequently, the predictions are clustered based on their genomic location. Overlapping predictions on the same strand yield a common cluster. For each cluster, the prediction with the highest GeMoMa score is selected. Non-identical predictions overlapping the high-scoring prediction with at least a user-specified percentage of borders (i.e., splice sites, start and stop codon, cf. common border filter) are treated as alterna- tive transcripts. Predictions that have completely identical borders to any previously selected prediction are removed and only listed in the GFF attribute field alternative. All filtered predictions of a cluster are assigned to one gene with a generic gene name. Finally, GAF checks for nested GeMoMa using RNA-seq GeMoMa predicts protein-coding genes utilizing the gen- eral conservation of protein-coding genes on the level of their amino acid sequence and on the level of their intron positions, i.e., the locations of exon-exon boundaries in CDSs [7]. To this end, sequences of (partially) protein- coding exons are extracted from well-annotated reference genomes. Individual exons are then matched to loci on the target genome using tblastn [15], matches are adjusted for proper splice sites, start codons and stop codons, respec- tively, and joined to full, protein-coding genes models. In this process, the conserved dinucleotides GT and GC for donor splice sites, and AG for acceptor splice sites have been used for the identification of splice sites border- ing matches to the (partially) protein-coding exons of the reference transcripts. The improved version of GeMoMa may now also include experimental splice site evidence extracted from mapped RNA-seq data to improve the accuracy of splice site and, hence, exon annotation. We visualize the extended GeMoMa pipeline in Fig. 1. GeMoMa allows for computing and ranking multiple predictions per reference transcript, but does not fil- ter these predictions. Predictions of different reference transcripts might be highly overlapping or even identi- cal, especially if the reference transcripts are from the same gene family. Since GeMoMa 1.4, the default param- eters for number of predictions and contig threshold have been changed which might lead to an increased number of highly overlapping or identical predictions. In addi- tion, it might be beneficial to run GeMoMa starting from multiple reference species to broaden the scope of tran- scripts covered by the predictions. However, these may also result in redundant predictions for, e.g., orthologs or paralogs stemming from the different reference species considered. To handle such situations, the new module GeMoMa annotation filter (GAF) of the improved version Starting from mapped RNA-seq data, the module Extract RNA-seq evidence (ERE) allows for extracting introns and, if user-specified, read coverage of genomic regions. GeMoMa filters these introns using a user- specified minimal number of split reads within the Page 3 of 12 Keilwagen et al. BMC Bioinformatics (2018) 19:189 Fig. 1 GeMoMa workflow. Blue items represent input data sets, green boxes represent GeMoMa modules, while grey boxes represent external modules. The GeMoMa Annotation Filter allows to combine predictions from different reference species and produces the final output. RNA-seq data is optional Fig. 1 GeMoMa workflow. RNA-seq pipelines Computational pipelines have been used to infer gene annotation from RNA-seq data produced by next genera- tion sequencing methods. Dozens of tools and tool com- binations have been proposed. Here, we focus on the short read mapper TopHat2 [17], the transcript assemblers Cufflinks [1] and StringTie [2], and the coding sequence predictor TransDecoder [16]. Based on the transcript assemblers, we build two RNA-seq pipelines following the instructions in [11]. MAKER2 predictions Recently, we have shown that GeMoMa outperforms state-of-the-art homology-based gene predictors [7]. We are not aware of any homology-based gene predic- tion program that allows for incorporating of RNA-seq Keilwagen et al. BMC Bioinformatics (2018) 19:189 Page 4 of 12 with the previous benchmark study, we use the manually curated gene set of Wormbase. data. Hence, we provide predictions of MAKER2 using the same reference proteins as GeMoMa for a mini- mal comparison. Internally, MAKER2 uses exonerate [5] for homology-based gene prediction. We run MAKER2 with default parameters except protein2genome=1, and genome and protein set to the respective input files. In addition, we run MAKER2 using (i) RNA-seq data in form of Trinity 2.4 transcripts (-jaccard_clip) [16], (ii) homology in form of proteins of one related refer- ence species, and (iii) ab-initio gene prediction in form of Augustus 3.3 [4]. In this case, we run MAKER2 with default parameters except genome, est, protein, and augustus_species, which have been set to the corre- sponding species. For comparison, we run Maker2 with the same parameter settings but using the GeMoMa pre- dictions for protein_gff instead of using protein. For the analysis of barley, we use the latest genome assembly and gene annotation [14]. As reference species, we choose A. thaliana [22], B. distachyon [23], O. sativa [24], and S. italica [25] (Additional file 1: Table S2). In addition to genome assembly and gene annotation, we also used RNA-seq data from four different public available data sets (ERP015182, ERP015986, SRP063318, SRP071745). Reads were mapped and assembled using Hisat2 and StringTie [26]. As reference annotation, we used the union of high and low confidence annotation. As independent evidence for validating GeMoMa pre- dictions in the nematode species and barley, we use ESTs and cDNAs. While Wormbase provides coordinates for best BLAT matches, we adapt the pipeline and download all available EST from NCBI and map them to the genome using BLAT [27]. Results and discussion Benchmark The comparison of different software pipelines is often critical as a) specific parameters settings might be cru- cial for good results and b) different input might be used. For these reasons, we designed the benchmark as follows. First, we use publicly available gene predictions results. Second, we limit the number of reference species to one in the initial study. We used GeMoMa for predicting the gene annota- tions of A. thaliana, C. elegans, D. melanogaster, and S. pombe. In Table 1, we summarize the performance of BRAKER1, MAKER2, and CodingQuarry as reported in Hoff et al. [11], as well as the performance of GeMoMa with and without RNA-seq evidence, purely RNA-seq- based pipelines and various MAKER2 predictions. The results of CodingQuarry reported by Hoff et al. [11] devi- ate substantially from those originally reported by Testa et al. [10]. We find that the performance of CodingQuarry is highly sensitive to RNA-seq processing, whereas the performance of GeMoMa is barely affected (Additional file 1: Table S5). For all comparisons, we provide sen- sitivity (Sn) and specificity (Sp) for the categories gene, transcript, and exon, respectively [28]. In addition, we compare CodingQuarry with GeMoMa for S. cerevisiae (Additional file 1: Table S6). Data For the benchmark studies, we consider target species and their genome versions as specified in the BRAKER1 supplement. For the homology-based prediction by GeMoMa, we choose one closely related reference species per target species that are sequenced and annotated [13, 18–20]. For these species, we consider the latest genome versions available (Additional file 1: Table S1). For the analysis of C. elegans, we use the man- ually curated gene set of C. briggsae provided by Wormbase. In addition, we use the experimental evi- dence from RNA-seq data referenced in the BRAKER1 publication. For the analysis of the four nematode species, C. brenneri, C. briggsae, C. japonica, and C. remanei, we use the genome assembly and gene annotation of Wormbase WS257 [13]. We choose the model organism C. elegans as reference species (Additional file 1: Table S2). In addition to genome assembly and gene annotation, we also use publicly available RNA-seq data of these four nematode species, which have been mapped by Worm- base using STAR [21]. We used a minimum intron size of 25 bp, a maximum intron size of 15Kb, specify that only reads mapping once or twice on the genome are reported, and alignments are reported only if their ratio of mis- matches to mapped length is less than 0.02. In accordance First, we compare the two purely homology-based predictions, namely on the one hand side MAKER2 using exonerate and on the other hand side GeMoMa without RNA-seq data. In all cases, we use the same reference species and reference proteins. We find that MAKER2 using only homologous proteins has a higher exon speci- ficity than GeMoMa without RNA-seq data for C. elegans, while the opposite is true for all other categories and target species. Page 5 of 12 Keilwagen et al. BMC Bioinformatics (2018) 19:189 Table 1 Benchmark results on the BRAKER1 test sets MAKER2+ (exonerate) GeMoMa+ without RNA-seq data GeMoMa+ with RNA-seq data RNAseq- Cufflinks RNAseq- StringTie BRAKER1∗ MAKER2∗ CodingQuarry∗ MAKER2+ (exonerate, Trinity, Augustus) MAKER2+ (GeMoMa, Trinity, Augustus) Arabidopsis thaliana (ref. A. Keilwagen et al. BMC Bioinformatics (2018) 19:189 Keilwagen et al. BMC Bioinformatics (2018) 19:189 Page 6 of 12 Keilwagen et al. BMC Bioinformatics (2018) 19:189 Hence, we compare GeMoMa to combined gene pre- diction approaches. Specifically, we compare the perfor- mance of GeMoMa using RNA-seq evidence to BRAKER1 in Fig. 2, which provides the best overall performance in [11]. We find that GeMoMa performs better than BRAKER1 for the categories gene and transcript with the exception of gene and transcript sensitivity for C. elegans. Interestingly, we find the biggest improvements for D. melanogaster where gene/transcript sensitivity and specificity increases between 18.2 and 27.7. For the exon category, we find a less clear picture. In total, we observe the worst results for C. elegans where the sensitivity for all three categories decreases between 3.2 and 13.2, while the specificity increases only between 2.2 and 8.6. Notably, we generally find the worst gene/transcript sensitivity and specificity for C. elegans compared with the other target species considering the best performance of all tools. Second, we additionally consider RNA-seq data. MAKER2 does not allow for combining RNA-seq evi- dence and homology-based predictions without using any ab-initio gene predictor. In contrast, GeMoMa allows for additionally using intron position conservation and RNA-seq data. For this reason, we compare the perfor- mance of GeMoMa with and without RNA-seq evidence (Table 1). We find that sensitivity and specificity in almost all cases increases by up to 13.9 with only two exceptions for transcript specificity of A. thaliana and D. melanogaster which decreases by at most 0.4. Hence, we summarize that RNA-seq evidence improves the sen- sitivity and specificity of GeMoMa and should be used if available. Third, we compare the performance of GeMoMa using RNA-seq evidence to that of purely RNA-seq-based pipelines, namely Cufflinks and StringTie (Table 1). We find for all four species that GeMoMa using RNA-seq evidence outperforms purely RNA-seq-based pipelines. Interestingly, purely RNA-seq-based pipelines also yield the worst gene/transcript sensitivity and specificity for C. elegans. Comparing the results based on different transcript assemblers, we find that the results based on StringTie are better than those based on Cufflinks for A. thaliana and C. elegans, while the opposite is true for S. pombe. For D. melanogaster, both pipelines per- form comparably. Additional RNA-seq reads increasing the coverage might improve the performance of purely RNA-seq-based pipelines but could also improve the per- formance of GeMoMa. Data lyrata) Gene Sn 44.0 61.3 66.5 28.9 35.9 64.4 51.3 NA 56.9 57.9 Gene Sp 47.8 65.7 71.3 47.9 59.1 52.0 52.5 NA 65.7 67.8 Transcript Sn 37.5 52.2 57.2 26.6 33.7 55.0 43.5 NA 48.3 49.1 Transcript Sp 47.8 65.7 65.3 35.6 48.3 50.9 52.5 NA 65.7 67.8 Exon Sn 70.0 79.3 80.6 58.1 60.8 82.9 76.1 NA 81.8 82.1 Exon Sp 81.9 86.6 87.5 81.9 87.1 79.0 76.1 NA 87.5 88.6 Caenorhabditis elegans (ref. C. briggsae) Gene Sn 26.2 39.6 49.1 18.7 22.6 55.0 41.0 NA 40.5 47.3 Gene Sp 38.0 49.9 63.8 29.1 36.1 55.2 30.8 NA 51.5 56.4 Transcript Sn 21.0 30.7 39.8 16.2 20.0 43.0 31.3 NA 31.4 36.2 Transcript Sp 38.0 49.9 58.7 24.1 30.1 53.2 30.8 NA 51.5 56.4 Exon Sn 50.3 64.2 67.1 54.4 59.1 80.2 69.4 NA 70.5 75.2 Exon Sp 82.6 81.5 87.5 81.3 84.1 85.3 62.3 NA 85.6 86.7 Drosophila melanogaster (ref. D. simulans) Gene Sn 64.3 78.2 83.1 55.7 55.2 64.9 55.2 NA 61.5 64.0 Gene Sp 69.2 81.6 87.1 71.3 73.5 59.4 46.3 NA 69.6 71.9 Transcript Sn 44.1 52.9 65.0 48.7 49.0 46.1 38.5 NA 42.7 44.3 Transcript Sp 69.2 81.6 81.2 60.1 65.7 57.9 46.3 NA 69.6 71.9 Exon Sn 69.0 76.3 80.0 67.8 66.2 75.0 66.5 NA 74.3 76.3 Exon Sp 89.1 92.0 93.3 85.4 88.3 81.7 66.9 NA 88.0 89.1 Schizosaccharomyces pombe (ref. S. octosporus) Gene Sn 49.2 76.4 79.2 69.0 65.8 77.4 42.8 79.7 71.6 74.6 Gene Sp 59.9 84.6 88.0 93.8 92.5 80.5 68.7 72.6 88.1 89.1 Transcript Sn 49.2 76.4 79.2 69.0 65.8 77.4 42.8 79.7 71.6 74.6 Transcript Sp 59.9 84.6 87.6 80.5 71.3 76.5 68.7 72.6 88.1 89.1 Exon Sn 56.1 81.6 83.1 77.2 77.7 83.2 50.1 79.6 79.2 81.2 Exon Sp 73.3 88.6 91.9 87.6 81.7 83.2 71.4 81.7 92.0 92.6 The target species are given in multi-column rows. The same reference species, which is given in brackets, is used for all tools using homology-based gene prediction indicated by plus. The asterisks indicates that the performance of BRAKER1, MAKER2 and CodingQuarry is given as reported in [11]. The highest value per line is depicted in bold-face Keilwagen et al. BMC Bioinformatics (2018) 19:189 In summary, we find that the gene predictors MAKER2, BRAKER1, CodingQuarry and GeMoMa, and the tran- script assemblers Cufflinks and StringTie often perform quite well on exon level. The main difference becomes evident on transcript and gene level, where exons need to be combined correctly (Table 1) as reported earlier [29, 30]. Homology-based gene predictors might benefit from experimentally validated and manually curated ref- erence transcripts guiding the prediction of transcripts in the target organism. Although GeMoMa performed well, it is not able to pre- dict genes that do not show any homology to a protein in the reference species, while ab-initio gene predictors might fail in other cases. As both types of approaches have their specific advantages, users will probably use combi- nations of different gene predictors in practice to obtain a comprehensive gene annotation. Summarizing these three observations, we find that GeMoMa performs better than purely homology-based or purely RNA-seq-based pipelines and that including RNA-seq data improves the performance of GeMoMa. A. thaliana C. elegans D. melanogaster S. pombe Difference to result of BRAKER1 −10 0 10 20 30 Gene sensitivity Gene specificity Transcript sensitivity Transcript specificity Exon sensitivity Exon specificity Fig. 2 Benchmark results. The y-axis depicts the difference between the GeMoMa with RNA-seq data and the BRAKER1 performance Page 7 of 12 Page 7 of 12 Keilwagen et al. BMC Bioinformatics (2018) 19:189 In addition, we performed a small runtime study for the two main time-consuming steps of the pipeline to demon- strate that GeMoMa is reasonably fast (Additional file 1: Table S7). the inclusion of all available evidence and that per- formance is decreased if some important evidence is missed [9]. Furthermore, we compare GeMoMa using RNA- seq evidence with MAKER2 using RNA-seq evidence, homology-based and ab-initio gene prediction. In some cases, it is hard to compare these results as sensitivity of one tool is higher than the sensitivity of the other tool and the opposite is true for specificity. In machine learning, recall, also known as sensitivity, and precision, which is called specificity in the context of gene prediction evaluation [31], are combined into a single scalar value called F1 measure [32] that can be compared more easily. Keilwagen et al. BMC Bioinformatics (2018) 19:189 We combined sensitivity and specificity resulting in an F1 measure for each evaluation level gene, transcript and exon (Additional file 1 – Table S4) We find that in many cases GeMoMa using RNA-seq evidence outper- forms MAKER2. The reason for this observation might be that RNA-seq data and homology based gene predic- tion is used in MAKER2 to train ab-initio gene predictors, in this case Augustus. With the recommended parameter setting, homology-based gene predictions are not directly used for the final prediction and doing so might further improve performance. Combined gene prediction pipelines Combined gene prediction pipelines, as for instance MAKER2, use RNA-seq evidence, homology-based and ab-initio methods for predicting final gene models. MAKER2 uses exonerate by default for homology-based gene prediction. However, MAKER2 also provides the possibility to use other homology-based gene predictors instead of exonerate (cf. parameter protein_gff). For this reason, we compare the performance of MAKER2 using either exonerate or GeMoMa for homology based gene prediction (Table 1). In addition, we use Augustus as ab- initio gene prediction program and Trinity transcripts in MAKER2. We find that MAKER2 using GeMoMa performs better than MAKER2 using exonerate for all species and all measure. The improvement varies between 0.3% and 6.8% with clearly the biggest improvement for C. elegans. In addition, we find that the MAKER2 performance is substantially improved compared to the performance of the the previously reported MAKER2 predictions, either purely based on proteins (cf. Table 1, column MAKER2+ (exonerate)) or as reported in [11] (cf. Maker2∗). These other predictions do not utilize all available sources of information as they either ignore RNA-seq data and ab-initio gene prediction or homology to proteins of related species. Based on this observation, we agree that combined gene prediction pipelines benefit from Analysis of nematode species The relatively poor results for C. elegans in the bench- mark study, might be due to insufficiencies in the current C. briggsae annotation. Hence, we decided to scruti- nize the Wormbase annotation of four nematode species comprising C. brenneri, C. briggsae, C. japonica, and C. remanei based on the model organism C. elegans. We compare GeMoMa predictions with manually curated CDS from Wormbase. Based on RNA-seq evidence, we collect multi-coding-exon predictions of GeMoMa with tie=1 and compare these to the annotation as depicted in Fig. 4. y The new GAF module of GeMoMa allows for com- bining the predictions based on different reference organisms. The combined predictions may be filtered by number of reference species with perfect support (#evi- dence), as indicated by the dashed line. We find that combining multiple reference organisms improves predic- tion performance and stability. Depending on the number of supporting reference organisms required, gene speci- ficity and gene sensitivity may be balanced according to the needs of a specific application. We observe that (i) gene sensitivity increases but specificity decreases when requiring support from at least one reference organ- ism, whereas (ii) gene specificity increases but sensitivity decreases severely filtering for perfect support from all eight reference species. In summary, the inclusion of mul- tiple reference species may yield an improved prediction performance for GeMoMa using the GAF module, where we suggest to filter predictions for support by at least two but not necessarily all reference species. In summary, we find between 6 749 differences for C. briggsae and 12 903 for C. brenneri (cf. Fig. 4). The most interesting category are new multi-coding-exon pre- dictions, which vary between 53 for C. briggsae and 1 974 for C. brenneri. The largest category are GeMoMa pre- dictions that missed exons compared to annotated CDSs, which vary between 2 340 for C. japonica and 4 220 for C. remanei. We additionally filter the transcripts showing differ- ences to obtain a smaller, more conservative set of high- confidence predictions. First, we filter new multi-coding exon GeMoMa predictions for tpc=1 obtaining between 39 and 996 for C. briggsae and C. brenneri, respectively. Second, we filter GeMoMa predictions that have differ- ent splice sites compared to highly overlapping annotated transcripts, contain new exons, have missing exons, or have new and missing exons for tie<1 of the overlapping annotation. Influence of reference species Utilizing different fly species from FlyBase [33], we scru- tinize the influence of different or multiple reference species on the performance of GeMoMa using RNA-seq data (Additional file 1: Table S8). In Fig. 3, we depict gene sensitivity and gene specificity for eight different reference species indicated by points. We find that performance Fig. 3 Gene sensitivity and specificity for D. melanogaster using different or multiple reference species in GeMoMa. The points correspond to the eight reference species. In addition, the dashed line indicates the usage of multiple reference species. Using multiple reference species allows for filtering identical predictions from several reference as indicated by the numbers Fig. 3 Gene sensitivity and specificity for D. melanogaster using different or multiple reference species in GeMoMa. The points correspond to the eight reference species. In addition, the dashed line indicates the usage of multiple reference species. Using multiple reference species allows for filtering identical predictions from several reference as indicated by the numbers Page 8 of 12 Keilwagen et al. BMC Bioinformatics (2018) 19:189 varies with the reference species. In this specific case, D. sechellia and D. persimilis yield the worst results for sin- gle reference-based predictions. This observation might be related to the fact that genome assembly of D. sechellia and D. persimilis is of lower quality [34], while the genome of D. simulans has been updated [35] later. Besides these two outliers, the performance of the different fly species as reference species for D. melanogaster in GeMoMa cor- relates with their evolutionary distance [36]. Generally speaking, the closer a reference species is related to the target species D. melanogaster, the better is the perfor- mance in terms of gene sensitivity and specificity. Hence, we speculate that two requirements must be met to have a good reference species. First, the evolutionary distance between reference and target species should be small and second, the genome assembly and annotation of the reference species should be comprehensive and of high quality. supported by at least two of the eight reference species. In addition, we find 9 single-coding-exon predictions that do not overlap with any annotated transcript, have a tpc of 1 and are supported by at least two of the five reference species. In summary, those genes supported by multiple reference organisms or additional RNA-seq data might be promising candidates for extending the existing genome annotation of D. melanogaster. Analysis of nematode species Predictions that do not overlap with any annotated CDS are depicted in yellow, predictions that differ from annotated CDSs only in splice sites are depicted in green, predictions that have additional exons compared to annotated CDSs are depicted in turquoise, predictions that missed some exons compared to annotated CDSs are depicted in blue, predictions with additional and missing exons compared to annotated CDSs are depicted in pink, predictions that only differ in the start of the CDS compared to annotated CDS are depicted in red, and any other category is depicted in gray Fig. 4 Summary of difference for GeMoMa predictions with tie=1. The relaxed evaluation (left panel) depicts differences between GeMoMa predictions and annotation without any filter on the annotation, while the conservative evaluation (right panel) applies additional filters for the annotation (cf. main text). Predictions that do not overlap with any annotated CDS are depicted in yellow, predictions that differ from annotated CDSs only in splice sites are depicted in green, predictions that have additional exons compared to annotated CDSs are depicted in turquoise, predictions that missed some exons compared to annotated CDSs are depicted in blue, predictions with additional and missing exons compared to annotated CDSs are depicted in pink, predictions that only differ in the start of the CDS compared to annotated CDS are depicted in red, and any other category is depicted in gray Fig. 4 Summary of difference for GeMoMa predictions with tie=1. The relaxed evaluation (left panel) depicts differences between GeMoMa predictions and annotation without any filter on the annotation, while the conservative evaluation (right panel) applies additional filters for the annotation (cf. main text). Predictions that do not overlap with any annotated CDS are depicted in yellow, predictions that differ from annotated CDSs only in splice sites are depicted in green, predictions that have additional exons compared to annotated CDSs are depicted in turquoise, predictions that missed some exons compared to annotated CDSs are depicted in blue, predictions with additional and missing exons compared to annotated CDSs are depicted in pink, predictions that only differ in the start of the CDS compared to annotated CDS are depicted in red, and any other category is depicted in gray For both evaluations, we find that the predictions for C. briggsae are in better accordance with the annotation than the predictions of the remaining three nematode species. Analysis of nematode species We obtain between 100 and 1 079 predic- tions with different splice-site, between 42 and 786 predic- tions containing new exons, between 548 and 1 431 predictions with missing exons, and between 284 and 1 191 predictions with new and missing exons. Finally, for GeMoMa predictions that differ in the start codon compared to the annotation, we filter for tpc=1 of the GeMoMa prediction and tpc<1 for the annotation obtain- ing between 14 and 149 for C. brenneri and C. remanei, respectively. In summary, we obtain between 1 065 pre- dictions differing from the annotation for C. briggsae and 4 735 predictions for C. brenneri, respectively (cf. Fig. 4) using these strict criteria. Despite the overall reduction of transcripts considered, GeMoMa predictions that missed exons compared to annotated CDSs are the largest cate- gory for all four nematode species. Furthermore, we check whether GeMoMa allows for identifying new transcripts in D. melanogaster that do not overlap with any annotated transcript but are sup- ported by RNA-seq data. First, we check whether we could identify transcripts based on the GeMoMa predic- tions using D. simulans as reference organism. We find 35 multi-coding-exon predictions that do not overlap with any annotated transcript but have a tie of 1, i.e., all introns are supported by split reads in the RNA-seq data (see “Methods”). In addition, we find 15 single-coding-exon predictions that do not overlap with any annotated tran- script but have a tpc of 1, i.e., that are fully covered by mapped RNA-seq reads. Second, we check whether we could identify transcripts that are supported by at least two of the eight reference species (cf. above). We find 14 multi-coding-exon predictions that do not over- lap with any annotated transcript, obtain a tie of 1 and are Keilwagen et al. BMC Bioinformatics (2018) 19:189 Page 9 of 12 Fig. 4 Summary of difference for GeMoMa predictions with tie=1. The relaxed evaluation (left panel) depicts differences between GeMoMa predictions and annotation without any filter on the annotation, while the conservative evaluation (right panel) applies additional filters for the annotation (cf. main text). Analysis of nematode species One possible explanation might be that the anno- tation of C. briggsae has recently been updated using RNA-seq data (Gary Williams, personal communication), while the annotation of C. japonica is based on Augustus (Erich Schwartz, personal communication) and the anno- tation of the other two nematodes are NGASP sets from multiple ab-initio gene prediction programs [37]. For C. japonica, we find the second best results, although C. japonica is phylogenetically more distantly related to C. elegans than the remaining two nematodes [38]. This is additional evidence that the annotation pipeline employed has a decisive influence on the quality and completeness of the annotation. in finding isoforms missed by traditional prediction methods. Analysis of barley Complementary to the studies in animals in the last sub- section, we used GeMoMa to predict the annotation of protein-coding genes in barley (Hordeum vulgare). Based on the benchmark results for D. melanogaster, we used several reference organisms to predict the gene annota- tion using GeMoMa and GAF and finally obtain 75 484 transcript predictions. Most of the predictions showed a good overlap with the annotation (F1 ≥0.8). Never- theless, 27 204 out of these 75 484 predictions had little (F1 <0.8) or no overlap with high or low confidence gene annotations. However, thousands of the transcripts contained in the official annotation do not have start or stop codons [14], which renders an exact comparison of predictions with perfect or at least very good overlap unreasonable. In addition, we checked for C. brenneri whether the GeMoMa predictions partially overlap with cDNAs or ESTs mapped to the C. brenneri genome. In 472 cases, the prediction overlaps with a cDNA or EST, but not with the annotation. In 364 out of these 472 cases, the prediction has tie=1. To evaluate the predictions, we man- ually checked about 9% (43) of the predicted missing genes with tie=1. Based on RNA-seq data, protein homol- ogy, cDNA/ESTs and manual curation, 95% were genuine new isoforms which have been missed in the original C. brenneri gene set. This shows that GeMoMa is valuable Hence, we focus on 19 619 predictions with no overlap with any annotated transcript (Table 2). Scrutinizing these predictions, we find 1 729 single-coding-exon predictions that are completely covered by RNA-seq reads (tpc=1) but that are not contained in the annotation. Out of these, 367 are partially supported by best BLAT matches of ESTs to the genome. In addition, we analyzed multi-coding-exon predictions and find 2 821 predictions that obtain tie=1, Page 10 of 12 Keilwagen et al. Analysis of barley BMC Bioinformatics (2018) 19:189 Table 2 Predictions that do not overlap with any high or low confidence annotation a) Single-coding-exon predictions #evidence tpc = 0 0 < tpc < 1 tpc = 1 1 1 971 (11) 878 (14) 1 005 (137) 2 204 (19) 158 (8) 299 (55) 3 200 (16) 126 (5) 257 (92) 4 91 (17) 43 (9) 168 (83) 2 466 (63) 1 205 (36) 1 729 (367) b) Multi-coding-exon predictions #evidence tie = 0 0 < tie < 1 tie = 1 1 9 671 (287) 942 (211) 1 681 (775) 2 283 (36) 86 (32) 456 (196) 3 155 (31) 64 (43) 382 (223) 4 142 (57) 55 (37) 302 (196) 10 251 (411) 1 147 (323) 2 821 (1 390) The numbers in parenthesis depict those predictions that are partially supported by any best BLAT hit of ESTs Table 2 Predictions that do not overlap with any high or low confidence annotation We also find that the performance depends on the evo- lutionary distance betwen reference and target organism. However, prediction performance also depends on sev- eral further aspects including i) the quality of the target genome (assembly), ii) the number of reference organisms available and iii) especially the quality of the reference annotation(s) itself. Hence, we recommend to balance between evolutionary distance and (expected) quality of the reference annotation when selecting reference species for GeMoMa. The integration of RNA-seq data into GeMoMa might help to overcome wrongly annotated splice sites in the reference species in some cases. However, missing or wrongly additional annotated exons in the reference anno- tation might still lead to partially wrong gene model predictions in the target species. The benefit of RNA-seq data, however, also depends on the quality and amount of sequenced reads, on the diversity (tissues, conditions) of the sequenced samples, and on the library type, where stranded libraries should be more informative than non- stranded ones. In addition, GeMoMa uses RNA-seq data currently only to refine homologous genes models and not to identify transcribed gene models that do not show any homology. Hence, GeMoMa should be used in com- bination with other gene predictors allowing for purely RNA-seq-based or ab-initio gene predcitions. Exemplar- ily, we demonstrate that GeMoMa helps to improve the performance of combined gene predictor pipelines as for instance MAKER2. Analysis of barley stating that each predicted intron is supported by at least one split read from mapped RNA-seq data. Out of these, 1 390 are partially supported by best BLAT matches of ESTs to the genome. Besides predictions that are well supported by RNA-seq data, we also observe thousands of predictions that are not (tpc = 0 or tie = 0) or only partially (0 < tpc < 1 or 0 < tie < 1) supported by RNA-seq. Despite no or only partial RNA-seq support, we find that 833 are partially supported by best BLAT matches of ESTs to the genome. Notably, model organisms have been used as target organisms in this benchmark, whereas they would typi- cally be used as reference organisms in real applications. Hence, the performance of homology-based gene pre- diction programs might be underestimated. In summary, we recommend to use homology-based gene prediction using RNA-seq data as implemented in GeMoMa when- ever high-quality gene annotations of related species are available. Alternatively, we can utilize the number of reference organisms that support a prediction (#evidence) to fil- ter the predictions as noted for D. melanogaster. This approach will decrease sensitivity, but increase specificity obtaining predictions with a high confidence. Although, we find the most predictions with #evidence = 1, we also find about 3 500 predictions with #evidence > 1, more than 1 100 of these predictions are additionally supported by RNA-seq data or ESTs. Interestingly, we find that GeMoMa works especially well for D. melanogaster in the benchmark study com- pared to the performance of its competitors. One possible reason could be that Flybase used homology and RNA-seq data besides other evidence to infer the gene annotation [19]. In contrast, we find the worst results in C. elegans in the benchmark study, which might be related to the fact that the C. elegans gene set contains many rare isoform community submissions whereas C. briggsae was anno- tated by a large scale gene predictions effort based on RNA-seq. Conclusions Summarizing the methods and results, we present an extension of GeMoMa that allows for the incorporation of RNA-seq data into homology-based gene prediction utilizing intron position conservation. Comparing the performance of GeMoMa with and without RNA-seq evi- dence, we demonstrate for all four organism included in the benchmark that RNA-seq evidence improves the per- formance of GeMoMa. GeMoMa performs equally well or better than BRAKER1, MAKER2, CodingQuarry, and purely RNA-seq-based pipelines on the benchmark data sets including plants, animals and fungi. Scrutinizing the annotation in Wormbase, we pre- dicted protein-coding transcripts for four nematode species based on the annotation of the model organism C. elegans. We find that a substantial part of the GeMoMa predictions is either missing, marked as modification Page 11 of 12 Keilwagen et al. BMC Bioinformatics (2018) 19:189 of annotated transcripts or alternative transcripts. Espe- cially for the three nematodes, C. brenneri, C. japonica and C. remanei, that are annotated solely using ab-initio gene prediction, we find a large part of the annotation that is marked as questionable or missing. This may give an indication, why homology-based gene prediction for C. elegans shows less good performance in the benchmark study. The GeMoMa predictions of the four nematodes will be included in Wormbase in the upcoming releases. Furthermore, GeMoMa will be included in the WormBase gene curation process and trialled for strain annotation. Ethics approval and consent to participate Not applicable. Ethics approval and consent to participate Not applicable. Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. Acknowledgements We thank Katharina Hoff for providing the BRAKER1 benchmark data sets, Carson Holt for assisting the MAKER2 comparison, Gil dos Santos for his comments on the quality of the Drosophila genome assemblies, Erich Schwartz for his comments on C. japonica, Alison Testa for her help with CodingQuarry, Gary Williams for his comments on C. briggsae, and Thomas Berner for technical assistance. We thank the open access publication fund of Martin Luther University Halle–Wittenberg for funding article-processing charges. 10. Testa AC, Hane JK, Ellwood SR, Oliver RP. CodingQuarry: highly accurate hidden Markov model gene prediction in fungal genomes using RNA-seq transcripts. BMC Genomics. 2015;16(1):170. https://doi.org/10.1186/ s12864-015-1344-4. 11. Hoff KJ, Lange S, Lomsadze A, Borodovsky M, Stanke M. BRAKER1: Unsupervised RNA-Seq-Based Genome Annotation with GeneMark-ET and AUGUSTUS. Bioinformatics. 2016;32(5):767. https://doi.org/10.1093/ bioinformatics/btv661. References 1. Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, Salzberg SL, Wold BJ, Pachter L. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation,. Nat Biotechnol. 2010;28(5):511–5. https://doi. org/10.1038/nbt.1621. GeMoMa provides a user-friendly documentation and can be use as command line tool or through the Galaxy workflow management system [39] providing its own Galaxy integration (Fig. 1). GeMoMa is freely avail- able under GNU GPL3 at http://www.jstacs.de/index.php/ GeMoMa. g 2. Pertea M, Pertea GM, Antonescu CM, Chang T-C, Mendell JT, Salzberg SL. StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat Biotech. 2015;33(3):290–5. https://doi.org/10.1038/ nbt.3122. 2. Pertea M, Pertea GM, Antonescu CM, Chang T-C, Mendell JT, Salzberg SL. StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat Biotech. 2015;33(3):290–5. https://doi.org/10.1038/ nbt.3122. 3. Solovyev V, Kosarev P, Seledsov I, Vorobyev D. Automatic annotation of eukaryotic genes, pseudogenes and promoters. Genome Biol. 2006;7(1):10. https://doi.org/10.1186/gb-2006-7-s1-s10. 3. Solovyev V, Kosarev P, Seledsov I, Vorobyev D. Automatic annotation of eukaryotic genes, pseudogenes and promoters. Genome Biol. 2006;7(1):10. https://doi.org/10.1186/gb-2006-7-s1-s10. Availability of data and materials Not applicable. Accession numbers of publicly available data used in this study are listed in Additional file 1. 12. Lomsadze A, Burns PD, Borodovsky M. Integration of mapped rna-seq reads into automatic training of eukaryotic gene finding algorithm. Nucleic Acids Res. 2014;42(15):119. https://doi.org/10.1093/nar/gku557. Abbreviations avgCov: Average coverage; cDNA: Complementary DNA; CDS: Coding sequence; ERE: Extract RNA-seq evidence; EST: Expressed sequence tag; GAF: GeMoMa annotation filter; GeMoMa: Gene Model Mapper; iAA: Identical amino acids; tae: Transcript acceptor evidence; tde: Transcript donor evidence; tie: Transcript intron evidence; tpc: Transcript percentage coverage; minCov: Minimal coverage; minSplitReads: Minimum number of split reads; pAA: Positive-scoring amino acids 7. Keilwagen J, Wenk M, Erickson JL, Schattat MH, Grau J, Hartung F. Using intron position conservation for homology-based gene prediction. Nucleic Acids Res. 2016;44(9):89. https://doi.org/10.1093/nar/gkw092. g g 8. Wang Z, Gerstein M, Snyder M. RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet. 2009;10(1):57–63. https://doi.org/10. 1038/nrg2484. 8. Wang Z, Gerstein M, Snyder M. RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet. 2009;10(1):57–63. https://doi.org/10. 1038/nrg2484. 9. Holt C, Yandell M. MAKER2: an annotation pipeline and genome-database management tool for second-generation genome projects. BMC Bioinformatics. 2011;12(1):491. https://doi.org/10.1186/ 1471-2105-12-491. Author details 1Institute for Biosafety in Plant Biotechnology, Julius Kühn-Institut (JKI) - Federal Research Centre for Cultivated Plants, D-06484, Quedlinburg, Germany. 2European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, CB10 1SD, Cambridge, UK. 3Plant Genome and Systems Biology, Helmholtz Center Munich - German Research Center for Environmental Health, D-85764, Neuherberg, Germany. 4Institute of Computer Science, Martin Luther University Halle–Wittenberg, D-06120, Halle (Saale), Germany. Furthermore, we predicted protein-coding transcripts for barley using four reference species and find several hundreds of predictions that are not included in the refer- ence annotation but are supported by RNA-seq data, ESTs or multiple reference species. Hence, we conclude that these are valuable predictions harboring additional barley genes. These predictions will be incorporated in the new barley annotation. Received: 20 November 2017 Accepted: 14 May 2018 Received: 20 November 2017 Accepted: 14 May 2018 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Additional files p g g 4. Stanke M, Diekhans M, Baertsch R, Haussler D. Using native and syntenically mapped cDNA alignments to improve de novo gene finding. Bioinformatics. 2008;24(5):637. https://doi.org/10.1093/bioinformatics/ btn013. 4. Stanke M, Diekhans M, Baertsch R, Haussler D. Using native and syntenically mapped cDNA alignments to improve de novo gene finding. Bioinformatics. 2008;24(5):637. https://doi.org/10.1093/bioinformatics/ btn013. Additional file 1: Supplementary Tables and Figures. Table S1: Data used for the BRAKER1 benchmark. Table S2: Data for Wormbase study. Table S3: Data for barley annotation. Table S4: F1-measure on the BRAKER1 test sets. Table S5: CodingQuarry and GeMoMa results for S. pombe using the original read mappings from [10, 11]. Table S6: CodingQuarry and GeMoMa results for S. cerevisiae. Table S7: GeMoMa runtime. Table S8: GeMoMa performance for D.melanogaster. (PDF 147 kb) 5. Slater G, Birney E. Automated generation of heuristics for biological sequence comparison. BMC Bioinformatics. 2005;6(1):31. https://doi.org/ 10.1186/1471-2105-6-31. 5. Slater G, Birney E. Automated generation of heuristics for biological sequence comparison. BMC Bioinformatics. 2005;6(1):31. https://doi.org/ 10.1186/1471-2105-6-31. 6. She R, Chu JS-C, Uyar B, Wang J, Wang K, Chen N. genBlastG: using BLAST searches to build homologous gene models. Bioinformatics. 2011;27(15):2141–3. https://doi.org/10.1093/bioinformatics/btr342. http://bioinformatics.oxfordjournals.org/content/27/15/2141.full.pdf+ html. Keilwagen et al. BMC Bioinformatics (2018) 19:189 Steijger T, Abril JF, Engström PG, Kokocinski F, Hubbard TJ, Guigó R, Harrow J, Bertone P, Consortium R, et al. Assessment of transcript reconstruction methods for RNA-seq. Nat Methods. 2013;10(12):1177–84. 15. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215(3):403–10. https://doi.org/10. 1016/S0022-2836(05)80360-2. 30. Conesa A, Madrigal P, Tarazona S, Gomez-Cabrero D, Cervera A, McPherson A, Szcze´sniak MW, Gaffney DJ, Elo LL, Zhang X, Mortazavi A. A survey of best practices for RNA-seq data analysis. Genome Biol. 2016;17(1):13. https://doi.org/10.1186/s13059-016-0881-8. 16. Haas BJ, Papanicolaou A, Yassour M, Grabherr M, Blood PD, Bowden J, Couger MB, Eccles D, Li B, Lieber M, MacManes MD, Ott M, Orvis J, Pochet N, Strozzi F, Weeks N, Westerman R, William T, Dewey CN, Henschel R, LeDuc RD, Friedman N, Regev A. De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat Protocols. 2013;8(8):1494–512. https://doi.org/10.1038/nprot.2013.084. 2016;17(1):13. https://doi.org/10.1186/s13059-016-0881-8. 31. Burset M, Guigó R. Evaluation of gene structure prediction programs. Genomics. 1996;34(3):353–67. https://doi.org/10.1006/geno.1996.0298. 32. Powers DMW. Evaluation: From precision, recall and F-measure to ROC, informedness, markedness & correlation. J Mach Learn Technol. 2011;2(1):37–63. 17. Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R, Salzberg SL. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 2013;14(4):R36. https://doi.org/ 10.1186/gb-2013-14-4-r36. 33. Gramates LS, Marygold SJ, Santos Gd, Urbano J-M, Antonazzo G, Matthews BB, Rey AJ, Tabone CJ, Crosby MA, Emmert DB, Falls K, Goodman JL, Hu Y, Ponting L, Schroeder AJ, Strelets VB, Thurmond J, Zhou P. FlyBase at 25: looking to the future. Nucleic Acids Res. 2017;45(D1):663–71. https://doi.org/10.1093/nar/gkw1016. 18. Rawat V, Abdelsamad A, Pietzenuk B, Seymour DK, Koenig D, Weigel D, Pecinka A, Schneeberger K. Improving the annotation of arabidopsis lyrata using rna-seq data. PLOS ONE. 2015;10(9):1–12. https://doi.org/10. 1371/journal.pone.0137391. 34. Clark AG, Eisen MB, Smith DR, Bergman CM, Oliver B, Markow TA, Kaufman TC, Kellis M, Gelbart W, Iyer VN, et al. Evolution of genes and genomes on the Drosophila phylogeny. Nature. 2007;450(7167):203–18. 19. Matthews BB, dos Santos G, Crosby MA, Emmert DB, St Pierre SE, Gramates LS, Zhou P, Schroeder AJ, Falls K, Strelets V, Russo SM, Gelbart WM, The FlyBase Consortium. Gene model annotations for drosophila melanogaster: Impact of high-throughput data. G3: Genes Genomes Genet. 2015;5(8):1721–36. https://doi.org/10.1534/g3.115. 018929. http://www.g3journal.org/content/5/8/1721.full.pdf. 35. Hu TT, Eisen MB, Thornton KR, Andolfatto P. Keilwagen et al. BMC Bioinformatics (2018) 19:189 Tuli MA, Van Auken K, Wang D, Wang X, Williams G, Wright A, Yook K, Berriman M, Kersey P, Schedl T, Stein L, Sternberg PW. Wormbase 2016: expanding to enable helminth genomic research. Nucleic Acids Res. 2016;44(D1):774. https://doi.org/10.1093/nar/gkv1217. 25. Bennetzen JL, Schmutz J, Wang H, Percifield R, Hawkins J, Pontaroli AC, Estep M, Feng L, Vaughn JN, Grimwood J, Jenkins J, Barry K, Lindquist E Hellsten U, Deshpande S, Wang X, Wu X, Mitros T, Triplett J, Yang X, Ye C-Y, Mauro-Herrera M, Wang L, Li P, Sharma M, Sharma R, Ronald PC, Panaud O, Kellogg EA, Brutnell TP, Doust AN, Tuskan GA, Rokhsar D, Devos KM. Reference genome sequence of the model plant Setaria. Nat Biotechnol. 2012;30(6):555–61. https://doi.org/10.1038/nbt.2196. Tuli MA, Van Auken K, Wang D, Wang X, Williams G, Wright A, Yook K, Berriman M, Kersey P, Schedl T, Stein L, Sternberg PW. Wormbase 2016: expanding to enable helminth genomic research. Nucleic Acids Res. 2016;44(D1):774. https://doi.org/10.1093/nar/gkv1217. 14. Mascher M, Gundlach H, Himmelbach A, Beier S, Twardziok SO, Wicker T, Radchuk V, Dockter C, Hedley PE, Russell J, Bayer M, Ramsay L, Liu H, Haberer G, Zhang X-Q, Zhang Q, Barrero RA, Li L, Taudien S, Groth M, Felder M, Hastie A, Šimková H, Staˇnková H, Vrána J, Chan S, MuñozAmatriaín M, Ounit R, Wanamaker S, Bolser D, Colmsee C, Schmutzer T, Aliyeva-Schnorr L, Grasso S, Tanskanen J, Chailyan A, Sampath D, Heavens D, Clissold L, Cao S, Chapman B, Dai F, Han Y, Li H, Li X, Lin C, McCooke JK, Tan C, Wang P, Wang S, Yin S, Zhou G, Poland JA, Bellgard MI, Borisjuk L, Houben A, Doležel J, Ayling S, Lonardi S, Kersey P, Langridge P, Muehlbauer GJ, Clark MD, Caccamo M, Schulman AH, Mayer KFX, Platzer M, Close TJ, Scholz U, Hansson M, Zhang G, Braumann I, Spannagl M, Li C, Waugh R, Stein N. A chromosome conformation capture ordered sequence of the barley genome. Nature. 2017;544(7651):427–33. https://doi.org/10.1038/nature22043. 26. Pertea M, Kim D, Pertea GM, Leek JT, Salzberg SL. Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown. Nat Protocols. 2016;11(9):1650–67. https://doi.org/10.1038/ nprot.2016.095. 27. Kent WJ. BLAT–the BLAST-like alignment tool. Genome Res. 2002;12(4): 656–64. https://doi.org/10.1101/gr.229202. Article published online before March 2002. 28. Keibler E, Brent MR. Eval: A software package for analysis of genome annotations. BMC Bioinformatics. 2003;4(1):50. https://doi.org/10.1186/ 1471-2105-4-50. 29. Keilwagen et al. BMC Bioinformatics (2018) 19:189 A second-generation assembly of the Drosophila simulans genome provides new insights into patterns of lineage-specific divergence. Genome Res. 2013;23(1):89–98. https://doi.org/10.1101/gr.141689.112. http://genome.cshlp.org/content/ 23/1/89.full.pdf+html. 36. Singh ND, Larracuente AM, Sackton TB, Clark AG. Comparative genomics on the drosophila phylogenetic tree. Annu Rev Ecol Evol Syst. 2009;40(1): 459–80. https://doi.org/10.1146/annurev.ecolsys.110308.120214. 20. RhindN, Chen Z, Yassour M, Thompson DA, Haas BJ, Habib N, Wapinski I, Roy S, Lin MF, Heiman DI, Young SK, Furuya K, Guo Y, Pidoux A, Chen HM, Robbertse B, Goldberg JM, Aoki K, Bayne EH, Berlin AM, Desjardins CA, Dobbs E, Dukaj L, Fan L, FitzGerald MG, French C, Gujja S, Hansen K, Keifenheim D, Levin JZ, Mosher RA, Müller CA, Pfiffner J, Priest M, Russ C, Smialowska A, Swoboda P, Sykes SM, Vaughn M, Vengrova S, Yoder R, Zeng Q, Allshire R, Baulcombe D, Birren BW, Brown W, Ekwall K, Kellis M, Leatherwood J, Levin H, Margalit H, Martienssen R, Nieduszynski CA, Spatafora JW, Friedman N, Dalgaard JZ, Baumann P, Niki H, Regev A, Nusbaum C. Comparative functional genomics of the fission yeasts. Science. 2011;332(6032):930–6. https://doi.org/10.1126/science.1203357. http://science.sciencemag.org/content/332/6032/930.full.pdf. 37. Coghlan A, Fiedler TJ, McKay SJ, Flicek P, Harris TW, Blasiar D, nGASP Consortium, Stein LD. ngasp–the nematode genome annotation assessment project. BMC Bioinformatics. 2008;9:549. https://doi.org/10. 1186/1471-2105-9-549. 38. Kiontke KC, Félix M-A, Ailion M, Rockman MV, Braendle C, Pénigault J-B, Fitch DH. A phylogeny and molecular barcodes for caenorhabditis, with numerous new species from rotting fruits. BMC Evol Biol. 2011;11(1):339. https://doi.org/10.1186/1471-2148-11-339. 39. Afgan E, Baker D, van den Beek M, Blankenberg D, Bouvier D, Cech M, Chilton J, Clements D, Coraor N, Eberhard C, Grüning B, Guerler A, Hillman-Jackson J, Von Kuster G, Rasche E, Soranzo N, Turaga N, Taylor J, Nekrutenko A, Goecks J. The galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2016 update. Nucleic Acids Res. 2016;44(W1):3. https://doi.org/10.1093/nar/gkw343. 21. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, Gingeras TR. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29(1):15. https://doi.org/10.1093/bioinformatics/ bts635. 22. Lamesch P, Berardini TZ, Li D, Swarbreck D, Wilks C, Sasidharan R, Muller R, Dreher K, Alexander DL, Garcia-Hernandez M, Karthikeyan AS, Lee CH, Nelson WD, Ploetz L, Singh S, Wensel A, Huala E. The Arabidopsis Information Resource (TAIR): improved gene annotation and new tools. Nucleic Acids Res. 2012;40(D1):1202. https://doi.org/10.1093/nar/gkr1090. 23. International Brachypodium Initiative. Genome sequencing and analysis of the model grass Brachypodium distachyon,. Nature. 2010;463(5):763–8. https://doi.org/10.1038/nature08747. 24. Authors’ contributions JK and JG implemented the software, designed and performed benchmark studies. JK, FH, MP, SOT and JG contributed to data analysis and interpretation, and to writing the manuscript. All authors read and approved the final manuscript. 13. Howe KL, Bolt BJ, Cain S, Chan J, Chen WJ, Davis P, Done J, Down T, Gao S, Grove C, Harris TW, Kishore R, Lee R, Lomax J, Li Y, Muller H-M, Nakamura C, Nuin P, Paulini M, Raciti D, Schindelman G, Stanley E, 13. Howe KL, Bolt BJ, Cain S, Chan J, Chen WJ, Davis P, Done J, Down T, Gao S, Grove C, Harris TW, Kishore R, Lee R, Lomax J, Li Y, Muller H-M, Nakamura C, Nuin P, Paulini M, Raciti D, Schindelman G, Stanley E, Page 12 of 12 Page 12 of 12 Keilwagen et al. BMC Bioinformatics (2018) 19:189 Keilwagen et al. BMC Bioinformatics (2018) 19:189 Ouyang S, Zhu W, Hamilton J, Lin H, Campbell M, Childs K, Thibaud-Nissen F, Malek R. L, Lee Y, Zheng L, Orvis J, Haas B, Wortman J, Buell CR. The tigr rice genome annotation resource: improvements and new features. Nucleic Acids Res. 2007;35(suppl_1):883. https://doi.org/10. 1093/nar/gkl976.
https://openalex.org/W2890252264	https://europepmc.org/articles/pmc6143602?pdf=render	English	null	Highly-sensitive optical organic vapor sensor through polymeric swelling induced variation of fluorescent intensity	Nature communications	2,018	cc-by	11,677	1 CAS Key Laboratory of Bio-inspired Materials and Interfacial Science, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, 100190 Beijing, China. 2 Engineering Research Center of Special Engineering Plastics Ministry of Education, Jilin University, 130012 Changchun, China. 3 Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 130022 Changchun, China. 4 School of Material Science and Engineering, Nanyang Technological University, Singapore 639798, Singapore. 5 Department of Chemistry, Beijing Normal University, 100875 Beijing, China. 6 The Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Department of Chemistry, Tsinghua University, 100084 Beijing, China. 7 Beijing Advanced Innovation Center for Biomedical Engineering, Beihang University, 100191 Beijing, China. Correspondence and requests for materials should be addressed to X.Z. (email: xqzhang@mail.ipc.ac.cn) or to Y.W. (email: wuyuchen@iccas.ac.cn) ARTICLE Results Fabrication of 1D polymer/AIE molecule arrays. A microgroove-structured silicon pillar template is prepared by photolithography and deep-reactive ion etching (DRIE). A selective modiﬁcation of low-surface-energy perﬂuoroalkyl silane (FAS) molecule is manipulated onto micropillar sidewalls of the as-prepared template, then yielding the asymmetric-wettability micropillar template with lyophobic sidewalls and lyophilic tops (Detailed descriptions and illustrations of the template fabrication and modiﬁcation as shown in Supplementary Methods, Supple- mentary Fig. 1, 2). This asymmetric-wettability micropillar tem- plate with the depth of 15 μm is crucial for the directional regulation of liquid ﬂuids, formation of capillary bridges, and controlled deposition of 1D polymer/AIE molecule array. To fabricate 1D polymer/AIE molecule array, a thin-ﬁlm dispersion of polymer and AIE molecular solution is sandwiched between the micropillars template and quartz glass to form structure of capillary-bridge-mediated assembly system (Fig. 1a and Supplementary Fig. 3). Owing to the Laplace pressure induced by the asymmetric wettability of micropillars, the three- phase contact line (TCL) is pinned onto tops of micropillars, then leading to the formation of liquid menisci with guiding a vertical dewetting. With development of liquid menisci, the continuous liquid ﬁlm splits into a series of isolated capillary bridges on the tops of micropillars (Fig. 1b). After further dewetting of capillary bridges, the controlled deposition of the mixture of polymer and AIE molecules was driven by the ordered and directional capillary ﬂows. Finally, 1D polymer/AIE molecule array with uniform composition and precise alignment is fabricated (Fig. 1c). The 1D polymer array fabricated by the capillary-bridge-mediated method performs a high morphological and crystallographic quality (Supplementary Fig. 4 and Note 1). p y p Optical organic vapor sensors with aligned one-dimensional (1D) structure have many advantages, such as higher sensitivity, spatial resolution, and rapid response, as compared to those based on ﬁlms and random ﬁber networks22. The difﬁculty in manip- ulating and aligning 1D array hinders the development of high- performance optical sensors. Innovations regarding the superwettability-based interfacial chemistry involved in chemical reaction and nanofabrication have attracted signiﬁcant attention in energy, environment, and health areas23–25. Controllable dewetting process has become an effective strategy to manipulate 1D array formation26–29. Aggregation-induced emission (AIE) molecules are nonemissive or provide weak ﬂuorescence in dis- persed state, but they become highly ﬂuorescent in aggregated state, which are unique comparing to traditional solvatochromic dyes30–34. Results If we combine 1D polymeric structure and AIE molecule together, polymer/AIE molecule microwires could be fabricated as optical organic vapor sensor. After exposure of polymer/AIE molecule microwires to organic vapor, polymeric swelling occurs and changes the aggregated state of AIE mole- cules, which will induce variation of ﬂuorescent intensity. In this case, the change of ﬂuorescent signal should be more obvious and sensitive than that of traditional solvatochromic dyes with wavelength shifts. More importantly, regulating the side chains of polymers can introduce speciﬁc molecular interaction between polymers and organic vapors, which can provide the opportunity to improve the discrimination and sensitivity of the sensors. We employ the scanning electron microscope (SEM), ﬂuorescence microscope, and atom force microscope (AFM) to characterize the morphology and composition of as- fabricated large-area 1D array as shown in Fig. 1d–g. SEM image of 1D array depicts the patterned high-quality micro- structures with large area (ca. 160 × 120 μm2), precise position and ordered alignment. The ﬂuorescent micrograph shows the strong uniform green ﬂuorescent emission, which illustrates the homodisperse of AIE molecules in the 1D polymer array (Supplementary Fig. 5 and Note 2). A zoom-in SEM image and AFM image exhibit a typical 1D microstructure with smooth surface and straight boundary (ca. 2.0 μm in width and ca. 320 nm in height). g The morphology of micropillar templates is considerable for the fabrication quality of 1D array, and the optimized micropillar template is 5 μm in width and 5 μm in gap. In comparison, 1D polymer/AIE molecule array regulated by the micropillar-structured template with different widths (2 μm and 10 μm) and gaps (2 μm and 10 μm) are formed with irregular and discontinuous morphology (Supplementary Fig. 6). The used polymer is polyethersulfone (PES), and the AIE molecule is 9,10-bis[(N-propylphenothiazin-3-yl)vinyl]- anthracene (AnPh3). The AIE feature of AnPh3 is characterized and shown in Supplementary Fig. 7. The concentration of PES and AnPh3 in dimethylformamide (DMF) is 0.30 g L−1 and 0.13 g L−1, respectively, and the AnPh3 content in the PES/ AnPh3 microwires is 30%. The diameter of obtained PES/ AnPh3 microwires is 1.92 μm. Here, we use a controllable dewetting strategy, termed capillary-bridge-mediated assembly, to fabricate polymer/AIE molecule microwires array as highly sensitive optical organic vapor sensor. The mechanism for organic vapor sensing is through polymeric swelling-induced variation of ﬂuorescent intensity. The prepared microwires array shows good reprodu- cibility and stability upon exposure to organic vapors. ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06101-8 A A rtiﬁcial organic vapor sensor array relays on a ﬁnite number of less selective receptors to generate a unique response pattern, yielding responses to a variety of dif- ferent analytes and providing a ﬁngerprint that allows classiﬁca- tion and identiﬁcation of the analytes1–5. Among them, optical sensor arrays provide an efﬁcient and sensitive approach for rapid detection and identiﬁcation of organic vapor based on colori- metric or ﬂuorescent changes quantiﬁed by spectrum6–8. In general, an optical sensor array must contain an active center and a chemo-responsive dye, which probes the chemical properties of analytes through strong chemical interactions rather than simple physical adsorption9,10. Solvatochromic dye is one of the most important chemo-responsive dyes based on the solvent polarity variation responsible for optical changes11–15. Of particular interest for optical organic vapor sensor, the use of polymers doped with a solvatochromic dye (Nile red) as a reporter through polymeric swelling by absorbing analytes has been constructed for optical ﬁber sensors16–20. However, the limitation is that the weak interactions, such as the absorption into polymers or physisorp- tion onto surfaces, usually lead to lower sensitivity of the optical organic vapor sensor. During polymeric swelling, solvatochromic shifts of the ﬂuorescent dyes are typically undetectable below 0.1% of the saturation vapor pressure of an organic solvent21. Thus, development of new types of optical organic vapor sensor with alternative mechanism to replace traditional solvatochromic shift type represents an important and highly demanded issue to improve the sensitivity of optical sensor. distinguish similar organic vapors of benzene and toluene. We believe this strategy of fabricating polymer/AIE molecule micro- wires array will provide new route to construct optical organic vapor sensor with high sensitivity. Highly-sensitive optical organic vapor sensor through polymeric swelling induced variation of ﬂuorescent intensity Xiangyu Jiang1, Hanfei Gao1, Xiqi Zhang 1, Jinhui Pang2, Yunqi Li 3, Kan Li1, Yuchen Wu1, Shuzhou Li 4, Jia Zhu 5, Yen Wei6 & Lei Jiang1,7 Xiangyu Jiang1, Hanfei Gao1, Xiqi Zhang 1, Jinhui Pang2, Yunqi Li 3, Kan Li1, Yuchen Wu1, Shuzhou Li 4, Jia Zhu 5, Yen Wei6 & Lei Jiang1,7 Traditional optical organic vapor sensors with solvatochromic shift mechanisms have lower sensitivity due to weak intermolecular interactions. Here, we report a general strategy to prepare a higher sensitivity optical organic vapor sensor through polymeric swelling-induced variation of ﬂuorescent intensity. We combine one-dimensional polymeric structures and aggregation-induced emission (AIE) molecules together to form a polymer/AIE microwires array as a sensor. The prepared sensors based on different commercial polymers can suc- cessfully classify and identify various organic vapors. Among them, the poly(vinyl butyral)/ AIE microwires array can detect methanol vapor as low as 0.05% of its saturation vapor pressure. According to the theory of like dissolves like, we further fabricate a polymer/AIE microwires array derived from designable polyethersulfones, through regulating their side chains, to distinguish similar organic vapors of benzene and toluene. Both experimental and theoretical simulation results reveal that speciﬁc molecular interactions between the poly- ethersulfones and organic vapors can improve the speciﬁc recognition performance of the sensors. 1 CAS Key Laboratory of Bio-inspired Materials and Interfacial Science, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, 100190 Beijing, China. 2 Engineering Research Center of Special Engineering Plastics Ministry of Education, Jilin University, 130012 Changchun, China. 3 Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 130022 Changchun, China. 4 School of Material Science and Engineering, Nanyang Technological University, Singapore 639798, Singapore. 5 Department of Chemistry, Beijing Normal University, 100875 Beijing, China. 6 The Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Department of Chemistry, Tsinghua University, 100084 Beijing, China. 7 Beijing Advanced Innovation Center for Biomedical Engineering, Beihang University, 100191 Beijing, China. Correspondence and requests for materials should be addressed to X.Z. (email: xqzhang@mail.ipc.ac.cn) or to Y.W. (email: wuyuchen@iccas.ac.cn) 1 NATURE COMMUNICATIONS \| (2018) 9:3799 \| DOI: 10.1038/s41467-018-06101-8 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06101-8 320 nm in height). The scale bar is 2 μm. The PES is employed, and the AIE molecule is AnPh3 calculations are carried out. Comparing and contrasting the interaction energies, from which the AIE dimers or the isolate AIE molecule and the monomer of polymer, the stability of intermolecular structure can be the logical order of respective models. From the results of AIE dimers and AIE/polymer (Supplementary Table 1, 2 and Fig. 2c), when the functional of wB97XD and B3LYP-D3(BJ) are adopted, the ΔEAIE/polymer, which are −0.7452 eV and −0.8388 eV, are larger than the interaction energy of AIE dimers. As a consequence, the stronger interaction between AIE and the monomer of polymer is proven, as well as the corresponding structure can be more stable. Moreover, AFM data for the PES/AnPh3 microwires before and after exposing the sensor in acetone vapor are provided in Fig. 2d–f. The result evidences the polymer swelling with volume enlargement of the microwires, as the width of microwires increases by 3.85 ± 0.34% and the height increases by 3.31 ± 0.23% after exposing the sensor in acetone vapor. Therefore, the proposed mechanism of the ﬂuorescent intensity variation after exposure of polymer/AIE molecule microwires to the organic vapor is shown in Fig. 2d. The absorption of acetone vapor leads to swelling of the PES/AnPh3 microwires36, which increases the intermolecular distance of AnPh3 molecules due to the stronger interaction between AnPh3 and PES, leading to the dispersion of AnPh3 molecules and resulting in the decreased ﬂuorescent intensity. Proposed mechanism for organic vapor sensing. To prove the feasibility of polymer/AIE molecule microwires array as optical organic vapor sensor, we ﬁrst investigate the response perfor- mance of polymer/non-AIE molecule microwires array to organic vapor (Supplementary Fig. 8 and Note 3). The non-AIE molecule is 10-propyl-phenothiazine-3,7-dicarbaldehyde (Ph3A2), which is synthesized from N-propyl-phenothiazine35. The non-AIE fea- ture of Ph3A2 is characterized and shown in Supplementary Fig. 7. Fluorescent emission spectra of PES/Ph3A2 microwires before (0%) and after (50 and 100%) exposure to acetone vapor are shown in Fig. 2a, 100% stands for the vapor pressure of acetone under ambient condition, and 50% represents half of the vapor pressure of acetone under ambient condition. The result shows almost no change in the ﬂuorescent intensity of PES/Ph3A2 microwires after exposure to acetone vapor. The inserted ﬂuor- escent images in Fig. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06101-8 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06101-8 d e f g 400 200 Height (nm) 0 0 2 Width (μm) 4 Quartz glass Organic solution Silicon template Structure of capillary-bridge-mediated assembly system a b c d e f g Dewetting of organic solution to form capillary bridges Capillary bridges 1D polymer/AIE arrays Formation of 1D polymer/AIE arrays 400 200 Height (nm) 0 0 2 Width (μm) 4 Fig. 1 Fabrication of 1D polymer/AIE molecule microwires array. a–c Scheme showing the capillary-bridge-mediated assembly method for assembly of 1D polymer/AIE molecules microwires arrays. A micropillar-structured template, with lyophobic sidewalls and lyophilic tops, is employed. Then, a thin-ﬁlm dispersion of polymer/AIE molecule solution is sandwiched between the micropillar template and quartz glass substrate to form the structure of capillary- bridge-mediated assembly system. Dewetted liquid ﬁlm is gradually divided into capillary bridges, which ﬁnally generate 1D polymer/AIE molecule microwires array. d SEM image and e ﬂuorescent image of large-area 1D polymer/AIE molecule microwires array fabricated on quartz glass. The scale bars are 20 μm and 5 μm for (d) and e, respectively. f Zoom-in SEM image of (d), and g AFM image showing an individual 1D microstructure with uniform composition, smooth surface, and straight boundary (ca. 2.0 μm in width and ca. 320 nm in height). The scale bar is 2 μm. The PES is employed, and the AIE molecule is AnPh3 d a a b Dewetting of organic solution to form capillary bridges Capillary bridges b e c capillary bridges 1D polymer/AIE arrays Formation of 1D polymer/AIE arrays c Formation of 1D polymer/AIE arrays Fig. 1 Fabrication of 1D polymer/AIE molecule microwires array. a–c Scheme showing the capillary-bridge-mediated assembly method for assembly of 1D polymer/AIE molecules microwires arrays. A micropillar-structured template, with lyophobic sidewalls and lyophilic tops, is employed. Then, a thin-ﬁlm dispersion of polymer/AIE molecule solution is sandwiched between the micropillar template and quartz glass substrate to form the structure of capillary- bridge-mediated assembly system. Dewetted liquid ﬁlm is gradually divided into capillary bridges, which ﬁnally generate 1D polymer/AIE molecule microwires array. d SEM image and e ﬂuorescent image of large-area 1D polymer/AIE molecule microwires array fabricated on quartz glass. The scale bars are 20 μm and 5 μm for (d) and e, respectively. f Zoom-in SEM image of (d), and g AFM image showing an individual 1D microstructure with uniform composition, smooth surface, and straight boundary (ca. 2.0 μm in width and ca. Results Array- based polymer/AIE molecule microwires are fabricated originat- ing from eight commercial polymers, and successfully classify and identify different organic vapors. Through molecular structure regulation of the designed polymers, we further fabricate different types of polymer/AIE molecule microwires array based on six synthetic polyethersulfones with different side chains, to NATURE COMMUNICATIONS \| (2018) 9:3799 \| DOI: 10.1038/s41467-018-06101-8 \| www.nature.com/naturecommunications 2 ARTICLE NATURE COMMUNICATIONS \| (2018) 9:3799 \| DOI: 10.1038/s41467-018-06101-8 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06101-8 2a also intuitively reveal no response phe- nomenon of PES/Ph3A2 microwires when exposed to acetone vapor. In comparison, PES/AnPh3 microwires array demonstrates obvious response performance to acetone vapor (Fig. 2b). Under the circumstance of acetone vapor, the ﬂuorescent intensity of PES/AnPh3 microwires array decreases to 71% of the original state under half of the saturated vapor pressure of acetone, and 53% of the original state under the saturated vapor pressure of acetone. The inserted ﬂuorescent images in Fig. 2b also indicate the response phenomenon of PES/AnPh3 microwires array with variation of ﬂuorescent intensity. This result convinces us that polymer/AIE molecule microwires array possesses obvious response performance to organic vapor. y Moreover, we have used four commercial AIE molecules with varied polarity (TPE, TPE-CHO and TPE-OH) and emission (TPE and TPETPAFN) to construct PES/commercial AIE molecule microwires array for organic vapors detection. Fluor- escent emission spectra of PES/commercial AIE molecule microwires before (0%) and after (50 and 100%) exposures to acetone vapor are shown in Supplementary Fig. 9, which indicates To investigate the mechanism for organic vapor sensing, density functional theory implemented in the Gaussian 09 package are performed. To determine the interaction energy of AIE dimers and AIE/polymer, density functional theory (DFT) NATURE COMMUNICATIONS \| (2018) 9:3799 \| DOI: 10.1038/s41467-018-06101-8 \| www.nature.com/naturecommunications 3 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06101-8 800 a b c d e f 600 600 400 Intensity (a.u.) 200 520 Before swelling 300 250 200 150 100 Height (nm) Height (nm) 50 0 –50 –100 2 4 Width (μm) 6 2 4 Width (μm) 6 300 350 250 200 150 100 50 0 –50 ca. 2.08 μm ca. 2.17 μm After swelling 560 Wavelength (nm) 600 400 Intensity (a.u.) 200 0 500 550 Wavelength (nm) Interaction energy ΔEAIE/AIE1 (–0.6868 eV) ΔEAIE/AIE3 (–0.2122 eV) ΔEAIE/AIE2 (–0.1226 eV) ΔEAIE/polymer (–0.7452 eV) Polymer/non-AIE Polymer/AIE N S S N S N O O Ph3A2 0% 50% 100% 0% 50% 100% 600 0% 100% 0% 100% Fig. 2 Proposed mechanism of polymer/AIE molecule microwires for organic vapor sensing. Fluorescent emission spectra of a PES/non-AIE molecule and b PES/AIE molecule microwires before (0%) and after (50 and 100%) exposure to acetone vapor. Inserted photos are ﬂuorescent images of PES/non-AIE molecule and PES/AIE molecule microwires before (0%) and after (100%) exposure to acetone vapor. All scale bars are 5 μm. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06101-8 100% is the vapor pressure of acetone under ambient condition, and 50% is half of the vapor pressure of acetone under ambient condition. The used polymer is PES, the non-AIE molecule is Ph3A2, and the AIE molecule is AnPh3. The molecular structures of Ph3A2 and AnPh3 are shown as the insets. Both ﬂuorescent excitation wavelengths are 440 nm. c Optimized geometry structures and interaction energies of AIE dimers and AIE/polymer for DFT models, proving the stronger interaction of AIE/polymer than that of AIE dimers. d The proposed mechanism of ﬂuorescent intensity variation after exposure of polymer/AIE molecule microwires to organic vapor. The absorption of acetone vapor causes swelling of the PES/AnPh3 microwires, and increases the intermolecular distance of AnPh3 molecules due to the stronger interaction between AnPh3 and PES, leading to the dispersion of AnPh3 molecules and decreased ﬂuorescent intensity. The used polymer is PES, and AIE molecule is AnPh3. The ﬁgures below represent AFM topographies of PES/AnPh3 microwires before (left) and after (right) exposure to saturated acetone vapor, indicating polymeric swelling increases the width of microwires (by 3.85 ± 0.34%, error bars, s.d. n = 20) after exposing the sensor in saturated acetone vapor. e, f Height diagram of PES/AnPh3 microwires, indicating polymeric swelling increases the height of microwires (by 3.31 ± 0.23%, error bars, s.d. n = 20) after exposing the sensor in saturated acetone vapor of PES/AnPh3 microwires with different diameters are shown in the inset of Fig. 3a. The diameter dependence of the relative variations of the ﬂuorescent intensity, ΔI/I0, shows downward trend with the increase of diameter of the microwires, where I0 is deﬁned as the baseline ﬂuorescent intensity of the PES/AnPh3 microwires prior to exposure to acetone vapor, and ΔI represents the ﬂuorescent intensity change of these microwires after and before exposure to acetone vapor. In comparison, ΔI/I0 shows almost constant with the increase of diameter of the PES/non-AIE molecule microwires (Non-AIE molecule is Ph3A2). It should be mentioned that the PES/AnPh3 microwires array with a diameter of 1.23 μm has the maximal error bar, and it is found that the microwires array with a diameter of 1.23 μm is inhomogeneous and partially broken (Supplementary Fig. 11a). The ﬂuorescent image of PES/AnPh3 microwires is provided to explain the large error bar of the ﬂuorescence of microwires with diameter 1.23 μm (Supplementary Fig. 11b). NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06101-8 100% is the vapor pressure of acetone under ambient condition, and 50% is half of the vapor pressure of acetone under ambient condition. The used polymer is PES, the non-AIE molecule is Ph3A2, and the AIE molecule is AnPh3. The molecular structures of Ph3A2 and AnPh3 are shown as the insets. Both ﬂuorescent excitation wavelengths are 440 nm. c Optimized geometry structures and interaction energies of AIE dimers and AIE/polymer for DFT models, proving the stronger interaction of AIE/polymer than that of AIE dimers. d The proposed mechanism of ﬂuorescent intensity variation after exposure of polymer/AIE molecule microwires to organic vapor. The absorption of acetone vapor causes swelling of the PES/AnPh3 microwires, and increases the intermolecular distance of AnPh3 molecules due to the stronger interaction between AnPh3 and PES, leading to the dispersion of AnPh3 molecules and decreased ﬂuorescent intensity. The used polymer is PES, and AIE molecule is AnPh3. The ﬁgures below represent AFM topographies of PES/AnPh3 microwires before (left) and after (right) exposure to saturated acetone vapor, indicating polymeric swelling increases the width of microwires (by 3.85 ± 0.34%, error bars, s.d. n = 20) after exposing the sensor in saturated acetone vapor. e, f Height diagram of PES/AnPh3 microwires, indicating polymeric swelling increases the height of microwires (by 3.31 ± 0.23%, error bars, s.d. n = 20) after exposing the sensor in saturated acetone vapor 800 a 600 400 Intensity (a.u.) 200 0 500 550 Wavelength (nm) Polymer/non-AIE N S O O Ph3A2 0% 50% 100% 600 0% 100% b 600 400 Intensity (a.u.) 200 520 560 Wavelength (nm) 600 Polymer/AIE S N S N 0% 50% 100% 0% 100% b a c Interaction energy ΔEAIE/AIE1 (–0.6868 eV) ΔEAIE/AIE3 (–0.2122 eV) ΔEAIE/AIE2 (–0.1226 eV) ΔEAIE/polymer (–0.7452 eV) d e f Before swelling 300 250 200 150 100 Height (nm) Height (nm) 50 0 –50 –100 2 4 Width (μm) 6 2 4 Width (μm) 6 300 350 250 200 150 100 50 0 –50 ca. 2.08 μm ca. 2.17 μm After swelling d c f Fig. 2 Proposed mechanism of polymer/AIE molecule microwires for organic vapor sensing. Fluorescent emission spectra of a PES/non-AIE molecule and b PES/AIE molecule microwires before (0%) and after (50 and 100%) exposure to acetone vapor. Inserted photos are ﬂuorescent images of PES/non-AIE molecule and PES/AIE molecule microwires before (0%) and after (100%) exposure to acetone vapor. All scale bars are 5 μm. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06101-8 d Variations of the ﬂuorescent intensity of the PES/AnPh3 microwires upon 100 sequential exposures to acetone vapor, proving good reproducibility and stability of the sensor 0.8 a b 0.6 0.6 0.4 0.2 Increasing diameter 0.0 0 20 AIE or non-AIE molecules content (%) 40 60 80 100 PES/AIE PES/non-AIE PES/AIE PES/non-AIE 0.4 0.2 ΔI/I0 ΔI/I0 0.0 1 2 3 Diameter (μm) 4 5 b 0.6 0.4 0.2 Increasing diameter 0.0 0 20 AIE or non-AIE molecules content (%) 40 60 80 100 PES/AIE PES/non-AIE ΔI/I0 b 0.8 a 0.6 PES/AIE PES/non-AIE 0.4 0.2 ΔI/I0 0.0 1 2 3 Diameter (μm) 4 5 c PES/AIE PES/non-AIE ΔI/I0 101 100 10–1 10–2 10–3 10–4 0.0 0.2 0.4 P/P* 0.6 0.8 1.0 d 0 20 40 60 80 90 Reversible cycles Intensity (a.u.) 92 94 96 98 100 d c Intensity (a.u.) Reversible cycles tics of polymer/AIE molecule microwires. a The diameter dependence of the relative variations of the ﬂuorescent Fig. 3 Sensor response characteristics of polymer/AIE molecule microwires. a The diameter dependence of the relative variations of the ﬂuorescent intensity, ΔI/I0 (where I0 is deﬁned as the baseline ﬂuorescent intensity of the PES/AnPh3 microwires prior to exposure to acetone vapor, and ΔI represents the ﬂuorescent intensity change of these microwires after and before exposure to acetone vapor), shows downward trend with the increase of diameter of the PES/AIE microwires, but shows almost constant in the case of PES/non-AIE microwires. The used polymer is PES, the AIE molecule is AnPh3, and the non-AIE molecule is Ph3A2. Insets are SEM images of PES/AnPh3 microwires with different diameters, all scale bars are 5 μm. Error bars, s d. (n = 50). b The curve illustrates the dependence of ΔI/I0 on the AIE molecules content or the non-AIE molecules content, which indicates the trend of increasing at ﬁrst and then decreasing with the increase of AnPh3 content of the microwires, but keeps almost constant with the increase of Ph3A2 content Error bars, s.d. (n = 50). c ΔI/I0 for the PES/AnPh3 microwires or PES/Ph3A2 microwires in response to varying partial pressures, P, of acetone vapor. P* is the vapor pressure of acetone under ambient conditions. The result indicates that, in the case of PES/AnPh3 microwires, ΔI/I0 enhances during increasing the partial pressure of acetone with a nonlinear response at higher acetone concentration, while the ΔI/I0 has a slight increase in the case of PES/ Ph3A2 microwires. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06101-8 The discontinuous and broken microwires will lead to decreased ﬂuorescent intensity at the discontinuous area. In contrast, the broken point of the micro- wires will induce increased ﬂuorescent intensity (Supplementary that all of these commercial AIE molecules can be used for organic vapors detection. In the case of acetone detection, the sensor of PES/TPE microwires has the most obvious relative variations of the ﬂuorescent intensity (Supplementary Fig. 9a). Increasing molecular polarity of AIE molecule or using longer wavelength of AIE molecule has less obvious relative variations of the ﬂuorescent intensity (Supplementary Fig. 9b–d). Sensor response characteristics. To further investigate the sensor response characteristics of PES/AnPh3 microwires, we prepare a series of PES/AnPh3 microwires with different diameters by controlling the concentration of PES and AnPh3 solution. All of AnPh3 contents in the PES/AnPh3 microwires are designed as 30%. By changing the concentrations of PES in DMF from 0.10 to 1.10 g L−1, the diameters of PES/AnPh3 microwires increase gradually, demonstrating diameters of 1.23 μm, 1.92 μm, 2.64 μm, 3.77 μm, 4.58 μm, and 5.00 μm for 0.10 g L−1, 0.30 g L−1, 0.50 g L−1, 0.70 g L−1, 0.90 g L−1, and 1.10 g L−1 of the PES concentration, respectively (Supplementary Fig. 10). SEM images NATURE COMMUNICATIONS \| (2018) 9:3799 \| DOI: 10.1038/s41467-018-06101-8 \| www.nature.com/naturecommunications 4 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06101-8 d Variations of the ﬂuorescent intensity of the PES/AnPh3 microwires upon 100 sequential exposures to acetone vapor, proving good reproducibility and stability of the sensor Fig. 11b, d). These two factors will lead to ﬂuctuations in the ﬂuorescent signal and cause large error bar of the ﬂuorescent intensity. When we tried to fabricate the microwires array with smaller diameter (ca. 0.55 μm), it was found that the microwires had more discontinuous morphologies and severe broken microwires (Supplementary Fig. 11c, d). As the PES/AnPh3 microwires array with diameter of 1.92 μm exhibits most obvious relative variations of the ﬂuorescent intensity in response to acetone vapor among these as-prepared PES/AnPh3 microwires, it is selected to investigate the sensor response characteristics in detail. Figure 3b shows the dependence of the relative variations of the ﬂuorescent intensity (ΔI/I0) on the AIE molecules content or non-AIE molecules content, which indicates the trend of increasing at ﬁrst and then decreasing with the increase of AnPh3 content of the microwires, but keeps almost constant with the increase of Ph3A2 content. The maximum value of ΔI/I0 occurs when the AnPh3 content of the microwires reaches 30%. Fig- ure 3c shows relative variations of the ﬂuorescent intensity (ΔI/I0) for the PES/AnPh3 microwires or PES/Ph3A2 microwires in response to varying partial pressures, P, of acetone vapor. P* is the vapor pressure of acetone under ambient conditions. The exposure times for different concentrations of acetone are sufﬁ- cient for the microwires to realize maximum ﬂuorescent change. The result indicates that, in the case of PES/AnPh3 microwires, the relative variations of the ﬂuorescent intensity are enhanced during increasing the partial pressure of acetone, and exhibits a nonlinear response at higher acetone concentration, indicating the microwires require longer polymer swelling procedure to cross through its percolation threshold. At low acetone vapor concentration, the PES/AnPh3 microwires exhibit a good linear dependency between relative variations of the ﬂuorescent inten- sity and vapor partial pressure. Figure 3c also indicates that the ΔI/I0 has a slight increase during increasing the partial pressure of acetone in the case of PES/Ph3A2 microwires. Moreover, sensor response time at different concentration of acetone vapor was measured by in situ PL spectrum37. The result indicates the Fig. 11b, d). These two factors will lead to ﬂuctuations in the ﬂuorescent signal and cause large error bar of the ﬂuorescent intensity. When we tried to fabricate the microwires array with smaller diameter (ca. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06101-8 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06101-8 0.8 a b c d 0.6 0.6 0.4 0.2 Increasing diameter 0.0 0 0 20 40 60 80 90 Reversible cycles Intensity (a.u.) 92 94 96 98 100 20 AIE or non-AIE molecules content (%) 40 60 80 100 PES/AIE PES/non-AIE PES/AIE PES/non-AIE PES/AIE PES/non-AIE 0.4 0.2 ΔI/I0 ΔI/I0 ΔI/I0 0.0 101 100 10–1 10–2 10–3 10–4 1 0.0 0.2 0.4 P/P* 0.6 0.8 1.0 2 3 Diameter (μm) 4 5 Fig. 3 Sensor response characteristics of polymer/AIE molecule microwires. a The diameter dependence of the relative variations of the ﬂuorescent intensity, ΔI/I0 (where I0 is deﬁned as the baseline ﬂuorescent intensity of the PES/AnPh3 microwires prior to exposure to acetone vapor, and ΔI represents the ﬂuorescent intensity change of these microwires after and before exposure to acetone vapor), shows downward trend with the increase of diameter of the PES/AIE microwires, but shows almost constant in the case of PES/non-AIE microwires. The used polymer is PES, the AIE molecule is AnPh3, and the non-AIE molecule is Ph3A2. Insets are SEM images of PES/AnPh3 microwires with different diameters, all scale bars are 5 μm. Error bars, s. d. (n = 50). b The curve illustrates the dependence of ΔI/I0 on the AIE molecules content or the non-AIE molecules content, which indicates the trend of increasing at ﬁrst and then decreasing with the increase of AnPh3 content of the microwires, but keeps almost constant with the increase of Ph3A2 content. Error bars, s.d. (n = 50). c ΔI/I0 for the PES/AnPh3 microwires or PES/Ph3A2 microwires in response to varying partial pressures, P, of acetone vapor. P* is the vapor pressure of acetone under ambient conditions. The result indicates that, in the case of PES/AnPh3 microwires, ΔI/I0 enhances during increasing the partial pressure of acetone with a nonlinear response at higher acetone concentration, while the ΔI/I0 has a slight increase in the case of PES/ Ph3A2 microwires. NS \| (2018) 9:3799 \| DOI: 10.1038/s41467-018-06101-8 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06101-8 For the quantitative analysis of dependence of ﬂuorescent emission intensity on the environmental RH, we measured in situ photoluminescence spectrum at RH of 25, 50 and 75%, which displays the equivalent intensity of 1D microstructures with RH changing. To further study the resistance of PES/AnPh3 microwires to water, AFM is employed for the quantitative characterization of their morphologies in order to infer the swelling property of microwires in water. Typical AFM images of the 1D microstructures (ca. 2 μm in width and ca. 320 nm in height) at RH of 25, 50, and 75% all depict the very small swelling ratio of <2%. According to the measurement and calculation of ten PES/AnPh3 microwires, the statistical distribution of their swelling ratios is plotted as shown in Supplementary Fig. 13 (0.83 ± 0.08%, 1.60 ± 0.10%, 1.75 ± 0.07% at RH of 25%, 50%, and 75%, respectively). We suggest that the water adsorption of polymer/ AIE molecule tends to be saturated with the increase of environmental RH. Owing to the hydrophobic nature of the nonpolar polymer molecules, the water adsorption of polymer/ AIE molecule microwires is very low even though the environ- mental RH is high. In conclusion, we demonstrate the detection performance of 1D array vapor sensors is not affected by environmental humidity. Array-based organic vapors sensing. As each individual PES/ AnPh3 microwires array has characteristic relative variations of the ﬂuorescent intensity, such data will be useful only in a simple environment such as a single known organic vapor. In order to be applicable to more complex detection, more cross-reactive sen- sors array will be required to obtain the data. To this end, ﬂuorescent intensity variation data are obtained for arrays of various polymer/AnPh3 microwires during exposure to seven organic vapors (Supplementary Fig. 14). In this respect, eight commercial polymers are employed to fabricate different poly- mer/AnPh3 microwires, including poly(styrene-co-allyl alcohol), poly(α-methylstyrene), poly(vinyl chloride-co-vinyl acetate), poly (vinyl acetate), poly(carbonate bisphenol A), poly(ether sulfone), poly(methyl methacrylate), and poly(vinyl butyral). The sensor arrays of these polymer/AnPh3 microwires are performed to conﬁrm the classiﬁcation capability among the following seven organic vapors, including toluene, methanol, formaldehyde, hexane, ethanol, benzene, and acetone. As the detection values of different sensors are highly correlated, principal component analysis (PCA) is used to classify different types of organic vapors38. PCA is a statistical method for exploring high- dimensional data sets by reducing the dimensions to the few principal components. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06101-8 Next, for each new observation, we identify the new observation belongs to the group which has the highest weight in ﬁve-nearest points in the training data set. The result is shown in Fig. 4b (plotted in sky-blue color), which demonstrates that our proposed method can classify and identify different organic vapors well, and the classiﬁcation rate is 100%. Furthermore, four commercial AIE molecules with varied polarity (TPE, TPE-CHO, and TPE- OH) and emission (TPE and TPETPAFN) are used to construct PES/commercial AIE molecule microwires array for array-based organic vapors sensing. PCA plots calculated from the ﬂuorescent intensity variation data obtained for arrays of eight polymer/ commercial AIE molecule microwires during exposure to seven organic vapors are provided in Supplementary Fig. 15, indicating different types of the organic vapors are also well separated with these microwires array. This result further veriﬁes the universality of this polymer swelling strategy for commercial AIE molecules. Figure 4c shows relative variations of the ﬂuorescent intensity (ΔI/I0) for the poly(vinyl butyral)/AnPh3 microwires in response to low methanol vapor concentration, demonstrating a good linear dependency with low limit of detection of 100 ppm, which equals to 0.05% of the saturation vapor pressure of methanol, revealing the sensing performance is more sensitive than the traditional optical organic vapor sensor with solvatochromic shift mechanism. In addition, the comparative data using a solvato- chromic dye (Ph3A) to construct PES/Ph3A microwires with the same experimental condition, but based on wavelength-shift approach, are provided in Supplementary Fig. 16. The result shows that the ﬂuorescent emission spectra of the PES/Ph3A microwires have no wavelength-shift or variation of the ﬂuor- escent intensity, which proves the wavelength-shift approach is not applicable to this polymeric swelling method. y y p g p To demonstrate the stability of PES/AnPh3 microwires in the presence of water, we test the ﬂuorescent emission and swelling measurements by the immersion in the water fog with different relative humidities (Supplementary Fig. 13). The PES/AnPh3 microwires were carefully placed on a sample stage and investigated under different relative humidities (RH) of 25, 50 and 75% (by an ultrasonic humidiﬁer using Milli-Q deionized water). In situ top-view ﬂuorescent microscopy observations were employed under different RH values, which illustrate the same intensity of ﬂuorescent emission and the very small swelling of PES/AnPh3 microwires at RH of 25, 50, and 75%. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06101-8 PCA plots calculated from the ﬂuorescent intensity variation data obtained for arrays of eight polymer/ commercial AIE molecule microwires during exposure to seven organic vapors are provided in Supplementary Fig. 15, indicating different types of the organic vapors are also well separated with these microwires array. This result further veriﬁes the universality of this polymer swelling strategy for commercial AIE molecules. Figure 4c shows relative variations of the ﬂuorescent intensity (ΔI/I0) for the poly(vinyl butyral)/AnPh3 microwires in response to low methanol vapor concentration, demonstrating a good linear dependency with low limit of detection of 100 ppm, which equals to 0.05% of the saturation vapor pressure of methanol, revealing the sensing performance is more sensitive than the traditional optical organic vapor sensor with solvatochromic shift mechanism. In addition, the comparative data using a solvato- chromic dye (Ph3A) to construct PES/Ph3A microwires with the same experimental condition, but based on wavelength-shift approach, are provided in Supplementary Fig. 16. The result shows that the ﬂuorescent emission spectra of the PES/Ph3A microwires have no wavelength-shift or variation of the ﬂuor- escent intensity, which proves the wavelength-shift approach is not applicable to this polymeric swelling method. sensor response times are 1.89 s, 2.08 s, 2.14 s, and 2.26 s for 25%, 50%, 75%, and 100% of acetone vapor concentration, respectively (Supplementary Fig. 12). Figure 3d shows variations of the ﬂuorescent intensity of the PES/AnPh3 microwires upon 100 sequential exposures to acetone vapor, proving good repro- ducibility and stability for optical organic vapor sensor. reveals that 94.3% of the variation in the data can be captured by the top two principal components, PC1 and PC2, which account for the variances of 73.7% and 20.6%, respectively (Fig. 4a). The PCA result indicates that the top two principal components are enough for the classiﬁcation, and also shows different types of the organic vapors are well separated. To verify our proposed clas- siﬁcation method, we measure another two sets of data from these seven organic vapors, and evaluate whether the sensors can cor- rectly identify the organic vapors through the location of vapors in the PCA plot. We apply the ﬁve-nearest estimation based on the top two principal components for classiﬁcation. Speciﬁcally, we ﬁrst calculate the top two principal components score of the new observations and plot the new observations in the ﬁgure. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06101-8 0.55 μm), it was found that the microwires had more discontinuous morphologies and severe broken microwires (Supplementary Fig. 11c, d). As the PES/AnPh3 microwires array with diameter of 1.92 μm exhibits most obvious relative variations of the ﬂuorescent intensity in response to acetone vapor among these as-prepared PES/AnPh3 microwires, it is selected to investigate the sensor response characteristics in detail. Figure 3b shows the dependence of the relative variations of the ﬂuorescent intensity (ΔI/I0) on the AIE molecules content or non-AIE molecules content, which indicates the trend of increasing at ﬁrst and then decreasing with the increase of AnPh3 content of the microwires, but keeps almost constant with the increase of Ph3A2 content. The maximum value of ΔI/I0 occurs when the AnPh3 content of the microwires reaches 30%. Fig- ure 3c shows relative variations of the ﬂuorescent intensity (ΔI/I0) NATURE COMMUNICATIONS \| (2018) 9:3799 \| DOI: 10.1038/s41467-018-06101-8 \| www.nature.com/naturecommunications 5 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06101-8 reveals that 94.3% of the variation in the data can be captured by the top two principal components, PC1 and PC2, which account for the variances of 73.7% and 20.6%, respectively (Fig. 4a). The PCA result indicates that the top two principal components are enough for the classiﬁcation, and also shows different types of the organic vapors are well separated. To verify our proposed clas- siﬁcation method, we measure another two sets of data from these seven organic vapors, and evaluate whether the sensors can cor- rectly identify the organic vapors through the location of vapors in the PCA plot. We apply the ﬁve-nearest estimation based on the top two principal components for classiﬁcation. Speciﬁcally, we ﬁrst calculate the top two principal components score of the new observations and plot the new observations in the ﬁgure. Next, for each new observation, we identify the new observation belongs to the group which has the highest weight in ﬁve-nearest points in the training data set. The result is shown in Fig. 4b (plotted in sky-blue color), which demonstrates that our proposed method can classify and identify different organic vapors well, and the classiﬁcation rate is 100%. Furthermore, four commercial AIE molecules with varied polarity (TPE, TPE-CHO, and TPE- OH) and emission (TPE and TPETPAFN) are used to construct PES/commercial AIE molecule microwires array for array-based organic vapors sensing. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06101-8 The pendant groups include 4-aminophenyl, 4-carboxyphenyl, 4-methylphenyl, m-dimethylphenyl, 4-ethylphenyl and 4-butylphenyl groups. e Experimental result of benzene and toluene identiﬁcation with polymer/ AnPh3 microwires (blue column), and theoretical simulation result of variation of Hansen solubility parameters distance (ΔRa, red column). The used polymer is one of the six synthetic PESs. Experimental result indicates the polymer/AnPh3 microwires derived from 4-methylphenyl-containing PES has the best distinction between benzene and toluene. Theoretical simulation result is in good agreement with the experimental result. Error bars, s.d. (n = 50). f Relative variations of the ﬂuorescent intensity (ΔI/I0) for the polymer/AnPh3 microwires derived from 4-methylphenyl-containing PES in response to the mixed benzene/toluene vapor with different toluene content. The result demonstrates a good linear dependency. Error bars, s.d. (n = 50) sensing and similar organic vapors identiﬁcation. a PCA plot calculated from the ﬂuorescent intensity variation data r/AnPh3 microwires during exposure to seven organic vapors, as presented by the ﬁrst two principal axes of PC1 and olymers include poly(styrene-co-allyl alcohol), poly(α-methylstyrene), poly(vinyl chloride-co-vinyl acetate), poly(vinyl Fig. 4 Array-based organic vapors sensing and similar organic vapors identiﬁcation. a PCA plot calculated from the ﬂuorescent intensity variation data obtained for arrays of eight polymer/AnPh3 microwires during exposure to seven organic vapors, as presented by the ﬁrst two principal axes of PC1 and PC2. The used eight commercial polymers include poly(styrene-co-allyl alcohol), poly(α-methylstyrene), poly(vinyl chloride-co-vinyl acetate), poly(vinyl t t ) l ( b t bi h l A) l ( th lf ) l ( th l th l t ) l ( i l b t l) d th AIE l l i A Ph Th PCA 4-butylphenyl-containing PES exhibits minimum discriminative performance of benzene and toluene. This experimental result proves regulating the side chains of polymers and introducing speciﬁc molecular interaction between polymers and organic vapors will improve the discrimination of the sensors. Moreover, theoretical simulation is carried out to investigate the inﬂuence of side chains modiﬁcation of synthetic PESs for the similar organic vapors identiﬁcation. We conduct Hansen solubility parameters (HSP) combined with the spatial exclusion volume effect of the molecular groups. HSP contain three components: dispersion, polar, and hydrogen bonding. Variations of HSP distance (ΔRa) are calculated to compare the molecular interaction of PES- toluene and PES-benzene. The calculation results indicate the PES/AnPh3 microwires array derived from 4-methylphenyl- containing PES has the largest ΔRa, and shows best distinction performance between toluene and benzene vapors. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06101-8 Samples can be plotted, making it possible to visually assess similarities and differences between samples and determine whether samples can be grouped. The PCA algorithm Similar organic vapors identiﬁcation. Toluene is an important raw material for organic chemical industry that is widely used as solvent and gasoline additive. The current production of toluene is relatively surplus, and a considerable amount of toluene is used for alkylation and disproportionation to produce benzene39, 40. Therefore, identiﬁcation of the toluene content in the mixed toluene/benzene vapors is of great signiﬁcance. To identify toluene content in the mixed toluene/benzene vapors, we further prepare different types of polymer/AnPh3 microwires based on six synthetic polyethersulfones (PESs), according to the theory of like dissolves like. The designed PES polymers contain pendant groups with different side chains, including 4-aminophenyl, 4- carboxyphenyl, 4-methylphenyl, m-dimethylphenyl, 4-ethylphe- nyl, and 4-butylphenyl groups (Fig. 4d). By regulating the molecular structure of synthetic PES, the as-prepared polymer/ AnPh3 microwires show obvious distinction for similar organic vapors of toluene and benzene. As shown in Fig. 4e, the polymer/ AnPh3 microwires array derived from 4-methylphenyl-containing PES has the best distinction between benzene and toluene. In comparison, the polymer/AnPh3 microwires array derived from NATURE COMMUNICATIONS \| (2018) 9:3799 \| DOI: 10.1038/s41467-018-06101-8 \| www.nature.com/naturecommunications 6 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06101-8 NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06101-8 In another word, theoretical simulation result of variation of ΔRa is in good agreement with the relative variations of the ﬂuorescent intensity (ΔI/I0) in the experimental result, which further evidences the 4-butylphenyl-containing PES exhibits minimum discriminative performance of benzene and toluene. This experimental result proves regulating the side chains of polymers and introducing speciﬁc molecular interaction between polymers and organic vapors will improve the discrimination of the sensors. Moreover, theoretical simulation is carried out to investigate the inﬂuence of side chains modiﬁcation of synthetic PESs for the similar organic vapors identiﬁcation. We conduct Hansen solubility parameters (HSP) combined with the spatial exclusion volume effect of the molecular groups. HSP contain three components: dispersion, polar, and hydrogen bonding. Variations of HSP distance (ΔRa) are calculated to compare the molecular interaction of PES- toluene and PES-benzene. The calculation results indicate the PES/AnPh3 microwires array derived from 4-methylphenyl- containing PES has the largest ΔRa, and shows best distinction performance between toluene and benzene vapors. In another word, theoretical simulation result of variation of ΔRa is in good agreement with the relative variations of the ﬂuorescent intensity (ΔI/I0) in the experimental result, which further evidences the theory of like dissolves like is applicable for this strategy. According to Hansen solubility parameter and the theory of like dissolves like, we can easily ﬁnd the most effective functional groups. From this we know that when our system needs to test a speciﬁc organic solvent, it only needs to regulate the side chains of polymers according to the structure of solvent molecules. Finally, we determine relative variations of the ﬂuorescent intensity (ΔI/ I0) for the polymer/AnPh3 microwires derived from 4- methylphenyl-containing PES polymer in response to the mixed benzene/toluene vapors with different toluene content. The result demonstrates good linear dependency at the whole scope of toluene content (Fig. 4f). NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06101-8 c Relative variations of the ﬂuorescent intensity (ΔI/I0) for the poly(vinyl butyral)/AnPh3 microwires in response to methanol vapor in low concentration, demonstrating a good linear dependency with low limit of detection of 100 ppm. Error bars, s.d. (n = 20). d The molecular structures of six synthetic PESs with different pendant groups. The pendant groups include 4-aminophenyl, 4-carboxyphenyl, 4-methylphenyl, m-dimethylphenyl, 4-ethylphenyl and 4-butylphenyl groups. e Experimental result of benzene and toluene identiﬁcation with polymer/ AnPh3 microwires (blue column), and theoretical simulation result of variation of Hansen solubility parameters distance (ΔRa, red column). The used polymer is one of the six synthetic PESs. Experimental result indicates the polymer/AnPh3 microwires derived from 4-methylphenyl-containing PES has the best distinction between benzene and toluene. Theoretical simulation result is in good agreement with the experimental result. Error bars, s.d. (n = 50). f Relative variations of the ﬂuorescent intensity (ΔI/I0) for the polymer/AnPh3 microwires derived from 4-methylphenyl-containing PES in response to the mixed benzene/toluene vapor with different toluene content. The result demonstrates a good linear dependency. Error bars, s.d. (n = 50) Fig. 4 Array-based organic vapors sensing and similar organic vapors identiﬁcation. a PCA plot calculated from the ﬂuorescent intensity variation data obtained for arrays of eight polymer/AnPh3 microwires during exposure to seven organic vapors, as presented by the ﬁrst two principal axes of PC1 and PC2. The used eight commercial polymers include poly(styrene-co-allyl alcohol), poly(α-methylstyrene), poly(vinyl chloride-co-vinyl acetate), poly(vinyl acetate), poly(carbonate bisphenol A), poly(ether sulfone), poly(methyl methacrylate), poly(vinyl butyral), and the AIE molecule is AnPh3. The PCA algorithm reveals that 94.3% of the variance in the data can be captured by PC1 and PC2, and shows that different types of the organic vapors are well separated. b Veriﬁcation of the proposed classiﬁcation method. Another two sets of data (sky-blue color) from these seven organic vapors are measured and evaluated whether the sensors can correctly identify organic vapors through the location of vapors in the PCA plot. The result reveals our proposed method can classify and identify different organic vapors well. c Relative variations of the ﬂuorescent intensity (ΔI/I0) for the poly(vinyl butyral)/AnPh3 microwires in response to methanol vapor in low concentration, demonstrating a good linear dependency with low limit of detection of 100 ppm. Error bars, s.d. (n = 20). d The molecular structures of six synthetic PESs with different pendant groups. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06101-8 Another two sets of data (sky-blue color) from these seven organic vapors are measured and evaluated whether the sensors can correctly identify organic vapors through the location of vapors in the PCA plot The result reveals our proposed 0.4 0.2 a 0.0 –0.2 PC2 (20.6%) PC1 (73.7%) –0.4 –0.6 –0.5 0.0 0.5 1.0 Acetone Toluene Methanol Formaldehyde Hexane Ethanol Benzene b 0.4 0.2 0.0 –0.2 PC2 (20.6%) –0.4 –0.6 PC1 (73.7%) –0.5 0.0 0.5 1.0 Acetone Toluene Methanol Formaldehyde Hexane Ethanol Benzene c 0.6 0.4 0.2 ΔI/I0 0.0 0 200 400 y = 0.000569x – 0.0498 R2 = 0.9788 Concentration (ppm) 600 800 1000 b c Concentration (ppm) f 0.5 0.4 0.3 0.2 ΔI/I0 0 20 40 60 Toluene content (%) 80 100 y = 0.1770x + 0.2659 R2 = 0.9924 5 1.5 1.0 0.5 0.0 4 3 ΔI /I0 ΔRa ΔRa 1 2 3 4 Material number 5 6 d e ΔI /I0 1 2 3 4 5 6 O O O S O R R NH2 COOH n d e f 0.5 1.5 1.0 0.5 0.0 0.4 0.3 0.5 0.4 0.3 0.2 ΔI /I0 ΔRa ΔRa ΔI /I0 ΔI/I0 1 2 3 4 5 6 O O O S O R R NH2 COOH n 1 2 3 4 Material number 5 6 0 20 40 60 Toluene content (%) y = 0.1770x + 0.2659 R2 = 0.9924 f d e Material number Toluene content (%) Fig. 4 Array-based organic vapors sensing and similar organic vapors identiﬁcation. a PCA plot calculated from the ﬂuorescent intensity variation data obtained for arrays of eight polymer/AnPh3 microwires during exposure to seven organic vapors, as presented by the ﬁrst two principal axes of PC1 and PC2. The used eight commercial polymers include poly(styrene-co-allyl alcohol), poly(α-methylstyrene), poly(vinyl chloride-co-vinyl acetate), poly(vinyl acetate), poly(carbonate bisphenol A), poly(ether sulfone), poly(methyl methacrylate), poly(vinyl butyral), and the AIE molecule is AnPh3. The PCA algorithm reveals that 94.3% of the variance in the data can be captured by PC1 and PC2, and shows that different types of the organic vapors are well separated. b Veriﬁcation of the proposed classiﬁcation method. Another two sets of data (sky-blue color) from these seven organic vapors are measured and evaluated whether the sensors can correctly identify organic vapors through the location of vapors in the PCA plot. The result reveals our proposed method can classify and identify different organic vapors well. NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06101-8 ARTICLE 0.4 0.2 a b c d e f 0.0 –0.2 PC2 (20.6%) PC1 (73.7%) –0.4 –0.6 0.4 0.6 0.5 1.5 1.0 0.5 0.0 0.4 0.3 0.4 0.2 0.5 0.4 0.3 0.2 ΔI/I0 ΔI /I0 ΔRa ΔRa ΔI /I0 ΔI/I0 0.0 0.2 0.0 –0.2 PC2 (20.6%) –0.4 –0.6 –0.5 1 2 3 4 5 6 O O O S O R R NH2 COOH 0.0 0.5 n 1.0 PC1 (73.7%) –0.5 0.0 0.5 1.0 1 2 3 4 Material number 5 6 0 20 40 60 Toluene content (%) 80 100 0 200 400 y = 0.000569x – 0.0498 R2 = 0.9788 y = 0.1770x + 0.2659 R2 = 0.9924 Concentration (ppm) 600 800 1000 Acetone Toluene Methanol Formaldehyde Hexane Ethanol Benzene Acetone Toluene Methanol Formaldehyde Hexane Ethanol Benzene Fig. 4 Array-based organic vapors sensing and similar organic vapors identiﬁcation. a PCA plot calculated from the ﬂuorescent intensity variation data obtained for arrays of eight polymer/AnPh3 microwires during exposure to seven organic vapors, as presented by the ﬁrst two principal axes of PC1 and PC2. The used eight commercial polymers include poly(styrene-co-allyl alcohol), poly(α-methylstyrene), poly(vinyl chloride-co-vinyl acetate), poly(vinyl acetate), poly(carbonate bisphenol A), poly(ether sulfone), poly(methyl methacrylate), poly(vinyl butyral), and the AIE molecule is AnPh3. The PCA algorithm reveals that 94.3% of the variance in the data can be captured by PC1 and PC2, and shows that different types of the organic vapors are well separated. b Veriﬁcation of the proposed classiﬁcation method. Discussion In this work, we prepared polymer/AIE molecule microwires array as highly sensitive optical organic vapor sensor, which adopts a new mechanism of polymeric swelling-induced variation of ﬂuorescent intensity. The prepared optical sensor of PES/AnPh3 NATURE COMMUNICATIONS \| (2018) 9:3799 \| DOI: 10.1038/s41467-018-06101-8 \| www.nature.com/naturecommunications 7 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06101-8 microwires array achieves the optimal response performance with a diameter of 1.92 μm and the AnPh3 content of 30%, and shows good reproducibility and stability upon exposure to organic vapors. We have further fabricated organic vapor sensors from various polymer/AnPh3 microwires array originated from eight commercial polymers, and successfully classiﬁed and identiﬁed seven organic vapors, demonstrating high-speciﬁc recognition performance and good separation efﬁciency according to the PCA result. More importantly, through molecular structure regulation of the designed PESs, we fabricated different types of polymer/ AnPh3 microwires array based on six synthetic PESs to distinguish similar organic vapors of benzene and toluene. Both experimental and theoretical simulation results prove introducing speciﬁc molecular interaction between polymers and organic vapors will improve the discrimination and sensitivity of the sensors. We expect this strategy of fabricating polymer/AIE molecule micro- wires array will not only open new route to construct novel optical organic vapor sensor with higher sensitivity, but also be used to design heterogeneous optical arrays for liquid sensing. microwires array achieves the optimal response performance with a diameter of 1.92 μm and the AnPh3 content of 30%, and shows good reproducibility and stability upon exposure to organic vapors. We have further fabricated organic vapor sensors from various polymer/AnPh3 microwires array originated from eight commercial polymers, and successfully classiﬁed and identiﬁed seven organic vapors, demonstrating high-speciﬁc recognition performance and good separation efﬁciency according to the PCA result. More importantly, through molecular structure regulation of the designed PESs, we fabricated different types of polymer/ AnPh3 microwires array based on six synthetic PESs to distinguish similar organic vapors of benzene and toluene. Both experimental and theoretical simulation results prove introducing speciﬁc molecular interaction between polymers and organic vapors will improve the discrimination and sensitivity of the sensors. We expect this strategy of fabricating polymer/AIE molecule micro- wires array will not only open new route to construct novel optical organic vapor sensor with higher sensitivity, but also be used to design heterogeneous optical arrays for liquid sensing. Characterization. Discussion A contact angle system (Dataphysics, OCA20, Germany) was employed for the static contact angle measurements of asymmetric-wettability micropillar template, and all angle data were achieved by the measurements in ten different positions of the same sample. The structures of PES/AnPh3 microwires array were investigated by scanning electron microscopy (SEM, JEOL, JSM-6700F, Japan) at an accelerating voltage of 3.0 kV. A Nanoscope IIIa instrument (Bruker, ICON2-SYS, Germany) was carried out for AFM measurements of the microwires. To certify the crystallinity of 1D polymer arrays, Grazing incidence wide-angle X- ray scattering (GIWAXS) was performed on XEUSS SAXS/WAXS system. In the system, the incidence angle is 0.2°, and the distance between sample and detector is 122 mm. Fluorescence microscopy images of the polymer/AIE molecule microwires array were obtained using an optical microscope (Vision Engineering Co., UK) coupled to a charge-coupled device (CCD) camera and connected to a desktop computer. The optical measurement was characterized under ambient conditions, using a spectroﬂuorophotometer (RF-5301-PC; Shimadzu, Kyoto, Japan) with a clean and conﬁned container. For demonstrating the uniform distribution of AIE dye molecules in microwires, a confocal laser scanning microscope (Nikon, N-C2- SIM, Japan) was carried out. The slit widths of both excitation and emission were 5 nm. Proton nuclear magnetic resonance (1H NMR) spectra were measured on a JEOL 400 MHz spectrometer, d6-DMSO was used as solvent and tetramethylsilane as the internal standard. Interaction energy calculation of AIE dimers and AIE/polymer. All calculations were performed using DFT implemented in the Gaussian 09 package43. The geometry optimization were performed at B3LYP level using the 6-31G(d, p) basis sets and the single-point energy were calculated at wB97XD/6-31G(d, p) and B3LYP-D3(BJ)/6-31G (d, p) levels, which including dispersion corrections44 to obtain the energy of interac- tion. Due to the skeleton of AIE molecule (AnPh3) is similar as AnPh45, we used the conformation of AnPh to accelerate the computations. Since there are three different neighbors around an AnPh molecule in AnPh crystal, three AnPh dimers are selected according to AnPh crystal. The geometries of AIE dimers and AIE/polymer were optimized. All structures corresponding to true energy minima were veriﬁed by the frequency calculations, which contained only positive frequencies. The single-point energies were then calculated to obtain the energy of interaction, which can be calcu- lated as, ΔE = E(AB) −E(A) −E(B) between two molecules A and B in dimers or complex AB44, where E denotes the single-point energy of respective species. Methods b i i Fabrication of asymmetric-wettability micropillar template. Silicon wafers (10 cm diameter, N doped, <100> oriented, 525 μm thick) were structured by a pho- tolithography method and deep-reactive ion etching with employing a direct laser writing apparatus, then operated resist stripping for selective modiﬁcation. First, tops of micropillars were protected by a SU-8 layer. Second, modiﬁers with low- surface energy were evaporated onto the as-protected templates. Finally, asymmetric-wettability micropillar templates were fabricated after removing the SU-8 layer (See Supplementary Methods). Fabrication of PES/AnPh3 microwires array. The AIE molecule of AnPh3 was synthesized from tetraethyl anthracene-9,10-diylbis(methylene) diphosphonate and 10-propyl-10H-phenothiazine-3-carbaldehyde41 (See Supplementary Meth- ods). Capillary-bridge-mediated assembly method was used to fabricate polymer/ AIE molecule microwires array42 (Supplementary Note 4). First, the as-prepared asymmetric-wettability micropillar template was employed. Then, 10 μL dispersion solution of polymer and AIE molecules (dissolved in DMF) is dropped onto the micropillars template and covered by a quartz glass to form the capillary-bridge- mediated assembly system. This assembly system was kept at a temperature of 25 °C and humidity of 60% to control the dewetting in a mild environment. With the evaporation of solvents, the continuous liquid ﬁlm was divided into a series of capillary bridges by the controlled dewetting. High-quality polymer/AIE molecule microwires were ﬁnally generated on the quartz glass by the regulated deposition and molecular packing in the conﬁned space of capillary bridges. All of AnPh3 contents in the PES/AnPh3 microwires should be equal to 30%. To obtain PES/ AnPh3 microwires with different diameters, we chose the PES (AnPh3) solutions at the concentrations of 0.10 (0.043), 0.30 (0.13), 0.50 (0.21), 0.70 (0.30), 0.90 (0.39), and 1.10 (0.47) g L−1. Calculation of Hansen solubility parameters distance. The Hansen solubility parameters (HSP) distance between two molecules, conventionally called Ra, is the measure of how alike they are. The famous formula is: Calculation of Hansen solubility parameters distance. The Hansen solubility parameters (HSP) distance between two molecules, conventionally called Ra, is the measure of how alike they are. The famous formula is: Ra2 ¼ 4 δD1 δD2 ð Þ2þ δP1 δP2 ð Þ2þ δH1 δH2 ð Þ2 ð1Þ ð1Þ The three components are dispersion (δD), polar (δP), and hydrogen bonding (δH). Methods b i i Calculation of variation of Hansen solubility parameters distance (ΔRa) is following the formula below: ΔRa ¼ Ra B ð Þ Ra T ð Þ ð2Þ ð2Þ Ra(B) is the HSP distance between the pendant group in the synthetic PES and benzene, and Ra(T) is the HSP distance between the pendant group in the synthetic PES and toluene. Hansen solubility parameters, the value of these parameters is acquired by checking the “Hansen Solubility Parameters: A User’s Handbook”. Preparation of commercial polymer/AIE microwires array. The procedures of preparing polymer/AIE molecule microwires array with commercial polymers are similar to that of fabrication of PES/AnPh3 microwire arrays. The AIE molecule was AnPh3. Eight commercial polymers, including poly(styrene-co-allyl alcohol), poly(α-methylstyrene), poly(vinyl chloride-co-vinyl acetate), poly(vinyl acetate), poly(carbonate bisphenol A), poly(ether sulfone), poly(methyl methacrylate), and poly(vinyl butyral) were used in this work. These commercial polymers were dissolved in tetrahydrofuran (THF), acetone, acetone, acetone, acetone, DMF, phenol, and methanol, respectively. The concentrations of these commercial polymers in the corresponding solvent were 0.30 g L−1, 0.40 g L−1, 0.40 g L−1, 0.50 g L−1, 0.40 g L−1, 0.30 g L−1, 0.40 g L−1, and 0.25 g L−1, respectively. The con- centrations of AnPh3 were designed as 0.13 g L−1, 0.17 g L−1, 0.17 g L−1, 0.21 g L −1, 0.17 g L−1, 0.13 g L−1, 0.17 g L−1, and 0.11 g L−1, respectively, to make all of AnPh3 contents in the polymer/AIE molecule microwires equal to 30%. The dia- meters of the polymer/AIE molecule microwires with different commercial poly- mers were ca. 2 μm. Discussion To determine the interaction energy of AIE dimers and AIE/polymer, DFT calculations were carried out. AnPh single-crystal structure is shown in Supplementary Fig. 17, and the optimized geometry structures and interaction energies are shown in Supplementary Table 1, 2. Data availability The authors declare that all the data supporting the ﬁndings of this study are available within the paper and its Supplementary Information or from the corresponding author on reasonable request. Received: 7 March 2018 Accepted: 3 August 2018 NATURE COMMUNICATIONS \| (2018) 9:3799 \| DOI: 10.1038/s41467-018-06101-8 \| www.nature.com/naturecommunications ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/s41467-018-06101-8 5. Finger, T. E., Bartel, D. L., Shultz, N., Goodson, N. B. & Greer, C. A. 5HTR3A- driven GFP labels immature olfactory sensory neurons. J. Comp. Neurol. 525, 1743–1755 (2017). 35. Sun, X. et al. Novel electroactive and photoactive molecular materials based on conjugated donor−acceptor structures for optoelectronic device applications. J. Phys. Chem. B 109, 10786–10792 (2005). 6. Rakow, N. A. & Suslick, K. S. A colorimetric sensor array for odour visualization. Nature 406, 710 (2000). y 36. Champeau, M. et al. In situ FTIR micro-spectroscopy to investigate polymeric ﬁbers under supercritical carbon dioxide: CO2 sorption and swelling measurements. J. Supercrit. Fluids 90, 44–52 (2014). 7. Walt, D. R. Bead-based ﬁber-optic arrays. Science 287, 451–452 (2000). 7. Walt, D. R. Bead-based ﬁber-optic arrays. Science 287, 4 37. Xue, P. et al. Aggregation-induced emission nanoﬁber as a dual sensor for aromatic amine and acid vapor. J. Mater. Chem. C 5, 11532 (2017). 8. Lin, H., Jang, M. & Suslick, K. S. Preoxidation for colorimetric sensor array 8. Lin, H., Jang, M. & Suslick, K. S. Preoxidation for colorimetric sensor array detection of VOCs. J. Am. Chem. Soc. 133, 16786–16789 (2011). 8. Lin, H., Jang, M. & Suslick, K. S. Preoxidation for colorim 8. Lin, H., Jang, M. & Suslick, K. S. Preoxidation for colorimetric sensor array detection of VOCs. J. Am. Chem. Soc. 133, 16786–16789 (2011). 9. You, L., Zha, D. & Anslyn, E. V. Recent advances in supramolecular analytical chemistry using optical sensing. Chem. Rev. 115, 7840–7892 (2015). 38. Jurs, P., Bakken, G. & McClelland, H. Computational methods for the analysis of chemical sensor array data from volatile analytes. Chem. Rev. 100, 2649–2678 (2000). sabe-Desmonts, L., Reinhoudt, D. N. & Crego-Calama, M. Desig 10. Basabe-Desmonts, L., Reinhoudt, D. N. & Crego-Calama, M. Design of ﬂuorescent materials for chemical sensing. Chem. Soc. Rev. 36, 993–1017 (2007). 39. Kubů, M. et al. Three-dimensional 10-ring zeolites: the activities in toluene alkylation and disproportionation. Catal. Today 259, 97–106 (2016). 11. Reichardt, C. Solvents and solvent effects: an introduction. Org. Process Res. Dev. 11, 105–113 (2007). 40. Gallego, E. M. et al. “Ab initio” synthesis of zeolites for preestablished catalytic reactions. Science 355, 1051–1054 (2017). 41. Zhang, X. et al. Inﬂuence of alkyl length on properties of piezoﬂuorochromic aggregation induced emission compounds derived from 9, 10-bis[(N- alkylphenothiazin-3-yl)vinyl]anthracene. Tetrahedron 70, 924–929 (2014). 12. Katritzky, A. R. et al. Author contributions 21. Askim, J. R., Mahmoudi, M. & Suslick, K. S. Optical sensor arrays for chemical sensing: the optoelectronic nose. Chem. Soc. Rev. 42, 8649–8682 (2013). L.J., X.Z. and Yc.W. conceived the original idea. X.J., H.G., Yc.W., and X.Z. designed and performed the experiments. J.P. synthesized the polyethersulfones. Y.L., J.Z., and S.L. conducted the theoretical simulation. K.L. drew the scheme. X.J., X.Z., H.G., Yc.W. and Y.W. analyzed the data. X.Z. wrote the original manuscript, Yc.W. and L.J. revised it. All authors discussed the results and commented on the manuscript. 22. LaFratta, C. N. & Walt, D. R. Very high density sensing arrays. Chem. Rev. 108, 614–637 (2008). 23. Wang, S., Liu, K., Yao, X. & Jiang, L. Bioinspired surfaces with superwettability: new insight on theory, design, and applications. Chem. Rev. 115, 8230–8293 (2015). Additional information 24. Wen, L., Tian, Y. & Jiang, L. Bioinspired super-wettability from fundamental research to practical applications. Angew. Chem. Int. Ed. 54, 3387–3399 (2015). 25. Su, B., Tian, Y. & Jiang, L. Bioinspired interfaces with superwettability: from materials to chemistry. J. Am. Chem. Soc. 138, 1727–1748 (2016). 24. Wen, L., Tian, Y. & Jiang, L. Bioinspired super-wettability from fundamental research to practical applications. Angew. Chem. Int. Ed. 54, 3387–3399 (2015). Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467- 018-06101-8. p pp g , ( ) 25. Su, B., Tian, Y. & Jiang, L. Bioinspired interfaces with superwettability: from materials to chemistry. J. Am. Chem. Soc. 138, 1727–1748 (2016). Competing interests: The authors declare no competing interests. 26. Huang, J., Kim, F., Tao, A. R., Connor, S. & Yang, P. Spontaneous formation of nanoparticle stripe patterns through dewetting. Nat. Mater. 4, 896–900 (2005). Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ 27. Han, W., Byun, M., Li, B., Pang, X. & Lin, Z. A simple route to hierarchically assembled micelles and inorganic nanoparticles. Angew. Chem. Int. Ed. 51, 12588–12592 (2012). Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. 28. Van Hameren, R. et al. Macroscopic hierarchical surface patterning of porphyrin trimers via self-assembly and dewetting. Science 314, 1433–1436 (2006). 29. Jiang, X. et al. Bioinspired 1D superparamagnetic magnetite arrays with magnetic ﬁeld perception. Adv. Mater. 28, 6952–6958 (2016). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. 30. ARTICLE Quantitative measures of solvent polarity. Chem. Rev. 104, 175–198 (2004). 13. Stitzel, S. E., Aernecke, M. J. & Walt, D. R. Artiﬁcial noses. Annu. Rev. Biomed. Eng. 13, 1–25 (2011). 42. Feng, J. et al. “Capillary-bridge lithography” for patterning organic crystals toward mode-tunable microlaser arrays. Adv. Mater. 29, 1603652 (2017). 14. Buss, C. E. & Mann, K. R. Synthesis and characterization of Pt(CN-p-(C2H5) C6H4)2(CN)2, a crystalline vapoluminescent compound that detects vapor- phase aromatic hydrocarbons. J. Am. Chem. Soc. 124, 1031–1039 (2002). 43. Frisch, M. J. et al. Gaussian 09, Revision A.1 (Gaussian, Inc., Wallingford, CT, 2009). 15. Hudson, Z. M. et al. Probing the structural origins of vapochromism of a triarylboron-functionalized platinum (II) acetylide by optical and multinuclear solid-state NMR spectroscopy. Inorg. Chem. 50, 3447–3457 (2011). 44. Grimme, S. et al. Dispersion-corrected mean-ﬁeld electronic structure methods. Chem. Rev. 116, 5105–5154 (2016). 45. Zhang, X. et al. Piezoﬂuorochromic properties and mechanism of an aggregation-induced emission enhancement compound containing N-hexyl- phenothiazine and anthracene moieties. J. Phys. Chem. B 115, 7606–7611 (2011). 16. Dickinson, T. A., White, J., Kauer, J. S. & Walt, D. R. A chemical-detecting system based on a cross-reactive optical sensor array. Nature 382, 697 (1996). l b h ( ) y p y 17. Walt, D. R. Fibre optic microarrays. Chem. Soc. Rev. 39, 38–50 (2010) 18. Levitsky, I., Krivoshlykov, S. G. & Grate, J. W. Rational design of a Nile Red/ polymer composite ﬁlm for ﬂuorescence sensing of organophosphonate vapors using hydrogen bond acidic polymers. Anal. Chem. 73, 3441–3448 (2001). Acknowledgements This work was ﬁnancially funded by the National Key R&D program of China (2016YFA0200803), the National Natural Science Foundation of China (51603211, 21633014, 21421061, and 51673107), and the 111 Project (B14009). This work was also supported by the China Postdoctoral Science Foundation (2017m611006) and Nationa Postdoctoral Program for Innovative Talents (BX201600174). work was ﬁnancially funded by the National Key R&D program of Ch g y g y 19. Li, D., Mills, C. A. & Cooper, J. M. Microsystems for optical gas sensing incorporating the solvatochromic dye Nile Red. Sens. Actuat. B Chem. 92, 73–80 (2003). (2016YFA0200803), the National Natural Science Foundation of China (51603211, supported by the China Postdoctoral Science Foundation (2017m611006) and National Postdoctoral Program for Innovative Talents (BX201600174). 20. White, J., Kauer, J. S., Dickinson, T. A. & Walt, D. R. Rapid analyte recognition in a device based on optical sensors and the olfactory system. Anal. Chem. 68, 2191–2202 (1996). References 1. Albert, K. J. et al. Cross-reactive chemical sensor arrays. Chem. Rev. 100, 2595–2626 (2000). Preparation of synthetic PES/AIE microwires array. The procedures of pre- paring polymer/AIE molecule microwires array with synthetic polyethersulfones are similar to that of fabrication of PES/AnPh3 microwires array. The six synthetic polyethersulfones contain different pendant groups, including m-dimethylphenyl, 4-aminophenyl, 4-carboxyphenyl, 4-methylphenyl, 4-ethylphenyl, and 4- butylphenyl groups. The synthetic methods of these six polyethersulfones are shown in the supplementary methods. 2. Mori, K., Nagao, H. & Yoshihara, Y. The olfactory bulb: coding and processing of odor molecule information. Science 286, 711–715 (1999). 3. Green, E. et al. A rational approach to minimal high-resolution cross-reactive arrays. J. Am. Chem. Soc. 128, 15278–15282 (2006). 3. Green, E. et al. A rational approach to minimal high-resolution cross-reactive arrays. J. Am. Chem. Soc. 128, 15278–15282 (2006). y 4. Vosshall, L. B. & Stocker, R. F. Molecular architecture of smell and taste in Drosophila. Annu. Rev. Neurosci. 30, 505–533 (2007). 4. Vosshall, L. B. & Stocker, R. F. Molecular architecture of smell and taste in Drosophila. Annu. Rev. Neurosci. 30, 505–533 (2007). NATURE COMMUNICATIONS \| (2018) 9:3799 \| DOI: 10.1038/s41467-018-06101-8 \| www.nature.com/naturecommunicatio 8 Additional information Li, D., Qin, W., Xu, B., Qian, J. & Tang, B. Z. AIE nanoparticles with high stimulated emission depletion efﬁciency and photobleaching resistance for long-term super-resolution bioimaging. Adv. Mater. 29, 1703643 (2017). 31. Cheng, Y. et al. Multiscale humidity visualization by environmentally sensitive ﬂuorescent molecular rotors. Adv. Mater. 29, 1703900 (2017). 32. Zhang, X. et al. Aggregation induced emission-based ﬂuorescent nanoparticles: fabrication methodologies and biomedical applications. J. Mater. Chem. B 2, 4398–4414 (2014). 33. Wang, J. et al. Ionization and anion−π+ interaction: a new strategy for structural design of aggregation-induced emission luminogens. J. Am. Chem. Soc. 139, 16974–16979 (2017). 34. Peng, H.-Q. et al. Dramatic differences in aggregation-induced emission and supramolecular polymerizability of tetraphenylethene-based stereoisomers. J. Am. Chem. Soc. 139, 10150–10156 (2017). NATURE COMMUNICATIONS \| (2018) 9:3799 \| DOI: 10.1038/s41467-018-06101-8 \| www.nature.com/naturecommunications 9
https://openalex.org/W3209030457	https://www.nature.com/articles/s42005-021-00733-x.pdf	English	null	Spatially-resolved electronic structure of stripe domains in IrTe2 through electronic structure microscopy	Communications physics	2,021	cc-by	5,945	1 State Key Laboratory of Low-Dimensional Quantum Physics and Department of Physics, Tsinghua University, Beijing 100084, P. R. China. 2 Synchrotron SOLEIL, Université Paris-Saclay, L’Orme des Merisiers, Saint Aubin-BP 48, 91192 Gif-sur-Yvette Cedex, France. 3 Madrid Institute of Materials Science (ICMM), Spanish Scientiﬁc Research Council (CSIC), Cantoblanco, E-28049 Madrid, Spain. 4 Department of Mechanical Engineering and Tsinghua-Foxconn Nanotechnology Research Center, Tsinghua University, Beijing 100084, P. R. China. 5 Frontier Science Center for Quantum Information, Beijing 100084, P. R. China. ✉email: syzhou@mail.tsinghua.edu.cn COMMUNICATIONS PHYSICS \| (2021) 4:229 \| https://doi.org/10.1038/s42005-021-00733-x \| www.nature.com/commsphys Results and discussion S ti ll l d l t Results and discussion Spatially resolved electronic structures of different domains. Figure 1d, e shows two representative NanoARPES spectra measured along the Γ-K direction (Fig. 1c) of IrTe2 at 80 K from two domains A and B, and they are strikingly different. While the dispersion in domain A is relatively simple with strong intensity at energies from −2.5 to −0.5 eV, the dispersion in domain B shows weak intensity starting from −1 eV to the Fermi energy (EF) with many weaker bands near EF. In addition, compared to domain A, there is an additional band near the K point (marked by box 2 in Fig. 1e). Spatially resolved intensity maps (Fig. 1f, g) integrated over box 1 and 2 allow to directly visualize the spatial distribution of these two types of domains with size of a few to tens of micrometers. Since the sample is a high-quality single crystal with a well-deﬁned crystal orientation, the observation of IrTe2 exhibits an intriguing stripe phase with separated domains at low temperature, where the electronic structure in the stripe phase has remained elusive. Upon cooling, it undergoes a ﬁrst-order phase transition from trigonal (1T with P3m1 symmetry, see Fig. 1b) to triclinic structure (P1) around 280 K, accompanied by complex stripe phase20 with periods of (3n + 2) × 1 × (3n + 2) (n = 1, 2, 3, ...) in the bulk20,21 and (3n + 2) × 1 on the surface22,23. Recent scanning tunneling microscopy and ARPES study on strained IrTe2 shows 6 × 1 phase can be stabilized by strain24. Suppressing the stripe phase by doping or intercalation20,25 leads to emergence of superconductivity. To understand the stripe phase, different sce- narios have been proposed including Fermi surface nesting20 or saddle point26 induced charge density wave, crystal ﬁeld effect27, a b Te Ir Te K M Γ 0 1 -1 0 -1 -2 E-EF (eV) ky (Å-1) d e 0 1 -1 ky (Å-1) Domain A Domain B Γ K K Γ K K f g 1 2 Max. Min. Intensity in Box 1 Intensity in Box 2 c 0 -1 -2 A A B A A B 10 um 10 um E(k,r) Light e- Fig. 1 Resolving the distinct electronic structures of separated domains in IrTe2 by NanoARPES with energy-, momentum-, and space-resolved information. a A schematic drawing of NanoARPES and MicroARPES. Results and discussion S ti ll l d l t The light beam is focused onto the sample by a Fresnel zone plate or a lens down to a few μm or even 100 nm scale and generate the photoelectrons which is measured by the analyzer. b, c Crystal structure of IrTe2 and the corresponding Brillouin zone above the stripe transition temperature. d, e Two characteristic dispersions observed in domains A and B (as labeled in panels f and g) measured at photon energy of 100 eV with p-polarization and temperature of 80 K. f Spatially resolved intensity map integrated over box 1 of panel d. g Spatially resolved intensity map integrated over box 2 of panel e. 2 0 1 -1 0 -1 -2 E-EF (eV) ky (Å-1) d Domain A Γ K K 1 e 0 1 -1 ky (Å-1) Domain B Γ K K 2 Max. Min. 0 -1 -2 a b Te c E(k,r) Light e- a b Te Ir Te K M Γ c E(k,r) Light e- g Intensity in Box 2 A A B 10 um f Intensity in Box 1 A A B 10 um Intensity in Box 2 Intensity in Box 1 g Fig. 1 Resolving the distinct electronic structures of separated domains in IrTe2 by NanoARPES with energy-, momentum-, and space-resolved information. a A schematic drawing of NanoARPES and MicroARPES. The light beam is focused onto the sample by a Fresnel zone plate or a lens down to a few μm or even 100 nm scale and generate the photoelectrons which is measured by the analyzer. b, c Crystal structure of IrTe2 and the corresponding Brillouin zone above the stripe transition temperature. d, e Two characteristic dispersions observed in domains A and B (as labeled in panels f and g) measured at photon energy of 100 eV with p-polarization and temperature of 80 K. f Spatially resolved intensity map integrated over box 1 of panel d. g Spatially resolved intensity map integrated over box 2 of panel e. Fig. 1 Resolving the distinct electronic structures of separated domains in IrTe2 by NanoARPES with energy-, momentum-, and space-resolved information. a A schematic drawing of NanoARPES and MicroARPES. The light beam is focused onto the sample by a Fresnel zone plate or a lens down to a few μm or even 100 nm scale and generate the photoelectrons which is measured by the analyzer. Spatially-resolved electronic structure of stripe domains in IrTe2 through electronic structure microscopy Ch h B 1 H Zh 1 Qi Li1 Sh h Zh 1 H i Zh 1 K D 1 K Zh 1 Changhua Bao 1, Hongyun Zhang1, Qian Li1, Shaohua Zhou1, Haoxiong Zhang1, Ke Deng1, Kenan Zhang1, Laipeng Luo1, Wei Yao 1, Chaoyu Chen2, José Avila 2, Maria C. Asensio 3, Yang Wu4 & Sh Zh 1 5✉ Phase separation in the nanometer- to micrometer-scale is characteristic for correlated materials, for example, high temperature superconductors, colossal magnetoresistance manganites, Mott insulators, etc. Resolving the electronic structure with spatially-resolved information is critical for revealing the fundamental physics of such inhomogeneous systems yet this is challenging experimentally. Here by using nanometer- and micrometer-spot angle- resolved photoemission spectroscopies (NanoARPES and MicroARPES), we reveal the spatially-resolved electronic structure in the stripe phase of IrTe2. Each separated domain shows two-fold symmetric electronic structure with the mirror axis aligned along 3 equivalent directions, and 6 × 1 replicas are clearly identiﬁed. Moreover, such electronic structure inhomogeneity disappears across the stripe phase transition, suggesting that electronic phase with broken symmetry induced by the 6 × 1 modulation is directly related to the stripe phase transition of IrTe2. Our work demonstrates the capability of NanoARPES and MicroARPES in elucidating the fundamental physics of phase-separated materials. MMUNICATIONS PHYSICS \| (2021) 4:229 \| https://doi.org/10.1038/s42005-021-00733-x \| www.nature.com/commsphys 1 ARTICLE COMMUNICATIONS PHYSICS \| https://doi.org/10.1038/s42005-021-00733-x COMMUNICATIONS PHYSICS \| https://doi.org/10.1038/s42005-021-00733-x B B y focusing the beam size down to a few μm or even 100 nm scale by a Fresnel zone plate1–3 (for synchrotron light source) or a lens4 (for laser source), nanometer- and micrometer-spot angle-resolved photoemission spectroscopies (NanoARPES1–3 and MicroARPES4, Fig. 1a) provide two important advantages over conventional ARPES which has a typical beam size of 50–100 μm. Firstly, it allows to measure the electronic structure of small sam- ples, which has been demonstrated in atomically thin ﬂakes5–7 or samples with mixed crystal orientations8,9. Secondly and more importantly, for phase-separated materials which consist of multiple domains with distinct electronic structures10–14, the newly added spatial-resolving capability provides new opportunities to reveal the intrinsic electronic structure of individual domain and the evolution of the phase separation across the phase transition. Such informa- tion cannot be obtained by conventional ARPES, which is, however, indispensable for understanding the fundamental physics of phase- separated materials. Recently, NanoARPES and MicroARPES have been applied to probe the electronic structure of individual domain in CeSb15 and Fe-based superconductors16–19 by utilizing the spectroscopic capability of ARPES. Combining the advantages of both microscopic and spectroscopic capabilities of NanoARPES and MicroARPES will allow for direct visualization of separated domains with spatially resolved information and the evolution of domains across the phase transition, thereby further elucidating the complex physics of phase-separated materials. dimerization28,29, local bonding30,31, and lattice deformation32. Obtaining the electronic structure of the stripe phase is critical for disentangling the puzzling physics. Despite extensive investigations, previous ARPES measurements on IrTe226,32–38 have been obtained by averaging over different domains, and the intrinsic electronic structure of each individual stripe domain and its temperature evolution across the phase transition remain elusive. Here, by using NanoARPES and MicroARPES, we resolve the separated domains and electronic structure of individual stripe domain in IrTe2. Each separated domain shows two-fold sym- metric electronic structure with the mirror axis aligned along 3 equivalent directions, and 6 × 1 reconstructions are clearly identiﬁed both in the Fermi surface map and the dispersion, suggesting 6 × 1 stripe phase. Moreover, such electronic structure inhomogeneity disappears across the stripe phase transition, suggesting that electronic phase with broken symmetry induced by the 6 × 1 modulation is directly related to the stripe phase transition of IrTe2. Our work demonstrates the power of NanoARPES and MicroARPES in elucidating the physics across the phase transition. COMMUNICATIONS PHYSICS \| https://doi.org/10.1038/s42005-021-00733-x We note that in principle 5 × 1 or 8 × 1 would also be compatible with the two-fold symmetric Fermi surface; however, signatures of 5 × 1 or 8 × 1 replicas have not been resolved experimentally, suggesting that those domains do not have signiﬁcant contribu- tion to the dispersions. gg 2 y g To further investigate the electronic structure of these separated domains, we map out the full three-dimensional electronic dispersions for each domain. Figure 2a, b shows the spatially resolved intensity maps measured on two representative samples where separated domains are clearly observed. Figure 2c–j shows the intensity maps at EF and −0.2 eV from four different domains. All these intensity maps clearly reveal the two-fold symmetry of the electronic structure with the symmetry axis aligned along three equivalent Γ–M directions at angles of 0∘, 120∘, and −120∘ (indicated by red solid arrows), which is in sharp contrast to previous ARPES measurements26,32–38 where spatial averaging gives rise to apparently three-fold symmetric electronic structure. Therefore, the strikingly different dispersions in Fig. 1 originate from different orientations of the mirror symmetry axes. Here, the observation of two-fold symmetric electronic structure in a three- fold symmetric crystal conﬁrms the broken symmetry in the stripe phase, and the space- and momentum-resolving capability allows to reveal the intrinsic electronic structure of each individual domain. With the capability to resolve the electronic structure of each individual domain, we can now investigate the intrinsic electronic structure and the nature of the stripe phase. The two-fold symmetric Fermi surface map (Fig. 3a) shows replica oval pockets around the Γ point translated by a scattering wave vectors of 1/3 a* (Fig. 3b) where a* is the reciprocal lattice vector. More replica pockets can be distinguished away from the Γ point translated by 1/6 a* which ﬁt well with the extra weak bands as indicated by the gray arrow in Fig. 3a. The replica pockets are also identiﬁed in the dispersion images shown in Fig. 3c, e and can be observed more clearly in the momentum distribution curves (MDCs) shown in Fig. 3d, f. We note that ﬁne features are observed inside the replica oval pocket translated by 1/3 a* from Γ. These features are absent in the oval pocket at Γ, yet their existence can be conﬁrmed by zooming in the intensity map (Fig. COMMUNICATIONS PHYSICS \| https://doi.org/10.1038/s42005-021-00733-x ARTICLE -120° 0° 1 0 -1 1 0 -1 1 0 -1 kx (Å-1) ky (Å-1) e EF -0.2 eV 1 0 -1 1 0 -1 g i Domain 2 Domain 3 Domain 4 0° 120° a 20 um Sample 1 2 3 b Sample 2 10 um 1 4 1 0 -1 c Domain 1 f h j d Max. Min. Fig. 2 Visualizing the stripe phases with three different orientations. a, b Spatially resolved intensity maps integrated over box 1 in Fig. 1d. c–j Fermi surface and intensity maps at −0.2 eV of corresponding domains in panels a, b with Brillouin zone of 1 × 1 (gray hexagon). a 20 um Sample 1 2 3 b Sample 2 b b Sample 2 10 um 1 4 Fig. 2 Visualizing the stripe phases with three different orientations. a, b Spatially resolved intensity maps integrated over box 1 in Fig. 1d. c–j Fermi surface and intensity maps at −0.2 eV of corresponding domains in panels a, b with Brillouin zone of 1 × 1 (gray hexagon). separated domains with different electronic structures therefore suggests that IrTe2 is an intrinsically inhomogeneous material. momentum directions displaced by 1/3 a* as indicated by dotted lines in Fig. 3b shows a good agreement yet with sharper peaks for MicroARPES, further conﬁrming the electronic reconstruc- tion with a scattering vector of 1/3 a. The spatially resolved intensity map measured by MicroARPES in Fig. 3h also shows separated domains with domain size up to hundred micrometers. The electronic reconstruction of 1/6 a is also observed in the zoom-in Fermi surface (Fig. 3k, also see Supplementary Fig. 8 and Supplementary Note 4), dispersion image (Fig. 3l) and corre- sponding MDCs (Fig. 3m) in the MicroARPES data. We have performed ﬁne spatial scan on 5 different samples using MicroARPES where the domain size varies; however, the dispersions of individual domain remain the same (see Supple- mentary Figs. 1, 2 and 7 and Supplementary Note 1), suggesting that the measured dispersions are independent of the domain size. By combining NanoARPES and MicroARPES measure- ments, we reveal the electronic reconstructions of 1/6 and 1/3 a, which suggests that the two-fold symmetric electronic structure is likely associated with the 6 × 1 reconstruction in the stripe phase. Results and discussion S ti ll l d l t b, c Crystal structure of IrTe2 and the corresponding Brillouin zone above the stripe transition temperature. d, e Two characteristic dispersions observed in domains A and B (as labeled in panels f and g) measured at photon energy of 100 eV with p-polarization and temperature of 80 K. f Spatially resolved intensity map integrated over box 1 of panel d. g Spatially resolved intensity map integrated over box 2 of panel e. COMMUNICATIONS PHYSICS \| (2021) 4:229 \| https://doi.org/10.1038/s42005-021-00733-x \| www.nature.com/commsphys 2 COMMUNICATIONS PHYSICS \| (2021) 4:229 \| https://doi.org/10.1038/s42005-021-00733-x \| www.nature.com/commsphys COMMUNICATIONS PHYSICS \| https://doi.org/10.1038/s42005-021-00733-x Guides for the pockets (solid curves) and replicas (dotted curves) translated by q1/3 and q1/6 are overplotted in b. c–f Dispersion images (c, e) along the direction as indicated in a and corresponding momentum distribution curves (MDC) (d, f). g High-resolution zoom-in Fermi surface map by MicroARPES. h Spatially resolved intensity map by integrating over momentum range of ky = (−0.1 Å−1, 0.1 Å−1) and energy range of E = (−0.05 eV, 0). i, j NanoARPES (i) and MicroARPES (j) dispersion images along the directions as indicated in b. The white curves are extracted dispersions from MicroARPES spectrum (j) and overplotted to NanoARPES spectrum in i. k Zoom-in Fermi surface map by MicroARPES. Solid curves are guides for the pockets and dotted curves are the replicas translated by q1/6. l, m Dispersion images (l) along the direction as indicated in k and corresponding MDCs (m). 80 K 130 K 200 K 220 K 240 K 260 K 300 K Warming Up a b j 180 K 80 K c d e f g h i Max. Min. Fig. 4 Spatially resolved MicroARPES intensity maps reveal the temperature dependence of the separated domains measured with laser source at photon energy of 6.2 eV. a–h The evolution of spatially resolved MicroARPES intensity maps upon warming by integrating over momentum range of ky = (−0.1 Å−1, 0.1 Å−1) and energy range of E = (−0.05 eV, 0). The scale bar is 200 μm. i Optical image of the sample. j Spatially resolved MicroARPES intensity map at 80 K after cooling back. 200 K d 130 K Warm b 80 K a i j f j 80 K Ma M 220 K e j 300 K h 240 K f 260 K g 80 K f h Fig. 4 Spatially resolved MicroARPES intensity maps reveal the temperature dependence of the separated domains measured with laser source at photon energy of 6.2 eV. a–h The evolution of spatially resolved MicroARPES intensity maps upon warming by integrating over momentum range of ky = (−0.1 Å−1, 0.1 Å−1) and energy range of E = (−0.05 eV, 0). The scale bar is 200 μm. i Optical image of the sample. j Spatially resolved MicroARPES intensity map at 80 K after cooling back. Figure 5a–c further shows the temperature evolution of the dispersion and intensity map measured in a single domain. COMMUNICATIONS PHYSICS \| https://doi.org/10.1038/s42005-021-00733-x 3g) near Γ point using our home-built MicroARPES system with a laser source at 6.2 eV (compared to 100 eV used in synchrotron-based NanoARPES measurements) with better energy and momentum resolution. A comparison of NanoARPES and MicroARPES dispersion images (Fig. 3i, j) measured along two equivalent Temperature evolution of the spatially resolved intensity map. To conﬁrm that such spatial inhomogeneity is directly related to the stripe phase transition, we perform temperature-dependent MicroARPES measurement. Figure 4a–h shows spatially resolved intensity maps measured at temperatures from 80 K to 300 K. Separated domains on the order of tens to hundreds of micro- meters with different intensity contrast are clearly observed below the stripe transition temperature. Remarkably, above the stripe phase transition temperature, the spatial intensity map becomes much more homogeneous at 300 K (Fig. 4h) similar to its optical image. After cooling back to 80 K, the spatial inhomogeneity appears again but with different distribution, suggesting that its distribution is related to history (Fig. 4j). The observation of the stripe domains and its disappearance at high temperature pro- vides direct evidence that the inhomogeneous electronic structure is an intrinsic property of the low-temperature stripe phase. 3 NICATIONS PHYSICS \| (2021) 4:229 \| https://doi.org/10.1038/s42005-021-00733-x \| www.nature.com/commsphys COMMUNICATIONS PHYSICS \| https://doi.org/10.1038/s42005-021-00733-x COMMUNICATIONS PHYSICS \| https://doi.org/10.1038/s42005-021-00733-x ARTICLE Max. Min. Fig. 3 Electronic reconstructions of 1/6 and 1/3 revealed by NanoARPES and MicroARPES. a, b Fine Fermi surface maps measured by NanoARPES. Guides for the pockets (solid curves) and replicas (dotted curves) translated by q1/3 and q1/6 are overplotted in b. c–f Dispersion images (c, e) along the direction as indicated in a and corresponding momentum distribution curves (MDC) (d, f). g High-resolution zoom-in Fermi surface map by MicroARPES. h Spatially resolved intensity map by integrating over momentum range of ky = (−0.1 Å−1, 0.1 Å−1) and energy range of E = (−0.05 eV, 0). i, j NanoARPES (i) and MicroARPES (j) dispersion images along the directions as indicated in b. The white curves are extracted dispersions from MicroARPES spectrum (j) and overplotted to NanoARPES spectrum in i. k Zoom-in Fermi surface map by MicroARPES. Solid curves are guides for the pockets and dotted curves are the replicas translated by q1/6. l, m Dispersion images (l) along the direction as indicated in k and corresponding MDCs (m). Fig. 3 Electronic reconstructions of 1/6 and 1/3 revealed by NanoARPES and MicroARPES. a, b Fine Fermi su of 1/6 and 1/3 revealed by NanoARPES and MicroARPES. a, b Fine Fermi surface maps measured by NanoARPES. Fig. 3 Electronic reconstructions of 1/6 and 1/3 revealed by NanoARPES and MicroARPES. a, b Fine Fermi surface maps measured by NanoARPES. Guides for the pockets (solid curves) and replicas (dotted curves) translated by q1/3 and q1/6 are overplotted in b. c–f Dispersion images (c, e) along the direction as indicated in a and corresponding momentum distribution curves (MDC) (d, f). g High-resolution zoom-in Fermi surface map by MicroARPES. h Spatially resolved intensity map by integrating over momentum range of ky = (−0.1 Å−1, 0.1 Å−1) and energy range of E = (−0.05 eV, 0). i, j NanoARPES (i) and MicroARPES (j) dispersion images along the directions as indicated in b. The white curves are extracted dispersions from MicroARPES spectrum (j) and overplotted to NanoARPES spectrum in i. k Zoom-in Fermi surface map by MicroARPES. Solid curves are guides for the pockets and dotted curves are the replicas translated by q1/6. l, m Dispersion images (l) along the direction as indicated in k and corresponding MDCs (m). Fig. 3 Electronic reconstructions of 1/6 and 1/3 revealed by NanoARPES and MicroARPES. a, b Fine Fermi surface maps measured by NanoARPES. COMMUNICATIONS PHYSICS \| (2021) 4:229 \| https://doi.org/10.1038/s42005-021-00733-x \| www.nature.com/commsphys COMMUNICATIONS PHYSICS \| https://doi.org/10.1038/s42005-021-00733-x 80 K 130 K 200 K 220 K 240 K 300 K Warming Up 180 K 0.0 -0.1 -0.2 E-EF (eV) P1 a TC1 TC2 b c -0.1 eV 0.1 0.0 kx (Å-1) -0.1 0.1 0.0 -0.1 0.1 0.0 -0.1 0.1 0.0 -0.1 0.1 0.0 -0.1 0.1 0.0 -0.1 0.1 0.0 -0.1 ky (Å-1) 0.2 0.1 0.0 -0.1 0.1 0.0 ky (Å-1) -0.1 0.1 0.0 -0.1 0.1 0.0 -0.1 0.1 0.0 -0.1 0.1 0.0 -0.1 0.1 0.0 -0.1 0.1 0.0 -0.1 -120° 120° 0° d e f g Max. Min. Max. Min. Fig. 5 Evolution of the electronic structure with temperature measured at a photon energy of 6.2 eV. a Evolution of the spatial image during warming by integrating over momentum range of ky = (−0.1 Å−1, 0.1 Å−1) and energy range of E = (−0.05 eV, 0). The scale bar is 200 μm. b Evolution of the dispersion at kx = 0 during warming. c Evolution of intensity maps at −0.1 eV during warming. d–f Three equivalent orientations of the stripes with 6 × 1 reconstruction in the ab plane with dimers formed by Ir atoms. g A schematic drawing showing a mixture of 6 × 1 stripe regions with different orientations (red, blue, and yellow) and other regions (gray area). 80 K 130 K 200 K 220 K 240 K 300 K Warming Up 180 K 0.0 -0.1 -0.2 E-EF (eV) P1 a TC1 TC2 b 0.1 0.0 k (Å-1) -0.1 0.1 0.0 -0.1 0.1 0.0 -0.1 0.1 0.0 -0.1 0.1 0.0 -0.1 0.1 0.0 -0.1 0.1 0.0 -0.1 Max. Min. b c -0.1 eV 0.1 0.0 kx (Å-1) -0.1 0.1 0.0 -0.1 0.1 0.0 -0.1 0.1 0.0 -0.1 0.1 0.0 -0.1 0.1 0.0 -0.1 ky (Å-1) 0.2 0.1 0.0 -0.1 y ( ) 0° d 120° e g -120° f 0° d g Fig. 5 Evolution of the electronic structure with temperature measured at a photon energy of 6.2 eV. a Evolution of the spatial image during warming by integrating over momentum range of ky = (−0.1 Å−1, 0.1 Å−1) and energy range of E = (−0.05 eV, 0). The scale bar is 200 μm. b Evolution of the dispersion at kx = 0 during warming. c Evolution of intensity maps at −0.1 eV during warming. Conclusion In summary, the energy-, momentum-, and space-resolving capability of NanoARPES and MicroARPES allows to visualize the separated domains and reveal the intrinsic and inhomoge- neous electronic structure in the stripe phase of IrTe2. Replica bands with 1/6 a wave vector (or 6 × 1 modulation) are identiﬁed in the dispersion which resembles the 6 × 1 reconstruction of the high temperature electronic state (see Supplementary Fig. 6 and Supplementary Note 3). We note that the period of the phase is strongly related to the Ir–Ir dimer concentration, and different dimer concentration leads to complex (3n + 2) × 1 stripe per- iod. At the highest dimer concentration21,39, this corresponds to the 6 × 1 electronic ground state (Fig. 5d–f). Here by directly revealing the electronic structure of each individual domain using NanoARPES and MicroARPES, we show that the 6 × 1 stripe phase is indeed the electronic ground state and the 6 × 1 modulation is directly related to the stripe phase transi- tion of IrTe2. Our work resolves the puzzle in the electronic ARPES measurement. MicroARPES measurements have been performed in the home laboratory at Tsinghua University with fourth harmonic generation light source. The photon energy is set to 6.2 eV with p-polarization. The energy resolution was set to 15 meV. The beam size is 15 μm. The sample was measured in a working vacuum at greater than 7 × 10−11 Torr. Surface sensitive NanoARPES measurements were performed at the beamline ANTARES of the synchrotron SOLEIL3 at France with a beam size of 150 nm. The photon energy is 100 eV. The energy and angular resolution were set to 25 meV and 0.1 deg, respectively. Methods S l Sample growth. High-quality IrTe2 single crystal was grown by self-ﬂux method. Ir pellet (99.95%, Alfa Aesar) and Te ingot (99.99%, Alfa Aesar) in an atomic ratio of 5:95 were mixed together and sealed in an evacuated silica ampoule. The mixture was heated up to 900 ∘C ﬁrst and kept for several hours, then to 1150 ∘C and kept for two days, ﬁnally cooled down to 920 ∘C in several hours with a low rate. Liquid Te was separated from IrTe2 single crystal by centrifugation. COMMUNICATIONS PHYSICS \| https://doi.org/10.1038/s42005-021-00733-x d–f Three equivalent orientations of the stripes with 6 × 1 reconstruction in the ab plane with dimers formed by Ir atoms. g A schematic drawing showing a mixture of 6 × 1 stripe regions with different orientations (red, blue, and yellow) and other regions (gray area). structure of the stripe phase of IrTe2, and we envision that the application of NanoARPES and MicroARPES to other phase- separated systems can yield important information on the intrinsic underlying physics. inhomogeneity is directly related to the different orientations of the stripe phases, and there is a coexistence of both 6 × 1 stripe domains with different stripe orientations (Fig. 5d–g) and other mixed domains (gray area in Fig. 5g) as schematically shown in Fig. 5g. COMMUNICATIONS PHYSICS \| https://doi.org/10.1038/s42005-021-00733-x Sharp dispersions near the Γ point are observed at low temperature and they disappear at 240 K and above. As was discussed above, the sharp dispersions are associated with the 6 × 1 reconstruction, and their disappearance indicates a phase transition from 6 × 1 to other reconstructions at Tc123. Further warming leads to another transition at Tc2 near 300 K, which corresponds to the transition to 1 × 1 phase. Similar evolution of the Fermi surface maps and dispersions is also observed for other domains but with a rotation angle of 120∘(see Supplementary Fig. 3 and Supplementary Note 2). In addition, broad dispersions and Fermi surface maps are also observed in some other locations (see Supplementary Fig. 4), suggesting that there are also other regions in addition to the 6 × 1 stripe, which is possibly caused by the small percentage of coexisting 8 × 1 domain as revealed in LEED measurement shown in Supplementary Fig. 5. Therefore, temperature- dependent MicroARPES measurements show that the spatial 4 ARTICLE COMMUNICATIONS PHYSICS \| https://doi.org/10.1038/s42005-021-00733-x COMMUNICATIONS PHYSICS \| (2021) 4:229 \| https://doi.org/10.1038/s42005-021-00733-x \| www.nature.com/commsphys References y 33. Ootsuki, D. et al. Electronic structure reconstruction by orbital symmetry breaking in IrTe2. J. Phys. Soc. Jpn. 82, 093704 (2013). 1. Dudin, P. et al. Angle-resolved photoemission spectroscopy and imaging with a submicrometre probe at the SPECTROMICROSCOPY-3.2L beamline of Elettra. J. Synchrotron Radiat. 17, 445–450 (2010). breaking in IrTe2. J. Phys. Soc. Jpn. 82, 093704 (2013). 34. Lee, H. et al. Electronic reconstruction on dimerized IrTe2. Europhys. Lett. 120, 47003 (2017). 2. Bostwick, A., Rotenberg, E., Avila, J. & Asensio, M. C. Zooming in on electronic structure: NanoARPES at SOLEIL and ALS. Synchrotron Radiat. News 25, 19–25 (2012). 35. Ootsuki, D. et al. A novel one-dimensional electronic state at IrTe2 surface. J. Phys. Soc. Jpn. 86, 123704 (2017). y p 36. Ootsuki, D. et al. Interplay between spin-orbit interaction and stripe-type charge-orbital order of IrTe2. J. Phys. Chem. Solids 128, 270–274 (2018). 3. Avila, J. & Asensio, M. C. First NanoARPES user facility available at SOLEIL: an innovative and powerful tool for studying advanced materials. Synchrotron Radiat. News 27, 24–30 (2014). 37. Monney, C. et al. Robustness of the charge-ordered phases in IrTe2 against photoexcitation. Phys. Rev. B 97, 2 (2018). 4. Schwirr, E. et al. Applications for ultimate spatial resolution in LASER based μ-ARPES: a FeSe case study. AIP Conf. Proc. 2054, 040017 (2019). 38. Rumo, M. et al. Examining the surface phase diagram of IrTe2 with photoemission. Phys. Rev. B 101, 235120 (2020). μ y f 5. Wilson, N. R. et al. Determination of band offsets, hybridization, and exciton binding in 2D semiconductor heterostructures. Sci. Adv. 3, 1601832 (2017). 39. Li, Q. et al. Bond competition and phase evolution on the IrTe2 surface. Nat. Commun. 5, 5358 (2014). 6. Zhang, H. et al. Resolving deep quantum-well states in atomically thin 2H- MoTe2 ﬂakes by nanospot angle-resolved photoemission spectroscopy. Nano Lett. 18, 4664–4668 (2018). Acknowledgements 7. Katoch, J. et al. Giant spin-splitting and gap renormalization driven by trions in single-layer WS2/h-BN heterostructures. Nat. Phys. 14, 355–359 (2018). This work is supported by the National Key R&D Program of China (Grants No. 2020YFA0308800, 2016YFA0301004), National Natural Science Foundation of China (Grants No. 11725418 and 11427903), Tsinghua University Initiative Scientiﬁc Research Program and Tohoku-Tsinghua Collaborative Research Fund, and Beijing Advanced Innovation Center for Future Chip (ICFC). We acknowledge SOLEIL for provision of synchrotron radiation facilities. 8. Bao, C. et al. Stacking-dependent electronic structure of trilayer graphene resolved by nanospot angle-resolved photoemission spectroscopy. Nano Lett. 17, 1564–1568 (2017). 9. Yao, W. et al. Quasicrystalline 30∘twisted bilayer graphene as an incommensurate superlattice with strong interlayer coupling. Proc. Natl. Acad. Sci. 115, 6928–6933 (2018). Author contributions 10. Uehara, M., Mori, S., Chen, C. & Cheong, S. Percolate phase separation underlies colossal magnetoresistance in mixed-valence manganites. Nature 399, 560 (1999). 11 Pan S H et al Microscopic electronic inhomogeneity in the high T Shuyun Z. conceived the research project. C.B. and Hongyun Z. performed the NanoARPES measurements and analyzed the data. C.B., Q.L., Shaohua Z., L.L., K.D., and W.Y. performed the laser-based MicroARPES measurements and analyzed the data. Shuyun Z. conceived the research project. C.B. and Hongyun Z. performed the NanoARPES measurements and analyzed the data. C.B., Q.L., Shaohua Z., L.L., K.D., and W.Y. performed the laser-based MicroARPES measurements and analyzed the data. Haoxiong Z., K.Z., and Y.W. grew and characterized the samples. C.C., J.A., and M.C.A. provided support for the NanoARPES experiments. C.B. and Shuyun Z. wrote the manuscript, and all authors commented on the manuscript. 11. Pan, S. H. et al. Microscopic electronic inhomogeneity in the high-Tc superconductor Bi2Sr2CaCu2O8+x. Nature 413, 282 (2001). 11. Pan, S. H. et al. Microscopic electronic inhomogeneity in the high superconductor Bi2Sr2CaCu2O8+x. Nature 413, 282 (2001). Haoxiong Z., K.Z., and Y.W. grew and characterized the samples. C.C., J.A., and M.C.A. provided support for the NanoARPES experiments. C.B. and Shuyun Z. wrote the manuscript, and all authors commented on the manuscript. 12. Dagotto, E. Complexity in strongly correlated electronic systems. Science 309, 257 (2005). 13. Qazilbash, M. et al. Mott transition in VO2 revealed by infrared spectroscopy and nano-imaging. Science 318, 1750 (2007). 14. Lai, K. et al. Mesoscopic percolating resistance network in a strained manganite thin ﬁlm. Science 329, 190 (2010). Received: 17 July 2021; Accepted: 4 October 2021; Received: 17 July 2021; Accepted: 4 October 2021; 30. Cao, H. et al. Origin of the phase transition in IrTe2: structural modulation and local bonding instability. Phys. Rev. B 88, 115122 (2013). g y y 31. Saleh, G. & Artyukhin, S. First-principles theory of phase transitions in IrTe2. J. Phys. Chem. Lett. 11, 2127–2132 (2020). y 32. Kim, K. et al. Origin of ﬁrst-order-type electronic and structural transitions in IrTe2. Phys. Rev. Lett. 114, 136401 (2015). p g The authors declare no competing interests. 15. Kuroda, K. et al. Devil’s staircase transition of the electronic structures in CeSb. Nat. Commun. 11, 2888 (2020). Reprints and permission information is available at http://www.nature.com/reprints Reprints and permission information is available at http://www.nature.com/reprints g y 20. Yang, J. J. et al. Charge-orbital density wave and superconductivity in the strong spin-orbit coupled IrTe2:Pd. Phys. Rev. Lett. 108, 116402 (2012). Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. 21. Pascut, G. L. et al. Series of alternating states with unpolarized and spin- polarized bands in dimerized IrTe2. Phys. Rev. B 90, 195122 (2014). 22. Hsu, P.-J. et al. Hysteretic melting transition of a soliton lattice in a commensurate charge modulation. Phys. Rev. Lett. 111, 266401 (2013). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. g y 23. Chen, C. et al. Surface phases of the transition-metal dichalcogenide IrTe2. Phys. Rev. B 95, 094118 (2017). 24. Nicholson, C. W. et al. Uniaxial strain-induced phase transition in the 2D topological semimetal IrTe2. Commun. Mater. 2, 1–8 (2021). 25. Kamitani, M. et al. Superconductivity in CuxIrTe2 driven by interlayer hybridization. Phys. Rev. B 87, 180501 (2013). y y 26. Qian, T. et al. Structural phase transition associated with van Hove singularity in 5d transition metal compound IrTe2. New J. Phys. 16, 123038 (2014). 27. Ootsuki, D. et al. Orbital degeneracy and peierls instability in the triangular- lattice superconductor Ir1−xPtxTe2. Phys. Rev. B 86, 014519 (2012). 28. Pascut, G. L. et al. Dimerization-induced cross-layer quasi-two-dimensionality in metallic IrTe2. Phys. Rev. Lett. 112, 086402 (2014). y 29. Eom, M. J. et al. Dimerization-induced Fermi-surface reconstruction in IrTe2. Phys. Rev. Lett. 113, 266406 (2014). Data availability The data that support the plots within this paper and other ﬁndings of this study are available from the corresponding author upon reasonable request. The data that supports the ﬁndings of this study are available within the article. COMMUNICATIONS PHYSICS \| (2021) 4:229 \| https://doi.org/10.1038/s42005-021-00733-x \| www.nature.com/commsphys 5 Additional information 16. Watson, M. D. et al. Probing the reconstructed Fermi surface of antiferromagnetic BaFe2As2 in one domain. npj Quantum Materials 4, 36 (2019). Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s42005-021-00733-x. Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s42005-021-00733-x. 17. Ma, J. et al. Spatial nematic ﬂuctuation in BaFe2(As1−xPx)2 revealed by spatially and angle-resolved photoemission spectroscopy. Phys. Rev. B 101, 094515 (2020). Correspondence and requests for materials should be addressed to Shuyun Zhou. 18. Rhodes, L. C., Watson, M. D., Haghighirad, A. A., Evtushinsky, D. V. & Kim, T. K. Revealing the single electron pocket of FeSe in a single orthorhombic domain. Phys. Rev. B 101, 235128 (2020). Peer review information Communications Physics thanks the anonymous reviewers for their contribution to the peer review of this work. y 19. Chen, Y. et al. Visualization of the electronic phase separation in superconducting KxFe2−ySe2. Nano Res. 14, 823–828 (2021). 19. Chen, Y. et al. Visualization of the electronic phase separation in superconducting KxFe2−ySe2. Nano Res. 14, 823–828 (2021). 20. Yang, J. J. et al. Charge-orbital density wave and superconductivity in the strong spin-orbit coupled IrTe2:Pd. Phys. Rev. Lett. 108, 116402 (2012). © The Author(s) 2021 Reprints and permission information is available at http://www.nature.com/reprints © The Author(s) 2021 6 COMMUNICATIONS PHYSICS \| (2021) 4:229 \| https://doi.org/10.1038/s42005-021-00733-x \| www.nature.com/commsphys
https://openalex.org/W2063283037	https://europepmc.org/articles/pmc3245229?pdf=render	English	null	Induction of Bcl-2 Expression by Hepatitis B Virus Pre-S2 Mutant Large Surface Protein Resistance to 5-Fluorouracil Treatment in Huh-7 Cells	PloS one	2,011	cc-by	11,225	Jui-Hsiang Hung1, Yen-Ni Teng2, Lily Hui-Ching Wang3, Ih-Jen Su4, Clay C. C. Wang5, Wenya Huang6, Kuan-Han Lee7, Kuan-Ying Lu3, Lyu-Han Wang3 1 Department of Biotechnology, Chia Nan University of Pharmacy and Science, Tainan, Taiwan, 2 Department of Biological Sciences and Technology, National University of Tainan, Tainan, Taiwan, 3 Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan, 4 National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Tainan, Taiwan, 5 Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, School of Pharmacy, Los Angeles, California, United States of America, 6 Department of Medical Technology, College of Medicine, National Cheng Kung University, Tainan, Taiwan, 7 Institute of Pharmaceutical Science, Chia Nan University of Pharmacy and Science, Tainan, Taiwan Abstract di h k h b d b h f h l l d b Funding: This work has been supported by a research grant from the National Science Council on Taiwan (NSC 100-2320-B-041-007), and in part by grant CN9911 to J-HH from Chia-Nan University of Pharmacy & Science, Tainan , Taiwan. 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: hung86@mail.chna.edu.tw Abstract Background: Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with poor prognosis due to resistance to conventional chemotherapy and limited efficacy of radiotherapy. Our previous studies have indicated that expression of Hepatitis B virus pre-S2 large mutant surface antigen (HBV pre-S2D) is associated with a significant risk of developing HCC. However, the relationship between HBV pre-S2D protein and the resistance of chemotherapeutic drug treatment is still unclear. Methodology/Principal Findings: Here, we show that the expression of HBV pre-S2D mutant surface protein in Huh-7 cell significantly promoted cell growth and colony formation. Furthermore, HBV pre-S2D protein increased both mRNA (2.760.5-fold vs. vehicle, p = 0.05) and protein (3.260.3-fold vs. vehicle, p = 0.01) levels of Bcl-2 in Huh-7 cells. HBV pre-S2D protein also enhances Bcl-2 family, Bcl-xL and Mcl-1, expression in Huh-7 cells. Meanwhile, induction of NF-kB p65, ERK, and Akt phosphorylation, and GRP78 expression, an unfolded protein response chaperone, were observed in HBV pre-S2D and HBV pre-S-expressing cells. Induction of Bcl-2 expression by HBV pre-S2D protein resulted in resistance to 5-fluorouracil treatment in colony formation, caspase-3 assay, and cell apoptosis, and can enhance cell death by co-incubation with Bcl-2 inhibitor. Similarly, transgenic mice showed higher expression of Bcl-2 in liver tissue expressing HBV pre-S2D large surface protein in vivo. Conclusion/Significance: Our result demonstrates that HBV pre-S2D increased Bcl-2 expression which plays an important role in resistance to 5-fluorouracil-caused cell death. Therefore, these data provide an important chemotherapeutic strategy in HBV pre-S2D-associated tumor. Citation: Hung J-H, Teng Y-N, Wang LH-C, Su I-J, Wang CCC, et al. (2011) Induction of Bcl-2 Expression by Hepatitis B Virus Pre-S2 Mutant Large Surface Protein Resistance to 5-Fluorouracil Treatment in Huh-7 Cells. PLoS ONE 6(12): e28977. doi:10.1371/journal.pone.0028977 Editor: Simon Afford, University of Birmingham, United Kingdom Received August 9, 2011; Accepted November 18, 2011; Published December 22, 2011 Copyright: 2011 Hung et al. This is an open-access article distributed under the terms of the Creative Commons Attributi unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: 2011 Hung 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. PLoS ONE \| www.plosone.org Cell culture and chemicals Huh-7 and cell line was obtained from ATCC (Manasses, VA, USA). Huh-7 pre-S2D and Huh-7 pre-S cell lines were generated from Huh-7. NeHepLxHT and NeHepLxHT pre-S2D cell lines were gift from Dr. Lily Hui-Ching Wang. NeHepLxHT cell line was derived by exogenous expression of human telomerase reverse transcriptase (hTERT) in normal human neonatal hepatocytes. These cell lines were maintained at 37uC in a 5% CO2 atmosphere in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, and 100 mg/ml streptomycin (Invitrogen, Ground Island, NY, USA). Culture medium was replaced every 2 days. ECL Western blot detection system was purchased from Millipore (Billerica, MA, USA). Anti- GRP78 was purchased from Transduction Laboratories. Anti-Bcl- 2, anti-Bcl-xL, anti-Mcl-1, anti-Bax, anti-Bak, anti-p-Ser311-p65, anti-p65, anti-MCL-1 anti-Bcl-xL, anti-Bax, anti-Bak, anti-ERK, anti-phospho-p44/42 MAPK, Akt, and p-Thr308-Akt antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-p-Ser276-p65, anti-rabbit IgG-horseradish peroxidase (HRP) conjugates, and rabbit anti-mouse IgG-HRP conjugates antibodies were purchased from Cell Signaling (Beverly, MA, USA). Anti-HA antibody and Bcl-2 inhibitor II, YC137 were purchased from Calbiochem (San Diego, CA, USA). HCC is one of the most common malignancies worldwide with poor prognosis due to resistance to conventional chemotherapy and limited efficacy of radiotherapy. A major challenge in the systemic treatment of HCC is cellular resistance to conventional cytotoxic agents. In addition, many studies have indicated that alternation of gene expression is correlated with drug resistance, such as overexpression of protein kinase C, EGF receptor, c- erbB2, c-ras and c-Bcl-2 in tumor cells [27,28,29,30]. Previous studies have indicated a high-level expression of Bcl-2 in many tumor tissues, including HCC [31,32]. The Bcl-2 gene family is a group of apoptosis-related genes which have been studied extensively. Bcl-2 protein overexpression is associated with drug resistance and poor clinical outcome in cancer patients. Apoptosis is mainly executed by proteases of caspase family, whereby members of Bcl-2 family, such as Bcl-2, Bcl-xL, or Mcl-1, inhibit caspase activation [33,34]. The proteins of Bcl-2 family are important regulators of apoptosis induced by a wide array of stimuli, including chemotherapeutic drugs. Plasmid and Stable Clone Cell Lines Construction Plasmid pIRES, pIRES-preS-HA, and pIRES-pre-S2D-HA were gifts from Dr. Wenya Huang. Huh-7 cells were transfected with pIRES, pIRES-preS-HA, and pIRES-pre-S2D-HA plasmids using Invitrogen LipofectAMINE 2000 reagent according to the manufacturer’s protocol. Cells were then selected by G418 for 2 weeks. Introduction understood [8,9]. HBV encodes three envelope proteins in the pre-S/S open reading frame that are named large, middle, and small surface proteins. The expression of HBV surface proteins is related to liver tumor development and a number of truncated surface gene mutants with a partially deleted pre-S region were already identified [10,11,12,13,14]. One of the major mutant types is the deletion of the pre-S2 region (pre-S2D). These pre-S2D mutants are becoming increasingly prevalent in serum and liver tissues of patients with chronic HBV infection and HCC [15,16,17]. Hepatocellular carcinoma (HCC) is a common malignancy affecting approximately one million people worldwide annually [1]. It is one of the most common causes of cancer morbidity and mortality in Asia and Africa. Hepatocellular neoplasms develop regularly from preneoplastic foci of altered hepatocytes, and hepatocellular cancer occurs both sporadically and in relation to chronic viral infection [2], environmental exposure [3], extensive alcohol intake [4], transgenic oncogenes [5,6,7] and alternative causes of hepatic cirrhosis. HBV is considered a major etiological factor in the development of HCC. Chronic HBV carriers have a greater than 100 fold increased relative risk of developing HCC, although the oncogenic mechanisms of HBV are not completely HBV surface mutant pre-S2D gene is deleted in approximately nucleotides 2 to 55 of the pre-S2 region and often contains a point mutation in the start codon of the pre-S2 region. The pre-S2D type of HBV large surface mutant protein is predominant in December 2011 \| Volume 6 \| Issue 12 \| e28977 1 December 2011 \| Volume 6 \| Issue 12 \| e28977 PLoS ONE \| www.plosone.org Induction of Bcl-2 by HBV Pre-S2 Mutant Protein therapeutic strategy of HBV pre-S2D large surface protein containing tumor cells. hepatocellular carcinoma patients with HBV infection [18,19]. Based on epidemiologic studies, HBV carriers who presented with the pre-S2D mutant protein in serum had worse disease outcomes than those who did not [20]. Overexpression of pre-S2D large surface proteins has been demonstrated in the induction of endoplasmic reticulum (ER) stress [21]. Unfolded proteins in the endoplasmic reticulum activate several signaling pathways that are referred to as the unfolded protein responses (UPR). UPR pathway has three components in mammalian cells: basic leucine zipper transcription factor ATF6, IRE1 RNA-processing enzyme, and ER localized kinase (PERK). Previous studies have indicated that activation of NF-kB is through calcium release, reactive oxygen species production, IRE1, and PERK signal pathway during ER stress [22,23,24,25,26]. Cell culture and chemicals The expression of HBV large surface proteins in pIRES- pre-S and pIRES-pre-S2D stable clone cell line were confirmed by Western blotting. From our previous study, HBV pre-S2D large surface protein expression in hepatocytes has been correlated with hepatocarcin- ogenesis. For patients with cancer who are from HBV-endemic areas, routine screening for HBsAg before cytotoxic chemotherapy should be performed, and there was a trend of poorer survival for patients who had developed severe hepatitis during chemotherapy [35]. Furthermore, a recent study indicated that a pre-chemo- therapy for high HBV viral levels is associated with poorer survival in HCC patients with chronic HBV infection [36], and the occurrences of HBV pre-S2D large surface proteins in HBV cancer patients are about 30% in Taiwan [18]. On the other hand, previous study has also indicated Bcl-2 protein expression in acute and chronic hepatitis, cirrhosis and hepatocellular carcinoma [37]. However, the relationship between HBV pre-S2D large surface protein and Bcl-2 expression or drug resistance is still unclear. Therefore, in this study, we employed HBV pre-S2D large surface protein expressing cells to analyze altered expressed proteins in hepatoma Huh-7 and immortalized hepatocyte NeHepLxHT cell lines. We identified induction of Bcl-2 family overexpression by HBV pre-S2D large surface protein in Huh-7 cells. Furthermore, HBV pre-S2D large surface protein expressing cell was more resistant to 5-fluorouracil treatment. However, the ability of 5- fluorouracil resistance was significantly decreased by co-incubating 5-fluorouracil with Bcl-2 inhibitor. We found that HBV large surface protein and HBV pre-S2D large surface protein enhanced Bcl-2 family expression and resistance to chemotherapeutic 5- fluorouracil treatment. Taken together, these results suggest that co-treatment of Bcl-2 inhibitor might be important for chemo- Introduction We have also characterized the NF-kB response and found that NF-kB was activated through multiple pathways, including calcium signaling and pp38 kinase [21]. Activation of NF-kB by ER stress leads to induction of many cellular genes that are largely anti-apoptosis in function. Statistical Analysis Results were presented as the mean 6 S.D., and statistical comparisons were made using the Student’s t test. Significance was defined at the p,0.05 or 0.01 levels. Colony formation assay For colony formation, Huh-7 and stable transfectant cells were seeded at 1000 per well in 6-well flat-bottomed plates and incubated in 10% FBS–supplemented DMEM for 24 h. The cells were treated with 5-fluorouracil as dose indicated and 0.25 mM 5- fluorouracil plus 1 or 5 mM Bcl-2 inhibitor (Bcl-2 inhibitor II, YC137) for 24 h. The culture medium was replenished, and cells were maintained at 37uC for 14 days with medium changed every other day. Grown colonies were fixed with 3.7% formaldehyde and stained with crystal violet. The number of cell colonies was determined directly on each well. Semi-quantitative RT-PCR The cells were washed with cold PBS and then harvested. Total RNA was extracted using Trizol Reagent (Invitrogen, Ground Island, NY, USA). The cDNA was reverse-transcribed from 1 mg of total RNA using oligo(dT) primers and Moloney murine leukemia-virus transcriptase. The following primer sequences were employed to detect Bcl-2 transcripts: sense (59-GCACC- CACTCCCTTCATACAAT-39) and antisense (59-ACGCAGGT- TACATTCGTCTTCC-39. PCR reaction was performed as follows: reverse transcription at 42uC for 60 min and denaturation at 72uC for 2 min; then amplification for 30 cycles at 94uC for 30 s, annealing at 56uC for 30 s, and extension at 72uC for 30 s, followed by a terminal elongation step at 72uC for 10 min and a final holding stage at 4uC). Meanwhile, the same amount of cDNA was amplified for 25 cycles using specific glyceraldehyde-3- phosphate dehydrogenase primers: 59-TGAAGGTCGGTGT- GAACGGATTTGGC-39 (sense) and 59-CATGTAGGCCAT- GAGGTCCACCAC-39 (antisense). The products were visualized after electrophoresis on a 1.5% agarose gel containing ethi- dium bromide. The signal level of the bands was quantified densitometrically. Immunohistochemical Staining Immunohistochemical staining was performed on depar-affi- nized tissue sections of formalin-fixed material. For immunohis- tochemical staining, 4-mm-thick paraffin sections were stained with mouse anti-preS or anti-Bcl-2. The primary antibodies and working dilutions were as follows: HBsAg (1:500, 7H11; gift from Dr. Wenya Huang), Bcl-2 (1:100, polyclonal N-19; Santa Cruz Biotechnology, Santa Cruz, CA). Detection was done with streptavidin-biotinylated peroxidase-conjugated reagents (LSAB+ kit; DAKO, Carpinteria, CA). A biotinylated anti-mouse second- ary antibody (DAKO) was then applied and incubated with peroxidase-conjugated streptoavidin, chromogenized by 3-amino- 9-ethylcarbazole. Annexin V/propidium iodide assay For assessment of apoptosis, both floating and adherent cells were collected and analyzed. Briefly, 56105 cells per dish were plated onto 6-cm dishes and incubated at 37uC for 16 h. The cells were treated with 5-fluorouracil alone or plus Bcl-2 inhibitor for 24 h. The cells were washed twice with PBS and collected by trypsinization. After centrifugation at 4006 g for 5 min at room temperature, the cells were stained with Annexin V and propidium iodide (1 mg/mL). The cell apoptosis distributions were deter- mined on a FACScort flow cytometer and analyzed by ModFitLT V3.0 software program. Transgenic Mice Tissue Protein Extraction The transgenic mouse liver tissues were gifts from Dr. Ih-Jen Su. The pre-S2D transgenic mice were constructed by injection of pre-S2D gene fragment into the male pronucleus of fertilized mouse ova. Microinjection was performed in Fvb/n mice. After 12 months, liver tissue from pre-S2D transgenic mice was homogenized in RIPA buffer (50 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mg/ml aprotinin, leupeptin, and pepstatin, 1 mM Na3VO4, and 1 mM NaF). Homogenates were centrifuged at 15,0006g for 10 min at 4uC, and the supernatants were collected. Total protein concentrations of the tissue lysates were quantified using the Micro BCATM protein assay reagent kit following the manufac- turer’s instructions. Results Expression of pre-S and pre-S2D proteins in Huh-7 cell To establish the expression of HBV pre-S large surface protein and HBV pre-S2D large surface protein in hepatoma Huh-7 cell line. HBV large surface wild type HBS gene and mutant HBS gene were constructed into pIRES plasmid (Fig. 1A). Huh-7 cells were transfected with pIRES, pIRES- pre-S-HA, and pIRES-pre-S2D-HA plasmids, and the trans- fectants were selected with G418. The expression of HBV large surface proteins in pIRES-pre-S2D-HA and pIRES-pre-S-HA stable clone cell lines were confirmed by Western blotting. The result shows that HBV pre-S large surface protein and HBV pre-S2D large surface protein can be expressed in Huh-7 cells (Fig. 1B). Western blot analysis y The cell lysates were collected with RIPA lysis buffer (50 mM Tris-cl pH 7.4. 150 mM NaCl. 1% NP40. 0.25% Na-deoxycho- late. 1 mM PMSF, 1 mM EDTA; 5 mg/ml Aprotinin.) containing protease inhibitors (1 mM PMSF, 1 mM orthovanadate, 1 mM EDTA, and 10 mg/mL: leupeptin). Protein concentrations of cell lysates were measured using a Micro BCA protein assay reagent kit (Pierce, Rockford, IL, USA). To the cell lysate, the same volume of SDS-PAGE sample loading buffer [100 mmol/L Tris- HCl, 4% SDS, 5% b-mercaptoethanol, 20% glycerol, and 0.1% bromphenol blue (pH 6.8)] was added, and the cell lysates were boiled for 10 min. Equal amounts of proteins were resolved in 10 or 12% SDS-polyacrylamide gels and transferred to nitrocellulose membranes using a semidry transfer cell. The blotted membrane was washed twice with TBS containing 0.1% Tween 20 (TBST; 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween-20). After blocking with TBST containing 5% nonfat milk for 1 h, the membranes were probed with the following antibodies against: HA, b-actin (43 Kda), GRP78 (78 kDa), Bcl-2 (26 kDa), p65 (65 kDa), p-Ser276-p65 (65 kDa), p-Ser311-p65 (65 kDa), Bcl-xL (30 kDa)), MCL-1 (40 kDa), Bax (23 kDa), Bak (30 kDa), Akt (62 kDa), pAkt (62 kDa), ERK (44 kDa), and pERK (44 kDa) antibodies in 1% TBST nonfat milk at 4uC overnight. The membrane was washed thrice with TBST for a total of 15 min. The secondary anti-mouse IgG-HRP conjugates or anti-rabbit PLoS ONE \| www.plosone.org December 2011 \| Volume 6 \| Issue 12 \| e28977 PLoS ONE \| www.plosone.org 2 Induction of Bcl-2 by HBV Pre-S2 Mutant Protein Induction of Bcl-2 by HBV Pre-S2 Mutant Protein IgG-HRP conjugates (1:2,000 dilutions) was subsequently incu- bated with the membrane for 1 h at room temperature and was washed extensively for 50 min with TBST. The blots were visualized with the enhanced chemiluminescence (GE, Pittsburgh, PA, USA), and according to the manufacturer’s instructions. The blots were developed with the ECL-Western blot detection system according to the manufacturer’s instruction. Cell viability Cell viability was assessed using the trypan blue staining assay in three replicates. Huh-7 and stable transfectant cells were seeded at 16104 per well in 24-well flat-bottomed plates and incubated in 10% FBS–supplemented DMEM for 24 h. Cells were treated with 0.1, 0.25, and 0.5 mM 5-fluorouracil in the same medium. Controls received the DMSO vehicle at a concentration same as that in drug-treated cells. After 4 days, cell viability was determined by trypan blue exclusion and microscopy examination. Analysis of caspase-3 activity Caspase-3 activity was determined using PE active caspase-3 apoptosis kit (BD Pharmingen). Briefly, Huh7 V, pre-S, and pre- S2D (56105) cells in 10-cm dishes were subjected to different drug treatments as indicated for 72 h and were resuspended cells in 0.5 ml Cytofix/Cytoperm solution for 20 min on ice. Further- more, cells were incubates in 100 ml of Perm/Wash buffer containing 20 ml caspase-3 antibody for 30 min at room temperature. Each sample was then added with 400 ml Perm/ Wash buffer, and caspase-3 activity signals were analyzed by flow cytometry. PLoS ONE \| www.plosone.org December 2011 \| Volume 6 \| Issue 12 \| e28977 3 Induction of Bcl-2 by HBV Pre-S2 Mutant Protein Figure 1. Expression of HBV pre-S2D large surface protein in Huh-7 cells. Schematic representation of various lengths of HBV surface protein. (A) The pre-S represents full length (384 a.a). S represents C-terminal of pre-S2D (158–384 a.a.) Numbers in each construct correspond to amino acid positions. The arrow represents the point mutation of pre-S2 start codon from ATG to ATA. Gray box indicates pre-S2 deletion region. (B) Huh-7 cells were transfected with pIRES-pre-S2D-HA (pre-S2D), pIRES-pre-S-HA (pre-S), or pIRES vector control (V). The expression of pre-S2D protein and pre-S in Huh-7 cells were analyzed by Western blotting with antibody for HA. b-actin was used as a loading control. doi:10.1371/journal.pone.0028977.g001 reconfirm the induction of Bcl-2 expression by pre-S2D proteins, we analyzed three additional clones of pre-S2D stable cell lines for the levels of Bcl-2 expression. The result shown that increased of Bcl-2 protein expression was also observed in those three additional preS-S2D stable cell lines (Fig. 3E). Furthermore, to evaluate the event of induction of Bcl-2 expression by pre-S2D proteins manner in human hepatocyte, Bcl-2 expression level was determined in NeHepLxHT and NeHepLxHT pre-S2D cell lines. The result indicated that expression of Bcl-2 was increased by pre-S2D proteins in human hepatocyte NeHepLxHT cells (Fig. 3F). The pre-S and pre-S2D large surface proteins enhance cell growth in Huh-7 cell line The characteristics of pre-S and pre-S2D large surface proteins in Huh-7 cells are investigated for cell growth rate. The stable cell lines of pre-S and pre-S2D large surface proteins were confirmed using trypan blue staining assay and colony formation assay. The result indicated that the growth rate of pre-S and pre-S2D stable cell lines increased at 72–96 h (Fig. 2A). Furthermore, in order to eliminate the possibility that pre-S and pre-S2D large surface proteins affect the colony-forming ability in Huh-7 cell line, we determined the number colonies in Huh-7 cells cultured for 14 days. As shown in Fig. 2B, Huh-7 pre-S2D cells produced the largest number of colonies, 39967. Huh-7 pre-S cells produced fewer colonies, 30766, and Huh-7 V cells produced the smallest number of colonies, 24265 (Fig. 2B). Induction of NF-kB, Akt and ERK phosphorylation by HBV pre-S2D and pre-S large surface protein Previous studies have indicated that expression of viral protein can cause ER stress and NF-kB activation [21,38,39]. Our previous results also demonstrated that ER stress induced NF-kB activation and nuclear localization. Therefore, to investigate the effect of pre-S2D and pre-S large surface proteins on ER stress and NF-kB activation in Huh-7 cells, the expression of GRP78 and NF-kB phosphorylation were analyzed by Western blotting. The expression of GRP78, an unfolded protein response chaperone, was enhanced by HBV pre-S2D and pre-S large surface proteins. Phosphorylation of NF-kB at ser276 and ser311 sites was increased in HBV pre-S2D and pre-S-expressing cells (Fig. 5A). In addition, the ERK MAP kinase and PI3-kinase/Akt pathways are major intracellular signaling modules, which are known to regulate diverse cellular processes including cell proliferation, survival and malignant transformation. The ERK and Akt phosphorylation status were determined by Western blotting. The result shows that induction of ERK and Akt phosphorylation were observed in Huh-7 pre-S2D and pre-S cell lines (Fig. 5B). doi:10.1371/journal.pone.0028977.g001 Increased Bcl-2 family expression in Huh-7 pre-S2D large surface protein cell line Induction of Bcl-2 expression by pre-S2D proteins was observed in Huh-7 cells. Furthermore, expression of Bcl-2 family was also estimated in Huh-7 V, pre-S2D, and pre-S cell lines, and the data indicated that Bcl-2 family, Bcl-xL and MCL-1, were increased in Huh-7 pre-S2D cells (Fig. 4A). For example, the Bc-xL and MCL- 1 expression levels in Huh-7 pre-S2D cells were enhanced to about 2.5 and 3-fold compared with Huh-7 V cells (Fig. 4B). Notably, Bcl-xL expression levels were also induced to about 2-fold in Huh- 7 pre-S cells. Figure 1. Expression of HBV pre-S2D large surface protein in Huh-7 cells. Schematic representation of various lengths of HBV surface protein. (A) The pre-S represents full length (384 a.a). S represents C-terminal of pre-S2D (158–384 a.a.) Numbers in each construct correspond to amino acid positions. The arrow represents the point mutation of pre-S2 start codon from ATG to ATA. Gray box indicates pre-S2 deletion region. (B) Huh-7 cells were transfected with pIRES-pre-S2D-HA (pre-S2D), pIRES-pre-S-HA (pre-S), or pIRES vector control (V). The expression of pre-S2D protein and pre-S in Huh-7 cells were analyzed by Western blotting with antibody for HA. b-actin was used as a loading control. doi:10.1371/journal.pone.0028977.g001 PLoS ONE \| www.plosone.org December 2011 \| Volume 6 \| Issue 12 \| e28977 Differential susceptibility of Huh-7 V, Huh-7 pre-S2D, and Huh-7 pre-S cell lines to 5-fluorouracil-induced cell death (B) Huh-7 stable cell lines were analyzed for colony formation ability by colony formation assay, and colony formation was scored after 7 days. The number of colonies in the graphs was representative of three independent experiments (lower panel). Data represent the mean 6 SD (n = 3). Significant differences (, P,0.05 and , P,0.01) between the control and experimental group are marked with an asterisks. doi:10.1371/journal.pone.0028977.g002 Figure 2. HBV pre-S2D large surface protein increases cell growth and colony formation. (A) The effect of HBV large surface protein on Huh-7 growth rate was determined and cells were maintained in FBS–supplemented DMEM for 4 days, and the number of cells was assessed by trypan blue staining assay. (B) Huh-7 stable cell lines were analyzed for colony formation ability by colony formation assay, and colony formation was scored after 7 days. The number of colonies in the graphs was representative of three independent experiments (lower panel). Data represent the mean 6 SD (n = 3). Significant differences (, P,0.05 and , P,0.01) between the control and experimental group are marked with an asterisks. doi:10.1371/journal.pone.0028977.g002 compared with Huh-7 V or Huh-7 pre-S (Fig. 6B). Furthermore, the effect of 5-fluorouracil on these three cell lines was evaluated by colony formation assay (Fig. 6C). The number of colonies was quantified from violet staining and Huh-7 pre-S2D exhibited largest number of colonies after 5-fluorouracil treatment (Fig. 6D). It is noteworthy that Huh-7 pre-S cell produced more colonies than Huh-7 V cell. To assess the effect of 5-fluorouracil on cell apoptosis, caspase-3 activity was also determined. Flow cytometric analysis of caspase-3 activity showed that 5-fluorouracil treatment was significantly increased caspase-3 activity in Huh-7 V and Figure 3. Elevated Bcl-2 expression in HBV pre-S2D cells. (A) Total RNA was isolated from Huh-7 -V, Huh-7 pre-S2D, and Huh-7 pre-S cells and then subjected to RT-PCR analysis with Bcl-2 and G3APDH specific primers. (B) For Huh-7 -V, Huh-7 pre-S2D, and Huh-7 pre-S cells, the relative levels of Bcl-2 mRNA were quantified. The data represent the mean of Bcl-2 mRNA expression level from three independent experiments. Columns, mean; bars, SD (n = 3). Significant differences (, P,0.01) between the control and experimental group are marked with an asterisks. (C) The cell lysates collected from three stable clones, and cell lysates were analyzed by Western blotting analysis with antibodies for hemagglutinin (HA) tag, Bcl-2, and b-actin. Differential susceptibility of Huh-7 V, Huh-7 pre-S2D, and Huh-7 pre-S cell lines to 5-fluorouracil-induced cell death p y , p , Huh-7 pre-S cell lines to 5-fluorouracil-induced cell death The in vitro antitumor efficacy of 5-fluorouracil in three human HCC stable cell lines, Huh-7 V, Huh-7 pre-S2D, and Huh-7 pre-S were evaluated. As these three stable cell lines harbor vector only and different HBV large surface proteins, these stable cell lines were treated with 5-fluorouracil at different doses indicated. To investigate whether HBV pre-S2D large protein was involved in cytotoxic drug resistance mechanisms, the cell viability was determined by trypan blue staining assay and colony formation. As shown in Figure 5A, the 72-hour 5-fluorouracil treatment resulted in dose-dependent, progressive morphological changes from flat to round in Huh-7 V and Huh-7 pre-S cells. In contrast, only minor morphologic changes in Huh-7 pre-S2D cells were observed after 72-hours 5-fluorouracil treatment (Fig. 6A). They showed differential susceptibility to the antiproliferative effect of 5- fluorouracil. Huh-7 pre-S2D cells exhibited better cell survival Many studies have indicated that overexpression of Bcl-2 family was correlated with chemotherapeutic drug resistance. To observe the effect of pre-S2D and pre-S large surface proteins on Bcl-2 family expression in Huh-7 cells, the expression of Bcl-2 was determined by RT-PCR and Western blotting assay for Huh- 7 V, Huh-7 pre-S2D, and Huh-7 pre-S cell line with specific Bcl- 2 primer and Bcl-2 antibody. First, the total RNA was extracted from three Huh-7 stable cell lines, and RT-PCR to quantify the Bcl-2 mRNA. High expression of Bcl-2 mRNA was observed in hepatoma cells that express the deletion forms of HBV large surface proteins (Fig. 3A and Fig. 3B). Furthermore, the result shows that the expression levels of Bcl-2 protein were significantly increased in Huh-7 pre-S2D cell. For example, expression level of Bcl-2 in Huh-7 pre-S2D cell was enhanced to about 3-fold compared with that in Huh-7 V cell (Fig. 3C and Fig. 3D). To December 2011 \| Volume 6 \| Issue 12 \| e28977 PLoS ONE \| www.plosone.org 4 Induction of Bcl-2 by HBV Pre-S2 Mutant Protein Figure 2. HBV pre-S2D large surface protein increases cell growth and colony formation. (A) The effect of HBV large surface protein on Huh-7 growth rate was determined and cells were maintained in FBS–supplemented DMEM for 4 days, and the number of cells was assessed by trypan blue staining assay. Differential susceptibility of Huh-7 V, Huh-7 pre-S2D, and Huh-7 pre-S cell lines to 5-fluorouracil-induced cell death (D) The levels of Bcl-2 protein in the graphs were representative of three independent experiments (lower panel). Columns, mean; bars, SD (n = 3). Significant differences (, P,0.01) between the control and experimental group are marked with an asterisks. (E) Furthermore, expression level of Bcl-2 was determined by using Western blotting in three individual pre-S2D stable cell lines. (F) Expression level of Bcl-2 was enhanced by pre- S2D in the immortalized human hepatocyte cell line. Total cell lysate from NeHep and NeHep-pre-S2D were determined by Western blotting analysis with Bcl-2, pre-S, and b-actin specific antibosies. doi:10.1371/journal.pone.0028977.g003 Figure 3. Elevated Bcl-2 expression in HBV pre-S2D cells. (A) Total RNA was isolated from Huh-7 -V, Huh-7 pre-S2D, and Huh-7 pre-S cells and then subjected to RT-PCR analysis with Bcl-2 and G3APDH specific primers. (B) For Huh-7 -V, Huh-7 pre-S2D, and Huh-7 pre-S cells, the relative levels of Bcl-2 mRNA were quantified. The data represent the mean of Bcl-2 mRNA expression level from three independent experiments. Columns, mean; bars, SD (n = 3). Significant differences (, P,0.01) between the control and experimental group are marked with an asterisks. (C) The cell lysates collected from three stable clones, and cell lysates were analyzed by Western blotting analysis with antibodies for hemagglutinin (HA) tag, Bcl-2, and b-actin. (D) The levels of Bcl-2 protein in the graphs were representative of three independent experiments (lower panel). Columns, mean; bars, SD (n = 3). Significant differences (*, P,0.01) between the control and experimental group are marked with an asterisks. (E) Furthermore, expression level of Bcl-2 was determined by using Western blotting in three individual pre-S2D stable cell lines. (F) Expression level of Bcl-2 was enhanced by pre- S2D in the immortalized human hepatocyte cell line. Total cell lysate from NeHep and NeHep-pre-S2D were determined by Western blotting analysis with Bcl-2, pre-S, and b-actin specific antibosies. doi:10.1371/journal.pone.0028977.g003 December 2011 \| Volume 6 \| Issue 12 \| e28977 PLoS ONE \| www.plosone.org 5 Induction of Bcl-2 by HBV Pre-S2 Mutant Protein Figure 4. The pre-S2D large surface protein induced expression of Bcl-2 family proteins. (A) Total cell lysate was isolated from Huh-7 -V, Huh-7 pre-S2D, and Huh-7 pre-S cells and then subjected to Western blotting analysis with Bcl-xL, Mcl-1, Bax, and Bad specific antibodies. (B) For Huh- 7 -V, Huh-7 pre-S2D, and Huh-7 pre-S cells, the relative levels of Bcl-xL, Mcl-1, Bax, and Bad protein were quantified. Bcl-2 inhibitor decreased colony formation and increased caspase-3 activity in Huh-7 pre-S2D cells under 5-fluorouracil treatment Bcl-2 inhibitor decreased colony formation and increased caspase-3 activity in Huh-7 pre-S2D cells under 5-fluorouracil treatment We observed that induction of Bcl-2 expression was enhanced by HBV pre-S2D large protein in Huh-7 cells, and the cells were resistant to 5-fluorouracil treatment. To determine whether the induction Bcl-2 expression by HBV pre-S2D large protein was corrected with resistant to 5-fluorouracil in Huh-7 cells, the combined effect of Bcl-2 inhibitor (Bcl-2 Inhibitor II, YC137) and 5-fluorouracil was evaluated by colony formation assay, caspase-3 activity assay. First of all, cells were exposed to 5-fluorouracil agent or a combination of 5-fluorouracil with Bcl-2 inhibitor II, YC137. After incubation, the number of colonies was determined by staining with crystal violet (Fig. 7A). The data show us that when co- incubated 0.25 mM 5-fluorouracil with 1 mM or 5 mM Bcl-2 inhibitor, it significantly decreased colony formation ability in Huh- 7 pre-S2D and Huh-7 pre-S cells (Fig. 7B). However, in Huh-7 pre- S2D cell line, the colony formation ability was more dramatically decreased by co-treatment 5-fluorouracil with Bcl-2 inhibitor II Y137. In addition, we also observed changes in Huh-7 pre-S2D cell morphology after 5-fluorouracil with or without Bcl-2 inhibitor treatment. As shown in Figure 7C, the 72-hours 5-fluorouracil treatment, there were only minor morphologic changes after 72- hours 5-fluorouracil treatment in Huh-7 pre-S2D cells. In contrast, when a combination of 5-fluorouracil with Bcl-2 inhibitor Y II YC137, progressive morphological changes from flat to round in Huh-7 pre-S2D cells. Moreover, we examined the effect of 5- fluorouracil or a combination of 5-fluorouracil with Bcl-2 inhibitor II Y137 on modulating the activity of caspase-3 in Huh7 pre-S2D cells by using flow cytometric analysis. As shown in Figure 7D, exposure to 5-fluorouracil with or without Bcl-2 inhibitor II Y137 led to stimulation of caspase-3 activity (Fig. 7D). For example, 5- fluorouracil caused 3-fold increase in caspase-3 activity at 0.25 mM. However, when co-incubation of 5-fluorouracil with Bcl-2 inhibitor Figure 5. Expression of pre-S2D large surface protein increased ER stress and NF-kB, ERK, and Akt phosphorylation in Huh-7 cells. (A) (B) Cells were maintained in FBS–supplemented DMEM and cell lysates were obtained by RIPA lysis buffer. The cell lysates were determined by Western blotting using antibodies specific for GRP78, NF-kB p65, p-Ser276 p65, p-Ser311 p65, ERK, p-ERK, Akt, p-AKTand b-actin. doi:10.1371/journal.pone.0028977.g005 Figure 5. Differential susceptibility of Huh-7 V, Huh-7 pre-S2D, and Huh-7 pre-S cell lines to 5-fluorouracil-induced cell death The data represent the mean of Bcl-xL, Mcl-1, Bax, and Bad expression level from three independent experiments. Columns, mean; bars, SD (n = 3). Significant differences (, P,0.05) between the control and experimental group are marked with an asterisks. doi:10.1371/journal.pone.0028977.g004 Figure 4. The pre-S2D large surface protein induced expression of Bcl-2 family proteins. (A) Total cell lysate was isolated from Huh-7 -V, Huh-7 pre-S2D, and Huh-7 pre-S cells and then subjected to Western blotting analysis with Bcl-xL, Mcl-1, Bax, and Bad specific antibodies. (B) For Huh- 7 -V, Huh-7 pre-S2D, and Huh-7 pre-S cells, the relative levels of Bcl-xL, Mcl-1, Bax, and Bad protein were quantified. The data represent the mean of Bcl-xL, Mcl-1, Bax, and Bad expression level from three independent experiments. Columns, mean; bars, SD (n = 3). Significant differences (, P,0.05) between the control and experimental group are marked with an asterisks. doi:10.1371/journal.pone.0028977.g004 Huh-7 pre-S cells and, to a lesser extent, Huh-7 pre-S2D cells (Fig. 6E). For example, 5-fluorouracil caused 4.2-fold and 3.5-fold increases in caspase-3 activity at 0.25 mM treatment for Huh-7 V and Huh-7 pre-S cell lines. However, caspase-3 activity in Huh-7 pre-S2D cells, there is only 2.5-fold increased after 5-fluorouracil treatment (Fig. 6F). Bcl-2 inhibitor decreased colony formation and increased caspase-3 activity in Huh-7 pre-S2D cells under 5-fluorouracil treatment (A) The morphologic changes after a 96-hour 0.1 mM, 0.25 mM and 0.5 mM 5-fluorouracil treatment of Huh-7 V, Huh-7 pre-S2D, and Huh-7 Pre-S cells. The cells were followed by photography under phase-contrast magnification. (B) Cytotoxicity of 5-fluorouracil to Huh-7 V, Huh-7 pre-S2D and Huh-7 pre-S cells were analyzed by trypan blue staining assay. Columns, mean of three independent experiments; bars, SD (n = 3). Significant differences (, P,0.05) between the control and experimental group are marked with an asterisks (C) Cell viability after treatment with 5-FU. Huh-7 V, Huh-7 pre-S2D and Huh-7 pre-S cells (16103) were seeded in 6-well plate, as described in Materials and Methods. Cells were then incubated for another 10 days, and the colony formation ability of three cell lines were evaluated using a crystal violet assay determined by crystal violet in response to 5-fluorouracil. (D) A quantitative measure of colony formation was determined by crystal violet and the number of colonies in the graphs was representative of three independent experiments (lower panel). Columns, mean; bars, SD (n = 3). Significant differences (, P,0.01 and , P,0.001) between the control and experimental group are marked with an asterisks. (E) Flow cytometric analysis the effect of 5-fluorouracil on increasing caspase-3 activity in Huh7 V, Huh-7 pre-S2D, and Huh- 7 pre-S cells, increased caspase-3 activity was observed in Huh7 V, Huh-7 pre-S2D, and Huh-7 pre-S cells, after 72-h exposure to 5-Fluorouracil. (F) The relative caspase-3 activities, normalized to DMSO control, at the indicated 0.25 mM of 5-fluorouracil. Columns, mean of three independent experiments; bars, SD (n = 3). Significant differences (, P,0.05) between the control and experimental group are marked with an asterisks. doi:10.1371/journal.pone.0028977.g006 2 family expression status in treated Huh-7 pre-S2D cells (Fig. 7I). The result indicated that the combination of 5-fluorouracil with Bcl-2 inhibitor reduced expression level of Bcl-2, Bcl-xL and Mcl- 1. In addition, we also evaluated the effects of 5-fluorouracil with or without Bcl-2 inhibitor on phosphorylation status of NF-kB, AKT and ERK in Huh-7 pre-S2D cells. As shown in Fig. 7J, NF- kB, Akt and ERK phosphorylation did not affect by 5-fluorouracil or combination treatment. II Y137, caspase-3 activity was enhanced to 4-fold in Huh-7 pre- S2D cells compared with Huh-7 pre-S2D control cells (Fig. 7E). Hepatitis B Virus Mutant Large Surface Protein Can Induce Bcl-2 in Vivo To confirm further that deletion forms of mutant HBV large surface proteins can induce Bcl-2 in vivo, we created transgenic mice that express the pre-S2 deletion form of HBV large surface protein under the control of its native promoter. The expression of HBV large surface protein was detected in the liver (Fig. 8A). Elevated expression of Bcl-2 was observed in liver of pre-S2D transgenic mice (Fig. 8B). Furthermore, to examine the event of increased Bcl-2 expression by pre-S2D in hepatocytes, both control and pre-S2D transgenic mice liver tissues were analyzed by The combination of 5-fluorouracil and Bcl-2 inhibitor induces the concomitant inhibition of Bcl-2, Bcl-xL and Mcl-1 expression Bcl-2 inhibitor decreased colony formation and increased caspase-3 activity in Huh-7 pre-S2D cells under 5-fluorouracil treatment In addition, to evaluate early apoptosis and late apoptosis (or necrosis), the effects of 5-fluorouracil or a combination of 5-fluorouracil with Bcl-2 inhibitor II Y137 on Huh-7 pre-S2D cells was determined by Annexin V assay (Fig. 7F), which measures the transfer of phosphatidylserine from the inner to the outer membrane of cells and can detect both early and late apoptotic cells. As shown in Figure 7G and H, the early apoptosis and late apoptosis (necrosis) was significantly induced in Huh-7 pre-S2D cells after incubation of 0.25 mM 5-fluorouracil with or without Bcl-2 inhibitor for 24, 48, and 72 h. For example, the late apoptosis (necrosis) indices of 5- fluorouracil or a combination 5-fluorouracil with Bcl-2 inhibitor II Y137 in 72 h treatment were 25 and 46%, respectively. Bcl-2 inhibitor decreased colony formation and increased caspase-3 activity in Huh-7 pre-S2D cells under 5-fluorouracil treatment Expression of pre-S2D large surface protein increased ER stress and NF-kB, ERK, and Akt phosphorylation in Huh-7 cells. (A) (B) Cells were maintained in FBS–supplemented DMEM and cell lysates were obtained by RIPA lysis buffer. The cell lysates were determined by Western blotting using antibodies specific for GRP78, NF-kB p65, p-Ser276 p65, p-Ser311 p65, ERK, p-ERK, Akt, p-AKTand b-actin. b doi:10.1371/journal.pone.0028977.g005 PLoS ONE \| www.plosone.org December 2011 \| Volume 6 \| Issue 12 \| e28977 6 Induction of Bcl-2 by HBV Pre-S2 Mutant Protein Figure 6. Expression of pre-S2D contributes to 5-fluorouracil treatment in Huh-7 cells. (A) The morphologic changes after a 96-hour 0.1 mM, 0.25 mM and 0.5 mM 5-fluorouracil treatment of Huh-7 V, Huh-7 pre-S2D, and Huh-7 Pre-S cells. The cells were followed by photography under phase-contrast magnification. (B) Cytotoxicity of 5-fluorouracil to Huh-7 V, Huh-7 pre-S2D and Huh-7 pre-S cells were analyzed by trypan blue staining assay. Columns, mean of three independent experiments; bars, SD (n = 3). Significant differences (, P,0.05) between the control and experimental group are marked with an asterisks (C) Cell viability after treatment with 5-FU. Huh-7 V, Huh-7 pre-S2D and Huh-7 pre-S cells (16103) were seeded in 6-well plate, as described in Materials and Methods. Cells were then incubated for another 10 days, and the colony formation ability of three cell lines were evaluated using a crystal violet assay determined by crystal violet in response to 5-fluorouracil. (D) A quantitative measure of colony formation was determined by crystal violet and the number of colonies in the graphs was representative of three independent experiments (lower panel). Columns, mean; bars, SD (n = 3). Significant differences (, P,0.01 and *, P,0.001) between the control and experimental group are marked with an asterisks. (E) Flow cytometric analysis the effect of 5-fluorouracil on increasing caspase-3 activity in Huh7 V, Huh-7 pre-S2D, and Huh- 7 pre-S cells, increased caspase-3 activity was observed in Huh7 V, Huh-7 pre-S2D, and Huh-7 pre-S cells, after 72-h exposure to 5-Fluorouracil. (F) The relative caspase-3 activities, normalized to DMSO control, at the indicated 0.25 mM of 5-fluorouracil. Columns, mean of three independent experiments; bars, SD (n = 3). Significant differences (, P,0.05) between the control and experimental group are marked with an asterisks. doi:10.1371/journal.pone.0028977.g006 Figure 6. Expression of pre-S2D contributes to 5-fluorouracil treatment in Huh-7 cells. The combination of 5-fluorouracil and Bcl-2 inhibitor induces the concomitant inhibition of Bcl-2, Bcl-xL and Mcl-1 expression To clarify the mechanism by which Bcl-2 inhibitor enhanced the antitumor activities of the 5-fluorouracil, we examined the Bcl- PLoS ONE \| www.plosone.org December 2011 \| Volume 6 \| Issue 12 \| e28977 7 Induction of Bcl-2 by HBV Pre-S2 Mutant Protein Figure 7. Expression of Bcl-2 contributes to 5-fluorouracil resistance in Huh-7 pre-S2D cells. (A) Huh-7 V, Huh-7 pre-S2D and Huh-7 pre-S cells (16103) were seeded in 6-well plates. Cells were treated with 5-fluorouracil with or without Bcl-2 inhibitor, as described in Materials and Methods, and then incubated for another 10 days, and the colony formation ability of three cell lines were evaluated using a crystal violet assay determined by crystal violet in response to 5-fluorouracil. (B) A quantitative measure of colony formation was determined by crystal violet and the number of colonies in the graphs was representative of three independent experiments (lower panel). Data represent the mean 6 SD (n = 3). Significant differences (, P,0.01 and , P,0.001) between the control and experimental group are marked with an asterisks. (C) The morphologic changes after a 72-hour 0.25 mM 5-fluorouracil with or without Bcl-2 inhibitor treatment of Huh-7 pre-S2D cells. (D) The effect of 5-fluorouracil or combination with Bcl-2 inhibitor on caspase-3 activity in Huh-7 pre-S2D cell line. Enzymatic activity of caspase-3 was determined by caspase-3 antibody, as described under ‘‘materials and methods’’. (E) The relative caspase-3 activities, normalized to DMSO control, at the indicated 0.25 mM 5- fluorouracil or with 5 mM Bcl-2 inhibitor. Columns, mean of three independent experiments; bars, SD (n = 3). Significant differences (*, P,0.001) between the control and experimental group are marked with an asterisks. (F) Both apoptosis and necrosis were involved in 5-Fluorouracil or combination with Bcl-2 inhibitor-induced cell death. Huh-7 pre-S2D cells were treated with 5-fluorourail with or without Bcl-2 inhibitor and cells were analyzed by annexin V assay. (G) (H) The ratio of apoptotic and necrotic cells, normalized to DMSO control, at the indicated 0.25 mM 5-fluorouracil or with 5 mM Bcl-2 inhibitor. Columns, mean of three independent experiments; bars, SD (n = 3). Significant differences (, P,0.05; *, P,0.001) between the control and experimental group are marked with an asterisks. (I) Downregulation of Bcl-2 family expression by combination 5-fluorouracil with Bcl-2 inhibitor. Cells were treated with 5-fluorourail with or without Bcl-2 inhibitor and cell lysates were obtained by RIPA lysis buffer. The combination of 5-fluorouracil and Bcl-2 inhibitor induces the concomitant inhibition of Bcl-2, Bcl-xL and Mcl-1 expression The cell lysates were determined by Western blotting using antibodies specific for Bcl-2, Bcl-xL, Mcl-1, Bax, Bak and b-actin. (J) The effect of 5-fluorouracil or combination with Bcl-2 inhibitor on Akt, ERK and NF-kB phosphorylation in Huh-7 pre-S2D cell line. The total lysates were analyzed by Western blotting using ERK, p-ERK, Akt, p-Akt, p65, p-ser276-p65, p-ser311-p65 and b-actin antibodies. Figure 7. Expression of Bcl-2 contributes to 5-fluorouracil resistance in Huh-7 pre-S2D cells. (A) Huh-7 V, Huh-7 pre-S2D and Huh-7 pre-S cells (16103) were seeded in 6-well plates. Cells were treated with 5-fluorouracil with or without Bcl-2 inhibitor, as described in Materials and Methods, and then incubated for another 10 days, and the colony formation ability of three cell lines were evaluated using a crystal violet assay determined by crystal violet in response to 5-fluorouracil. (B) A quantitative measure of colony formation was determined by crystal violet and the number of colonies in the graphs was representative of three independent experiments (lower panel). Data represent the mean 6 SD (n = 3). Significant differences (, P,0.01 and *, P,0.001) between the control and experimental group are marked with an asterisks. (C) The morphologic changes after a 72-hour 0.25 mM 5-fluorouracil with or without Bcl-2 inhibitor treatment of Huh-7 pre-S2D cells. (D) The effect of 5-fluorouracil or combination with Bcl-2 inhibitor on caspase-3 activity in Huh-7 pre-S2D cell line. Enzymatic activity of caspase-3 was determined by caspase-3 antibody, as described under ‘‘materials and methods’’. (E) The relative caspase-3 activities, normalized to DMSO control, at the indicated 0.25 mM 5- fluorouracil or with 5 mM Bcl-2 inhibitor. Columns, mean of three independent experiments; bars, SD (n = 3). Significant differences (, P,0.001) between the control and experimental group are marked with an asterisks. (F) Both apoptosis and necrosis were involved in 5-Fluorouracil or combination with Bcl-2 inhibitor-induced cell death. Huh-7 pre-S2D cells were treated with 5-fluorourail with or without Bcl-2 inhibitor and cells were analyzed by annexin V assay. (G) (H) The ratio of apoptotic and necrotic cells, normalized to DMSO control, at the indicated 0.25 mM 5-fluorouracil or with 5 mM Bcl-2 inhibitor. Columns, mean of three independent experiments; bars, SD (n = 3). Significant differences (, P,0.05; **, P,0.001) between the control and experimental group are marked with an asterisks. (I) Downregulation of Bcl-2 family expression by combination 5-fluorouracil with Bcl-2 inhibitor. The combination of 5-fluorouracil and Bcl-2 inhibitor induces the concomitant inhibition of Bcl-2, Bcl-xL and Mcl-1 expression Cells were treated with 5-fluorourail with or without Bcl-2 inhibitor and cell lysates were obtained by RIPA lysis buffer. The cell lysates were determined by Western blotting using antibodies specific for Bcl-2, Bcl-xL, Mcl-1, Bax, Bak and b-actin. (J) The effect of 5-fluorouracil or combination with Bcl-2 inhibitor on Akt, ERK and NF-kB phosphorylation in Huh-7 pre-S2D cell line. The total lysates were analyzed by Western blotting using ERK, p-ERK, Akt, p-Akt, p65, p-ser276-p65, p-ser311-p65 and b-actin antibodies. doi:10.1371/journal.pone.0028977.g007 ER stress, Akt and ERK phosphorylation were also observed. Moreover, resistant cells caused by expression of HBV pre-S2D large surface protein could be sensitized by Bcl-2 inhibitor. Bcl-2 inhibitor overcomes drug resistance to 5-fluorouracil in Huh-7 pre-S2D cells, which provides a new approach to the combina- tional therapy of HBV pre-S2D large surface protein-related HCC. Overall, our data provide evidence for relationship between Bcl-2 and HBV pre-S2D, and suggest that Bcl-2 gene is an important determinant of drug-induced apoptosis thereby modu- lating resistance to chemotherapy in HBV pre-S2D- containing cells. ER stress, Akt and ERK phosphorylation were also observed. Moreover, resistant cells caused by expression of HBV pre-S2D large surface protein could be sensitized by Bcl-2 inhibitor. Bcl-2 inhibitor overcomes drug resistance to 5-fluorouracil in Huh-7 pre-S2D cells, which provides a new approach to the combina- tional therapy of HBV pre-S2D large surface protein-related HCC. Overall, our data provide evidence for relationship between Bcl-2 and HBV pre-S2D, and suggest that Bcl-2 gene is an important determinant of drug-induced apoptosis thereby modu- lating resistance to chemotherapy in HBV pre-S2D- containing cells. immunohistochemical staining assay. A strong Bcl-2 expression was found in pre-S2D transgenic mice hepatocytes. These results altogether demonstrate that Pre-S mutant surface proteins can induce Bcl-2 expression in vitro and in vivo. PLoS ONE \| www.plosone.org Discussion The expression of Bcl-2 and HBV surface proteins in liver tissues of control and pre-S2D transgenic mice was analyzed by Immunohistochemical staining (6400). doi:10.1371/journal.pone.0028977.g008 repair and the multidrug resistance-associated protein, are well understood. An alternation of various regulatory genes in tumor cells may also affect cellular sensitivity to chemotherapeutic drug. The changes in genes expression involve a diverse group of gene products, including oncogenes, tumor suppressor genes, cell cycle regulators, transcription factors, DNA repair factors, growth factor receptors, and cell death regulators. The multiple mechanisms of intrinsic drug resistance are not thoroughly understood and may involve the expression of multiple genes during tumor progression. In this study, Bcl-2 expression was enhanced by HBV pre-S2D large surface protein and the consequence of Bcl-2 expression was associated with resistance to 5-fluorouracil in hepatoma cells. In addition, the result has shown that phosphorylation of NF-kB p65 was increased in Huh-7 pre-S2D and Huh-7 pre-S cell lines. Transcriptional activity of NF-kB is controlled by phosphorylation of p65 at multiple serine residues. Here we show that induction of p65 phosphorylation at Ser276 and Ser311 sites is induced in Huh-7 pre-S2D and Huh-7 pre-S cells. In our previous studies, we demonstrated that activation of NF-kB was enhanced by ER stress through PERK-eIF2a signal pathway, pp38 kinase and calcium signaling [21]. Activation of NF-kB regulates more than 100 gene expressions as far as presently known. These genes have been reported to be involved in diverse physiological conditions of cells, such as inflammation and immune response, cell proliferation, cell differentiation, and apoptosis [40]. The induction of NF-kB activation by HBV pre-S2D large surface protein may practically provide anti-apoptotic effect on chemotherapeutic treatment. Taken together, the mechanisms of HBV pre-S2D large protein in HCC are not fully understood and need further investigation. expression, induces anti-apoptotic gene expression [43], stimulates cytokine production [44], induces angiogenesis via VEGF, IL-8, PDGF [45], activation of various cell cycle genes [46], and enhances the expression of the multidrug resistance (MDR) protein and mediates chemoresistance of tumor cells [47]. On the other hand, previous studies have indicated that NF-kB p65 mRNA and protein overexpression were detected in 5-fluoroura- cil-resistant cancer cell lines [48]. The resistant cell lines had higher NF-kB DNA binding and transcriptional activity. Inhibi- tion of NF-kB activity can enhance the cytotoxicity of some chemotherapeutic drugs. In addition, induction ERK and Akt phosphorylation was observed in both Huh-7 pre-S and pre-S2D- expressing cells. Discussion In this study, we show that HBV pre-S2D large surface protein enhances Bcl-2 gene expression and alters chemotherapeutic drug resistance by a mechanism involving Bcl-2 proto-oncogene expression in human hepatoma cancer cells. Furthermore, expression of Bcl-2 was increased by pre-S2D proteins in human immortalized hepatocyte cells and pre-S2D transgenic mice liver tissue. In addition, Bcl-2 family, Bcl-xL and Mcl-1, were increased in Huh-7 pre-S2D cells. In Huh-7 pre-S2D and Huh-7 pre-S cells, Modulation of apoptosis may influence resistance to chemo- therapy, thus affecting the outcome of cancer treatment. Several mechanisms of chemotherapeutic drug resistance in tumors, such as overexpression of the multidrug resistance gene, increased DNA PLoS ONE \| www.plosone.org December 2011 \| Volume 6 \| Issue 12 \| e28977 December 2011 \| Volume 6 \| Issue 12 \| e28977 8 Induction of Bcl-2 by HBV Pre-S2 Mutant Protein Figure 8. Elevated Bcl-2 is associated with pre-S2D expression in vivo. (A) Bcl-2 expression was elevated in transgenic mice expressing pre-S2 deletion (D2) HBV large surface protein. The expression of Bcl-2 in liver tissues (control and pre-S2D) was determined by Western blotting with antibodies for pre-S, Bcl-2, and b-actin for three individual transgenic mice samples. The expression levels of Bcl-2 were quantified by using image J software. The data represent the mean of Bcl-2 protein expression level from three independent experiments. Columns, mean; bars, SD (n = 3). Significant differences (, P,0.05) between the control and experimental group are marked with an asterisks. (C) A marked increase of Bcl-2 expression in pre-S2D-expressing hepatocytes was observed. The expression of Bcl-2 and HBV surface proteins in liver tissues of control and pre-S2D transgenic mice was analyzed by Immunohistochemical staining (6400). doi:10.1371/journal.pone.0028977.g008 Figure 8. Elevated Bcl-2 is associated with pre-S2D expression in vivo. (A) Bcl-2 expression was elevated in transgenic mice expressing pre-S2 deletion (D2) HBV large surface protein. The expression of Bcl-2 in liver tissues (control and pre-S2D) was determined by Western blotting with antibodies for pre-S, Bcl-2, and b-actin for three individual transgenic mice samples. The expression levels of Bcl-2 were quantified by using image J software. The data represent the mean of Bcl-2 protein expression level from three independent experiments. Columns, mean; bars, SD (n = 3). Significant differences (*, P,0.05) between the control and experimental group are marked with an asterisks. (C) A marked increase of Bcl-2 expression in pre-S2D-expressing hepatocytes was observed. Discussion Taken together, these results may be able to explain that although protein level of Bcl-2 was not altered in Huh-7 pre-S cells, pre-S-expressing cells exhibited minor drug resistant to 5-fluorouracil treatment. repair and the multidrug resistance-associated protein, are well understood. An alternation of various regulatory genes in tumor cells may also affect cellular sensitivity to chemotherapeutic drug. The changes in genes expression involve a diverse group of gene products, including oncogenes, tumor suppressor genes, cell cycle regulators, transcription factors, DNA repair factors, growth factor receptors, and cell death regulators. The multiple mechanisms of intrinsic drug resistance are not thoroughly understood and may involve the expression of multiple genes during tumor progression. In this study, Bcl-2 expression was enhanced by HBV pre-S2D large surface protein and the consequence of Bcl-2 expression was associated with resistance to 5-fluorouracil in hepatoma cells. In addition, the result has shown that phosphorylation of NF-kB p65 was increased in Huh-7 pre-S2D and Huh-7 pre-S cell lines. Transcriptional activity of NF-kB is controlled by phosphorylation of p65 at multiple serine residues. Here we show that induction of p65 phosphorylation at Ser276 and Ser311 sites is induced in Huh-7 pre-S2D and Huh-7 pre-S cells. In our previous studies, we demonstrated that activation of NF-kB was enhanced by ER stress through PERK-eIF2a signal pathway, pp38 kinase and calcium signaling [21]. Activation of NF-kB regulates more than 100 gene expressions as far as presently known. These genes have been reported to be involved in diverse physiological conditions of cells, such as inflammation and immune response, cell proliferation, cell differentiation, and apoptosis [40]. The induction of NF-kB activation by HBV pre-S2D large surface protein may practically provide anti-apoptotic effect on chemotherapeutic treatment. Taken together, the mechanisms of HBV pre-S2D large protein in HCC are not fully understood and need further investigation. Our previous studies indicate that overexpression of pre-S2D large surface proteins have been demonstrated in the induction of endoplasmic reticulum (ER) stress [18], oxidative stress and DNA damage [49], COX-2 expression [21], cyclin A expression [50], degradation of p27Kip1 [51], vascular endothelial growth factor-A [12], interaction with a-acid glucosidase [52], and lipid upregula- tion [53]. These results suggested that expression of HBV large surface proteins, especially pre-S2D mutant, might be important for hepatocarcinogenesis. Similar to HBV pre-S2D large surface protein, Epstein-Barr virus latent membrane protein 1 has been shown to induce Bcl-2 expression [54]. PLoS ONE \| www.plosone.org December 2011 \| Volume 6 \| Issue 12 \| e28977 References 1. Motola-Kuba D, Zamora-Valde´s D, Uribe M, Me´ndez-Sa´nchez N (2006) Hepatocellular carcinoma. An overview. Ann Hepatol 5(1): 16–24. 19. Huy TT, Ushijima H, Win KM, Luengrojanakul P, Shrestha PK, et al. (2003) High prevalence of hepatitis B virus pre-s mutant in countries where it is endemic and its relationship with genotype and chronicity. J Clin Microbiol 41(12): 5449–5455. p p ( ) 2. Kao JH, Chen DS (2005) Changing disease burden of hepatocellular carcinoma in the Far East and Southeast Asia. Liver Int 25(4): 696–703. 3. Chen CJ, Wang LY, Lu SN, Wu MH, You SL, et al. (1996) Elevated aflatoxin exposure and increased risk of hepatocellular carcinoma. Hepatology 24(1): 38–42. 20. Chen BF, Liu CJ, Jow GM, Chen PJ, Kao JH, et al. (2006) High prevalence and mapping of pre-S deletion in hepatitis B virus carriers with progressive liver diseases. Gastroenterology 130(4): 1153–1168. 4. Thorgeirsson SS, Grisham JW (2002) Molecular pathogenesis of human hepatocellular carcinoma. Nat Genet 31(4): 339–346. 21. Hung JH, Su IJ, Lei HY, Wang HC, Lin WC, et al. (2004) Endoplasmic reticulum stress stimulates the expression of cyclooxygenase-2 through activation of NF-kappaB and pp38 mitogen-activated protein kinase. J Biol Chem 279(45): 46384–46392. 5. Lee JW, Soung YH, Kim SY, Lee HW, Park WS, et al. (2005) PIK3CA gene is frequently mutated in breast carcinomas and hepatocellular carcinomas. Oncogene 24(8): 1477–1480. 22. Pahl HL, Baeuerle PA (1996) Activation of NF-kappa B by ER stress requires both Ca2+ and reactive oxygen intermediates as messengers. FEBS Lett 392(2): 129–136. g ( ) 6. Feitelson MA, Sun B, Satiroglu Tufan NL, Liu J, Pan J, et al. (2002) Genetic mechanisms of hepatocarcinogenesis. Oncogene 21(16): 2593–2604. 7. Suzuki K, Hayashi N, Yukinori Y, Yoshihara H, Miyamoto Y, et al. (1994) Expression of the c-met protooncogene in human hepatocellular carcinoma. Hepatology 20(5): 1231–1236. 23. Kaneko M, Niinuma Y, Nomura Y (2003) Activation signal of nuclear factor- kappa B in response to endoplasmic reticulum stress is transduced via IRE1 and tumor necrosis factor receptor-associated factor 2. Biol Pharm Bull 26(7): 931–935. 8. Parkin DM, Pisani P, Ferlay J (1993) Prevalence of HPV in cytomorphologically normal cervical smears, as determined by the polymerase chain reaction, is age- dependent. Int J Cancer 53(6): 919–923. 24. Kaneko M, Takahashi T, Niinuma Y, Nomura Y (2004) Manganese superoxide dismutase is induced by endoplasmic reticulum stress through IRE1-mediated nuclear factor (NF)-kappaB and AP-1 activation. References Biol Pharm Bull 27(8): 1202–1206. p J ( ) 9. Arbuthnot P, Kew M (2001) Hepatitis B virus and hepatocellular carcinoma. Int J Exp Pathol 82(2): 77–100. 10. Chisari FV, Klopchin K, Moriyama T, Pasquinelli C, Dunsford HA, et al. (1989) Molecular pathogenesis of hepatocellular carcinoma in hepatitis B virus transgenic mice. Cell 59(6): 1145–56. 25. Hu P, Han Z, Couvillon AD, Kaufman RJ, Exton JH (2006) Autocrine tumor necrosis factor alpha links endoplasmic reticulum stress to the membrane death receptor pathway through IRE1alpha-mediated NF-kappaB activation and down-regulation of TRAF2 expression. Mol Cell Biol 26(8): 3071–3084. 11. Wang Y, Cui F, Lv Y, Li C, Xu X, et al. (2004) HBsAg and HBx knocked into the p21 locus causes hepatocellular carcinoma in mice. Hepatology 39(2): 318–324. 26. Jiang HY, Wek SA, McGrath BC, Scheuner D, Kaufman RJ, et al. (2003) Phosphorylation of the alpha subunit of eukaryotic initiation factor 2 is required for activation of NF-kappaB in response to diverse cellular stresses. Mol Cell Biol 23(16): 5651–5663. 12. Yang JC, Teng CF, Wu HC, Tsai HW, Chuang HC, et al. (2009) Enhanced expression of vascular endothelial growth factor-A in ground glass hepatocytes and its implication in hepatitis B virus hepatocarcinogenesis. Hepatology 49(6): 1962–1971. 27. Gavrielides MV, Frijhoff AF, Conti CJ, Kazanietz MG (2004) Protein kinase C and prostate carcinogenesis: targeting the cell cycle and apoptotic mechanisms. Curr Drug Targets 5(5): 431–443. 13. Chen CH, Hung CH, Lee CM, Hu TH, Wang JH, et al. (2007) Pre-S deletion and complex mutations of hepatitis B virus related to advanced liver disease in HBeAg-negative patients. Gastroenterology 133(5): 1466–1474. 28. Hisaka T, Yano H, Haramaki M, Utsunomiya I, Kojiro M (1999) Expressions of epidermal growth factor family and its receptor in hepatocellular carcinoma cell lines: relationship to cell proliferation. Int J Oncol 14(3): 453–460. 14. Sugauchi F, Ohno T, Orito E, Sakugawa H, Ichida T, et al. (2003) Influence of hepatitis B virus genotypes on the development of preS deletions and advanced liver disease. J Med Virol 70(4): 537–544. 29. Ross JS, Fletcher JA (1999) HER-2/neu (c-erb-B2) gene and protein in breast cancer. Am J Clin Pathol 112(1 Suppl 1): S53–67. 15. Fan YF, Lu CC, Chang YC, Chang TT, Lin PW, et al. (2000) Identification of a pre-S2 mutant in hepatocytes expressing a novel marginal pattern of surface antigen in advanced diseases of chronic hepatitis B virus infection. J Gastroenterol Hepatol 15(5): 519–528. Acknowledgments We thank our lab members for critical reading of the manuscript and Prof. Ming-Derg Lai (Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University) for assistance with manuscript editing. Author Contributions Conceived and designed the experiments: J-HH. Performed the experi- ments: J-HH. Analyzed the data: Y-NT. Contributed reagents/materials/ analysis tools: Y-NT K-YL L-HW LH-CW. Wrote the paper: I-JS CW WH K-HL. Conceived and designed the experiments: J-HH. Performed the experi- ments: J-HH. Analyzed the data: Y-NT. Contributed reagents/materials/ analysis tools: Y-NT K-YL L-HW LH-CW. Wrote the paper: I-JS CW WH K-HL. Discussion The up-regulating expression of the cellular oncogene bcl-2 by EBV virus latent membrane protein 1 protects infected B cells from programmed cell death. Therefore, induction of the cellular oncogene bcl-2 expression has been demonstrated in two different types of virus. The induction of the cellular oncogene bcl-2 by other virus-encoded proteins warrants further investiga- tion. In addition, recently, accumulating evidence has indicated In addition, other previous reports have indicated that constitutive NF-kB activation is found in approximately 15–20% of all cancers [41,42], and NF-kB has been associated with several aspects of tumorigenesis. NF-kB plays an important role in gene expression in cancer development. It inhibited proapoptotic gene December 2011 \| Volume 6 \| Issue 12 \| e28977 December 2011 \| Volume 6 \| Issue 12 \| e28977 PLoS ONE \| www.plosone.org 9 Induction of Bcl-2 by HBV Pre-S2 Mutant Protein that altered miRNA level resulted from mutation or aberrant expression is correlated with gene expression and various human cancers development. For example, miR15b and miR-16 were demonstrated to play a role in the development of MDR in gastric cancer cells by targeting the antiapoptotic gene BCL2 [55]. Alternation of miRNA level by pre-S and pre-S2D in hepatoma cells may also provide important role in resistance to chemother- apeutic drugs. estimate HBV DNA levels in chronic HBV cancer patients, the HBsAg DNA sequence should be analyzed before undergoing systemic chemotherapy. That information of HBV pre-S2D large surface protein with HBV chronic infection is an adverse factor for survival and may be associated with a higher incidence of severe hepatitis during chemotherapy. Taken together, these results may provide a novel chemotherapeutic strategy for HBV pre-S2D large surface protein tumor cells. Chemotherapy for the treatment of cancer became a clinical practice more than 50 years ago. Although the chemotherapy has successfully treated many types of tumors, including HCC, HCC are inherently chemotherapy-resistant tumors and are known to overexpress the drug-resistant genes. Thus, there is a clear need for developing effective, life-prolonging therapeutic strategies for the large number of HCC patients with advanced disease. In this study, we reported that induction of cellular oncogene bcl-2 expression by HBV pre-S2D large surface proteins increased drug resistance to 5-fluorouracil treatment and drug resistance can be significantly decreased by co-incubation with Bcl-2 inhibitor. Because the occurrences of HBV pre-S2D large surface proteins in HBV cancer patients are about 30% [18]. Therefore, beside to 1. Motola-Kuba D, Zamora-Valde´s D, Uribe M, Me´ndez-Sa´nchez N (2006) Hepatocellular carcinoma. An overview. Ann Hepatol 5(1): 16–24. Induction of Bcl-2 by HBV Pre-S2 Mutant Protein Induction of Bcl-2 by HBV Pre-S2 Mutant Protein 35. Yeo W, Mo FK, Chan SL, Leung NW, Hui P, et al. (2007) Hepatitis B viral load predicts survival of HCC patients undergoing systemic chemotherapy. Hepatology 45(6): 1382–1389. 46. Guttridge DC, Albanese C, Reuther JY, Pestell RG, Baldwin AS (1999) NF- kappaB controls cell growth and differentiation through transcriptional regulation of cyclin D1. Mol Cell Biol 19(8): 5785–5799. p gy ( ) 36. Yeo W, Chan PK, Ho WM, Zee B, Lam KC, et al. (2004) Lamivudine for the prevention of hepatitis B virus reactivation in hepatitis B s-antigen seropositive cancer patients undergoing cytotoxic chemotherapy. J Clin Oncol 22(5): 927–934. 47. Bourguignon LY, Xia W, Wong G (2009) Hyaluronan-mediated CD44 interaction with p300 and SIRT1 regulates beta-catenin signaling and NFkappaB-specific transcription activity leading to MDR1 and Bcl-xL gene expression and chemoresistance in breast tumor cells. J Biol Chem 284(5): 2657–2671. 37. Nakopoulou L, Stefanaki K, Vourlakou C, Manolaki N, Gakiopoulou H, et al. (1999) Bcl-2 protein expression in acute and chronic hepatitis, cirrhosis and hepatocellular carcinoma. Pathol Res Pract 195(1): 19–24. 48. Wang W, Cassidy J, O’Brien V, Ryan KM, Collie-Duguid E (2004) Mechanistic and predictive profiling of 5-Fluorouracil resistance in human cancer cells. Cancer Res 64(22): 8167–8176. 38. Kanda T, Yokosuka O, Nagao K, Saisho H (2006) State of hepatitis C viral replication enhances activation of NF-kB- and AP-1-signaling induced by hepatitis B virus X. Cancer Lett 234(2): 143–148. 49. Hsieh YH, Su IJ, Wang HC, Chang WW, Lei HY, et al. (2004) Pre-S mutant surface antigens in chronic hepatitis B virus infection induce oxidative stress and DNA damage. Carcinogenesis 25(10): 2023–2032. 39. Li S, Ye L, Yu X, Xu B, Li K, et al. (2009) Hepatitis C virus NS4B induces unfolded protein response and endoplasmic reticulum overload response- dependent NF-kappaB activation. Virology 391(2): 257–264. 50. Wang HC, Chang WT, Chang WW, Wu HC, Huang W, et al. (2005) Hepatitis B virus pre-S2 mutant upregulates cyclin A expression and induces nodular proliferation of hepatocytes. Hepatology 41(4): 761–70. 40. Ghosh S, Karin M (2002) Missing pieces in the NF-kappaB puzzle. Cell 109 Suppl: S81–96. 51. Hsieh YH, Su IJ, Wang HC, Tsai JH, Huang YJ, Chang WW, et al. (2007) Hepatitis B virus pre-S2 mutant surface antigen induces degradation of cyclin- dependent kinase inhibitor p27Kip1 through c-Jun activation domain-binding protein 1. Mol Cancer Res 5(10): 1063–1072. 41. References 30. Fisher TC, Milner AE, Gregory CD, Jackman AL, Aherne GW, et al. (1993) bcl- 2 modulation of apoptosis induced by anticancer drugs: resistance to thymidylate stress is independent of classical resistance pathways. Cancer Res 53(14): 3321–3326. J p ( ) 16. Fan YF, Lu CC, Chen WC, Yao WJ, Wang HC, et al. (2001) Prevalence and significance of hepatitis B virus (HBV) pre-S mutants in serum and liver at different replicative stages of chronic HBV infection. Hepatology 33(1): 277–286. 31. Takehara T, Liu X, Fujimoto J, Friedman SL, Takahashi H (2001) Expression and role of Bcl-xL in human hepatocellular carcinomas. Hepatology 34(1): 55–61. 32. Watanabe J, Kushihata F, Honda K, Mominoki K, Matsuda S, et al. (2002) Bcl- xL overexpression in human hepatocellular carcinoma. Int J Oncol 21(3): 515–519. 17. Abe K, Thung SN, Wu HC, Tran TT, Le Hoang P, et al. (2009) Pre-S2 deletion mutants of hepatitis B virus could have an important role in hepatocarcinogen- esis in Asian children. Cancer Sci 100(12): 2249–2254. 33. Adams JM, Cory S (1998) The Bcl-2 protein family: arbiters of cell survival. Science 281(5381): 1322–1326. 18. Wang HC, Wu HC, Chen CF, Fausto N, Lei HY, et al. (2003) Different types of ground glass hepatocytes in chronic hepatitis B virus infection contain specific pre-S mutants that may induce endoplasmic reticulum stress. Am J Pathol 163(6): 2441–2449. 34. Gross A, McDonnell JM, Korsmeyer SJ (1999) BCL-2 family members and the mitochondria in apoptosis. Genes Dev 13(15): 1899–1911. PLoS ONE \| www.plosone.org December 2011 \| Volume 6 \| Issue 12 \| e28977 December 2011 \| Volume 6 \| Issue 12 \| e28977 10 Induction of Bcl-2 by HBV Pre-S2 Mutant Protein Pikarsky E, Porat RM, Stein I, Abramovitch R, Amit S, et al. (2004) NF-kappaB functions as a tumour promoter in inflammation-associated cancer. Nature 431(7007): 461–466. p ( ) 52. Hung JH, Yan CW, Su IJ, Wang HC, Lei HY, Lin WC, et al. (2010) Hepatitis B virus surface antigen interacts with acid alpha-glucosidase and alters glycogen metabolism. Hepatol Res 40(6): 633–640. 42. Karin M, Greten FR (2005) NF-kappaB: linking inflammation and immunity to cancer development and progression. Nat Rev Immunol 5(10): 749–759. 43. Ravi R, Bedi A (2004) NF-kappaB in cancer–a friend turned foe. Drug Resist Updat 7(1): 53–67. p ( ) 53. Chang YS, Tsai CT, Huangfu CA, Huang WY, Lei HY, et al. (2011) ACSL3 and GSK-3b are essential for lipid upregulation induced by endoplasmic reticulum stress in liver cells. J Cell Biochem 2011 112(3): 881–893. 44. Matsusaka T, Fujikawa K, Nishio Y, Mukaida N, Matsushima K, et al. (1993) Transcription factors NF-IL6 and NF-kappa B synergistically activate transcrip- tion of the inflammatory cytokines, interleukin 6 and interleukin 8. Proc Natl Acad Sci U S A 90(21): 10193–10197. 54. Henderson S, Rowe M, Gregory C, Croom-Carter D, Wang F, et al. (1991) Induction of bcl-2 expression by Epstein-Barr virus latent membrane protein 1 protects infected B cells from programmed cell death. Cell 65(7): 1107–1115. 45. Huang S, Robinson JB, Deguzman A, Bucana CD, Fidler IJ (2000) Blockade of nuclear factor-kappaB signaling inhibits angiogenesis and tumorigenicity of human ovarian cancer cells by suppressing expression of vascular endothelial growth factor and interleukin 8. Cancer Res 60(19): 5334–5339. 55. Xia L, Zhang D, Du R, Pan Y, Zhao L, et al. (2008) miR-15b and miR-16 modulate multidrug resistance by targeting BCL2 in human gastric cancer cells. Int J Cancer 123(2): 372–379. PLoS ONE \| www.plosone.org December 2011 \| Volume 6 \| Issue 12 \| e28977 11
https://openalex.org/W3011411479	https://hal.inrae.fr/hal-02573763/file/CropProtection-07-2020.pdf	English	null	Analysis of interactions amongst shade trees, coffee foliar diseases and coffee yield in multistrata agroforestry systems	Crop protection	2,020	cc-by	12,817	To cite this version: Clémentine Durand-Bessart, Philippe Tixier, Alcide Quinteros, Federico Andreotti, Bruno Rapidel, et al.. Analysis of interactions amongst shade trees, coffee foliar diseases and coffee yield in multistrata agroforestry systems. Crop Protection, 2020, 133, pp.105137. 10.1016/j.cropro.2020.105137. hal- 02573763 Analysis of interactions amongst shade trees, coffee foliar diseases and coffee yield in multistrata agroforestry systems Clémentine Durand-Bessart, Philippe Tixier, Alcide Quinteros, Federico Andreotti, Bruno Rapidel, Camille Tauvel, Clementine Allinne Distributed under a Creative Commons Attribution 4.0 International License Corresponding author 17 Clémentine Durand-Bessart, Biogéosciences UMR CNRS/uB 6282, Université de 18 Bourgogne, 6 bd Gabriel, 21000 Dijon, France; + 33 (0)3 80 39 63 56; clementine.durand- 19 bessart@u-bourgogne.fr 20 © 2020 published by Elsevier. This manuscript is made available under the Elsevier user license https://www.elsevier.com/open-access/userlicense/1.0/ HAL Id: hal-02573763 https://hal.inrae.fr/hal-02573763v1 Submitted on 26 Nov 2021 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Version of Record: https://www.sciencedirect.com/science/article/pii/S0261219420300703 Manuscript_1d4cacb341e64767fc3b1905b24ee446 Version of Record: https://www.sciencedirect.com/science/article/pii/S0261219420300703 Manuscript_1d4cacb341e64767fc3b1905b24ee446 Analysis of interactions amongst shade trees, coffee foliar 1 diseases and coffee yield in multistrata agroforestry 2 systems 3 4 Clémentine Durand-Bessarta1, Philippe Tixierbc, Alcide Quinterosd, Federico 5 Andreottief, Bruno Rapidela, Camille Tauvela, Clémentine Allinnede 6 7 a CIRAD, UMR SYSTEM, Université de Montpellier, CIHEAM-IAMM, CIRAD, INRA, 8 Montpellier SupAgro, Montpellier, France 9 b GECO, Université de Montpellier, CIRAD, Montpellier, France 10 c CIRAD, UPR GECO, F-34398 Montpellier, France 11 d CIRAD, UMR System, Turrialba, Costa Rica 12 e CATIE, Agriculture, Livestock and Agroforestry Program, Turrialba, Costa Rica 13 f Laboratory of Geo-information Science and Remote Sensing, Wageningen University & 14 Research, 6708 PB Wageningen, The Netherlands 15 16 Corresponding author 17 Clémentine Durand-Bessart, Biogéosciences UMR CNRS/uB 6282, Université de 18 Bourgogne, 6 bd Gabriel, 21000 Dijon, France; + 33 (0)3 80 39 63 56; clementine.durand- 19 bessart@u-bourgogne.fr 20 21 Abstract 22 1 P t dd Biogéosciences UMR CNRS/ B 6282 U i ité d B 6 bd Abstract 22 1 Present address: Biogéosciences UMR CNRS/uB 6282, Université de Bourgogne, 6 bd Gabriel, 21000 Dijon, France; + 33 (0)3 80 39 63 56; clementine.durand-bessart@u- bourgogne.fr 1 Present address: Biogéosciences UMR CNRS/uB 6282, Université de Bourgogne, 6 bd Gabriel, 21000 Dijon, France; + 33 (0)3 80 39 63 56; clementine.durand-bessart@u- bourgogne.fr 1 Present address: Biogéosciences UMR CNRS/uB 6282, Université de Bourgogne, 6 bd Gabriel, 21000 Dijon, France; + 33 (0)3 80 39 63 56; clementine.durand-bessart@u- bourgogne.fr 1 © 2020 published by Elsevier. This manuscript is made available under the Elsevier user license https://www.elsevier.com/open-access/userlicense/1.0/ © 2020 published by Elsevier. This manuscript is made available under the Elsevier user license https://www.elsevier.com/open-access/userlicense/1.0/ In complex coffee-based agroforestry systems, quantifying the impact of shade trees on 23 coffee disease regulation and coffee yield is crucial for improving these systems and 24 designing more sustainable ones. To this end, we analyzed interactions amongst shade 25 trees, coffee plants (cv. Catimor), the coffee foliar disease complex and soil characteristics. 26 We studied systems characterized by 40 variables measured in 60 plots located on three 27 farms (monitored for 2 years) in Nicaragua. These variables characterized six system 28 components grouped in six statistical blocks: shade trees (shade percentage and species 29 abundancy), soil characteristics (fertility), foliar diseases, coffee plant characteristics (age 30 and size), coffee growth and yield. We used partial least square path modelling (PLS-PM), 31 i.e. a structural equation modelling approach used to understand and quantify interactions 32 between the six blocks. Shade trees (mostly the associated shade percentage) had direct 33 positive effects on foliar disease severity and incidence and soil quality, while having 34 negative effects on coffee growth and yield. Soil characteristics (carbon, nitrogen, litter index, 35 water infiltration potential) were negatively correlated with foliar diseases. An excessive 36 shade percentage then had an indirect negative effect on coffee growth and yield due to the 37 increased prevalence of foliar diseases. Finding the optimal shade cover can help reduce 38 foliar diseases and enhance coffee berry production. The ‘dose effect’ of shade cover must 39 also be considered because excessive shade, as well as lack of shade, have negative 40 impacts on coffee growth and yield. Overall, effective shade management requires an 41 analysis of trade-offs between soil quality, disease regulation and yield gains. Abstract 22 In conclusion, 42 PLS-PM turned out to be a good tool for studying agroecosystem networks and enabled us 43 to put forward some foliar disease management and coffee yield enhancement guidelines. 44 45 49 2 2 1. INTRODUCTION 50 Pests and diseases reduce coffee yields in Central American coffee-based agroforestry 51 systems by 15-30% (Cerda et al., 2015). Sustainable management of these diseases based 52 on agroecological processes, e.g. biological regulation optimization, is thus a key lever to 53 increase coffee yield while maintaining the environmental sustainability of the cropping 54 system. Trees associated with coffee plots support biological regulation to a major extent 55 through direct and indirect processes (Ratnadass et al. 2012). Direct regulation effects that 56 reduce diseases involve different processes, including: 1) dilution of host density, 2) 57 reduction of soil diseases by favoring beneficial microorganisms, 3) allelopathic effects, 4) 58 reservoir of natural enemies, and 5) creation of microclimates unfavourable for the diseases 59 (Ratnadass et al. 2012). Shade trees may have indirect beneficial effects on coffee plants, 60 mostly by enhancing coffee nutrition (Sauvadet et al. 2018). In complex agroecosystems with 61 high spatially heterogeneous plant diversity associated with coffee plants, unravelling the 62 direct and indirect effects of shade trees on all coffee crop systems is a great challenge. 63 64 Nicaragua is eighth largest C. arabica producing country in the world, with production 65 reaching 2.54 million kg in the 2017-2018 cycle. Coffee cropping has a huge socioeconomic 66 impact in this country, where 44 thousand coffee producers cultivate a total area of 1.5 67 million ha. Most of the farms grow coffee under agroforestry systems, and 97% of them are 68 less than 14 ha. Nicaraguan coffee-based agroforestry systems are known to be particularly 69 complex with a remarkable diversity of shade trees (Haggar et al., 2015). This diversity 70 includes species that produce goods for local markets, native forest species that are grown 71 mainly for timber, along with service tree species — mostly Fabaceae — that are planted to 72 provide shade while improving soil fertility and crop system sustainability (Barradas and 73 Fanjul, 1986; Vaast et al., 2005). The coffee rust outbreak that occurred in 2013–2014 led 74 farmers to replace the rust-sensitive cv. Caturra plants in their coffee plantations with rust- 75 resistant cv. Catimor plants (Libert Amico et al., 2019). However, Catimor cultivars are 76 1. INTRODUCTION Other diseases like brown-eye spot 78 (Cercospora coffeicola Berk. & Cooke), anthracnose (Colletotrichum sp.) and coffee thread 79 blight (Corticium kolegora) also affect cv. Catimor coffee plants (Waller et al., 2007). These 80 foliar diseases have negative effects on coffee growth and production, and interact with each 81 other as a disease complex depending on the coffee crop status and the microclimatic 82 conditions. A major way to improve disease management is to integrate the role of shade 83 trees on these foliar diseases (Avelino et al., 2018; Allinne et al., 2019). Indeed, ALS and 84 coffee thread blight are favored by high humidity when shade levels are elevated, unlike 85 brown-eye spot and anthracnose that tend to affect sun-grown coffee plants (Staver et al., 86 2001; Muller et al., 2008; Bedimo et al., 2012). Most Nicaraguan farmers do not have 87 sufficient financial resources to manage pests and diseases using pesticides (Bro et al., 88 2019). Understanding the relations within disease complexes that affect coffee plants and the 89 diversity of shade trees is crucial and requires an overview of the entire pathosystem. 90 91 particularly sensitive to the American leaf spot (ALS) disease caused by Mycena citricolor 77 (Sequeira 1958; Staver et al., 2001; Allinne et al., 2016). Other diseases like brown-eye spot 78 (Cercospora coffeicola Berk. & Cooke), anthracnose (Colletotrichum sp.) and coffee thread 79 blight (Corticium kolegora) also affect cv. Catimor coffee plants (Waller et al., 2007). These 80 foliar diseases have negative effects on coffee growth and production, and interact with each 81 other as a disease complex depending on the coffee crop status and the microclimatic 82 conditions. A major way to improve disease management is to integrate the role of shade 83 trees on these foliar diseases (Avelino et al., 2018; Allinne et al., 2019). Indeed, ALS and 84 coffee thread blight are favored by high humidity when shade levels are elevated, unlike 85 brown-eye spot and anthracnose that tend to affect sun-grown coffee plants (Staver et al., 86 2001; Muller et al., 2008; Bedimo et al., 2012). Most Nicaraguan farmers do not have 87 sufficient financial resources to manage pests and diseases using pesticides (Bro et al., 88 2019). Understanding the relations within disease complexes that affect coffee plants and the 89 diversity of shade trees is crucial and requires an overview of the entire pathosystem. 1. INTRODUCTION & Cooke), anthracnose (Colletotrichum sp.) and coffee thread 79 blight (Corticium kolegora) also affect cv. Catimor coffee plants (Waller et al., 2007). These 80 foliar diseases have negative effects on coffee growth and production, and interact with each 81 other as a disease complex depending on the coffee crop status and the microclimatic 82 conditions. A major way to improve disease management is to integrate the role of shade 83 trees on these foliar diseases (Avelino et al., 2018; Allinne et al., 2019). Indeed, ALS and 84 coffee thread blight are favored by high humidity when shade levels are elevated, unlike 85 brown-eye spot and anthracnose that tend to affect sun-grown coffee plants (Staver et al., 86 2001; Muller et al., 2008; Bedimo et al., 2012). Most Nicaraguan farmers do not have 87 sufficient financial resources to manage pests and diseases using pesticides (Bro et al., 88 2019). Understanding the relations within disease complexes that affect coffee plants and the 89 diversity of shade trees is crucial and requires an overview of the entire pathosystem. 90 91 In these complex agroforestry systems, shade trees do not just provide shade to the crop 92 system, they also have directly impact coffee growth and yield by increasing the leaf surface 93 and coffee quality (Vaast et al., 2005; Charbonnier et al., 2017). Conversely, shade trees and 94 coffee plants can compete for light and nutrients, especially under high shade tree density 95 conditions (Charbonnier et al., 2013). However, shade trees may also markedly alter the soil 96 characteristics, and in some cases improve soil fertility (Sauvadet et al., 2018). This is well- 97 known by farmers who often plant nitrogen-fixing trees (e.g. Inga spp.) to favor nitrogen 98 fixation (Cerdán et al., 2012). These interactions between shade trees and agroecosystem 99 processes are also driven by farmers through their pruning practices whereby the canopy is 100 opened and dead branches are left on the ground (Cerdán et al., 2012). This litter addition 101 around coffee plants may enhance soil fertility and promote the activity of beneficial soil 102 microorganisms (Sauvadet et al., 2018). Coffee plant resistance against foliar diseases is 103 dependent on these soil characteristics. Indeed, coffee plants growing in more fertile soil 104 particularly sensitive to the American leaf spot (ALS) disease caused by Mycena citricolor 77 (Sequeira 1958; Staver et al., 2001; Allinne et al., 2016). 1. INTRODUCTION 50 Pests and diseases reduce coffee yields in Central American coffee-based agroforestry 51 systems by 15-30% (Cerda et al., 2015). Sustainable management of these diseases based 52 on agroecological processes, e.g. biological regulation optimization, is thus a key lever to 53 increase coffee yield while maintaining the environmental sustainability of the cropping 54 system. Trees associated with coffee plots support biological regulation to a major extent 55 through direct and indirect processes (Ratnadass et al. 2012). Direct regulation effects that 56 reduce diseases involve different processes, including: 1) dilution of host density, 2) 57 reduction of soil diseases by favoring beneficial microorganisms, 3) allelopathic effects, 4) 58 reservoir of natural enemies, and 5) creation of microclimates unfavourable for the diseases 59 (Ratnadass et al. 2012). Shade trees may have indirect beneficial effects on coffee plants, 60 mostly by enhancing coffee nutrition (Sauvadet et al. 2018). In complex agroecosystems with 61 high spatially heterogeneous plant diversity associated with coffee plants, unravelling the 62 direct and indirect effects of shade trees on all coffee crop systems is a great challenge. 63 64 Nicaragua is eighth largest C. arabica producing country in the world, with production 65 reaching 2.54 million kg in the 2017-2018 cycle. Coffee cropping has a huge socioeconomic 66 impact in this country, where 44 thousand coffee producers cultivate a total area of 1.5 67 million ha. Most of the farms grow coffee under agroforestry systems, and 97% of them are 68 less than 14 ha. Nicaraguan coffee-based agroforestry systems are known to be particularly 69 complex with a remarkable diversity of shade trees (Haggar et al., 2015). This diversity 70 includes species that produce goods for local markets, native forest species that are grown 71 mainly for timber, along with service tree species — mostly Fabaceae — that are planted to 72 provide shade while improving soil fertility and crop system sustainability (Barradas and 73 Fanjul, 1986; Vaast et al., 2005). The coffee rust outbreak that occurred in 2013–2014 led 74 farmers to replace the rust-sensitive cv. Caturra plants in their coffee plantations with rust- 75 resistant cv. Catimor plants (Libert Amico et al., 2019). However, Catimor cultivars are 76 3 3 particularly sensitive to the American leaf spot (ALS) disease caused by Mycena citricolor 77 (Sequeira 1958; Staver et al., 2001; Allinne et al., 2016). Other diseases like brown-eye spot 78 (Cercospora coffeicola Berk. 1. INTRODUCTION 90 In these complex agroforestry systems, shade trees do not just provide shade to the crop 92 system, they also have directly impact coffee growth and yield by increasing the leaf surface 93 and coffee quality (Vaast et al., 2005; Charbonnier et al., 2017). Conversely, shade trees and 94 coffee plants can compete for light and nutrients, especially under high shade tree density 95 conditions (Charbonnier et al., 2013). However, shade trees may also markedly alter the soil 96 characteristics, and in some cases improve soil fertility (Sauvadet et al., 2018). This is well- 97 known by farmers who often plant nitrogen-fixing trees (e.g. Inga spp.) to favor nitrogen 98 fixation (Cerdán et al., 2012). These interactions between shade trees and agroecosystem 99 processes are also driven by farmers through their pruning practices whereby the canopy is 100 opened and dead branches are left on the ground (Cerdán et al., 2012). This litter addition 101 around coffee plants may enhance soil fertility and promote the activity of beneficial soil 102 microorganisms (Sauvadet et al., 2018). Coffee plant resistance against foliar diseases is 103 dependent on these soil characteristics. Indeed, coffee plants growing in more fertile soil 104 4 have higher regeneration properties and growth, which are key physiological resistance 105 factors (Ratnadass et al., 2012). Soil fertility also influences the quality and abundance of 106 coffee beans produced (Barel and Jacquet, 1994; Lin, 2010). 107 108 New tools are needed to study this network of interactions between different agroecosystem 109 components overall. We used a structural equation modelling (SEM) approach called partial 110 least square path modelling (PLS-PM) to gain further insight into the direct and indirect 111 effects of shade trees on coffee foliar diseases and coffee yield in Nicaraguan coffee-based 112 agroforestry systems. 113 Table 1. Description of the three farms where the study was conducted in the area around El Tuma-La 129 Dalia, Nicaragua. 130 4.2. STUDY SITES 116 The study took place in the Matagalpa region (Nicaragua’s main coffee production area) near 117 the village of El Tuma–La Dalia. We studied three small coffee farms from May 2016 to 118 February 2018 under conventional, low-input and organic disease management conditions. 119 The farms were chosen for their high shade tree diversity, with marked variability in the 120 proportion of shade, i.e. 49-85% (Table 1). These farms only grew non-certified Coffea 121 arabica (Rubiaceae) cv. Catimor plants. This genetic material was rust resistant (no evidence 122 of rust affection was observed during the experiment) but sensitive to American leaf spot 123 caused by Mycena citricolor (Allinne et al., 2016; Libert Amico et al, 2019). The farms were 124 located between 13°02’67.7’’N and 13°08’75.6’’N and between 85°61’42.7’’W and 125 85°71’48.3’’W, within a similar elevation range (650-850 m a.s.l.). The mean annual 126 temperature was 23°C, with annual precipitation ranging from 2,000 to 2,600 mm. The rainy 127 season in this region is between May and December (Amores Contreras, 2015). 128 Table 1. Description of the three farms where the study was conducted in the area around El Tuma-La 129 Dalia, Nicaragua. 130 Farm 5 5 Variables Unit 1 2 3 Farm description Community Yale 3 Hilipo 2 Aguas Amarillas GPS location (N, W) 13.08756, - 85.61427 13.03735,- 8571483 13.02677,- 85.67999 Elevation (m) 750-800 850 650 - 700 Area (ha) 5 3 3 Meteorological data Average temperature [min-max] (°C) 22.3 [19.2- 29] 21.9 [19.2- 28.4] 23.9 [21.1- 28.7] Cumulative rainfull (mm) 2600 2341 2132 Coffee plot description Average coffee age (year) 9 6 6 Coffee density (plants/ha) 8882 8620 8679 Average shade cover [min-max] (%) 72 [62-77] 65 [49-76] 80 [71-85] Average shade tree density (tree/ha) 360 350 487 Average shade species richness 30 18 37 Coffee crop managment Weeds / manual (3 x) manual (3x) chemical: glyphosate+ paraquat Diseases / copper (Bordeaux mixture) (1x) copper (Bordeaux mixture) (1x) carbendazim (2x) Pests / / / cipermetrina Fertilization / / biofertilizer (foliar) NPK (20-5-20) Variables 4.3. AGROFORESTRY SYSTEM CHARACTERIZATION 132 For each farm, we selected 20 circular plots (14 m dia.), centered on a shade tree and sorted 133 in four different situations, with five replicates each. The first three situations were based 134 around three common tree species spread on the farms, while the last situation was made 135 around a random tree, from another tree species. 4.3.1. SHADE TREE CHARACTERIZATION 144 All shade trees within the sampled area were identified according to their species and family. 145 Their characteristics (height (m), circumference (cm), leaf size) and their host status for ALS, 146 brown-eye spot and anthracnose were recorded (Boshier et al., 2009; Cerdán et al., 2012). 147 For all species, we also specified their main usage, and classified them in one or more 148 categories: wood, timber, fruit, shade, N-fixation, native and wild (Pineda, 2006; Boshier et 149 al., 2009; Román et al., 2012; Amores Contreras, 2015; Caceido, 2016). 150 We combined the variables describing each shade tree by performing a multiple factor 151 analysis (MFA) to cluster the shade-tree species within homogeneous groups based on the 152 previously described variables. MFA was performed with R software using the MFA function 153 from the FactoMineR package (Pagès, 2013). 154 We took hemispherical pictures to characterize the shade percentage at four different times: 156 November 2016, February 2017, June 2017 and September 2017. Hemispherical pictures 157 were taken above all selected coffee plants with a Nikon Coolpix 4500 equipped with a 158 fisheye converter (FC-E8 0.21x). These pictures were analyzed using Gap Light Analyzer 159 (GPA-V2) software to assess the shade percentage above each coffee tree (Frazer et al., 160 1999). The annual mean shade percentage was used for the analysis. 161 162 4.2. STUDY SITES 116 The common species were: 1) Cordia 136 alliodora (Ruiz & Pav.) Oken, laurel (Boraginaceae) a native forest species; 2) Inga 137 oerstediana Benth, guaba roja (Leguminosae) service plant species; 3) Musa spp. Jussieu, 138 guineo (Musaceae). 139 For each farm, we selected 20 circular plots (14 m dia.), centered on a shade tree and sorted 133 in four different situations, with five replicates each. The first three situations were based 134 around three common tree species spread on the farms, while the last situation was made 135 around a random tree, from another tree species. The common species were: 1) Cordia 136 alliodora (Ruiz & Pav.) Oken, laurel (Boraginaceae) a native forest species; 2) Inga 137 oerstediana Benth, guaba roja (Leguminosae) service plant species; 3) Musa spp. Jussieu, 138 guineo (Musaceae). 139 The distance between each plot was maximized. Inside each plot, the analysis included: (1) 140 four coffee plants selected randomly within 5 m of the central tree, and (2) all shade trees 141 taller than the coffee plants. 142 6 6 143 4.3.2. COFFEE TREE CHARACTERIZATION 163 The number of 178 dead branches per coffee plant after harvest is a proxy of primary yield loss (Cerda et 179 al., 2017). 180 181 4.3.3. COFFEE FOLIAR DISEASE CHARACTERIZATION 182 The measured foliar disease complex encompassed American leaf spot (ALS; Mycena 183 citricolor), brown-eye spot (Cercospora caffeicola Berk. & Cooke), anthracnose 184 (Colletotrichum sp.) and coffee thread blight (Corticium kolegora). We measured the severity 185 (i.e. the percentage of diseased leaves) and the incidence (i.e. the percentage of leaf area 186 affected by diseases) of these diseases on three branches of selected coffee plants. As ALS 187 is a major foliar disease, we decided to treat it separately from other diseases to gain clear 188 insight into the relationship between the agrosystem and the ALS incidence and severity. We 189 separately integrated ALS variation patterns and those of other diseases by calculating the 190 relative AUDPC based on the measurements obtained at four dates (May 2017, September 191 2017, December 2017, February 2018), which represented a complete disease development 192 cycle. 193 194 the middle and one at the top of each selected coffee tree). All meas 170 obtained at four times, representing a complete annual coffee 171 development cycle: beginning of the rainy season (May 2017), be 172 harvest period (September 2017), peak of the harvest (December 20 173 harvest (February 2018). All physiological variables were integrate 174 course by determining the area under the disease progress curve (A 175 frequently done for diseases (Simko and Piepho, 2011). 176 • Coffee yield was described by the number of fruiting branches per 177 number of fruiting nodes per plant as proxies of the accessible yield. 178 dead branches per coffee plant after harvest is a proxy of primary yield 179 al., 2017). 180 181 4.3.3. COFFEE FOLIAR DISEASE CHARACTERIZATION 182 The measured foliar disease complex encompassed American leaf spot 183 citricolor), brown-eye spot (Cercospora caffeicola Berk. & Cooke), 184 (Colletotrichum sp.) and coffee thread blight (Corticium kolegora). We measur 185 (i.e. the percentage of diseased leaves) and the incidence (i.e. the percentag 186 affected by diseases) of these diseases on three branches of selected coffee 187 is a major foliar disease, we decided to treat it separately from other disease 188 insight into the relationship between the agrosystem and the ALS incidence an 189 4.3.2. COFFEE TREE CHARACTERIZATION 163 The coffee plant variables were measured from May 2017 to February 2018, describing: 164 • Inherent coffee characteristics not affected by the local environment, such as age 165 (years) and circumference (cm). 166 • Inherent coffee characteristics not affected by the local environment, such as age 165 (years) and circumference (cm). 166 • Coffee vegetative growth, described by the total number of nodes per branch, the 167 number of new nodes per branch, the number of leaves per branch and the average 168 leaf area. We measured these variables on three branches (one at the bottom, one in 169 7 the middle and one at the top of each selected coffee tree). All measurements were 170 obtained at four times, representing a complete annual coffee physiological 171 development cycle: beginning of the rainy season (May 2017), beginning of the 172 harvest period (September 2017), peak of the harvest (December 2017) and post- 173 harvest (February 2018). All physiological variables were integrated over a time 174 course by determining the area under the disease progress curve (AUDPC), as is 175 frequently done for diseases (Simko and Piepho, 2011). 176 the middle and one at the top of each selected coffee tree). All measurements were 170 obtained at four times, representing a complete annual coffee physiological 171 development cycle: beginning of the rainy season (May 2017), beginning of the 172 harvest period (September 2017), peak of the harvest (December 2017) and post- 173 harvest (February 2018). All physiological variables were integrated over a time 174 course by determining the area under the disease progress curve (AUDPC), as is 175 frequently done for diseases (Simko and Piepho, 2011). 176 the middle and one at the top of each selected coffee tree). All measurements were 170 obtained at four times, representing a complete annual coffee physiological 171 development cycle: beginning of the rainy season (May 2017), beginning of the 172 harvest period (September 2017), peak of the harvest (December 2017) and post- 173 harvest (February 2018). All physiological variables were integrated over a time 174 course by determining the area under the disease progress curve (AUDPC), as is 175 frequently done for diseases (Simko and Piepho, 2011). 176 • Coffee yield was described by the number of fruiting branches per plant and the 177 number of fruiting nodes per plant as proxies of the accessible yield. 4.3.3. COFFEE FOLIAR DISEASE CHARACTERIZATION 182 The measured foliar disease complex encompassed American leaf spot (ALS; Mycena 183 citricolor), brown-eye spot (Cercospora caffeicola Berk. & Cooke), anthracnose 184 (Colletotrichum sp.) and coffee thread blight (Corticium kolegora). We measured the severity 185 (i.e. the percentage of diseased leaves) and the incidence (i.e. the percentage of leaf area 186 affected by diseases) of these diseases on three branches of selected coffee plants. As ALS 187 is a major foliar disease, we decided to treat it separately from other diseases to gain clear 188 insight into the relationship between the agrosystem and the ALS incidence and severity. We 189 separately integrated ALS variation patterns and those of other diseases by calculating the 190 relative AUDPC based on the measurements obtained at four dates (May 2017, September 191 2017, December 2017, February 2018), which represented a complete disease development 192 cycle. 193 8 8 Measurements for characterizing the soil in each coffee plot were obtained at the beginning 196 of the 2017 rainy season (between June and August). According to the protocol described by 197 Thoumazeau et al. (2019a, 2019b) and adapted by Andreotti (2018). The measured soil 198 characteristics included: 199 • The soil chemical composition: organic carbon (g/kg), pH, nitrogen percentage (N), 200 iron (Fe), potassium (K), magnesium (Mg), and phosphorus (P) in ppm. Soil organic 201 carbon and nitrogen are key soil components and are both indicators of soil fertility. 202 • The soil chemical composition: organic carbon (g/kg), pH, nitrogen percentage (N), 200 iron (Fe), potassium (K), magnesium (Mg), and phosphorus (P) in ppm. Soil organic 201 carbon and nitrogen are key soil components and are both indicators of soil fertility. 202 • The litter index accounted for the litter quantity and quality, which highly influences 203 soil fertility, the nutrient cycle while being the main carbon source for soil organisms 204 (Sauvadet et al., 2016). 205 • The Beerkan test was applied to measure the soil infiltration potential and generate 206 information on the water infiltration potential (Lassabatere et al., 2006). 207 • The cation exchange capacity (CEC), which expresses the capacity of a soil to retain 208 nutrients, was used as a soil fertility indicator (Chapman, 1965). 209 All three farms had the same soil physical characteristics, including a loamy sandy texture. 210 211 4.4. 4.3.3. COFFEE FOLIAR DISEASE CHARACTERIZATION 182 The PLS-PM method was 222 9 specifically chosen for its flexibility to manipulate datasets with numerous variables, and its 223 capacity to represent clearly complex interaction systems. 224 225 PLS-PM is a blend of two models: a measurement model and a structural model (Fig. 1). The 226 measurement model defines the relationships between observed variables and latent 227 variables inside blocks, with each block being represented by a latent variable and built with 228 observed variables (Fig. 1). The structural model investigates relationships between latent 229 variables using a linear regression approach. We performed this network analysis with R 230 software using the plspm function from the plspm package (Sanchez et al., 2013). 231 specifically chosen for its flexibility to manipulate datasets with numerous variables, and its 223 capacity to represent clearly complex interaction systems. 224 specifically chosen for its flexibility to manipulate datasets with numerous variables, and its 223 capacity to represent clearly complex interaction systems. 224 225 PLS-PM is a blend of two models: a measurement model and a structural model (Fig. 1). The 226 measurement model defines the relationships between observed variables and latent 227 variables inside blocks, with each block being represented by a latent variable and built with 228 observed variables (Fig. 1). The structural model investigates relationships between latent 229 variables using a linear regression approach. We performed this network analysis with R 230 software using the plspm function from the plspm package (Sanchez et al., 2013). 231 PLS-PM is a blend of two models: a measurement model and a structural model (Fig. 1). The 226 measurement model defines the relationships between observed variables and latent 227 variables inside blocks, with each block being represented by a latent variable and built with 228 observed variables (Fig. 1). The structural model investigates relationships between latent 229 variables using a linear regression approach. We performed this network analysis with R 230 software using the plspm function from the plspm package (Sanchez et al., 2013). 231 232 Figure 1. Scheme of the PLS-PM. Description of the measurement model (inside each block) and the 233 structural model (black arrows between blocks). Exogenous latent variables are just explanatory, while 234 endogenous are explanatory and explained (by other latent variable, either exogenous or 235 endogenous). Block 1 and 3 are explicative, while block 2 is formative. 236 237 232 Figure 1. Scheme of the PLS-PM. 4.3.3. COFFEE FOLIAR DISEASE CHARACTERIZATION 182 Description of the measurement model (inside each block) and the 233 structural model (black arrows between blocks). Exogenous latent variables are just explanatory, while 234 endogenous are explanatory and explained (by other latent variable, either exogenous or 235 endogenous). Block 1 and 3 are explicative, while block 2 is formative. 236 237 Figure 1. Scheme of the PLS-PM. Description of the measurement model (inside ea 233 4.3.3. COFFEE FOLIAR DISEASE CHARACTERIZATION 182 STATISTICAL ANALYSIS 212 Structural equation modelling (SEM) is particularly appropriate for gainign insight into 213 interactions between shade trees, soil, diseases and coffee plants. SEM analyses are able to 214 explain relationships between observed variables by clustering them as latent variables 215 representative of common concepts. Structural equation modelling analyses can be used to 216 understand complex systems (Hoyle, 2012; Vinzi and Trinchera, 2013) and was successfully 217 applied to analyse ecological regulations in agroforestry systems in banana and cocoa 218 plantation settings (Poeydebat et al. 2017; Oliveira et al., 2018). This type of analysis is 219 divided into two main types: the SEM-ML method based on the maximum likelihood (ML) and 220 the partial least square path modelling (PLS-PM) method based on simple regressions to 221 • The litter index accounted for the litter quantity and quality, which highly influences 203 soil fertility, the nutrient cycle while being the main carbon source for soil organisms 204 (Sauvadet et al., 2016). 205 • The Beerkan test was applied to measure the soil infiltration potential and generate 206 information on the water infiltration potential (Lassabatere et al., 2006). 207 • The cation exchange capacity (CEC), which expresses the capacity of a soil to retain 208 nutrients, was used as a soil fertility indicator (Chapman, 1965). 209 • The cation exchange capacity (CEC), which expresses the capacity of a soil to retain 208 nutrients, was used as a soil fertility indicator (Chapman, 1965). 209 All three farms had the same soil physical characteristics, including a loamy sandy texture. 210 211 Structural equation modelling (SEM) is particularly appropriate for gainign insight into 213 interactions between shade trees, soil, diseases and coffee plants. SEM analyses are able to 214 explain relationships between observed variables by clustering them as latent variables 215 representative of common concepts. Structural equation modelling analyses can be used to 216 understand complex systems (Hoyle, 2012; Vinzi and Trinchera, 2013) and was successfully 217 applied to analyse ecological regulations in agroforestry systems in banana and cocoa 218 plantation settings (Poeydebat et al. 2017; Oliveira et al., 2018). This type of analysis is 219 divided into two main types: the SEM-ML method based on the maximum likelihood (ML) and 220 the partial least square path modelling (PLS-PM) method based on simple regressions to 221 explain the latent variables (Vinzi and Trinchera, 2013). 4.5. MEASUREMENT MODEL BUILDING 238 Our measurement model contained six blocks including latent variables corresponding to the 239 measurement domains presented earlier, i.e. inherent coffee characteristics (hereafter simply 240 Our measurement model contained six blocks including latent variables corresponding to the 239 measurement domains presented earlier, i.e. inherent coffee characteristics (hereafter simply 240 10 called ‘coffee characteristics‘), shade trees, soil characteristics, foliar diseases, coffee growth 241 and coffee yield (Table 2). We built three blocks related to coffee plants because we 242 assumed that these blocks represent a specific aspect of coffee plants, and influence each 243 other. Each block was composed of a latent variable and its related observed variables 244 (Table 2). According to the method described by Sanchez (2013), the coffee characteristics, 245 shade trees, soil characteristics, foliar diseases and coffee growth blocks were reflective 246 because the variables observed inside each block were well correlated. Indeed, for each 247 reflective block, observed variables have to move in the same direction, and when a variable 248 increases or decreases, the others change in the same way (Sanchez, 2013). We verified 249 this condition by examining the unidimensionality of these blocks with Dillon-Goldstein’s rho. 250 A block is unidimensional when its rho value is higher than 0.7 (Sanchez, 2013). 251 Inversely, the coffee yield block was formative because the numbers of fruiting nodes and 252 fruiting branches were not closely correlated with the number of dead branches. As formative 253 blocks do not require highly correlated observed variables, the block unidimensionality is not 254 calculated. 255 256 Table 2. Description of blocks represented by their latent variable and observed variables. ALS is 257 American leaf spot of coffee; organic C is soil organic carbon; N is the nitrogen percentage; iron (Fe), 258 potassium (K), magnesium (Mg), and phosphorus (P) are in ppm; CEC is the cation exchange 259 capacity. 4.5. MEASUREMENT MODEL BUILDING 238 260 Latent variable Related observed variables Coffee characteristics Age, size (circumference) Shade trees Shade (%), abundance of services trees, fruit trees and timber trees Soil characteristics Litter index, Beerkan test, organic C, N, pH, Mg, Fe, K, CEC Diseases ALS severity, ALS incidence, other disease severity, other disease incidence Coffee growth Number of nodes, number of new nodes, number of leaves, leaf size, coffee heigh Coffee yield Number of fruiting branches, number of fruiting nodes, number of dead branches 261 called ‘coffee characteristics‘), shade trees, soil characteristics, foliar diseases, coffee growth 241 and coffee yield (Table 2). We built three blocks related to coffee plants because we 242 assumed that these blocks represent a specific aspect of coffee plants, and influence each 243 other. Each block was composed of a latent variable and its related observed variables 244 (Table 2). According to the method described by Sanchez (2013), the coffee characteristics, 245 shade trees, soil characteristics, foliar diseases and coffee growth blocks were reflective 246 because the variables observed inside each block were well correlated. Indeed, for each 247 reflective block, observed variables have to move in the same direction, and when a variable 248 increases or decreases, the others change in the same way (Sanchez, 2013). We verified 249 this condition by examining the unidimensionality of these blocks with Dillon-Goldstein’s rho. 250 A block is unidimensional when its rho value is higher than 0.7 (Sanchez, 2013). 251 called ‘coffee characteristics‘), shade trees, soil characteristics, foliar diseases, coffee growth 241 and coffee yield (Table 2). We built three blocks related to coffee plants because we 242 assumed that these blocks represent a specific aspect of coffee plants, and influence each 243 other. Each block was composed of a latent variable and its related observed variables 244 (Table 2). According to the method described by Sanchez (2013), the coffee characteristics, 245 shade trees, soil characteristics, foliar diseases and coffee growth blocks were reflective 246 because the variables observed inside each block were well correlated. Indeed, for each 247 reflective block, observed variables have to move in the same direction, and when a variable 248 increases or decreases, the others change in the same way (Sanchez, 2013). We verified 249 this condition by examining the unidimensionality of these blocks with Dillon-Goldstein’s rho. 250 A block is unidimensional when its rho value is higher than 0.7 (Sanchez, 2013). 4.5. MEASUREMENT MODEL BUILDING 238 Higher 264 coefficients indicated a higher influence on the block. 265 266 4.5. MEASUREMENT MODEL BUILDING 238 251 Inversely, the coffee yield block was formative because the numbers of fruiting nodes and 252 fruiting branches were not closely correlated with the number of dead branches. As formative 253 blocks do not require highly correlated observed variables, the block unidimensionality is not 254 calculated. 255 256 Table 2. Description of blocks represented by their latent variable and observed variables. ALS is 257 American leaf spot of coffee; organic C is soil organic carbon; N is the nitrogen percentage; iron (Fe), 258 potassium (K), magnesium (Mg), and phosphorus (P) are in ppm; CEC is the cation exchange 259 capacity. 260 Latent variable Related observed variables Coffee characteristics Age, size (circumference) Shade trees Shade (%), abundance of services trees, fruit trees and timber trees Soil characteristics Litter index, Beerkan test, organic C, N, pH, Mg, Fe, K, CEC Diseases ALS severity, ALS incidence, other disease severity, other disease incidence Coffee growth Number of nodes, number of new nodes, number of leaves, leaf size, coffee heig Coffee yield Number of fruiting branches, number of fruiting nodes, number of dead branches 261 Inversely, the coffee yield block was formative because the numbers of fruiting nodes and 252 fruiting branches were not closely correlated with the number of dead branches. As formative 253 blocks do not require highly correlated observed variables, the block unidimensionality is not 254 calculated. 255 256 Table 2. Description of blocks represented by their latent variable and observed variables. ALS is 257 American leaf spot of coffee; organic C is soil organic carbon; N is the nitrogen percentage; iron (Fe), 258 potassium (K), magnesium (Mg), and phosphorus (P) are in ppm; CEC is the cation exchange 259 capacity. 260 Latent variable Related observed variables Coffee characteristics Age, size (circumference) Shade trees Shade (%), abundance of services trees, fruit trees and timber trees Soil characteristics Litter index, Beerkan test, organic C, N, pH, Mg, Fe, K, CEC Diseases ALS severity, ALS incidence, other disease severity, other disease incidence Coffee growth Number of nodes, number of new nodes, number of leaves, leaf size, coffee height Coffee yield Number of fruiting branches, number of fruiting nodes, number of dead branches 261 261 11 We only kept observed variables that were correlated with the latent variable with a 262 regression coefficient higher than 0.5 (Sanchez, 2013). Inside each block, the regression 263 coefficient value explained how the observed variables influenced the latent variable. 4.1. CLUSTERING OF SHADE TREES 290 The MFA led to three groups of shade tree, i.e. timber, service and fruit trees. The timber 291 group was composed mostly of native and forest species intended for wood production. The 292 service group had shade-tree species, mostly N-fixing species, that were planted to improve 293 the soil quality and shade percentage, though some of these species were hosts of some 294 coffee foliar diseases like ALS. The fruit group was represented mostly by species producing 295 secondary fruit products sold in local markets or consumed locally. These groups were used 296 in the PLS-PM model to build the ‘shade trees’ block. 297 4.6. STRUCTURAL MODEL BUILDING 267 The 292 service group had shade-tree species, mostly N-fixing species, that were planted to improve 293 the soil quality and shade percentage, though some of these species were hosts of some 294 coffee foliar diseases like ALS. The fruit group was represented mostly by species producing 295 secondary fruit products sold in local markets or consumed locally. These groups were used 296 in the PLS-PM model to build the ‘shade trees’ block. 297 298 4.2. PLS-PM MODEL OUTPUT 299 The measurement model and structural model results shed light on the complex network of 300 interactions between shade trees, coffee plants, foliar diseases and soil characteristics. 301 While the measurement model showed the block compositions, the structural model revealed 302 the strength of the relation between them. The overall PLS-PM had a goodness-of-fit of 303 0.4971. 304 305 4.3. RELATIONSHIPS BETWEEN THE OBSERVED AND ASSOCIATED LATENT VARIABLES (WITHIN 306 BLOCK) 307 Finally, the goodness-of-fit test was used to evaluate the model robustness (Sanchez, 2013). 285 Finally, the goodness-of-fit test was used to evaluate the model robustness (Sanchez, 2013). 285 All statistical analyses were performed with the R 3.5.1 package (Team R Core, 2018) and 286 with an alpha level of 0.05. 287 All statistical analyses were performed with the R 3.5.1 package (Team R Core, 2018) and 286 with an alpha level of 0.05. 287 4.6. STRUCTURAL MODEL BUILDING 267 The relationship between blocks was defined in the structural model according to previous 268 studies (Fig. 2) (Allinne et al., 2016; Cerda et al., 2017). The latent variables shade trees and 269 coffee characteristics were not explained by the other blocks and threfore were exogenous, 270 while the latent variables soil, diseases, coffee growth and coffee yield were endogenous, 271 because they were explained by the other blocks (Fig. 2). 272 273 Figure 2. Pathways between the latent variables. Coffee characteristics and shade trees are 274 exogenous (i.e. only explanatory) (white), while soil characteristics, diseases, coffee growth and coffee 275 yield are endogenous (grey). 276 277 Figure 2. Pathways between the latent variables. Coffee characteristics and shade trees are 274 exogenous (i.e. only explanatory) (white), while soil characteristics, diseases, coffee growth and coffee 275 yield are endogenous (grey). 276 277 In order to validate our block, the PLS-PM model was used to calculate the R² coefficient of 278 each exogenous block that expressed the explained variability for each block. Other latent 279 variables better explained the block when the R² coefficients were high. The model 280 In order to validate our block, the PLS-PM model was used to calculate the R² coefficient of 278 each exogenous block that expressed the explained variability for each block. Other latent 279 variables better explained the block when the R² coefficients were high. The model 280 12 parameters were thus adjusted to have R² coefficients higher than 0.2, which is a moderately 281 low value (Sanchez, 2013). 282 The regression coefficient between blocks clarified the relationship between the block, i.e. 283 either positive or negative. 284 The regression coefficient between blocks clarified the relationship between the block, i.e. 283 either positive or negative. 284 either positive or negative. 284 Finally, the goodness-of-fit test was used to evaluate the model robustness (Sanchez, 2013). 285 All statistical analyses were performed with the R 3.5.1 package (Team R Core, 2018) and 286 with an alpha level of 0.05. 287 288 3. RESULTS 289 4.1. CLUSTERING OF SHADE TREES 290 The MFA led to three groups of shade tree, i.e. timber, service and fruit trees. The timber 291 group was composed mostly of native and forest species intended for wood production. 4.2. PLS-PM MODEL OUTPUT 299 The measurement model and structural model results shed light on the complex network of 300 interactions between shade trees, coffee plants, foliar diseases and soil characteristics. 301 While the measurement model showed the block compositions, the structural model revealed 302 the strength of the relation between them. The overall PLS-PM had a goodness-of-fit of 303 0.4971. 304 305 4.3. RELATIONSHIPS BETWEEN THE OBSERVED AND ASSOCIATED LATENT VARIABLES (WITHIN 306 BLOCK) 307 The measurement model and structural model results shed light on the complex network of 300 interactions between shade trees, coffee plants, foliar diseases and soil characteristics. 301 While the measurement model showed the block compositions, the structural model revealed 302 the strength of the relation between them. The overall PLS-PM had a goodness-of-fit of 303 0.4971. 304 4.3. RELATIONSHIPS BETWEEN THE OBSERVED AND ASSOCIATED LATENT VARIABLES (WITHIN 306 BLOCK) 307 13 The measurement model findings showed that all reflective blocks (‘coffee characteristics’, 308 ‘shade trees’, ‘soil characteristics’, ‘diseases’ and ‘coffee growth’) had a Dillon-Goldstein’s 309 rho higher than 0.7 (Fig. 3). The correlation coefficients between the observed and 310 associated latent variables were higher than 0.5, except for the ‘other-disease severity’ 311 variable in the diseases block (Fig. 3). Although the ‘other-disease severity’ variable had a 312 coefficient of 0.41, we decided to keep it in the model because it provided a better 313 representation of the pest complex in the system (Fig. 3). 314 The measurement model findings showed that all reflective blocks (‘coffee characteristics’, 308 ‘shade trees’, ‘soil characteristics’, ‘diseases’ and ‘coffee growth’) had a Dillon-Goldstein’s 309 rho higher than 0.7 (Fig. 3). The correlation coefficients between the observed and 310 associated latent variables were higher than 0.5, except for the ‘other-disease severity’ 311 variable in the diseases block (Fig. 3). Although the ‘other-disease severity’ variable had a 312 coefficient of 0.41, we decided to keep it in the model because it provided a better 313 representation of the pest complex in the system (Fig. 3). 314 The ‘coffee characteristics’ latent variable was well-explained by its two observed variables, 315 i.e. age (0.96) and circumference (0.91) (Fig. 3). From the ‘shade trees’ block, only the 316 observed variables of the mean shade percentage (M. shade, 0.94) and service group (Serv, 317 0.64) had a significant impact. 4.2. PLS-PM MODEL OUTPUT 299 Reflective blocks are represented by arrows going from the latent to the observed variables, 332 while the direction is reversed for formative blocks. The Dillon-Goldstein’s rho values are shown above 333 each block. Circ is the circumference of coffee plants; Age is age of coffee plants; Serv is the 334 abundance of service trees; M. shade is the mean shade percentage; C is soil organic carbon; Lit. 335 Index is the litter index; Beerkan is the Beerkan test results; N is soil nitrogen percentage; Inc. ALS is 336 the ALS incidence; Sev. ALS is the ALS severity; Sev. Other is the severity of the other foliar diseases; 337 Leaf nb is the number of leaves; Height is the height of coffee plants; New nod is the number of new 338 nodes; Nodes nb is the number of nodes; Dead br is the number of dead branches, Fruit. Nod is the 339 number of fruiting nodes; Fruit. Br is the number of fruiting branches. 340 341 Figure 3. Results of the measurement model representing the six blocks with their latent variables 329 (ovals) and observed variables (rectangles), and the correlation coefficient between each latent 330 variable and the observed variables. Exogenous blocks are shown in white and endogenous blocks in 331 grey. Reflective blocks are represented by arrows going from the latent to the observed variables, 332 while the direction is reversed for formative blocks. The Dillon-Goldstein’s rho values are shown above 333 each block. Circ is the circumference of coffee plants; Age is age of coffee plants; Serv is the 334 abundance of service trees; M. shade is the mean shade percentage; C is soil organic carbon; Lit. 335 Index is the litter index; Beerkan is the Beerkan test results; N is soil nitrogen percentage; Inc. ALS is 336 the ALS incidence; Sev. ALS is the ALS severity; Sev. Other is the severity of the other foliar diseases; 337 Leaf nb is the number of leaves; Height is the height of coffee plants; New nod is the number of new 338 nodes; Nodes nb is the number of nodes; Dead br is the number of dead branches, Fruit. Nod is the 339 number of fruiting nodes; Fruit. Br is the number of fruiting branches. 340 341 4.2. PLS-PM MODEL OUTPUT 299 The ‘soil characteristics’ latent variable was explained only by 318 the Beerkan test (0.83), organic carbon quantity (0.6) and nitrogen percentage (0.62), as well 319 as the litter index (0.68). The ‘soil characteristics’ parameter thus represented the soil fertility. 320 The ‘diseases’ block was mainly explained by the observed variables related to ALS severity 321 (0.94) and ALS incidence (0.94). The remaining observed variables that explained the ‘coffee 322 growth’ block were the height (0.6), number of nodes (0.87), number of new nodes (0.9) and 323 number of leaves (0.55). Inside the ‘coffee yield’ block, the number of fruiting branches per 324 tree was more significant (0.87), it was correlated with the number of fruiting nodes (0.76) per 325 branch and with the number of dead branches (0.56). 326 327 14 328 Figure 3. Results of the measurement model representing the six blocks with their latent variables 329 (ovals) and observed variables (rectangles), and the correlation coefficient between each latent 330 variable and the observed variables. Exogenous blocks are shown in white and endogenous blocks in 331 grey. Reflective blocks are represented by arrows going from the latent to the observed variables, 332 while the direction is reversed for formative blocks. The Dillon-Goldstein’s rho values are shown above 333 each block. Circ is the circumference of coffee plants; Age is age of coffee plants; Serv is the 334 abundance of service trees; M. shade is the mean shade percentage; C is soil organic carbon; Lit. 335 Index is the litter index; Beerkan is the Beerkan test results; N is soil nitrogen percentage; Inc. ALS is 336 the ALS incidence; Sev. ALS is the ALS severity; Sev. Other is the severity of the other foliar diseases; 337 Leaf nb is the number of leaves; Height is the height of coffee plants; New nod is the number of new 338 nodes; Nodes nb is the number of nodes; Dead br is the number of dead branches, Fruit. Nod is the 339 number of fruiting nodes; Fruit. Br is the number of fruiting branches. 340 328 Figure 3. Results of the measurement model representing the six blocks with their latent variables 329 (ovals) and observed variables (rectangles), and the correlation coefficient between each latent 330 variable and the observed variables. Exogenous blocks are shown in white and endogenous blocks in 331 grey. 4.4. RELATIONSHIPS BETWEEN BLOCKS 342 All endogenous blocks had an R² coefficient higher than 0.2, the diseases and ‘coffee yield’ 343 latent endogenous variables had an R² coefficient of between 0.2 and 0.5, the ‘soil 344 characteristics’ and ‘coffee growth’ latent endogenous variables had an R² higher than 0.5 345 (Fig. 4). 346 The ‘soil characteristics’ block was positively correlated with the ‘shade trees’ block (0.14), 347 the ‘coffee characteristic’ block (0.74) and the ‘coffee growth’ block (0.19), but negatively 348 The ‘soil characteristics’ block was positively correlated with the ‘shade trees’ block (0.14), 347 the ‘coffee characteristic’ block (0.74) and the ‘coffee growth’ block (0.19), but negatively 348 15 correlated with ‘coffee yield’ (-0.08) and ‘diseases’ (-0.1). ‘Diseases’ was positively correlated 349 with ‘coffee characteristics’ (0.33) and ‘shade trees’ (0.49). 350 correlated with ‘coffee yield’ (-0.08) and ‘diseases’ (-0.1). ‘Diseases’ was positively correlated 349 with ‘coffee characteristics’ (0.33) and ‘shade trees’ (0.49). 350 The ‘coffee growth’ and ‘coffee yield’ blocks were negatively correlated with ‘diseases’ (-0.02; 351 -0.12) and ‘shade trees’ (-0.25; -0.37). 352 The ‘coffee characteristics’ and ‘coffee growth’ had a positive correlation (0.54). The ‘coffee 353 yield’ block was negatively correlated with the ‘coffee characteristics’ and ‘coffee growth’ 354 blocks (-0.36; -0.06). 355 356 Figure 4. Results of the structural model representing the network of interactions between blocks, as 357 shown by significant paths. Each arrow represents shade with its regression coefficient: blue arrows 358 represent shade with a positive coefficient and red arrows with a negative coefficient. Endogenous 359 blocks (grey) are also represented by their R² coefficient, with the coefficient being null for exogenous 360 blocks (white). 361 362 356 356 Figure 4. Results of the structural model representing the network of interactions between blocks, as 357 shown by significant paths. Each arrow represents shade with its regression coefficient: blue arrows 358 represent shade with a positive coefficient and red arrows with a negative coefficient. Endogenous 359 blocks (grey) are also represented by their R² coefficient, with the coefficient being null for exogenous 360 blocks (white). 361 Figure 4. Results of the structural model representing the network of interactions between blocks, as 357 shown by significant paths. Each arrow represents shade with its regression coefficient: blue arrows 358 represent shade with a positive coefficient and red arrows with a negative coefficient. 4.4. RELATIONSHIPS BETWEEN BLOCKS 342 Endogenous 359 blocks (grey) are also represented by their R² coefficient, with the coefficient being null for exogenous 360 blocks (white). 361 4.1. ANALYSIS OF DIRECT EFFECTS AMONG THE SYSTEM COMPONENTS The soil characteristics block (soil fertility proxy) was positively correlated with the shade tree 365 block. This correlation meant that soil fertility was higher in plots with a larger shade 366 16 percentage and with a greater number of N-fixing trees, confirming the findings of the recent 367 study of Sauvadet et al. (2018). Indeed, the fertility under shade trees was increased by the 368 N-fixation capacity of the service trees as well as by the shade tree pruning practices (Beer 369 et al., 1997). The increased litter quality and quantity restored soil organic carbon and 370 promoted the development of vital soil microorganisms like bacterial-feeding nematodes 371 (Sauvadet et al., 2018). Most N-fixing tree species lose their leaves during the dry season; 372 those leaves are fast decomposing materials that represent a source of C and nutrients for 373 the soil (Tapia-Coral et al., 2005). Litter restitution thus improves and maintains the soil 374 activity and fertility during this season, which is crucial for coffee production (Wintgens, 375 2004). 376 The diseases block was positively correlated with shade trees, thus highlighting their 377 importance in disease management (Avelino et al., 2018). The model confirmed that ALS — 378 a major component of the block — was favored by high shade which induces high humidity 379 (Avelino et al., 2007). In addition, some species of the service tree group, especially Inga 380 spp. were ALS hosts (Granados Montero, 2015) and could be a significant source of 381 inoculum (Staver et al., 2001). 382 The negative correlation between shade trees and coffee growth and coffee yield suggested 383 that reducing the canopy openness limits the light available for coffee growth (DaMatta, 384 2004). Shade trees were generating 49-85% shade (73% on average), which was much 385 above the shade percentage found in most conventional coffee plantations. Although the 386 effect of the shade percentage on coffee growth is still quite controversial, higher growth 387 rates (up to the 40% threshold) are usually observed under shade (Charbonnier et al. 2017). 388 In our case, all plots were equal or above this threshold and a negative effect of shade on 389 coffee growth and yield was therefore expected. 390 The negative correlation between the ‘soil characteristics’ block and the ‘diseases’ block, 391 indicated that coffee plants growing in more fertile soils are less affected by foliar diseases. 4.1. ANALYSIS OF DIRECT EFFECTS AMONG THE SYSTEM COMPONENTS 392 Soil fertility could have induced a physiological resistance, as demonstrated with coffee rust 393 (Toniutti et al 2017). 394 17 395 The positive relation observed between coffee characteristics (bigger and older coffee plants) 396 and soil characteristics (fertility proxy) was probably related to the fact that soil quality was 397 higher in older plantations. This could be explained by the acceleration of carbon cycle 398 dynamics due to the increased organic matter input in the soil system, notably from litter fall 399 linked to increased biotic activity, as demonstrated in rubber plantations (Thoumazeau et al, 400 2019b). Moreover, old coffee plants were more pruned in the previous year and pruning 401 residue was left on the ground, thus increasing the soil organic matter (Gomez-Munoz et al. 402 2016). Pruning practices on older coffee plants could also explain why, despite the fact that 403 they were growing on more fertile soils and had the better growth, they had the lowest berry 404 production. Indeed, freshly pruned coffee plants first distribute their resources to promote 405 growth (Charbonnier et al. 2017). The positive correlation between the coffee plant age and 406 the disease incidence and severity illustrated that, besides their better growth and resistance 407 related to high fertility, older coffee plants were more sensitive to foliar diseases. 408 409 As expected, foliar-diseases had a negative effect on coffee growth and yield. Foliar 410 diseases reduced the leaf area available for photosynthesis process and did not allow plants 411 to recover and sprout leaves or new nodes (Waller et al., 2007). Higher disease incidence 412 and severity reduced fruiting production — this was the combined result of decreased 413 photosynthesis and reduced redistribution of resources from leaves to fruits (Cerda et al., 414 2017). 415 416 4.2. ANALYSIS OF INDIRECT EFFECTS OF THE SYSTEM COMPONENTS ON DISEASES, COFFEE 417 GROWTH AND YIELD 418 Here we review the indirect and antagonistic effects highlighted by the PLS-PM model. First, 419 a close relationship was noted between coffee characteristics, coffee growth and soil blocks. 420 As discussed previously, the plots with higher fertility were associated with older coffee 421 395 The positive relation observed between coffee characteristics (bigger and older coffee plants) 396 and soil characteristics (fertility proxy) was probably related to the fact that soil quality was 397 higher in older plantations. 4.1. ANALYSIS OF DIRECT EFFECTS AMONG THE SYSTEM COMPONENTS This could be explained by the acceleration of carbon cycle 398 dynamics due to the increased organic matter input in the soil system, notably from litter fall 399 linked to increased biotic activity, as demonstrated in rubber plantations (Thoumazeau et al, 400 2019b). Moreover, old coffee plants were more pruned in the previous year and pruning 401 residue was left on the ground, thus increasing the soil organic matter (Gomez-Munoz et al. 402 2016). Pruning practices on older coffee plants could also explain why, despite the fact that 403 they were growing on more fertile soils and had the better growth, they had the lowest berry 404 production. Indeed, freshly pruned coffee plants first distribute their resources to promote 405 growth (Charbonnier et al. 2017). The positive correlation between the coffee plant age and 406 the disease incidence and severity illustrated that, besides their better growth and resistance 407 related to high fertility, older coffee plants were more sensitive to foliar diseases. 408 409 As expected, foliar-diseases had a negative effect on coffee growth and yield. Foliar 410 diseases reduced the leaf area available for photosynthesis process and did not allow plants 411 to recover and sprout leaves or new nodes (Waller et al., 2007). Higher disease incidence 412 and severity reduced fruiting production — this was the combined result of decreased 413 photosynthesis and reduced redistribution of resources from leaves to fruits (Cerda et al., 414 2017). 415 416 4.2. ANALYSIS OF INDIRECT EFFECTS OF THE SYSTEM COMPONENTS ON DISEASES, COFFEE 417 GROWTH AND YIELD 418 Here we review the indirect and antagonistic effects highlighted by the PLS-PM model. First, 419 a close relationship was noted between coffee characteristics, coffee growth and soil blocks. 420 As discussed previously, the plots with higher fertility were associated with older coffee 421 As expected, foliar-diseases had a negative effect on coffee growth and yield. Foliar 410 diseases reduced the leaf area available for photosynthesis process and did not allow plants 411 to recover and sprout leaves or new nodes (Waller et al., 2007). Higher disease incidence 412 and severity reduced fruiting production — this was the combined result of decreased 413 photosynthesis and reduced redistribution of resources from leaves to fruits (Cerda et al., 414 2017). 415 Here we review the indirect and antagonistic effects highlighted by the PLS-PM model. 4.1. ANALYSIS OF DIRECT EFFECTS AMONG THE SYSTEM COMPONENTS First, 419 a close relationship was noted between coffee characteristics, coffee growth and soil blocks. 420 As discussed previously, the plots with higher fertility were associated with older coffee 421 18 plants, which had higher growth due to the pruning practices. However, for the same reason, 422 these coffee plants had the lowest berry production. 423 Although shade cover had antagonistic effects on the leaf diseases studied, with a high level 424 of ALS but a low level of brown-eye spot (Avelino et al, 2018), we found that the shade trees 425 favored foliar diseases overall. By increasing the soil fertility, they increased the coffee 426 resistance, thereby reducing the disease incidence and severity. Moreover, the negative 427 relationship between high shade cover and coffee growth and yield was direct but also 428 indirect via the foliar diseases fostered by the shade trees. Conversely, high shade cover 429 was also indirectly and positively related to high soil fertility, which increases the growth of 430 coffee plants and reinforces their resistance (Toniutti et al. 2017). 431 Finally, coffee production resulted from a set of factors derived from the direct and indirect 432 effects of all components. All effects within the agroecosystem must be taken into account to 433 achieve balanced foliar disease management. It is now essential to quantify the trade-off 434 between shade trees, soil characteristics, diseases, coffee growth and yield in order to 435 improve overall agroecosystem management, and above all coffee production. 436 437 4.3. PLS-PM TO UNDERPIN FUTURE INITIATIVES AND PROSPECTS 438 The PLS-PM findings had a goodness-of-fit of about 0.5, which is an average value. We 439 noted that all of the ‘soil characteristics’ and ‘coffee growth’ blocks were better explained 440 than others. Inversely, the ‘diseases’ and ‘coffee yield’ blocks were less well explained than 441 other blocks. 442 It would be interesting to integrate the herbaceous layer in the analysis so as to gain insight 443 into the ‘diseases’ and ‘coffee yield’ blocks. Recent studies showed that this herbaceous 444 layer also has an impact on the ALS incidence and severity (Granados Montero, 2015). The 445 extent of the incidence and severity of the herbaceous layer would directly affect the 446 incidence and severity of diseases on the coffee plants and indirectly the coffee yield via a 447 19 direct impact on dead branches. Adding the secondary loss, i.e. dead branches, in 2016 448 directly affected the number of available berry producing branches in 2017. 449 In future studies, it would also be interesting to integrate coffee growth from previous years to 450 take the biannual resource allocation of coffee plants into account. Another improvement 451 would be to integrate temperature fluctuations and precipitation patterns, which have a 452 marked impact on coffee tree growth and production (Charbonnier et al., 2017), as well as on 453 the degree of ALS incidence and severity (Avelino et al., 2007), but that will require larger 454 datasets. 455 5. CONCLUSION PLS-PM enabled us to study the network of interactions occurring within the agroecosystem, 458 including antagonistic effects of shade trees. First, shade trees had a negative effect on 459 coffee growth and yield and increased the foliar diseases incidence and severity, and 460 secondly, they increased soil fertility which in turn decreased the disease prevalence and 461 increased coffee growth. This holistic approach regarding the role of trees in the ecosystem 462 highlighted the need to consider the shade percentage quantitatively (an excess or lack of 463 shade negatively impacted coffee growth and yield). It will be essential to assess the trade- 464 offs between shade management, soil quality, disease regulation and yield gain when 465 designing cropping systems that optimize shade cover. 466 The authors declare that they have no conflicts of interest. 469 20 This research was financially supported by the STRADIV project (grant n° 1504-003) funded 472 by Agropolis Fondation. Special thanks to Marvin Rivera, Mariano Suazo and Adolfo Cajina, 473 the three farmers who kindly welcomed and guided us in their coffee plantations over the 2- 474 year study period. We are grateful to David Manlay for English revision. We thank the editors 475 and the anonymous reviewers for the comments we received that helped improving the 476 manuscript. 477 This research was financially supported by the STRADIV project (grant n° 1504-003) funded 472 by Agropolis Fondation. Special thanks to Marvin Rivera, Mariano Suazo and Adolfo Cajina, 473 the three farmers who kindly welcomed and guided us in their coffee plantations over the 2- 474 year study period. We are grateful to David Manlay for English revision. We thank the editors 475 and the anonymous reviewers for the comments we received that helped improving the 476 manuscript. 477 REFERENCES 479 Allinne, C., Boudrot, A., De Melo, E., Granados, E., Merle, I., Pico, J., Vonthron, S., Avelino, 480 J., 2019. Régulation des bioagresseurs du caféier par le couvert arboré au Costa 481 Rica. Agroforesterie et services écosystémiques en zone tropicale. 53-61. 482 Allinne, C., Savary, S., Avelino, J., 2016. Delicate balance between pest and disease 483 injuries, yield performance, and other ecosystem services in the complex coffee- 484 based systems of Costa Rica. Agriculture, Ecosystems & Environment 222, 1–12. 485 https://doi.org/10.1016/j.agee.2016.02.001 486 Amores Contreras, M., 2015. Contribución de los árboles en finca a los medios de vida de 487 familias rurales en dos sitios contrastantes de Nicaragua (master thesis). CATIE 488 A li J Alli C C d R Will L S S 2018 M l i l Di S 489 Avelino, J., Allinne, C., Cerda, R., Willocquet, L., Savary, S., 2018. Multiple Disease System 489 in Coffee: From Loss Assessment to Sustainable Management. Annual Review of 490 Phytopathology 56, 611-635. 491 Avelino, J., Cabut, S., Barboza, B., Barquero, M., Alfaro, R., Esquivel, C., Durand, J.-F., 492 Cilas, C., 2007. Topography and Crop Management Are Key Factors for the 493 Development of American Leaf Spot Epidemics on Coffee in Costa Rica. 494 Phytopathology 97, 1532–1542. https://doi.org/10.1094/PHYTO-97-12-1532 495 Barel, M., Jacquet, M., 1994. La qualité du café : ses causes, son appréciation, son 496 amélioration. Plantations, Recherche, Développement 1, 5–13. 497 Barradas, V.L., Fanjul, L., 1986. Microclimatic chacterization of shaded and open-grown 498 coffee (Coffea arabica L.) plantations in Mexico. Agricultural and Forest Meteorology 499 38, 101–112. https://doi.org/10.1016/0168-1923(86)90052-3 500 p g ( ) Beer, J., Muschler, R., Kass, D., Somarriba, E., 1997. Shade management in coffee and 501 cacao plantations. Agroforestry Systems 38, 139–164. 502 https://doi.org/10.1023/A:1005956528316 503 p g Boshier, D., Salvado, J., Detlefsen, G., 2009. Mesoamerican tree species: a source book for 504 farm planting and ecological restoration. Final Technical Report. 505 C id A 2016 Di id d l i d b d i i dif 06 Caceido, A., 2016. Diversidad y almacenamiento de carbono, en dos sitios con diferente 506 grado de intensificación de uso de suelo de Nicaragua. (No. Thesis C133). CATIE, 507 Turrialba (Costa Rica). 508 Caceido, A., 2016. Diversidad y almacenamiento de carbono, en dos sitios con diferente 506 grado de intensificación de uso de suelo de Nicaragua. (No. Thesis C133). CATIE, 507 Turrialba (Costa Rica). 508 Camargo P.D., Camargo, M.B.P.D., 2001. REFERENCES 479 Definição e esquematização das fases 509 fenológicas do cafeeiro arábica nas condições tropicais do Brasil. Bragantia 60, 65– 510 68. https://doi.org/10.1590/S0006-87052001000100008 511 Camargo P.D., Camargo, M.B.P.D., 2001. Definição e esquematização das fases 509 fenológicas do cafeeiro arábica nas condições tropicais do Brasil. Bragantia 60, 65– 510 68. https://doi.org/10.1590/S0006-87052001000100008 511 p g Cerda, R., Allinne, C., Krolczyk, L., Mathiot, C., Clement, E., Harvey, C.A., Aubertot, J.-N., 512 Tixier, P., Gary, C., Avelino, J., 2015. Ecosystem services provided by coffee 513 agroecosystems across a range of topo-climatic conditions and management 514 strategies. FSD5 Proceedings: Multi-Functional Farming Systems in a Changing 515 World. European Society of Agronomy. 516 p g Cerda, R., Allinne, C., Krolczyk, L., Mathiot, C., Clement, E., Harvey, C.A., Aubertot, J.-N., 512 Tixier, P., Gary, C., Avelino, J., 2015. Ecosystem services provided by coffee 513 agroecosystems across a range of topo-climatic conditions and management 514 strategies. FSD5 Proceedings: Multi-Functional Farming Systems in a Changing 515 World. European Society of Agronomy. 516 21 Cerda, R., Avelino, J., Gary, C., Tixier, P., Lechevallier, E., Allinne, C., 2017. Primary and 517 Secondary Yield Losses Caused by Pests and Diseases: Assessment and Modelling 518 in Coffee. PLOS ONE 12, e0169133. https://doi.org/10.1371/journal.pone.0169133 519 p g j p Cerdán, C.R., Rebolledo, M.C., Soto, G., Rapidel, B., Sinclair, F.L., 2012. Local knowledge 520 of impacts of tree cover on ecosystem services in smallholder coffee production 521 systems. Agricultural Systems 110, 119–130. 522 https://doi.org/10.1016/j.agsy.2012.03.014 523 Chapman, H.D., 1965. Cation-Exchange Capacity 1. Methods of Soil Analysis. Part 2. 524 Chemical and Microbiological Properties agronomymonogra, 891–901. 525 https://doi.org/10.2134/agronmonogr9.2.c6 526 p g g g Charbonnier, F., le Maire, G., Dreyer, E., Casanoves, F., Christina, M., Dauzat, J., Eitel, 527 J.U.H., Vaast, P., Vierling, L.A., Roupsard, O., 2013. Competition for light in 528 heterogeneous canopies: Application of MAESTRA to a coffee (Coffea arabica L.) 529 agroforestry system. Agricultural and Forest Meteorology 181, 152–169. 530 https://doi.org/10.1016/j.agrformet.2013.07.010 531 p g j g Charbonnier, F., Roupsard, O., Maire, G. le, Guillemot, J., Casanoves, F., Lacointe, A., 532 Vaast, P., Allinne, C., Audebert, L., Cambou, A., Clément‐Vidal, A., Defrenet, E., 533 Duursma, R.A., Jarri, L., Jourdan, C., Khac, E., Leandro, P., Medlyn, B.E., Saint‐ 534 André, L., Thaler, P., Meersche, K.V.D., Aguilar, A.B., Lehner, P., Dreyer, E., 2017. 535 Increased light-use efficiency sustains net primary productivity of shaded coffee 536 plants in agroforestry system. Plant, Cell & Environment 40, 1592–1608. REFERENCES 479 537 https://doi.org/10.1111/pce.12964 538 p g p DaMatta, F.M., 2004. Ecophysiological constraints on the production of shaded and 539 unshaded coffee: a review. Field Crops Research 86, 99–114. 540 https://doi.org/10.1016/j.fcr.2003.09.001 541 p g j Granados Montero, M., 2015. Estudio de la epidemiología y alternativas de manejo 542 agroecológico del ojo de gallo (Mycena citricolor) en cafeto bajo sistemas 543 agroforestales en Costa Rica (thesis). Universidad de Costa Rica. 544 g ( ) Descroix, F., Snoeck, J., 2008. Environmental Factors Suitable for Coffee Cultivation, in: 545 Coffee: Growing, Processing, Sustainable Production. Wiley-Blackwell, pp. 164–177. 546 https://doi.org/10.1002/9783527619627.ch6 547 Frazer, G.W., Kenneth, D., Lertzman, P., Charles, D., Canham, D., Sallaway, D.P., 548 Marinakis, D., 1999. Gap Light Analyzer (GLA), Version 2.0: Imaging software to 549 extract canopy structure and gap light transmission indices from true-colour fisheye 550 photographs, users manual and program documentation, Simon Fraser University, 551 Burnaby, British Columbia, and the Institute of Ecosystem Studies, Millbrook, New 552 York. ed. 553 Granados Montero MDM, 2015. Estudio de la epidemiologia y alternativas de manejo 54 agroecologico del ojo de gallo (Mycena citricolor) en cafeto bajo sistema 55 agroforestales en Costa-Rica (thesis). Ciudad Universitaria Rodrigo Facio: Costa 56 Rica. 57 Gómez-Muñoz, B., Valero-Valenzuela, J. D., Hinojosa, M. B., García-Ruiz, R. (2016). 558 Management of tree pruning residues to improve soil organic carbon in olive groves. 559 European journal of soil biology, 74, 104-113 560 p j gy Haggar, J., Asigbaase, M., Bonilla, G., Pico, J., Quilo, A., 2015. Tree diversity on sustainably 561 certified and conventional coffee farms in Central America. Biodivers Conserv 24, 562 1175–1194. https://doi.org/10.1007/s10531-014-0851-y 563 p g y Hoyle, R.H., 2012. Handbook of Structural Equation Modelling. Guilford Press. y q g Lin, B.B., 2010. The role of agroforestry in reducing water loss through soil evaporation and 565 crop transpiration in coffee agroecosystems. Agricultural and Forest Meteorology 150, 566 510–518. https://doi.org/10.1016/j.agrformet.2009.11.010 567 p p g y g 510–518. https://doi.org/10.1016/j.agrformet.2009.11.010 Libert Amico, A., Ituarte-Lima, C., Elmqvist, T., 2019. Learning from social–ecological crisis 568 for legal resilience building: multi-scale dynamics in the coffee rust epidemic. 569 Sustainability Science, 1-17. 570 22 Muller, R., Berry, D., Avelino, J., Bieysse, D., 2008. Coffee Diseases, in: Coffee: Growing, 571 Processing, Sustainable Production. Wiley-Blackwell, pp. 491–545. 572 https://doi.org/10.1002/9783527619627.ch18 573 p g Oliveira, P.H.G., Gama-Rodrigues, A.C., Gama-Rodrigues, E.F., Sales, M.V.S., 2018. Litter 574 and soil-related variation in functional group abundances in cacao agroforests using 575 structural equation modelling. Ecological Indicators 84, 254–262. 576 https://doi.org/10.1016/j.ecolind.2017.08.030 577 Pagès, J., 2013. Analyse factorielle multiple avec R. EDP Sciences. 578 g y p Pineda, A.G., 2006. Flora Util Ethnonotanica de Nicaragua. Gobierno de Nicaragua, 579 Ministerio del Ambiente y los Recursos Naturales. 580 Puech, C., Poggi, S., Baudry, J., Aviron, S., 2015. Do farming practices affect natural 581 enemies at the landscape scale? Landscape Ecol 30, 125–140. 582 https://doi.org/10.1007/s10980-014-0103-2 583 p g Ratnadass, A., Fernandes, P., Avelino, J., Habib, R., 2012. Plant species diversity for 584 sustainable management of crop pests and diseases in agroecosystems: a review. 585 Agron. Sustain. Dev. 32, 273–303. https://doi.org/10.1007/s13593-011-0022-4 586 Román, F., De Liones, R., Sautu, A., Deago, J., Hall, J.S., 2012. Guía para la propagación 587 de 120 especies de árboles nativos de Panamá y el neotrópico. Environmental 588 Leadership and Training Initiative--ELTI, Yale School of Forestry & Environmental 589 Studies, New Haven, CT. 590 Sanchez, G., 2013. PLS_Path_Modelling_with_R.pdf, Trowchez Editions. ed. Sanchez, G., Trinchera, L., Russolillo, G., 2013. Introduction to the R package plspm p g p p Sauvadet, M., Chauvat, M., Fanin, N., Coulibaly, S., Bertrand, I., 2016. Comparing the 593 effects of litter quantity and quality on soil biota structure and functioning: Application 594 to a cultivated soil in Northern France. Applied Soil Ecology 107, 261–271. 595 https://doi.org/10.1016/j.apsoil.2016.06.010 596 p g j p Sauvadet M, Den Meersche KV, Allinne C, et al., 2018. Shade trees have higher impact on 597 soil nutrient availability and food web in organic than conventional coffee agroforestry. 598 Science of The Total Environment 649, 1065-1074. 599 , Simko, I., Piepho, H.-P., 2011. The Area Under the Disease Progress Stairs: Calculation, 600 Advantage, and Application. Phytopathology 102, 381–389. 601 https://doi.org/10.1094/PHYTO-07-11-0216 602 p g Staver, C., Guharay, F., Monterroso, D., Muschler, R.G., 2001. Designing pest-suppressive 603 multistrata perennial crop systems: shade-grown coffee in Central America. 604 Agroforestry Systems 53, 151–170. Wintgens, J. N. (2004). Coffee: growing, processing, sustainable production. A guidebook for 632 growers, processors, traders, and researchers. WILEY-VCH Verlag GmbH & Co. 633 KGaA. 634 p p g y g 510–518. https://doi.org/10.1016/j.agrformet.2009.11.010 https://doi.org/10.1023/A:1013372403359 605 Tapia-Coral, S.C., Luizão, F.J., Wandelli, E., Fernandes, E.C.M., 2005. Carbon and nutrient 606 stocks in the litter layer of agroforestry systems in central Amazonia, Brazil. 607 Agroforest Syst 65, 33–42. https://doi.org/10.1007/s10457-004-5152-0 608 Thoumazeau, A., Bessou, C., Renevier, M.-S., Panklang, P., Puttaso, P., Peerawat, M., 609 Heepngoen, P., Polwong, P., Koonklang, N., Sdoodee, S., Chantuma, P., Lawongsa, 610 P., Nimkingrat, P., Thaler, P., Gay, F., Brauman, A., 2019a. Biofunctool®: a new 611 framework to assess the impact of land management on soil quality. Part B: 612 investigating the impact of land management of rubber plantations on soil quality with 613 the Biofunctool® index. Ecological Indicators 97, 429–437. 614 https://doi.org/10.1016/j.ecolind.2018.10.028 615 Thoumazeau, A., Bessou, C., Renevier, M.-S., Trap, J., Marichal, R., Mareschal, L., 616 Decaëns, T., Bottinelli, N., Jaillard, B., Chevallier, T., Suvannang, N., Sajjaphan, K., 617 Thaler, P., Gay, F., Brauman, A., 2019b. Biofunctool®: a new framework to assess 618 the impact of land management on soil quality. Part A: concept and validation of the 619 set of indicators. Ecological Indicators 97, 100–110. 620 https://doi.org/10.1016/j.ecolind.2018.09.023 621 p g j Toniutti, L., Breitler, J.-C., Etienne, H., Campa, C., Doulbeau, S., Urban, L., Lambot, C., 622 Pinilla, J.-C.H., Bertrand, B., 2017. Influence of Environmental Conditions and 623 Genetic Background of Arabica Coffee (C. arabica L) on Leaf Rust (Hemileia 624 vastatrix) Pathogenesis. Front. Plant Sci. 8. https://doi.org/10.3389/fpls.2017.02025 625 23 Vaast, P., Van Kanten, R., Siles, P., Dzib, B., Franck, N., Harmand, J.-M., Génard, M., 2005. 626 Shade: A key factor for coffee sustainability and quality. ASIC. 627 y y q y Vinzi, V.E., Trinchera, L., 2013. Modèles à équations structurelles, approches basées sur les 628 composantes, in: Modèles à variables latentes et modèles de mélange. p. 27. 629 Waller, J.M., Bigger, M., Hillocks, R.J., 2007. Coffee Pests, Diseases and Their 630 Management. CABI. 631 g Wintgens, J. N. (2004). Coffee: growing, processing, sustainable production. A guidebook for 632 growers, processors, traders, and researchers. WILEY-VCH Verlag GmbH & Co. 633 KGaA. 634 24
https://openalex.org/W2922024165	https://europepmc.org/articles/pmc6458411?pdf=render	English	null	Depression chains in seafloor of contrasting morphology, Atacama Trench margin: a comment on Marsh<i>et al.</i>(2018)	Royal Society open science	2,019	cc-by	4,627	royalsocietypublishing.org/journal/rsos royalsocietypublishing.org/journal/rsos Comment Cite this article: Purser A et al. 2019 Depression chains in seafloor of contrasting morphology, Atacama Trench margin: a comment on Marsh et al. (2018). R. Soc. open sci. 6: 182053. Autun Purser1, Helena Herr1,2, Simon Dreutter1, Boris Dorschel1, Ronnie N. Glud3,4, Laura Hehemann1, Ulrich Hoge1, Alan J. Jamieson5, Thomas D. Linley5, Heather A. Stewart6 and Frank Wenzho¨fer1,7 1Alfred Wegener Helmholz Institute of Polar and Marine Research, Bremerhaven, Germany 2Center of Natural History, University of Hamburg, Hamburg, Germany 3Department of Biology, University of Southern Denmark, 5230 Odense, Denmark 4Department of Ocean and Environmental Sciences, Tokyo University of Marine Science and Technology, Tokyo, Japan 5School of Natural and Environmental Sciences, Newcastle University, Newcastle Upon Tyne, UK 6British Geological Survey, Lyell Centre, Edinburgh, UK 7 Keywords: Keywords: marine mammals, ichnology, deep-diving mammals, Atacama Trench, sidescan sonar, towed camera sled This comment presents acoustic and visual data showing deep seafloor depression chains similar to those reported in Marsh et al. (R. Soc. open sci. 5: 180286), though from a different deep- sea setting. Marsh et al. present data collected during cruise JC120 from polymetallic nodule rich sites within the Clarion- Clipperton Fracture Zone (CCFZ), at water depths of between 3999 and 4258 m. Within this comment, we present data collected with equivalent acoustic and imaging devices on-board the RV Sonne (SO261—March/April 2018) from the Atacama Trench, approximately 4000 m depth, which shows comparable depression chains in the seafloor. In contrast with the CCFZ observations, our study area was wholly free of polymetallic nodules, an observation therefore weakening the ‘ballast collection’ by deep-sea diving mammals formation hypothesis discussed in their paper. We support their alternate hypothesis that if these features are indeed generated by deep-diving Author for correspondence: Autun Purser e-mail: autun.purser@awi.de The accompanying reply can be viewed at http:// dx.doi.org/10.1098/rsos.180286. & 2019 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited. Autun Purser1, Helena Herr1,2, Simon Dreutter1, Boris Dorschel1, Ronnie N. Glud3,4, Laura Hehemann1, Ulrich Hoge1, Alan J. Jamieson5, Thomas D. Linley5, Heather A. Stewart6 and Frank Wenzho¨fer1,7 http://dx.doi.org/10.1098/rsos.182053 http://dx.doi.org/10.1098/rsos.182053 Received: 30 November 2018 Accepted: 18 February 2019 Subject Category: Biology (whole organism) Subject Areas: ecology/behaviour Keywords: marine mammals, ichnology, deep-diving mammals, Atacama Trench, sidescan sonar, towed camera sled Author for correspondence: Autun Purser e-mail: autun.purser@awi.de The accompanying reply can be viewed at http:// dx.doi.org/10.1098/rsos.180286. Received: 30 November 2018 Accepted: 18 February 2019 1Alfred Wegener Helmholz Institute of Polar and Marine Research, Bremerhaven, Germany 2Center of Natural History, University of Hamburg, Hamburg, Germany 3Department of Biology, University of Southern Denmark, 5230 Odense, Denmark 4Department of Ocean and Environmental Sciences, Tokyo University of Marine Science and Technology, Tokyo, Japan 5School of Natural and Environmental Sciences, Newcastle University, Newcastle Upon Tyne, UK 5School of Natural and Environmental Sciences, Newcastle University, Newcastle Upon Tyne, UK 6British Geological Survey, Lyell Centre, Edinburgh, UK 7Max Planck Institute for Marine Microbiology, Bremen, Germany Subject Category: Biology (whole organism) g y y g 7Max Planck Institute for Marine Microbiology, Bremen, Germany AP, 0000-0001-5427-0151; HH, 0000-0002-5028-2419; SD, 0000-0002-0878-0780; BD, 0000-0002-3495-5927; RNG, 0000-0002-7069-893X; AJJ, 0000-0001-9835-2909; TDL, 0000-0002-6583-3105; HAS, 0000-0002-5590-6972; FW, 0000-0002-4621-0586 AP, 0000-0001-5427-0151; HH, 0000-0002-5028-2419; SD, 0000-0002-0878-0780; BD, 0000-0002-3495-5927; RNG, 0000-0002-7069-893X; AJJ, 0000-0001-9835-2909; TDL, 0000-0002-6583-3105; HAS, 0000-0002-5590-6972; FW, 0000-0002-4621-0586 5School of Natural and Environmental Sciences, Newcastle University, Newcastle Upon Tyne, UK 6British Geological Survey, Lyell Centre, Edinburgh, UK 7Max Planck Institute for Marine Microbiology, Bremen, Germany 2. Results At 2081905000 S 718002000 W on the eastern flank of the Atacama Trench, at depths of 3990–4140 m (cruise survey ID SO261/109-1, figure 1), a number of extended chains of depression features, reminiscent of those presented in Marsh et al. [2] were observed during an OFOBS survey of approximately 2.5 km length (figure 2 and electronic supplementary material, S1). By chance, one depression was passed directly over and imaged by still (figure 3) and video cameras (video frames mosaiced into figure 4). By using the tri-laser sizing system of the OFOBS and the PAPARA(ZZ)I 2.6 software [3], the imaged depression disturbance area was estimated to be 55+10 cm in width and 1.5–2 m in length. In the concurrently collected sidescan sonar data, the average spacing of the depressions in the most extensive chain imaged (presented in figure 2) was determined to be approximately 8.5 m, roughly the length of a Cuvier’s beaked whale. At one point a chain encountered a small ridge, the likely surface expression of a small fault (western section of figure 2), at which point the depression chain is offset by approximately 30 m before continuing in a roughly E–W direction. The sidescan data showed all depressions to be elongated in the direction of the depression chain. These depression chains weave across, in and out of the surveyed region, with at least one chain appearing to be in excess of 500 m length. A three- dimensional model of the imaged depression was generated using frames extracted from the concurrently collected video (electronic supplementary material, S2 and S3), from which an estimated maximum depression depth of approximately 15 cm was estimated. Further depressions were partially imaged by the camera systems at other locations (electronic supplementary material, S4). In some sections of cruise survey SO261/109-1, secondary chains of depressions bisect longer chains (as shown in figure 2 and the eastern section of S1). Even though they interweave, the course of all chains observed was roughly perpendicular to the depth contours (figure 1). As in the CCFZ data, we observed variations in the sharpness of the outlines of the depression features present on the Atacama Trench flank. Two chains of depressions with very sharp, comparable outlines interweave each other at the eastern extremity of S1, with a further pair of chains observed toward the west of S1 (shown in detail in figure 1). 2 megafauna, then they are more likely the resultant traces of infauna feeding or marks made during opportunistic capture of benthic fish/cephalopods. We observed these potential prey fauna with lander and towed camera systems during the cruise, with example images of these presented here. Both the SO261 and JC120 cruises employed high-resolution sidescan systems at deployment altitudes seldom used routinely until the last few years during scientific deep-sea surveys. Given that both cruises found these depression chains in contrasting physical regions of the East Pacific, they may have a more ubiquitous distribution than at just these sites. Thus, the impacts of cetacean foraging behaviour on deep seafloor communities, and the potential relevance of these prey sources to deep-diving species, should be considered. royalsocietypublishing.org/journal/rsos R. Soc. open sci. 6: 182053 1. Introduction and methodology During March/April 2018, the research vessel RV Sonne investigated the Atacama Trench, offshore the west coast of South America (Cruise SO261), as part of the multidisciplinary HADES European Research Council (HADES-ERC) study of deep trench systems. A towed sled (the Ocean Floor Observation and Bathymetry System (OFOBS[1])) incorporating cameras (still and video) and sidescan sonar was deployed at an altitude of 1.5–2 m above seafloor at water depths between 3500 and 6000 m at seven locations (figure 1). The still camera and sidescan systems were similar to those used by Marsh et al. [2], though the additional mounting of a video camera and a 50 cm spaced tri-laser sizing system on OFOBS allowed georeferenced video frames to be extracted for subsequent creation of three-dimensional seafloor models [1]. In addition to conducting OFOBS tows, baited HD camera landers were also deployed, collecting data on bait- attending fish and mobile fauna present at various depths within the surveyed area. During March/April 2018, the research vessel RV Sonne investigated the Atacama Trench, offshore coast of South America (Cruise SO261), as part of the multidisciplinary HADES European Research 2. Results The second of these chain pairs exhibited less distinct edges in the acoustic data. In the case of both these chain pairings, the spacing of the individual depressions, while uniform for a particular chain, differed from that of the companion chain by approximately 15%. Both OFOBS and baited camera landers recorded a range of fish (primarily the macrourids Coryphaenoides armatus and Coryphaenoides yaquinae, though also the ophidiid Bassozetus sp. and an unidentified Ipnopidae) at approximately 4000 m depth, and other typical deep-sea benthic fauna including crustacean, ophiuroid, holothurian, jellyfish and hemichordate species. Several benthic –20° 0° –20° –6000 –3000 –3000 –4000 Antofagasta Antofagasta 0 50 100 km Lima –40° –60° –80° –60° –22° –24° –72° –70° –68° (b) (a) Figure 1. (a) Location of the Atacama Trench and region of study. (b) Map showing the location of OFOBS deployments conducted during SO261. Cruise survey SO261/109-1, with image and acoustic data collected and the focus of the current study, is indicated by a black star. Open star symbols represent OFOBS deployments where only image data was collected successfully. –20° 0° –20° –6000 –3000 –3000 –4000 Antofagasta Antofagasta 0 50 100 km Lima –40° –60° –80° –60° –22° –24° –72° –70° –68° (b) (a) Fi 1 ( ) L i f h A T h d i f d (b) M h i h l i f OFOBS d l d d royalsocietypublishing.org/journal/rsos R. Soc. open sci. 6: 182053 Figure 1. (a) Location of the Atacama Trench and region of study. (b) Map showing the location of OFOBS deployments conducted during SO261. Cruise survey SO261/109-1, with image and acoustic data collected and the focus of the current study, is indicated by a black star. Open star symbols represent OFOBS deployments where only image data was collected successfully. 0 20°19¢48¢¢S 20°19¢54¢¢S 71°1¢0¢¢W 71°0¢45¢¢W depression directly imaged 50 100 m Figure 2. Georeferenced, processed sidescan data collected from a flight height of 1.5–2 m showing the chain of depressions reminiscent of those presented in Marsh et al. [2]. Individual depression features outlined by red and blue circles. Red arrow indicates the position of the image of seafloor given in figures 3 and 4. 71°0¢45¢¢W 71°0¢45¢¢W 71°1¢0¢¢W Figure 2. Georeferenced, processed sidescan data collected from a flight height of 1.5–2 m showing the chain of depressions reminiscent of those presented in Marsh et al. [2]. Individual depression features outlined by red and blue circles. 2. Results Red arrow indicates the position of the image of seafloor given in figures 3 and 4. incirrate octopi of unknown species were also imaged, reminiscent of those recently reported in Purser et al. [3] from comparable depth in the DISCOL region of the East Pacific. Additionally, indications of infauna activity were observed, such as burrows, mounds and signs of sediment disturbance by emergent fauna (figure 5). incirrate octopi of unknown species were also imaged, reminiscent of those recently reported in Purser et al. [3] from comparable depth in the DISCOL region of the East Pacific. Additionally, indications of infauna activity were observed, such as burrows, mounds and signs of sediment disturbance by emergent fauna (figure 5). 50 cm Figure 3. Image of depression feature taken from 1.5 m altitude. Lazer points (circled in red) have a 50 cm spacing. 0 10 20 m Figure 4. Two-dimensional georeferenced mosaic of OFOBS image frames (grey tint) and video frames (green tint) mapped directly onto sidescan derived bathymetric data. royalsocietypublishing.org/journal/rsos R. Soc. open sci. 6: 182053 4 50 cm Figure 3. Image of depression feature taken from 1.5 m altitude. Lazer points (circled in red) have a 50 cm spacing. royalsocietypublishing.org/journal/rsos R. So 4 royalsocietypublishing.org/journal/rsos R. Soc. open sci. 6: 182053 ure 3. Image of depression feature taken from 1.5 m altitude. Lazer points (circled in red) have a 50 cm spacing. 0 10 20 m Figure 4. Two-dimensional georeferenced mosaic of OFOBS image frames (grey tint) and video frames (green tint) mapped directly onto sidescan derived bathymetric data. 0 10 20 m Figure 4. Two-dimensional georeferenced mosaic of OFOBS image frames (grey tint) and video frames (green tint) mapped directly onto sidescan derived bathymetric data. 50 cm bar (a) (b) (c) (d) (e) ( f) 50 cm bar 50 cm 50 cm 15 cm 25 cm Figure 5. Mobile fauna observed at approximately 4000 m depth during SO261. (a) Bassozetus spp. (b,c) Coryphaenoides spp. (d) Crustacean on fish fall. (e) Ophiuroid on mound. (f ) Unknown incirrate octopus. (c) 50 cm 50 cm bar (a) (b) 50 cm bar (a) (b) (e) 25 cm (d) 50 cm ( f) 15 cm (e) ( f) (d) Figure 5. Mobile fauna observed at approximately 4000 m depth during SO261. (a) Bassozetus spp. (b,c) Coryphaenoides spp. (d) Crustacean on fish fall. (e) Ophiuroid on mound. (f ) Unknown incirrate octopus. 3.2. Formation hypotheses Like Marsh et al. [2], we find it difficult to assign an abiotic formation mechanism for these depression chains. However, unlike the CCFZ studyarea of Marsh et al., there are potentialmechanisms for fluidflow withinthe Atacama Trench setting with bend-related faulting within subduction trenches suggested to play a role in local fluid circulation [4]. Depressions, such as pockmarks related to fluid-flow processes, have diverse morphologies and can occur in both random and non-random distributions. Distribution of these crater- like depressions is controlled by underlying geological features such as faults or buried channels [5–7]. However, the depressions observed herein are contrary to the fault trend evident in these data (figure 2) suggesting that there is no underlying geological control related to the formation of these depression chains. We did not image any fish larger than 1 m, and the larger individuals we did observe were moving slowly above the seafloor (Coryphanoides armatus and C. yaquinae), not interacting with it in any notable way, on any occasion. No sediment excavation or disturbance by fish was apparent. We believe the overriding hypothesis that cetaceans are causing these depressions is greatly strengthened by our new data. Cetacean contact with the seafloor and seafloor cable infrastructure has been reported (at least in shallower regions of our area of research) since the nineteenth century, and particularly prominently in the eastern Pacific [8]. Both deep-diving sperm whales (Physeter macrocephalus) and Cuvier’s beaked whales (Ziphius cavirostris) are numerous in the region and eastern Pacific in general [9], Cuvier’s beaked whales are preferentially observed in regions with seafloor slope, such as the Atacama Trench margin [10]. As highlighted in Marsh et al. [2], no whales have yet been observed (i.e. tagged) diving to the seafloor depths associated with CCFZ or the region of the Atacama Trench investigated here. The maximum dive depth recorded in cetaceans thus far has been 2992 m by a Cuvier’s beaked whale [11]. These whales are probably physiologically capable of diving much deeper, as deep as 5000 m [12,13], and certainly spend extended periods close to the seafloor during foraging dives [14,15]. y p p g g g Marsh et al. [2] present a range of hypotheses as to why whales may be diving to abyssal depths and interacting with the seafloor; some of which are supported by our new observations, others weakened. 3.2. Formation hypotheses The idea that the whales forming the depression chains observed on the eastern flank of the Atacama Trench were seeking stones to consume, to function in the role of gastroliths, is highly unlikely, given the total absence in images of any surface solid material in this survey. Further, marine vertebrates with hydrofoil limbs (such as penguins, otariid pinnipeds and extinct plesiosaurs) have been suggested to use such material, rather than caudal finned cetaceans ([15] and references therein), such as the negatively buoyant beaked whales [16]. We also believe it unlikely that the active pursuit of a particular fish or cephalopod individual would result in the depression patterns observed. Our depression tracks formed continuous, unbroken, undulating and evenly spaced chains, whereas active pursuit may be expected to result in tracks with sudden changes of direction or variations in swimming speed (and therefore depression spacing). The chains we observed were also unlike the chaotic and intense ‘surface of the moon’ depressions reported for shallow bottlenose dolphin (Tursiops truncatus) feeding [17] or the short chains and parallel splayed arrays of elongated depressions as formed by benthic feeding gray whales (Eschrichtius robustus) [18,19]. The individual outline dimensions of the depressions do match almost exactly those observed in Woodside et al. [20]; the authors suggested these to have been made by Cuvier’s beaked whales in the Mediterranean. The depressions imaged during our study appear far less fresh, partially infilled with sediment and biodetritus, than the examples in [20] or indeed those in Marsh et al. [2]. We observed no clear central groove [20] in the depressions imaged on the Atacama Trench. Of the deep-diving whale species reported in the eastern Pacific, sperm whales and Cuvier’s beaked whales are the most widely reported, with information on diets for both species commonly published from opportunistic strandings from diverse and contrasting locations [21–24], possibly reflective of local dietary availabilities to individuals from these ubiquitous species rather than of firm dietary preferences. The wide range of fauna observed in the image data collected during our cruise include species occasionally found in the stomach contents of both Cuvier’s beaked and sperm whales, although generally sperm whales seem to prefer deep-sea squid from higher in the water column [24,25], despite spending approximately 50 min very close to the seafloor during documented dives [14]. Images of deep squid were captured during deployment of the OFOBS instrument. 3.1. Depression geomorphology and distribution The ultra-low deployment height of the sonar systems used by Marsh et al. [2] and ourselves seem to indicate that such depression chains in sediments of approximately 4000 m depth are potentially widespread geomorphological features (at least in the East Pacific) not detected by previous iterations of deep-sea survey equipment. Though the chains were broadly similar in spacing, size and form to those presented in Marsh et al. [2] within the CCFZ, the seafloor characteristics differed greatly in hard substrate availability, with our surveyed area of the Atacama Trench wholly free of polymetallic nodules, which were abundant in the CCFZ. of deep-sea survey equipment. Though the chains were broadly similar in spacing, size and form to those presented in Marsh et al. [2] within the CCFZ, the seafloor characteristics differed greatly in hard substrate availability, with our surveyed area of the Atacama Trench wholly free of polymetallic nodules, which were abundant in the CCFZ. 5 References 10. Ferguson MC, Barlow J, Reilly SB, Gerrodette T. 2006 Predicting Cuvier’s (Ziphius cavirostris) and Mesoplodon beaked whale population density from habitat characteristics in the eastern tropical Pacific Ocean. J. Cetacean Res. Manag. 7, 287–299. 1. Purser A et al. 2018 Ocean Floor Observation and Bathymetry System (OFOBS): a new towed camera/sonar system for deep-sea habitat surveys. IEEE J. Ocean. Eng. 44, 87–99. (doi:10. 1109/JOE.2018.2794095) the western continental margin of India: employing multibeam bathymetry and backscatter data. Mar. Pet. Geol. 10, 2107–2117. (doi:10.1016/j.marpetgeo.2010. 09.005) the western continental margin of India: employing multibeam bathymetry and backscatter data. Mar. Pet. Geol. 10, 2107–2117. (doi:10.1016/j.marpetgeo.2010. 09.005) the western continental margin of India: employing multibeam bathymetry and backscatter data. Mar. Pet. Geol. 10, 2107–2117. (doi:10.1016/j.marpetgeo.2010. 09.005) 6. Gafeira J, Dolan M, Monteys X. 2018 Geomorphometric characterization of pockmarks by using a GIS-based semi-automated toolbox. Geosciences 8, 154. (doi:10.3390/ geosciences8050154) 2. Marsh L, Huvenne VAI, Jones DOB. 2018 Geomorphological evidence of large vertebrates interacting with the seafloor at abyssal depths in a region designated for deep-sea mining. R. Soc. open sci. 5, 180286. (doi:10.1098/ rsos.180286) 11. Schorr GS, Falcone EA, Moretti DJ, Andrews RD. 2014 First long-term behavioral records from Cuvier’s beaked whales (Ziphius cavirostris) reveal record-breaking dives. PLOS ONE 9, e92633. (doi:10.1371/journal.pone.0092633) 7. Gay A, Lopez M, Cochonat P, Sultan N, Cauiquil E, Brigaud F. 2002 Sinuous pockmark belt as indicator of a shallow buried turbiditic channel on the lower slope of the Congo basin, West African margin. Geol. Soc. Lond. Spec. Publ. 216, 173–189. (doi:10.1144/GSL.SP.2003.216.01.12) 3. Purser A, Marcon Y, Hoving H-JT, Vecchione M, Piatkowski U, Eason D, Bluhm H, Boetius A. 2016 Association of deep-sea incirrate octopods with manganese crusts and nodule fields in the Pacific Ocean. Curr. Biol. 26, R1268–R1269. (doi:10.1016/j.cub.2016.10.052) 12. Cranford TW, McKenna MF, Soldevilla MS, Wiggins SM, Goldbogen JA, Shadwick RE, Krysl P, St JL, Hildebrand JA. 2008 Anatomic geometry of sound transmission and reception in Cuvier’s beaked whale (Ziphius cavirostris). Anat. Rec. Hoboken N.J. 291, 353–378. (doi:10. 1002/ar.20652) 8. Heezen BC. 1957 Whales entangled in deep sea cables. Deep Sea Res. 4, 105–115. (doi:10. 1016/0146-6313(56)90040-5) 4. Ranero CR, Phipps Morgan J, McIntosh K, Reichert C. 2003 Bending-related faulting and mantle serpentinization at the Middle America trench. Nature 425, 367–373. (doi:10.1038/ nature01961) 13. Tyack P, Johnson M, Soto N, Sturlese A, Madsen P. 2006 Extreme diving of beaked whales. J. Exp. Biol. 209, 4238–4253. (doi:10.1242/ jeb.02505) 9. royalsocietypublishing.org/journal/rsos R. Soc. open sci. 6: 182053 6 Data accessibility. The datasets supporting this article have been uploaded as part of the electronic supplementary material. All additional georeferenced sidescan and image-based data collected via OFOBS during cruise survey SO261/109-1 are available from PANGAEA in full, native resolution at https://doi.pangaea.de/10.1594/ PANGAEA.894733. Authors’ contributions. A.P., L.H., U.H. and F.W. collected the sidescan and image data. F.W. was principal investigator while R.N.G. was the scientific leader during SO261 and both conceived the study. S.D., B.D., L.H. and H.A.S. georeferenced and processed the geomorphological data. A.J.J. and T.L. collected and analysed the fish data. A.P. and H.H. analysed the combined dataset and prepared the manuscript. All authors actively contributed to the manuscript and gave their final approval for publication. p g pp p Competing interests. We have no competing interests. F di h SO f ld k f d d b h S C d d G (‘ h d d Competing interests. We have no competing interests. Competing interests. We have no competing interests. Funding. The SO261 cruise fieldwork was funded by the HADES-ERC Advanced Grant (‘Benthic diagenesis and microbiology of hadal trenches’ Grant agreement No. 669947) awarded to R.N.G. (University Southern Denmark). Ship-time on board the RV SONNE was financed by BMBF and was awarded to F.W. (AWI/MPI) M. Zabel (MARUM), and R.N.G. (University of Southern Denmark/Tokyo University). Antje Boetius oversaw the development of the OFOBS system, which was funded by ERC advanced grant ‘Abyss’ (Investigator grant no. 294757) and the FRAM project. Acknowledgements. We would like to thank the crew and scientific party of RV SONNE cruise SO261 for their able and enthusiastic assistance in the collection of the datasets discussed within this comment. H.A.S. publishes with the permission of the Executive Director of the British Geological Survey (United Kingdom Research and Innovation). Two anonymous reviewers are thanked for constructive review of the initial manuscript, input from which has improved this current draft. 3.2. Formation hypotheses Possibly opportunistic grazing of the benthic environment is made prior to a return to the surface, particularly if primary prey targets were not encountered during the dive, due to either absence of prey of any acoustic disturbance. Additionally, younger or stressed individuals may graze more readily on slow-moving benthic fauna than on fast- moving free-swimming prey higher in the water column. Foraging for a mixed, varied diet of infauna and fauna, without the active pursuit of prey individuals [21,26], would likely result in the extended depression chains observed on the Atacama Trench margin. Such opportunistic feeding, when conducted in regions containing small stones or nodules, may result in accidental consumption, potentially accounting for the occasional individuals found with such inorganic material within their stomachs [27,28]. From our data, the interweaving of depressions of similar age characteristics would support a loose group/small pod of foraging whales. Such roughly parallel chains of depressions can also be seen in fig. 3b of Marsh et al. [2] in the wider spatial data collected by their AUV system. Potentially, these depression chains then are merely the individual components of a more spatially extensive seafloor interaction made by a pod of foraging whales. y yp g g j 6 References Rendell L, Whitehead H, Escribano R. 2004 Sperm whale habitat use and foraging success off northern Chile: evidence of ecological links between coastal and pelagic systems. Mar. Ecol. Prog. Ser. 275, 289–295. (doi:10.3354/ meps275289) 5. Dandapath S, Chakraborty B, Karisiddaiah SM, Menezes A, Ranade G, Fernandes W, Naik DK, Raju KNP. 2010 Morphology of pockmarks along 14. Guerra M, Hickmott L, Van der Hoop J, Rayment W, Leunissen E, Slooten E, Moore M. 2017 Diverse foraging strategies by a marine top whale feeding. Ecology 66, 1965–1975. (doi:10.2307/2937392) predator: sperm whales exploit pelagic and demersal habitats in the Kaiko¯ura submarine canyon. Deep Sea Res. Part Oceanogr. Res. Pap. 128, 98–108. (doi:10.1016/j.dsr.2017.08.012) 15. Taylor MA. 1993 Stomach stones for feeding or buoyancy? The occurrence and function of gastroliths in marine tetrapods. Phil. Trans. R. Soc. Lond. B 341, 163–175. (doi:10. 1098/rstb.1993.0100) predator: sperm whales exploit pelagic and demersal habitats in the Kaiko¯ura submarine canyon. Deep Sea Res. Part Oceanogr. Res. Pap. 128, 98–108. (doi:10.1016/j.dsr.2017.08.012) 15. Taylor MA. 1993 Stomach stones for feeding or buoyancy? The occurrence and function of gastroliths in marine tetrapods. Phil. Trans. R. Soc. Lond. B 341, 163–175. (doi:10. 1098/rstb.1993.0100) predator: sperm whales exploit pelagic and demersal habitats in the Kaiko¯ura submarine canyon. Deep Sea Res. Part Oceanogr. Res. Pap. 128, 98–108. (doi:10.1016/j.dsr.2017.08.012) scale and relationship to migrations. J. Anim. Ecol. 65, 429–438. (doi:10.2307/5778) 25. Joyce TW, Durban JW, Claridge DE, Dunn CA, Fearnbach H, Parsons KM, Andrews RD, Ballance LT. 2017 Physiological, morphological, and ecological tradeoffs influence vertical habitat use of deep-diving toothed-whales in the Bahamas. PLoS ONE 12, e0185113. (doi:10. 1371/journal.pone.0185113) 7 25. 20. royalsocietypublishing.org/journal/rsos R. Soc. open sci. 6: 182053 20. Woodside JM, David L, Frantzis A, Hooker SK. 2006 Gouge marks on deep-sea mud volcanoes in the eastern Mediterranean: caused by Cuvier’s beaked whales? Deep Sea Res. Part Oceanogr. Res. Pap. 53, 1762–1771. (doi:10. 1016/j.dsr.2006.08.011) 15. 15. Taylor MA. 1993 Stomach stones for feeding or buoyancy? The occurrence and function of gastroliths in marine tetrapods. Phil. Trans. R. Soc. Lond. B 341, 163–175. (doi:10. 1098/rstb.1993.0100) 21. Santos MB, Pierce GJ, Herman J, Lo´pez A, Guerra A, Mente E, Clarke MR. 2001 Feeding ecology of Cuvier’s beaked whale (Ziphius cavirostris): a review with new information on the diet of this species. J. Mar. Biol. Assoc. U. K. 81, 687–694. (doi:10.1017/S0025315401004386) 26. 26. West KL, Walker WA, Baird RW, Mead JG, Collins PW. 2017 Diet of Cuvier’s beaked whales Ziphius cavirostris from the North Pacific and a comparison with their diet world-wide. Mar. Ecol. Prog. Ser. 574, 227–242. (doi:10.3354/ meps12214) 16. Miller P, Narazaki T, Isojunno S, Aoki K, Smout S, Sato K. 2016 Body density and diving gas volume of the northern bottlenose whale (Hyperoodon ampullatus). J. Exp. Biol. 219, 2458–2468. (doi:10.1242/jeb.137349) 17. Rossbach KA, Herzing DL. 1997 Underwater observations of benthic-feeding bottlenose dolphins (Tursiops truncatus) near Grand Bahama Island, Bahamas. Mar. Mammal Sci. 13, 498–504. (doi:10.1111/j.1748-7692.1997. tb00658.x) 22. Spitz J, Cherel Y, Bertin S, Kiszka J, Dewez A, Ridoux V. 2011 Prey preferences among the community of deep-diving odontocetes from the Bay of Biscay, Northeast Atlantic. Deep Sea Res. Part Oceanogr. Res. Pap. 58, 273–282. (doi:10.1016/j.dsr.2010.12.009) 27. Nemoto T, Nasu K. 1963 Stones and other aliens in the stomachs of sperm whales in the Bering Sea. Sci. Rep. Whales Res. Inst. 17, 83–91. 28. Walker WA, Mead JG, Brownell RL. 2002 Diets of Baird’s beaked whales, Berardius Bairdii, in the Southern Sea of Okhotsk and off the Pacific Coast of Honshu, Japan. Mar. Mammal Sci. 18, 902–919. (doi:10.1111/j.1748-7692.2002. tb01081.x) 18. Jones ML, Swartz SL, Leatherwood S. 2012 The gray whale: Eschrichtius robustus. Cambridge, MA: Academic Press. 23. Kenyon KW. 1961 Cuvier beaked whales stranded in the Aleutian Islands. J. Mammal. 42, 71–76. (doi:10.2307/1377244) 19. Oliver JS, Slattery PN. 1985 Destruction and opportunity on the sea floor: effects of gray 24. Whitehead H. 1996 Variation in the feeding success of sperm whales: temporal scale, spatial 24. Whitehead H. 1996 Variation in the feeding success of sperm whales: temporal scale, spatial
https://openalex.org/W2133092058	https://www.repository.cam.ac.uk/bitstream/1810/324027/1/The%20application%20of%20a%20monolithic%20triphenylphosphine%20reagent%20for%20conducting%20Ramirez%20gem-dibromoolefination%20reactions%20in%20flow.pdf	English	null	The application of a monolithic triphenylphosphine reagent for conducting Ramirez <i>gem</i>-dibromoolefination reactions in flow	Beilstein journal of organic chemistry	2,013	cc-by	5,834	Abstract The application of a monolithic form of triphenylphosphine to the Ramirez gem-dibromoolefination reaction using flow chemistry techniques is reported. A variety of gem-dibromides were synthesised in high purity and excellent yield following only removal of solvent and no further off-line purification. It is also possible to perform the Appel reaction using the same monolith and the rela- tionship between the mechanisms of the two reactions is discussed. The application of a monolithic form of triphenylphosphine to the Ramirez gem-dibromoolefination reaction using flow chemistry techniques is reported. A variety of gem-dibromides were synthesised in high purity and excellent yield following only removal of solvent and no further off-line purification. It is also possible to perform the Appel reaction using the same monolith and the rela- tionship between the mechanisms of the two reactions is discussed. * Corresponding author * Corresponding author Keywords: bromination; flow chemistry; Ramirez gem-dibromoolefination reaction; solid-supported reagent; triphenylphosphine monolith Full Research Paper Address: 1Innovative Technology Centre, Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, Cambridgeshire, CB2 1EW, U.K. and 2GlaxoSmithKline, Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, U.K. Email: Steven V. Ley* - svl1000@cam.ac.uk Introduction [8-13]. Reagents are typically supported on low-crosslinked gel- type or macroporous beads; however, these are characterised by poor mass transfer properties as well as presenting practical problems when used in packed beds in flow reactions due to changes in structure and morphology when subjected to solvents of varying polarity [14,15]. To avoid some of the prob- lems associated with using resin beads, monolithic supports have been developed for use in continuous-flow chemistry systems. Monoliths are a single continuous piece of uniformly porous material, prepared by precipitation polymerisation of a functionalised monomer [16-20]. The monolith internal struc- ture varies compared to bead-like supports, consisting of a The advantages of applying flow chemistry processing to organic synthesis have been extensively demonstrated in the literature, increasing the safety, efficiency and reproducibility of many organic chemistry reactions, causing this technology to be accepted as an important new tool to aid the modern research chemist [1-7]. Combining this enabling technology with solid- supported reagents and scavengers offers synergistic benefits over using the two technologies independently. Utilising polymer-supported reagents and scavengers to purify the flow stream permits telescoping of reaction sequences or facilitates direct isolation of pure compounds from flow reactors, removing the need for labour-intensive manual operations 1781 1781 Beilstein J. Org. Chem. 2013, 9, 1781–1790. recently stereospecific hydrogenolysis, Stille and Suzuki reac- tions have been used to further elaborate these useful products [43-45]. combination of large macropores for flow through passage, in combination with a network of smaller mesopores to allow diffusion towards the active sites. This combined geometry has been shown to result in superior chemical efficiency over tradi- tional supports by providing a shorter diffusion pathway to active sites via convective flow-through the macropores, as well as providing lower void volumes [16]. Practically, their rigid structure is secure over a wide range of solvents and under rea- sonable pressures compared to beads due to a high degree of cross linking, making them advantageous when applied to flow processes [21,22]. The triphenylphosphine oxide byproduct can often be difficult to remove from the reaction mixture, requiring extensive, time- consuming purification procedures to isolate the desired prod- uct in high purity. For Ramirez gem-dibromoolefination reac- tions, successful strategies have been developed to facilitate this separation through derivatising the triphenylphosphine (or its oxide) to achieve purification via filtration [46,47], as well as by immobilising the triphenylphosphine on a solid-support [48]. Introduction A polymer-supported equivalent of triphenylphosphine has also been successfully utilised by our group and by others in batch Wittig reactions [49,50], Mitsunobu and Staudinger aza-Wittig reactions [51,52], as well as many examples concerning the Appel reaction [51-57]. Originally monoliths were developed to facilitate the isocratic separation of peptides [17,23]; however, our group and others have shown interest in using monolithic supports to facilitate key chemical transformations [24-35]. The above advantages of using monolithic supports over traditional beads in flow chem- istry protocols can greatly facilitate the synthesis of fine chem- icals using these enabling technologies [36]. We have recently reported on the development of a monolithic triphenylphos- phine reagent and its application to the Staudinger aza-Wittig and Appel reactions in flow [37-40]. The immobilisation of tri- phenylphosphine in this manner allowed the facile production of a collection of pure compounds using flow chemistry tech- nologies with no need for further offline purification. Following the successful application of this monolith to the Appel reac- tion (the transformation of alkyl alcohols to the corresponding bromides), we wished to investigate the application of this monolith to the closely related Ramirez gem-dibromo- olefination reaction; the formation of gem-dibromoolefins from aldehydes or ketones. Following our success using a monolithic form of triphenyl- phosphine to facilitate the Appel reaction, we wanted to explore the use of this monolith for performing the Ramirez gem-di- bromoolefination reaction in flow. The monolithic form of tri- phenylphosphine should have improved flow characteristics compared to bead-based equivalents circumventing the prob- lems associated with using these solid-supported reagents in combination with flow techniques. Key intermediates for the Ramirez dibromoolefination reaction, 1 and 2 depicted in Scheme 1, are also known to be potential intermediates in the Appel reaction [58,59] and consequently we also wished to investigate the interplay between the two reaction mechanisms. Results and Discussion Formation of the triphenylphosphine monolith In 1962 Ramirez, Desai and McKelvie reported the formation of dibromophosphorane 1 and (dibromomethylene)triphenylphos- phorane (2) from the room temperature reaction of carbon tetrabromide with two equivalents of triphenylphosphine (Scheme 1) [41]. Addition of benzaldehyde then gave the desired gem-dibromoolefin, (2,2-dibromovinyl)benzene (3) in 84% yield. Triphenylphosphine oxide (4) was also isolated from the reaction as a byproduct. These gem-dibromoolefin products are particularly important intermediates in the one carbon homologation of an aldehyde into the corresponding terminal alkyne, known as the Corey–Fuchs reaction [42], and more Formation of the triphenylphosphine monolith The triphenylphosphine monoliths for the Ramirez reactions were formed using precipitation polymerisation of the phos- phine monomer 5 (Scheme 2). A polymerisation mixture of the triphenylphosphine monomer 5, cross-linking components divinylbenzene (6) and styrene (7) along with the porogen, 1-dodecanol (8), was heated to 50 °C until a homogeneous mix- ture was achieved. The initiator, dibenzoyl peroxide (9) was then added and the temperature maintained at 50 °C until this had completely dissolved. The mixture was then transferred to a glass column, the ends sealed with custom-made PTFE end Scheme 1: Formation of gem-dibromoolefin 3 from the reaction of carbon tetrabromide and triphenylphosphine as reported by Ramirez et al. [41]. 1: Formation of gem-dibromoolefin 3 from the reaction of carbon tetrabromide and triphenylphosphine as reported by Ramirez et al. [41]. 1782 Beilstein J. Org. Chem. 2013, 9, 1781–1790. Scheme 2: Formation of the triphenylphosphine monoliths. Scheme 2: Formation of the triphenylphosphine monoliths. Scheme 2: Formation of the triphenylphosphine monoliths. starting materials were then eluted from the monolith using a stream of dichloromethane at elevated temperature (60 °C). This polymerisation protocol consistently gave a low pressure drop across the monoliths for use in flow reactions. The mono- liths were calculated to have a phosphorus loading of 1.85 mmol of phosphorus per gram, resulting in approximately 4.63 mmol of phosphorus per monolith. pieces and heated to 92 °C for 48 hours using a Vapourtec R4 heating unit. This protocol can be clearly viewed in a video previously released by our group [40], however the Ramirez monoliths employ a higher ratio of styrene to divinylbenzene. This results in a lower proportion of crosslinking within the monolith, allowing greater flexibility in the backbone of the polymer whilst still maintaining desirable monolithic character- istics during flow reactions. Results and Discussion Formation of the triphenylphosphine monolith This greater flexibility has previ- ously been shown to assist with the formation of active species 1 and 2 in solid-supported triphenylphosphine beads, by allowing neighbouring group interactions between the tri- phenylphosphine residues. Loading the monolith to give the active Ramirez brominating species Interestingly the batch bromination of 3-phenylpropiolaldehyde (Table 1, entry 7) requires the addition of 2.5 equivalents of 2,6- lutidine [60], however pleasingly, this was not required when the substrate was brominated using the flow procedure. It was also possible to use the monolith on a series of heterocyclic sub- strates with high yields (Table 1, entries 8–10). However, nico- tinaldehyde (Table 1, entry 11) was found to give a reduced yield and unusually contamination of subsequent products formed using the same monolith was observed. X-ray crystal- lography and mass spectrometry confirmed that the product isolated was the hydrobromide salt of the desired gem-dibromo- olefin, presumably formed from an additional reaction with the monolith. The salt formed will coordinate to other ionic sites within the monolith, reducing the isolated yield and resulting in contamination of further products as it is slowly released from the column. Benzylic aldehydes containing electron withdrawing and donating groups on the phenyl ring (Table 1, entries 1–4) were transformed in high yield, as well as alkyl aldehydes (Table 1, entries 5 and 6). Unsurprisingly, aldehydes containing a phenol moiety were found to give little or no mass return as the pheno- lic hydroxy group reacted with the triphenylphosphine sites within the monolith, leaving the product bound to the polymer. Interestingly the batch bromination of 3-phenylpropiolaldehyde (Table 1, entry 7) requires the addition of 2.5 equivalents of 2,6- lutidine [60], however pleasingly, this was not required when the substrate was brominated using the flow procedure. It was also possible to use the monolith on a series of heterocyclic sub- strates with high yields (Table 1, entries 8–10). However, nico- tinaldehyde (Table 1, entry 11) was found to give a reduced yield and unusually contamination of subsequent products formed using the same monolith was observed. X-ray crystal- lography and mass spectrometry confirmed that the product isolated was the hydrobromide salt of the desired gem-dibromo- olefin, presumably formed from an additional reaction with the monolith. The salt formed will coordinate to other ionic sites within the monolith, reducing the isolated yield and resulting in contamination of further products as it is slowly released from the column. The formation of the active species was accompanied by a colour change, resulting in a bright yellow polymer (Figure 1, b). Each monolith was shown to have an active loading towards the Ramirez transformation of approximately 0.8 mmol. Loading the monolith to give the active Ramirez brominating species Loading the monolith with carbon tetrabromide to give the active species for the Ramirez gem-dibroomolefination reactions was found to proceed in a facile manner using a single pass protocol with the monolith being cooled to 0 °C (Scheme 3). Cooling the monolith by submerging it in an ice- water bath was found to be necessary to prevent the formation The resultant white polymer (see Figure 1, a) was cooled to room temperature and the end plugs exchanged with standard flow-through end pieces. The porogen and any unreacted Figure 1: a. An unfunctionalised triphenylphosphine monolith; b. Monolith after functionalisation with carbon tetrabromide at 0 °C; c. Monolith after complete consumption of the active Ramirez gem-dibromoolefination species; d. Monolith after complete consumption of the active Ramirez gem-di- bromoolefination species and the Appel brominating species. Figure 1: a. An unfunctionalised triphenylphosphine monolith; b. Monolith after functionalisation with carbon tetrabromide at 0 °C; c. Monolith after complete consumption of the active Ramirez gem-dibromoolefination species; d. Monolith after complete consumption of the active Ramirez gem-di- bromoolefination species and the Appel brominating species. 1783 Beilstein J. Org. Chem. 2013, 9, 1781–1790. Scheme 4: Flow synthesis of gem-dibromoolefins using the functional- ised triphenylphosphine monolith. of an inseparable side product, observed if reactions were performed at room temperature. Scheme 3: Functionalising the triphenylphosphine monolith to give the active Ramirez monolith using carbon tetrabromide. Scheme 3: Functionalising the triphenylphosphine monolith to give the active Ramirez monolith using carbon tetrabromide. Scheme 4: Flow synthesis of gem-dibromoolefins using the functional- ised triphenylphosphine monolith. Interestingly, an external source of triphenylphosphine was not required to form the solid-supported equivalents of active species 1 and 2, indicating that the polymer chains within the monolith have sufficient conformational freedom to allow neighbouring group interactions between triphenylphosphine sites. Any attempts to use a solution of triphenylphosphine to increase the active loading of the monolith was found to result in the formation of insoluble triphenylphosphine salts which crystallised and blocked the flow tubing downstream of the monolith. Benzylic aldehydes containing electron withdrawing and donating groups on the phenyl ring (Table 1, entries 1–4) were transformed in high yield, as well as alkyl aldehydes (Table 1, entries 5 and 6). Unsurprisingly, aldehydes containing a phenol moiety were found to give little or no mass return as the pheno- lic hydroxy group reacted with the triphenylphosphine sites within the monolith, leaving the product bound to the polymer. Loading the monolith to give the active Ramirez brominating species Although this is a relatively low active loading, this is not unex- pected as two equivalents of triphenylphosphine are required for the formation of one equivalent of the active Ramirez bromin- ating species. gem-dibromoolefination reactions in flow 2013, 9, 1781–1790. Table 2: α-Chiral aldehydes and ketones containing electron-withdrawing groups converted to the corresponding gem-dibromides using the triphenyl- phosphine monolith in flow. Table 2: α-Chiral aldehydes and ketones containing electron-withdrawing groups converted to the corresponding gem-dibromides using the triphenyl- phosphine monolith in flow. Entry Starting material Product Isolated yield (%)a 1 95 2 84 3 91 4 98 5 84b aReactions performed on a 0.2 mmol scale; breaction run at 0.10 mL/min with a previously unused monolith. Entry 84 aReactions performed on a 0.2 mmol scale; breaction run at 0.10 mL/min with a previously unused monolith. gem-dibromoolefination reactions in flow With the functionalised monolith in hand, it was then used to perform the Ramirez gem-dibromoolefination reaction in flow to transform aldehydes into their corresponding gem-dibromo- olefins. A 0.1 M solution of the aldehyde in dichloromethane was prepared and introduced into the flow system via the use of a sample loop. This solution was passed through the loaded monolith at a rate of 0.5 mL/min while the monolith was main- tained at 0 °C using a cooling bath (Scheme 4). The output was collected for 1 h 15 min and the solvent removed in vacuo to give complete conversion to the pure gem-dibromoolefin prod- uct without any further manipulation. A colour change was associated with the reaction, with the monolith changing from a bright yellow to dull dark yellow colour (Figure 1, b and c). A single monolith could be used for multiple transformations with no cross contamination between substrates run in sequential reactions through a single monolith (with the exception of the nicotinaldehyde substrate explained above). An important test of this methodology was the application to α-chiral aldehydes, to ensure that racemisation of the sensitive chiral centre is avoided in chiral structures (Table 2). A butane- 2,3-diacetal derived aldehyde (Table 2, entry 1) and a diastereo- This procedure was applied to a wide variety of aldehydes, giving the gem-dibromoolefin products in high yields and purity following only removal of the solvent by evaporation (Table 1). 1784 Beilstein J. Org. Chem. 2013, 9, 1781–1790. Table 1: Gem-dibromides prepared from the corresponding aldehydes using the triphenylphosphine monolith in flow. Table 1: Gem-dibromides prepared from the corresponding aldehydes using the triphenylphosphine monolith in flow. Entry Starting material Product Isolated yield (%)a 1 80 2 95 3 93 4 98 5 79 6 78b 7 83 8 97 9 91 10 87 11 41c aReactions performed on a 0.2 mmol scale; bproduct volatile, coutput collected for 2 hours rather than 1 h 15 min. Product Isolated yield ( 80 95 93 98 79 78b 83 97 91 87 41c ected for 2 hours rather than 1 h 15 min 98 79 5 78b 6 83 7 97 8 10 aReactions performed on a 0.2 mmol scale; bproduct volatile, coutput collected for 2 hours rather than 1 h 15 min. aReactions performed on a 0.2 mmol scale; bproduct volatile, coutput collected for 2 hours rather than 1 h 15 min. 1785 Beilstein J. Org. Chem. Utilising the loaded monolith for the Appel reaction in flow Entry Starting material Product Conversion after one pass (%)a Time required for full conversionb Isolated yield (%)c 1 100 – 82 2 100 – 95 3 13 14 h 30 min 90 aOne pass through the monolith at 0.5 mL/min, percentage conversion determined by 1H NMR analysis; bsubstrate recirculated through the monolith at 0.5 mL/min until full consumption of starting material indicated by TLC; creactions performed on a 0.2 mmol scale. Table 3: Alkyl bromides prepared from the corresponding alcohols using the triphenylphosphine monolith for the Appel reaction in flow. Entry Starting material Product 1 2 3 3 aOne pass through the monolith at 0.5 mL/min, percentage conversion determined by 1H NMR analysis; bsubstrate recirculated through the monolith at 0.5 mL/min until full consumption of starting material indicated by TLC; creactions performed on a 0.2 mmol scale. Scheme 5: Flow synthesis of bromides from the corresponding alco- hols using the functionalised triphenylphosphine monolith in the Appel reaction at 0 °C. anism through which the Appel reaction proceeds on solid- support. It is known that the Appel reaction can proceed either through intermediates 1 and 2 which are common to both the Ramirez and Appel reactions, or via the alternative pathway (Scheme 6) which only requires one equivalent of triphenyl- phosphine per molecule of carbon tetrabromide to give inter- mediate 13 (Scheme 6) [59]. It has been previously noted that intermediate 2, while not an active brominating agent in the Appel reaction, is known to assist in the formation of 10 by deprotonating the alcohol to form 11 [57]. However, it is thought that both possible pathways for the Appel reaction are utilised when using solid-supported triphenylphosphine due to the evidence for neighbouring-group interactions (the forma- tion of 1 and 2), along with site isolation effects ensuring the formation of 13. Scheme 5: Flow synthesis of bromides from the corresponding alco- hols using the functionalised triphenylphosphine monolith in the Appel reaction at 0 °C. The reactions reported below were therefore performed sequen- tially using a single monolith. Pleasingly, it was found that after exhausting the monolith of the gem-dibromoolefination active species through multiple Ramirez reactions, the monolith could then be used to successfully perform the Appel reaction in flow. Approximately 0.55 mmol of alcohol could be transformed into the corresponding alkyl bromide following approximately 0.80 mmol of successful gem-dibromoolefination reactions. Utilising the loaded monolith for the Appel reaction in flow meric aldehyde containing an acetonide (Table 2, entry 2) were successfully brominated using the flow protocol, being isolated in high yield with retention of stereochemistry as determined by 1H NMR. The method was then applied to an enantiopure alde- hyde (Table 2, entry 3) which could be transformed to the desired product in high yield [61]. The two active species formed during the Ramirez gem-di- bromoolefination reaction (1 and 2 in Scheme 1) are also known to be potential intermediates in the Appel reaction and we have previously shown that these monoliths can facilitate this forma- tion using similar conditions [39]. We wished to investigate the relationship between the two reactions and hoped to establish conditions to perform both reactions using a single protocol. Using a similar configuration to the Ramirez reactions in flow, a selection of alcohols were directed through the monolith loaded with carbon tetrabromide at 0 °C (Scheme 5). Gratify- ingly it was found that the monoliths prepared for the Ramirez gem-dibromoolefination reactions could be used directly for the Appel transformation, giving the bromide products in high yield and high purity following removal of the dichloromethane solvent (Table 3). Citronellol (Table 3, entry 1) and an indole derived alcohol (Table 3, entry 2), could be transformed in a facile manner using a single pass of the alcohol through the monolith at 0 °C, however the allyl alcohol (Table 3, entry 3) required recycling through the monolith to effect complete There is also precedent for performing Ramirez gem-dibromo- olefin reactions on carbonyl groups other than aldehydes, such as certain ketones activated using electron withdrawing groups [47]. A selection of these ketones were therefore subjected to the flow Ramirez reaction conditions (Table 2). Unsurprisingly, unactivated ketones such as cyclohexanone and benzophenone gave no conversion to the desired dibromide using the standard conditions. However, with some optimisation, an acyl cyanide (Table 2, entry 4) and a silyl protected ynone (Table 2, entry 5) could be converted to the desired gem-dibromoolefins respect- ively in high yields. Interestingly, it was found that full conver- sion could only be achieved for the silyl protected ynone using a low flow rate and a previously unused monolith, indicating some reduction in reactivity with each use of the monolith. 1786 Beilstein J. Org. Chem. 2013, 9, 1781–1790. Table 3: Alkyl bromides prepared from the corresponding alcohols using the triphenylphosphine monolith for the Appel reaction in flow. Conclusion We would like to thank the EPSRC and GlaxoSmithKline (Stevenage) for studentship support (KAR) and the BP 1702 Professorship (SVL) for financial support. We gratefully acknowledge Hokko Chemical Industry Co., Ltd. for their kind donation of diphenyl(4-vinylphenyl)phosphine. In summary, the monolithic form of triphenylphosphine recently described by our group [37-40] has been successfully applied to the Ramirez gem-dibromoolefination reaction in flow. The monolith was loaded with carbon tetrabromide at 0 °C using a single pass protocol to give the active brominating agent. This monolith was then utilised in the Ramirez reaction in flow, transforming a variety of different aldehydes to the corresponding gem-dibromoolefins in high yields and excellent purity following only removal of solvent. α-Chiral aldehydes were also successfully transformed, without racemisation of the stereocentre and two ketones bearing electron-withdrawing groups were converted into the desired dibromoolefins in high yield. It was further demonstrated that the same monoliths could be applied to the Appel reaction, giving a small selection of alkyl bromides in high yield and purity without further off-line 1. Webb, D.; Jamison, T. F. Chem. Sci. 2010, 1, 675–680. doi:10.1039/c0sc00381f 2. Wegner, J.; Ceylan, S.; Kirschning, A. Adv. Synth. Catal. 2012, 354, 17–57. doi:10.1002/adsc.201100584 3. Wenger, J.; Ceylan, S.; Kirschning, A. Chem. Commun. 2011, 47, 4583–4592. doi:10.1039/C0CC05060A 4. Hartman, R. L.; McMullen, J. P.; Jensen, K. F. Angew. Chem., Int. Ed. 2011, 50, 7502–7519. doi:10.1002/anie.201004637 Utilising the loaded monolith for the Appel reaction in flow When the Appel reaction was performed after the Ramirez reac- tion, the monolith once again changed colour from dull dark yellow to off-white (depicted in Figure 1, c and d). However, when the loaded monolith was first used for the Appel reaction, there was no conversion observed for a subsequent Ramirez gem-dibromoolefination, with only the starting aldehyde being recovered from the output. conversion. In batch, this reaction required low temperature conditions (−78 °C) and the presence of base to give an isol- ated yield of 78% [62], however this could be improved to 90% by performing this reaction in flow at 0 °C. Loading the mono- lith using the protocol described above was found to give an ap- proximate active loading of 0.6 mmol for the Appel reaction. Utilising one monolith for both reactions potentially broadens the synthetic utility of the supported reagent and so performing both reactions sequentially using a single monolith was investi- gated. It was anticipated that these studies into the interplay between the reactions might also assist to elucidate the mech- 1787 Beilstein J. Org. Chem. 2013, 9, 1781–1790. Scheme 6: Mechanisms for the Ramirez and Appel reactions [41,59]. Scheme 6: Mechanisms for the Ramirez and Appel reactions [41,59]. purification protocols. It was also shown that a single monolith could be used sequentially for Ramirez reactions and then the Appel reaction, but not in reverse order. This indicates that the Appel reaction consumes the Ramirez active brominating agent during the reaction. An alternative mechanistic pathway can ensue if the Appel reaction is performed subsequent to the Ramirez reaction. These results indicate that the Appel reaction consumes all of the active Ramirez species 2 (Scheme 6), preventing the progress of the Ramirez dibromoolefination. However, if this species is consumed through multiple Ramirez gem-dibromo- olefination reactions then an alternative brominating agent is utilised to perform the Appel reaction, or alternatively inter- mediate 2 is not required for the Appel mechanism using inter- mediate 1. This is supported by previous observations in the literature that indicate that the predominant pathway for the Appel reaction on solid-support is through intermediates 1 and 2 although overall both pathways are utilised [59]. The possi- bility of performing the Appel reaction following the use of the same monolith for the Ramirez gem-dibromoolefination reaction gives wider synthetic applications for this flow methodology. Supporting Information Supporting Information File 1 Experimental part. [http://www.beilstein-journals.org/bjoc/content/ supplementary/1860-5397-9-207-S1.pdf] References 1. Webb, D.; Jamison, T. F. Chem. Sci. 2010, 1, 675–680. doi:10.1039/c0sc00381f 2. Wegner, J.; Ceylan, S.; Kirschning, A. Adv. Synth. Catal. 2012, 354, 17–57. doi:10.1002/adsc.201100584 3. Wenger, J.; Ceylan, S.; Kirschning, A. Chem. Commun. 2011, 47, 4583–4592. doi:10.1039/C0CC05060A 4. Hartman, R. L.; McMullen, J. P.; Jensen, K. F. Angew. Chem., Int. Ed. 2011, 50, 7502–7519. doi:10.1002/anie.201004637 4. Hartman, R. L.; McMullen, J. P.; Jensen, K. F. Angew. Chem., Int. Ed. 2011, 50, 7502–7519. doi:10.1002/anie.201004637 1788 Beilstein J. Org. Chem. 2013, 9, 1781–1790. 5. Mason, B. P.; Price, K. E.; Steinbacher, J. L.; Bogdan, A. R.; McQuade, D. T. Chem. Rev. 2007, 107, 2300–2318. doi:10.1021/cr050944c 23.Švec, F.; Tennikova, T. B. Historical Review. In Monolithic Materials: Preparation, Properties and Applications; Švec, F.; Tennikova, T. B.; Deyl, Z., Eds.; Journal of Chromatography Library, Vol. 67; Elsevier Science B. V.: Amsterdam, The Netherlands, 2003; pp 1–15. Science B. V.: Amsterdam, The Netherlands, 2003; pp 1–15. 6. Ahmed-Omer, B.; Brandta, J. C.; Wirth, T. Org. Biomol. Chem. 2007, 5, 733–740. doi:10.1039/b615072a 24.Nikbin, N.; Ladlow, M.; Ley, S. V. Org. Process Res. Dev. 2007, 11, 458–462. doi:10.1021/op7000436 7. Baxendale, I. R.; Hayward, J. J.; Lanners, S.; Ley, S. V.; Smith, C. D. Heterogeneous Reactions. In Microreactors in Organic Synthesis and Catalysis; Wirth, T., Ed.; Wiley-VCH: Weinheim, Germany, 2008; pp 84–122. 25.Baumann, M.; Baxendale, I. R.; Ley, S. V.; Nikbin, N.; Smith, C. D. Org. Biomol. Chem. 2008, 6, 1587–1593. doi:10.1039/b801634h 26.Baumann, M.; Baxendale, I. R.; Martin, L. J.; Ley, S. V. Tetrahedron 2009, 65, 6611–6625. doi:10.1016/j.tet.2009.05.083 8. Myers, R. M.; Roper, K. A.; Baxendale, I. R.; Ley, S. V. The Evolution of Immobilized Reagents and their Application in Flow Chemistry for the Synthesis of Natural Products and Pharmaceutical Compounds. In Modern Tools for the Synthesis of Complex Bioactive Molecules; Cossy, J.; Arseniyadis, S., Eds.; John Wiley & Sons: Hoboken, NJ, U.S.A., 2012; pp 359–394. doi:10.1002/9781118342886.ch11 27.Lange, H.; Capener, M. J.; Jones, A. X.; Smith, C. J.; Nikbin, N.; Baxendale, I. R.; Ley, S. V. Synlett 2011, 869–873. doi:10.1055/s-0030-1259923 odern Tools for the Synthesis of Complex Bioactive Molecules; 28.Tripp, J. A.; Stein, J. A.; Svec, F.; Fréchet, J. M. J. Org. Lett. 2000, 2, 195–198. doi:10.1021/ol9912837 9. García-Verdugo, E.; Luis, S. V. Flow Processes Using Polymer-supported Reagents, Scavengers and Catalysts. In Chemical Reactions and Process under Flow Conditions; Luis, S. V.; Garcia-Verdugo, E., Eds.; Royal Society of Chemistry: Cambridge, U.K., 2009; pp 44–85. References doi:10.1039/9781847559739-00044 29.Kirschning, A.; Altwicker, C.; Dräger, G.; Harders, J.; Hoffmann, N.; Hoffmann, U.; Schönfeld, H.; Solodenko, W.; Kunz, U. Angew. Chem., Int. Ed. 2001, 40, 3995–3998. doi:10.1002/1521-3773(20011105)40:21<3995::AID-ANIE3995>3.0.CO ;2-V U.K., 2009; pp 44–85. doi:10.1039/9781847559739-00044 10.Baxendale, I. R.; Ley, S. V. Solid Supported Reagents in Multi-Step Flow Synthesis. In New Avenues to Efficient Chemical Synthesis: Emerging Technologies; Seeberger, P. H.; Blume, T., Eds.; Ernst Schering Foundation Symposium Proceedings, Vol. 2006/3; Springer-Verlag: Berlin, Germany, 2007; pp 151–185. doi:10.1007/2789_2007_033 30.Kunz, U.; Schönfeld, H.; Kirschning, A.; Solodenko, W. 30.Kunz, U.; Schönfeld, H.; Kirschning, A.; Solodenko, W. J. Chromatogr., A 2003, 1006, 241–249. doi:10.1016/S0021-9673(03)00556-9 merging Technologies; Seeberger, P. H.; Blume, T., Eds.; Ernst doi:10.1016/S0021-9673(03)00556-9 Schering Foundation Symposium Proceedings, Vol. 2006/3; 31.Solodenko, W.; Wen, H.; Leue, S.; Stuhlmann, F.; 31.Solodenko, W.; Wen, H.; Leue, S.; Stuhlmann, F.; Sourkouni-Argirusi, G.; Jas, G.; Schönfeld, H.; Kunz, U.; Kirschning, A. Eur. J. Org. Chem. 2004, 3601–3610. doi:10.1002/ejoc.200400194 Sourkouni-Argirusi, G.; Jas, G.; Schönfeld, H.; Kunz, U.; Kirschning, Springer-Verlag: Berlin, Germany, 2007; pp 151–185. doi:10.1007/2789_2007_033 11.Hodge, P. Ind. Eng. Chem. Res. 2005, 44, 8542–8553. 11.Hodge, P. Ind. Eng. Chem. Res. 2005, 44, 8542–8553. doi:10.1021/ie040285e 32.Mennecke, K.; Kirschning, A. Beilstein J. Org. Chem. 2009, 5, No. 21. doi:10.3762/bjoc.5.21 33.Burguete, M. I.; García-Verdugo, E.; Vicent, M. J.; Luis, S. V.; 12.Kirschning, A.; Solodenko, W.; Mennecke, K. Chem.–Eur. J. 2006, 12, 5972–5990. doi:10.1002/chem.200600236 33.Burguete, M. I.; García-Verdugo, E.; Vicent, M. J.; Luis, S. V.; Pennemann, H.; Graf von Keyserling, N.; Martens, J. Org. Lett. 2002, 4, 3947–3950. doi:10.1021/ol026805o Pennemann, H.; Graf von Keyserling, N.; Martens, J. Org. Lett. 2002 13.Baxendale, I. R.; Deeley, J.; Griffiths-Jones, C. M.; Ley, S. V.; 13.Baxendale, I. R.; Deeley, J.; Griffiths-Jones, C. M.; Ley, S. V.; Saaby, S.; Tranmer, G. K. Chem. Commun. 2006, 2566–2568. doi:10.1039/b600382f 4, 3947–3950. doi:10.1021/ol026805o 34.Altava, B.; Burguete, M. I.; García-Verdugo, E.; Luis, S. V.; Vicent, M. J. Green Chem. 2006, 8, 717–726. doi:10.1039/b603494b 4.Altava, B.; Burguete, M. I.; García-Verdugo, E.; Luis, S. V.; Vicent, M. J. Green Chem. 2006, 8, 717–726. doi:10.1039/b603494b 14.Sherrington, D. C. Chem. Commun. 1998, 2275–2286. doi:10.1039/a803757d 35.Bou-Hamdan, F. R.; Krüger, K.; Tauer, K.; McQuade, D. T.; Seeberger, P. H. Aust. J. Chem. 2013, 66, 213–217. doi:10.1071/CH12405 Seeberger, P. H. Aust. J. Chem. 2013, 66, 213–217. 40.Beilstein TV: "Conducting Appel reactions in flow using a triphenylphosphine monolith". http://www.beilstein.tv/tvpost/conducting-appel-reactions-in-flow-using- 40.Beilstein TV: "Conducting Appel reactions in flow using a a-triphenylphosphine-monolith/ (accessed June 30, 2013). 19.Buchmeiser, M. R. Polymer 2007, 48, 2187–2198. doi:10.1016/j.polymer.2007.02.045 19.Buchmeiser, M. R. Polymer 2007, 48 doi:10.1016/j.polymer.2007.02.045 19.Buchmeiser, M. R. Polymer 2007, 48, 2187–2198. 41.Desai, N. B.; McKelvie, N.; Ramirez, F. J. Am. Chem. Soc. 1962, 84, 1745–1747. doi:10.1021/ja00868a057 doi:10.1016/j.polymer.2007.02.045 42.Corey, E. J.; Fuchs, P. L. Tetrahedron Lett. 1972, 13, 3769–3772. doi:10.1016/S0040-4039(01)94157-7 20.Švec, F.; Huber, C. G. Anal. Chem. 2006, 78, 2100–2107. doi:10.1021/ac069383v doi:10.1071/CH12405 15.Svec, F.; Fréchet, J. M. J. Science 1996, 273, 205–211. doi:10.1126/science.273.5272.205 15.Svec, F.; Fréchet, J. M. J. Science 1996, 273, 205–211. 36.Sachse, A.; Galarneau, A.; Coq, B.; Fajula, F. New J. Chem. 2011, 35, 259–264. doi:10.1039/c0nj00965b doi:10.1126/science.273.5272.205 16.Peters, E. C.; Svec, F.; Fréchet, J. M. J. Adv. Mater. 1999, 11, 1169–1181. doi:10.1002/(SICI)1521-4095(199910)11:14<1169::AID-ADMA1169>3. 0.CO;2-6 37.Smith, C. J.; Smith, C. D.; Nikbin, N.; Ley, S. V.; Baxendale, I. R. Org. Biomol. Chem. 2011, 9, 1927–1937. doi:10.1039/c0ob00813c 38.Smith, C. J.; Nikbin, N.; Ley, S. V.; Lange, H.; Baxendale, I. R. Org. Biomol. Chem. 2011, 9, 1938–1947. doi:10.1039/c0ob00815j 17.Svec, F.; Fréchet, J. M. J. Anal. Chem. 1992, 64, 820–822. doi:10.1021/ac00031a022 39.Roper, K. A.; Lange, H.; Polyzos, A.; Berry, M. B.; Baxendale, I. R.; Ley, S. V. Beilstein J. Org. Chem. 2011, 7, 1648–1655. doi:10.3762/bjoc.7.194 18.Švec, F.; Fréchet, J. M. J. Rigid Macroporous Organic Polymer Monoliths Prepared by Free Radical Polymerization. In Monolithic Materials: Preparation, Properties and Applications; Švec, F.; Tennikova, T. B.; Deyl, Z., Eds.; Journal of Chromatography Library, Vol. 67; Elsevier Science B. V.: Amsterdam, The Netherlands, 2003; pp 19–50. 40.Beilstein TV: "Conducting Appel reactions in flow using a triphenylphosphine monolith". http://www.beilstein.tv/tvpost/conducting-appel-reactions-in-flow-using- doi:10.1002/anie.197508011 59.Tömösközi, I.; Gruber, L.; Radics, L. Tetrahedron Lett. 1975, 29, 59.Tömösközi, I.; Gruber, L.; Radics, L. Tetrahedron Lett. 1975, 29, 2473–2476. doi:10.1016/0040-4039(75)80041-4 2473–2476. doi:10.1016/0040-4039(75)80041-4 60.Métay, E.; Hu, Q.; Negishi, E.-i. Org. Lett. 2006, 8, 5773–5776. doi:10.1021/ol0623825 61.Sneddon, H. F.; Gaunt, M. J.; Ley, S. V. Org. Lett. 2003, 5, 1147–1150. doi:10.1021/ol034248f 62.Harding, S. L. Azadirachtin: towards a second generation synthesis. Ph.D. Thesis, University of Cambridge, U.K., 2011. Ph.D. Thesis, University of Cambridge, U.K., 2011. Beilstein J. Org. Chem. 2013, 9, 1781–1790. doi:10.1021/jo00151a010 51.Choi, M. K. W.; He, H. S.; Toy, P. H. J. Org. Chem. 2003, 68, 51.Choi, M. K. W.; He, H. S.; Toy, P. H. J. Org. Chem. 2003, 68, 9831–9834. doi:10.1021/jo035226+ 9831–9834. doi:10.1021/jo035226+ 52.Årstad, E.; Barrett, A. G. M.; Hopkins, B. T.; Köbberling, J. Org. Lett. 52.Årstad, E.; Barrett, A. G. M.; Hopkins, B. T.; K 2002, 1975–1977. doi:10.1021/ol026008q 53.Anilkumar, G.; Nambu, H.; Kita, Y. Org. Process Res. Dev. 2002, 6, 53.Anilkumar, G.; Nambu, H.; Kita, Y. Org. Process Res. Dev. 2002, 6, 190–191. doi:10.1021/op010094c 190–191. doi:10.1021/op010094c 54.Cainelli, G.; Contento, M.; Manescalchi, F.; Plessi, L. Synthesis 1983, 306–308. doi:10.1055/s-1983-30314 54.Cainelli, G.; Contento, M.; Manescalchi, F.; Plessi, L. Synthesis 19 55.Sherrington, D. C.; Craig, D. J.; Dagleish, J.; Domin, G.; Taylor, J.; Meehan, G. V. Eur. Polym. J. 1977, 13, 73–76. Meehan, G. V. Eur. Polym. J. 1977, 13, 73–76. doi:10.1016/0014-3057(77)90140-9 doi:10.1016/0014-3057(77)90140-9 56.Harrison, C. R.; Hodge, P. J. Chem. Soc., Chem. Commun. 1978, 813 815 doi:10 1039/C39780000813 56.Harrison, C. R.; Hodge, P. J. Chem. Soc 813–815. doi:10.1039/C39780000813 56.Harrison, C. R.; Hodge, P. J. Chem. Soc., Chem. Commu 813–815. doi:10.1039/C39780000813 57.Hodge, P.; Khoshdel, E. J. Chem. Soc., Perkin Trans. 1 1984, 195–198. doi:10.1039/P19840000195 57.Hodge, P.; Khoshdel, E. J. Chem. Soc., Perkin Trans. 1 198 195–198. doi:10.1039/P19840000195 58.Appel, R. Angew. Chem., Int. Ed. Engl. 1975, 12, 801–811. doi:10 1002/anie 197508011 58.Appel, R. Angew. Chem., Int. Ed. Engl. 1975, 12, 801–811. doi:10.1002/anie.197508011 58.Appel, R. Angew. Chem., Int. Ed. Engl. 1975, 12, 801–811. doi:10.1002/anie.197508011 doi:10.1016/S0040-4039(01)94157-7 21.Kunz, U.; Kirschning, A.; Wen, H.-L.; Solodenko, W.; Cecilia, R.; Kappe, C. O.; Turek, T. Catal. Today 2005, 105, 318–324. doi:10.1016/j.cattod.2005.06.046 43.Uenishi, J.; Kawahama, R.; Yonemitsu, O.; Tsuji, J. J. Org. Chem. 1996, 61, 5716–5717. doi:10.1021/jo961013r 43.Uenishi, J.; Kawahama, R.; Yonemitsu, O.; Tsuji, J. J. Org. Chem. Kappe, C. O.; Turek, T. Catal. Today 2005, 105, 318–324. 1996, 61, 5716–5717. doi:10.1021/jo961013r 44.Shen, W.; Wang, L. J. Org. Chem. 1999, 64, 8873–8879. doi:10.1021/jo991116k doi:10.1016/j.cattod.2005.06.046 22.Jas, G.; Kirschning, A. Chem.–Eur. J. 2003, 9, 5708–5723. doi:10.1002/chem.200305212 45.Shen, W. Synlett 2000, 737–739. doi:10.1055/s-2000-6626 46.Poupon, J.-C.; Boezio, A. A.; Charette, A. B. Angew. Chem., Int. Ed. 2006, 45, 1415–1420. doi:10.1002/anie.200503599 1789 Beilstein J. Org. Chem. 2013, 9, 1781–1790. 47.Fang, Y.-Q.; Lifchits, O.; Lautens, M. Synlett 2008, 413–417. doi:10.1055/s-2008-1032045 48.Hodge, P.; Khoshdel, E. React. Polym. 1985, 3, 143–150. 49.Bolli, M. H.; Ley, S. V. J. Chem. Soc., Perkin Trans. 1 1998, 49.Bolli, M. H.; Ley, S. V. J. Chem. Soc., 2243–2246. doi:10.1039/A803612H 49.Bolli, M. H.; Ley, S. V. J. Chem. Soc., Perkin Trans. 1 2243–2246. doi:10.1039/A803612H 49.Bolli, M. H.; Ley, S. V. J. Chem. Soc., Perkin Trans. 1 1998, 2243–2246. doi:10.1039/A803612H 2243–2246. doi:10.1039/A803612H 50.Bernard, M.; Ford, W. T. J. Org. Chem. 1983, 48, 326–332. 50.Bernard, M.; Ford, W. T. J. Org. Chem. 1983, 48, 326–332. doi:10.1021/jo00151a010 License and Terms License and Terms This is an Open Access article 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. 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https://openalex.org/W4297467622	https://afst.centre-mersenne.org/item/10.5802/afst.1583.pdf	English	null	Tangents to subsolutions -- existence and uniqueness, Part I	arXiv (Cornell University)	2,014	cc-by	33,712	ANNALES DE LA FACULTÉ DES SCIENCES Mathématiques F. REESE HARVEY AND H. BLAINE LAWSON Tangents to subsolutions: existence and uniqueness, Part I Tome XXVII, no 4 (2018), p. 777-848. <http://afst.cedram.org/item?id=AFST_2018_6_27_4_777_0> © Université Paul Sabatier, Toulouse, 2018, tous droits réservés. L’accès aux articles de la revue « Annales de la faculté des sci- ences de Toulouse Mathématiques » (http://afst.cedram.org/), implique l’accord avec les conditions générales d’utilisation (http://afst.cedram. org/legal/). Toute reproduction en tout ou partie de cet article sous quelque forme que ce soit pour tout usage autre que l’utilisation à ﬁn strictement personnelle du copiste est constitutive d’une infraction pénale. Toute copie ou impression de ce ﬁchier doit contenir la présente mention de copyright. <http://afst.cedram.org/item?id=AFST_2018_6_27_4_777_0> © Université Paul Sabatier, Toulouse, 2018, tous droits réservés. © Université Paul Sabatier, Toulouse, 2018, tous droits réservés. L’accès aux articles de la revue « Annales de la faculté des sci- ences de Toulouse Mathématiques » (http://afst.cedram.org/), implique l’accord avec les conditions générales d’utilisation (http://afst.cedram. org/legal/). Toute reproduction en tout ou partie de cet article sous quelque forme que ce soit pour tout usage autre que l’utilisation à ﬁn strictement personnelle du copiste est constitutive d’une infraction pénale. Toute copie ou impression de ce ﬁchier doit contenir la présente mention de copyright. cedram Article mis en ligne dans le cadre du Centre de diffusion des revues académiques de mathématiques http://www.cedram.org/ (*) Reçu le 20 juin 2016, accepté le 29 octobre 2016. (1) Department of Mathematics, RICE University, Houston, TX 77005-1982, USA — harvey@rice.edu (2) Department of Mathematics, Stony Brook University, Stony Brook, NY 11794-3651, USA — blaine@math.sunysb.edu Partially supported by the N.S.F.. Article proposé par Vincent Guedj. cedram Article mis en ligne dans le cadre du Centre de diffusion des revues académiques de mathématiques http://www.cedram.org/ Volume XXVII, no 4, 2018 pp. 777-848 Volume XXVII, no 4, 2018 pp. 777-848 Annales de la faculté des sciences de Toulouse F. Reese Harvey (1) and H. Blaine Lawson (2) F. Reese Harvey (1) and H. Blaine Lawson (2) ABSTRACT. — There is an interesting potential theory associated to each degen- erate elliptic, fully nonlinear equation f(D2u) = 0. These include all the potential theories attached to calibrated geometries. This paper begins the study of tangents to the subsolutions in these theories, a topic inspired by the results of Kiselman in the classical plurisubharmonic case. Fundamental to this study is a new invariant of the equation, called the Riesz characteristic, which governs asymptotic structures. The existence of tangents to subsolutions is established in general, as is the exis- tence of an upper semi-continuous density function. Two theorems establishing the strong uniqueness of tangents (which means tangents are always unique and are Riesz kernels) are proved. They cover all O(n)-invariant convex cone equations and their complex and quaternionic analogues, with the exception of the homogeneous Monge–Ampère equations, where uniqueness fails. They also cover a large class of geometrically deﬁned subequations which includes those coming from calibrations. A discreteness result for the sets where the density is ⩾c > 0 is also established in any case where strong uniqueness holds. A further result (which is sharp) asserts the Hölder continuity of subsolutions when the Riesz characteristic p satisﬁes 1 ⩽p < 2. Many explicit examples are examined. The second part of this paper [23] is devoted to the “geometric cases”. A Homo- geneity Theorem and an additional Strong Uniqueness Theorem are proved, and the tangents in the Monge–Ampère cases are completely classiﬁed. RÉSUMÉ. — Il existe une théorie du potentiel intéressante associée à chaque équa- tion, nonlinéaire et élliptique dégénérée, de la forme f(D2u) = 0. Ceci inclut toutes les théories du potentiel associées aux calibrations. Cet article commence l’étude des tangents aux sous-solutions dans ces théories, un sujet inspiré par l’oeuvre de Kiselman dans le cas pluri-potentiel classique. Fondamentale à notre étude est une nouvelle invariante, la caractéristique de Riesz, qui gouverne les structures asympto- tiques. L’existence de tangents aux sous-solutions est établie en général; on démontre aussi l’existence générale d’une fonction de densité, semi-continue supérieurement. – 777 – F. Reese Harvey and H. Blaine Lawson Deux théorèmes qui établissent l’unicité forte de tangents (i.e., tangents sont toujours unique et sont noyaux de Riesz) sont démontrés. Ils comprennent toutes les sous- équations qui sont des cones convexes et O(n)-invariants, ainsi que leurs analogues complexes et quatérnioniques, avec l’exception de l’équation de Monge–Ampère, pour laquelle l’unicité forte ne tient pas. Ils s’appliquent aussi à une grande classe de sous- équations déﬁnies géométriquement. Parmi elles sont toutes celles qui proviennent de calibrations. Un résultat de ﬁnitude locale, pour les ensembles de densité ⩾c > 0, est établi dans chaque cas où régit l’unicité forte. Selon un autre résultat, quand la carac- téristique de Riesz p satisfait 1 ⩽p < 2, alors toutes les functions sous-harmoniques sont Hölder-continues. On considère beaucoup d’exemples explicites. La deuxième partie de cet article [23] concerne les “cas géométriques”. On y établit un Théorème d’Homogénéité et un Théorème d’Unicité Forte. Aussi, les es- paces tangents pour les équations de Monge–Ampère (réelles, complexes et quater- nioniques) sont classiﬁés complètement. 1. Introduction The point of this paper is to introduce and study tangents for a wide class of degenerate elliptic, fully nonlinear equations of the form F(D2u) = 0 in Rn. It was inspired by Kieselman’s study [29] (cf. [30]) of tangents to plurisubharmonic functions in classical pluripotential theory. The aim is to develop techniques for studying the behavior, in particular the singular be- havior, of subsolutions: the upper semi-continuous functions u which satisfy F(D2u) ⩾0 in the viscosity sense. A number of quite general results are ob- tained. These include existence, uniqueness and “harmonicity” of tangents for a wide range of equations. Densities for subsolutions are deﬁned and shown to be upper semi-continuous, and a structure theorem is proved for the sets where the density is ⩾c > 0. A key to the analysis is the notion of the Riesz characteristic of the equation. This invariant is a real number p ⩾1 which governs the asymptotic behavior of singularities, and is easily computed in all of the examples, no matter how degenerate (see Sections 3 and 4). For this study we focus on the closed set F = {A ∈Sym2(Rn) : F(A) ⩾0} (cf. [14, 32]), and the operator F will play no role. This set is always assumed to have the following three properties: (1) (Positivity) F + P ⊂F where P ≡{A ⩾0}. (2) (ST-Invariance) F is invariant under a subgroup G ⊂O(n) which acts transitively on the sphere Sn−1 ⊂Rn. (3) (Cone Property) tF ⊂F for all t ⩾0. (1) (Positivity) (2) (ST-Invariance) (3) (Cone Property) tF ⊂F for all t ⩾0. A closed set F satisfying Positivity is called a subequation, and the viscosity F-subsolutions are called F-subharmonic functions. Each subequation F has – 778 – Tangents to subsolutions: existence and uniqueness, Part I its own potential theory ([14, 17]). For some of the results here, in addition to these three conditions, F is also assumed to be convex. In this case distribu- tion theory provides an alternate but equivalent foundation (Theorem 9.5) for subsolutions, which is helpful. The equations covered here include many classical examples coming from real, complex and calibrated geometry, such as the Monge–Ampère and Hes- sian equations. The reader is encouraged to glance at Section 4 for some basic examples. 1. Introduction At the time of the ﬁrst writing of this paper the authors were unaware of its connections to the important work of Armstrong, Sirakov and Smart [1]. They also studied conical subequations F ⊂Sym2(Rn) with the additional assumption that F is uniformly elliptic. This is a stringent assumption which eliminates many of the examples arising from geometry. They also studied only solutions (as opposed to the much more general subsolutions consid- ered here). On the other hand they do not assume invariance or convexity, which is extremely nice. There are also connections of our work to that of Labutin [33] who, like Armstrong, Sirakov and Smart, studied uniformly el- liptic equations. At the end of this introduction the overlap / lack of overlap is discussed in more detail. We begin the paper by introducing the algebraically deﬁned and easily computable Riesz characteristic pF for F, which determines much of the behavior of subsolutions examined here. The name comes from the fact that when p ≡pF is ﬁnite, the classical pth Riesz kernel Kp(\|x\|), where Kp(t) =      t2−p if 1 ⩽p < 2 log t if p = 2 − 1 tp−2 if 2 < p < ∞, (1.1) (1.1) is a solution of the non-linear equation F. In fact, every increasing radial solution is of the form ΘKp(\|x\|) + C for constants Θ ⩾0 and C ∈R. The signs in (1.1) have been chosen so that Kp(t) is always increasing. When p is ﬁnite, there is an associated tangential p-ﬂow on F-subharmonic functions u at each point x0, given for x0 = 0 by ur(x) = ( rp−2u(rx) if p ̸= 2 u(rx) −M(u, r) if p = 2, (1.2) (1.2) where where M(u, r) ≡sup \|x\|⩽r u. (1.3) (1.3) The tangents to u at 0 ∈Rn are deﬁned to be the set T0(u) cluster points of the ﬂow (1.2). When F is convex, these cluster points are taken – 779 – F. Reese Harvey and H. Blaine Lawson in L1 loc(Rn). When 1 ⩽pF < 2 (but F not necessarily convex), they can be taken in the local β-Hölder norm for β < 2 −p. In either case, U ∈ T0(u) if and only if there exists a sequence rj ↓0 such that urj →U (in the appropriate space). It is a basic result that tangents are always entire F-subharmonic functions on Rn. 1. Introduction In particular, the L1 loc-limits have unique upper semi-continuous representatives which are viscosity F-subsolutions (see Theorem 9.5(2)). A fundamental result, which is proved in Sections 11 and 15, is the following. Theorem 1.1 (Existence of Tangents). — If F is convex or if pF < 2, then tangents always exist. A natural question is whether tangents are actually solutions (as opposed to subsolutions). The answer is no (if pF ⩾2). Classical pluripotential theory provides (self) tangent examples with large singular sets. It also provides the remedy: an appropriate concept enlarging the space of (viscosity) solutions. An F-subharmonic function on Xopen ⊂Rn is called F-maximal if for each F-subharmonic function v on X and each compact subset K ⊂X, v ⩽u on X −K ⇒ v ⩽u on X. If u is F-maximal on X, then on any subdomain Y ⊂X where u is continu- ous, it is a viscosity solution (or “F-harmonic”). In particular, it is always the Perron function for its boundary values on any ball. A second fundamental result is the following (see Theorem 10.2 and Corollary 10.3). Theorem 1.2 (Maximality of Tangents). — If F is convex, then tan- gents are always maximal outside the origin in Rn. If pF < 2, then tangents are F-harmonic (maximal and continuous) outside the origin. Existence and regularity (in the weakened form of maximality) for tan- gents brings us to the natural question of uniqueness. Here there are several versions. We say that uniqueness of tangents holds for the subequation F if for every F-subharmonic function u deﬁned in a neighborhood of 0, there is exactly one tangent to u at 0. We say that strong uniqueness of tangents holds for F if for every such u, the unique tangent is Θ(u)Kp(\|x\|), with Θ(u) ⩾0. We say that strong uniqueness of tangents holds for F if for every such u, the unique tangent is Θ(u)Kp(\|x\|), with Θ(u) ⩾0. We say that homogeneity of tangents holds for F if every tangent to an F-subharmonic is ﬁxed by the tangential p-ﬂow (1.2). Since the ﬂow takes a tangent to u to another tangent to u, uniqueness of tangents implies homogeneity of tangents. – 780 – Tangents to subsolutions: existence and uniqueness, Part I Several important special cases where uniqueness holds are discussed in Section 12 (Propositions 12.2, 12.4 and 12.5). 1. Introduction One of the main results of this paper is the Strong Uniqueness Theo- rem in Section 13. Note that there is a natural action of the group O(n) on Sym2(Rn). The subequations F ⊂Sym2(Rn) which are O(n)-invariant are exactly those which are deﬁned in terms of the eigenvalues of the matrices A ∈Sym2(Rn). Every such subequation has a complex and quaternionic counterpart deﬁned on Cn and Hn by applying the same eigenvalue con- straints to the complex or quaternionic hermitian symmetric part of A. Theorem 1.3.I (Strong Uniqueness of Tangents I). — Suppose F is a convex O(n)-invariant subequation, or the complex or quaternionic counter- part of such an equation. Then, except for the three basic cases P, PC, PH, strong uniqueness of tangents holds for F. There do exist non-convex O(n)-invariant subequations of every Riesz characteristic for which strong uniqueness fails. See Example 13.15. There do exist non-convex O(n)-invariant subequations of every Riesz characteristic for which strong uniqueness fails. See Example 13.15. Theorem 1.3.I establishes strong uniqueness for a wide range of equations. These include the kth Hessian equations (k < n) and p-convexity equations (p real, 1 ⩽p ⩽n), the trace powers of the Hessian, equations coming from Gårding polynomials, and much more. Each of these has a complex and a quaternionic counterpart to which Theorem 1.3.I applies. However, there are many U(n)- and Sp(n)·Sp(1)-invariant subequations, arising from calibrations and Lagrangian geometry, which have no O(n)-invariant coun- terpart, so that Theorem 1.3.I does not apply. Results in these cases are provided by Theorems 1.3.II and 1.3.III below, which require a completely diﬀerent method of proof. Suppose F = F(G) is a subequation deﬁned by a compact subset G ⊂ G(p, Rn) of the Grassmannian of p-planes in Rn (see Example 4.4). Suppose F = F(G) is a subequation deﬁned by a compact subset G ⊂ G(p, Rn) of the Grassmannian of p-planes in Rn (see Example 4.4). Theorem 1.3.II (Strong Uniqueness II). — Fix p ⩾2 and n ⩾3. Then strong uniqueness of tangents to F(G)-subharmonic functions holds for: Theorem 1.3.II (Strong Uniqueness II). — Fix p ⩾2 and n ⩾3. 1. Introduction Then strong uniqueness of tangents to F(G)-subharmonic functions holds for: (1) Every compact SU(n)-invariant subset G ⊂GR(p, Cn) with the one exception G = GC(1, Cn), (1) Every compact SU(n)-invariant subset G ⊂GR(p, Cn) with the one exception G = GC(1, Cn), (2) Every compact Sp(n) · Sp(1)-invariant subset G ⊂GR(p, Hn) with three exceptions, namely the sets of real p-planes which lie in a quaternion line for p = 2, 3, 4 (when p = 4 this is GH(1, Hn)), (3) For p ⩾5, every compact Sp(n)-invariant subset G ⊂GR(p, Hn). This result is based on a companion theorem which has further appli- cations. Given G ⊂G(p, Rn) as above, we say that G has the transitiv- ity property if for any two vectors x, y ∈Rn there exist W1, . . . , Wk ∈G – 781 – F. Reese Harvey and H. Blaine Lawson with x ∈W1, y ∈Wk and dim(Wi ∩Wi+1) > 0 for all i = 1, . . . , k −1. The subequations attached to Lagrangian, Special Lagrangian, Associative, Coassociative, and Cayley geometries all have this property. with x ∈W1, y ∈Wk and dim(Wi ∩Wi+1) > 0 for all i = 1, . . . , k −1. The subequations attached to Lagrangian, Special Lagrangian, Associative, Coassociative, and Cayley geometries all have this property. with x ∈W1, y ∈Wk and dim(Wi ∩Wi+1) > 0 for all i = 1, . . . , k −1. The subequations attached to Lagrangian, Special Lagrangian, Associative, Coassociative, and Cayley geometries all have this property. Theorem 1.3.III (Strong Uniqueness III). — If G has the transitivity property, then strong uniqueness of tangents holds for all F(G)-subharmonic functions. Theorems 1.3.II and 1.3.III will be proved in Part II ([23]) of this paper. There homogeneity of tangents is proved ﬁrst, and then strong uniqueness is established. This method makes no use of uniform ellipticity, and has its roots in pluripotential theory, not viscosity theory. It is important to note that uniqueness of tangents does not always hold. In the basic case of convex functions (F = P) we have uniqueness, but strong uniqueness fails. For classical plurisubharmonic functions (the complex coun- terpart: F = PC), the uniqueness question was raised in [11] and answered in the negative by Kiselman [29], who actually characterized the sets which can arise as T0(u) for a plurisubharmonic function u in Cn. 1. Introduction In Part II of this paper a similar result is obtained for the quaternionic counterpart PH. The proof of Theorem 1.3.I involves several steps. The ﬁrst step is of a classical nature going back to standard potential theory for the Laplacian and used by Labutin and Armstrong–Sirakov–Smart in viscosity theory. In our formulation it involves various characterizations of radial F-harmonics. For example, a result (Theorems 2.4 and 2.7), straightforward in the smooth case, but which ﬁlls a gap in the literature, characterizes the radial viscosity subsolutions u(x) = ψ(\|x\|) as the subsolutions of the one-variable subequa- tion RF : ψ′′(r) + pF −1 r ψ′(r) ⩾0 (1.4) (1.4) This classical subequaton is reviewed in detail in Section 5. Several important facts are derived. For example, all subsolutions of (1.4) are continuous, which has the important consequence that if a radial function is F-maximal, then it is F-harmonic (a solution), and hence of the form ΘKp(\|x\|) + c. Another consequence of (1.4) is that quotients ψ(r)−ψ(t) K(r)−K(t) are jointly (or “doubly”) monotone. This can be applied to a general non-radial F-subsolution u by associating to u several radial functions which are also F-subharmonic (Lem- mas 6.1 and 6.2). The simplest is the maximum M(u, \|x\|) deﬁned by (1.3), which is a basic tool in [33, 35, 34] and [1]. We choose the following formu- lation (see Section 6). – 782 – Tangents to subsolutions: existence and uniqueness, Part I Theorem 1.4 (Double Monotonicity). — Let u be F-subharmonic in a neighborhood of the origin in Rn. Then M(u, r) −M(u, s) K(r) −K(s) is increasing in r and s. (1.5) (1.5) for all 0 < s < r where M is deﬁned. for all 0 < s < r where M is deﬁned. Furthermore, if F is convex, the same statement holds with M(u, r) re- placed by either S(u, r) ≡ S u(rσ) dσ or V (u, r) ≡ B u(rx) dx (1.6) S(u, r) ≡ S u(rσ) dσ or V (u, r) ≡ B u(rx) dx (1.6) (1.6) (the spherical or volume average) where B ≡{\|x\| ⩽1} is the unit ball, S ≡ ∂B is the unit sphere, and ﬄ S = 1 \|S\| ´ S denotes the average or “normalized” integral. This theorem has several immediate consequences for the functions Ψ(u,r) for Ψ = M, S, V . 1. Introduction In particular, it leads to the concept of densities (see Corol- lary 5.4). Definition 1.5. — Suppose u is F-subharmonic in a neighborhood of 0 ∈Rn. Then the M-density of u at 0 is the decreasing limit ΘM(u, 0) ≡lim s<r↓0 M(u, r) −M(u, s) K(r) −K(s) . When F is convex, there are also Ψ-densities ΘΨ(u, 0) ≡lim s<r↓0 Ψ(u, r) −Ψ(u, s) K(r) −K(s) . for Ψ = S and V as in (1.6). for Ψ = S and V as in (1.6). Elementary results concerning these densities are established in Lem- ma 5.5. When F is convex, each F subharmonic function is classically ∆-sub- harmonic, and so ∆u = µ ⩾0 (a positive measure). Thus we also have the standard “mass density” Θq(µ, 0) ≡lim r↓0 µ (Br(0)) α(q)rq where q = n −p. where q = n −p. In this convex case all of the densities for M, S, V and µ are universally related, and when p = 2 we have the further result that ΘM = ΘS = ΘV (see Propositions 7.1 and 7.2). In this convex case all of the densities for M, S, V and µ are universally related, and when p = 2 we have the further result that ΘM = ΘS = ΘV (see Propositions 7.1 and 7.2). As noted, tangents need not be unique. However, the averages of tangents are uniquely determined by the density alone, even in the most degenerate cases. This is step two in the proof of the Stong Uniqueness Theorem 1.3.I. It – 783 – F. Reese Harvey and H. Blaine Lawson is also the key step in the proof of existence (Theorem 1.1) and maximality (Theorem 1.2). In the classical case of pluripotential theory the Riesz characteristic is 2, and our next result, when p = 2, is an extension of the work of Kiselman [29]. Theorem 1.6 (Averages of Tangents). — Suppose F is convex and u is an F-subharmonic function deﬁned in a neighborhood of the origin in Rn. Let p = pF be the Riesz characteristic of F. If p ̸= 2, then each tangent U to u at 0 has averages M(r) = sup S U(rσ) = ΘM(u)K(r) , S(r) = S U(rσ) dσ = ΘS(u)K(r) , and V (r) = B U(rx) dx = ΘV (u)K(r) . 1. Introduction One completes the proof of Theorem 1.3.I by showing that for each tangent U and rotation g, we must have U = g∗U or otherwise one can produce a tangent which is not C1. As with most notions of density in analysis, we have the following. Theorem 1.8 (Upper Semi-Continuity of Density). — Suppose u is F- subharmonic on an open set X ⊂Rn. Then each of the densities ΘM(u, x), ΘS(u, x), ΘV (u, x) considered above is an upper semi-continuous function of x. Equivalently, for all c ⩾0 and each Θ as above, the sets considered above is an upper semi-continuous function of x. Equivalently, for all c ⩾0 and each Θ as above, the sets Ec ≡{x : Θ(u, x) ⩾c} are closed. We also note that by standard geometric measure theory cHn−p(Ec) ⩽µ(X). In many cases one can say much more about these high density sets Ec for c > 0. For classical plurisubharmonic functions in Cn a deep theorem, due to L. Hörmander, E. Bombieri and in its ﬁnal form by Siu ([3, 25, 40]), states that Ec is a complex analytic subvariety. One straightforwardly deduces from this result that for the 2-convexity subequation P2 in R2n the set Ec is discrete, since PC(J) ⊂P2 for all orthogonal (parallel) complex structures J on R2n. This very restrictive corollary has a quite general extension. Theorem 1.9 (Structure of High Density Sets). — Suppose strong uniqueness of tangents holds for F. Then for any F-subharmonic function u, the set Ec(u) is discrete. Theorem 1.9 is essentially sharp. Suppose Ωis a domain with strictly convex boundary. Given any ﬁnite subset E = {xj}N j=1 ⊂Ω, any set of numbers Θj > 0, j = 1, . . . , N, and any ϕ ∈C(∂Ω), there exists a unique continuous u : Ω→[−∞, ∞) such that (1) u is F-harmonic on Ω−E, (1) u is F-harmonic on Ω−E, (2) u ∂Ω= ϕ, and (2) u ∂Ω= ϕ, and ∂Ω (3) Θ(u, xj) = Θj for j = 1, . . . , N. ∂Ω (3) Θ(u, xj) = Θj for j = 1, . . . , N. 1. Introduction (1.7) (1.7) In particular, ΘΨ(U) = ΘΨ(u) for Ψ = M, S or V . (1.8) (1.8) When p = 2, all the densities of u and any tangent U to u at 0, agree, and will be simply denoted by Θ = Θ(u). Speciﬁcally, we have p y y ( ) p ﬁ y, Θ(u) = ΘM(U) = ΘS(U) = ΘV (U) = ΘM(u) = ΘS(u) = ΘV (u). (1.9) Moreover, the averages of a tangent U to u are given by M(r) = Θ log r, S(r) = Θ log r+ S U , and V (r) = Θ log r+ B U . (1.10) (1.9) This result about spherical averages of tangents has many applications, for example it is enough to prove maximality of tangents (see Theorem 8.2). This result about spherical averages of tangents has many applications, for example it is enough to prove maximality of tangents (see Theorem 8.2). Theorem 1.7 (Maximality Criterion). — Suppose F is convex and U is an F-subsolution on an annular region A about 0. If the spherical average S(U, \|x\|) is an increasing F-solution on A, then U is maximal on A. Some of the remaining steps in the proof of Theorem 1.3.I, which are given in detail in Section 12, can be outlined as follows. By applying the maximality criterion we conclude in Theorem 10.2 that all tangents are F- maximal. Now if F ′ is any subequation which contains F and has the same Riesz characteristic, then an F-tangent U to u is also an F ′-tangent to u. In the O(n)-invariant (and the other cases of Theorem 1.3.III) it is somewhat surprising that there is a simple convex subequation of characteristic p which contains all the others (Proposition 13.10). This largest subequation is very nice; in particular, it is uniformly elliptic. This, together with Theorem 8.7, shows that tangents are harmonic for this largest subequation, and that they – 784 – Tangents to subsolutions: existence and uniqueness, Part I are C1. One completes the proof of Theorem 1.3.I by showing that for each tangent U and rotation g, we must have U = g∗U or otherwise one can produce a tangent which is not C1. are C1. See Remark 14.2 for more details. The subequations with characteristic 1 ⩽p < 2 are very diﬀerent in nature from those where p ⩾2. They are discussed in detail in Section 15. In particular, the following is proved. – 785 – F. Reese Harvey and H. Blaine Lawson The Work of Armstrong, Sirakov and Smart In the very interesting paper [1] the authors also study conical sube- quations F ⊂Sym2(Rn) with the added assumption that F is uniformly elliptic. However, they do not assume invariance or convexity. An impor- tant part of their work (which is “automatic” in our case) proves the exis- tence and uniqueness of fundamental solutions: F-harmonic functions Φ on Rn −{0}, which are invariant under the ﬂow Φr(x) = rp−2Φ(rx) for some p ⩾1, p ̸= 2 and bounded from above or below. (When p = 2 the log enters as it does here.) They show the existence and uniqueness of two families of such solutions (up to positive scalars and additive constants) among all entire punctured F-harmonics with a one-sided bound. In our degenerate cases two fundamental solutions are not always available. In fact, they are if and only if both F and the dual eF have ﬁnite Riesz characteristics. (See Proposition 3.16 for a description of all such subequations.) One of the results in [1] is closely related to the work here. They prove existence and strong uniqueness of tangents to solutions of uniformly elliptic equations. That is, under their assumptions that F is conical and uniformly elliptic, they prove that: Any F-harmonic function deﬁned on Bϵ −{0} and bounded above (or below), has a unique tangent of the form ΘΦ for some Θ ⩾0 (see [1, (5.13), ﬀ.]). This paper addresses a much broader class of functions, namely subso- lutions to degenerate elliptic equations. Naturally the equations must be in some ways restricted, but the results apply to a wide range of geometrically interesting cases. Here it is shown that tangents exist and are maximal, and that maximal plus continuous implies F-harmonic. However, it is not true that maximal implies continuous in this general case. It fails for example for PC, as does uniqueness of tangents (not just strong uniqueness, see Kisel- man [29]). Said diﬀerently, the step from maximal to F-harmonic does not always hold in the degenerate subharmonic case, and it is somewhat surprising that strong uniqueness of tangents can actually be established for such a broad spectrum of interesting subequations with p ⩾2. We should add that the techniques used in proving strong uniqueness in the non-O(n)-invariant cases are substantially diﬀerent from those in the O(n)-case, and they appear in the sequel (Part II) to this paper. F. Reese Harvey and H. Blaine Lawson Theorem 1.10 (Hölder Continuity 1 ⩽p < 2). — Suppose F is a (not necessarily convex) subequation with Riesz characteristic 1 ⩽p < 2. Then each F-subharmonic function is locally Hölder continuous with exponent α ≡2 −p. Furthermore, if u is an F-subharmonic deﬁned in a neighborhood of 0 ∈Rn, then every sequence {urj}∞ j=1 with rj ↓0, has a subsequence which converges locally uniformly to an F-subharmonic function U on Rn. In fact for each 0 < β < 2 −p there exists a subsequence which converges locally in β-Hölder norm. Finally, when F is convex, this limit U is F-harmonic on Rn −{0}. For the kth Hessian equation the Riesz characteristic is p = n/k. For k > n/2, the Hölder continuity result for this subequation is a fundamental theorem of Trudinger and Wang [42], and their proof can be carried over to more general convex equations. However, we do not require convexity in Theorem 1.10. In Appendix A we examine the radial subequation for the “subaﬃne” case eP ≡{λmax ⩾0} and establish a basic dichotomy: the Increasing/Decreasing Lemma. In Appendix B we show that the subequation P(δ) ≡{A + δtr(A) ⩾0} is uniformly elliptic in the conventional sense. While in Section 4 we give a number of examples to which our the- ory applies, many more examples are given in the appendix to Part II. That appendix also constructs the maximal and minimal subequations of Riesz characteristic p (showing, in particular, that these largest and small- est subequations exist). There is a companion result describing the largest and smallest convex subequations of characteristic p. The largest is given in Proposition 13.10. The smallest is given in Lemma A.1 of Part II. It is worth noting that the main results in this paper (existence, strong uniqueness, maximality, etc.) apply to any subequation obtained by a linear change of variables, i.e. of the form gtFg for g ∈GLn(R) (where F is as assumed herein). This means for cone subequations F which are invariant under a conjugate subgroup g−1Gg where G ⊂O(n) acts transitively on Sn−1. Of course the notion of Riesz characteristic must be reformulated in this case, and the Riesz kernel Kp(\|x\|) must be replaced by its transform Kp(\|gx\|). – 786 – Tangents to subsolutions: existence and uniqueness, Part I The Work of Armstrong, Sirakov and Smart For the question of existence we need to assume convexity or that 1 ⩽ p < 2. This is quite reasonable since we are dealing with subsolutions and the equations are only degenerate elliptic. One needs a function space in – 787 – F. Reese Harvey and H. Blaine Lawson which to extract convergent subsequences just to get oﬀthe ground. These assumptions provide such spaces, namely L1 loc and Hölder. The work in [1] is related to earlier results of Labutin [33, 34, 35] who studied the Pucci extremal equations. He established among other things a removable singularity result and an extension of a classical result of Bôcher. In this work the classical Riesz kernels also play a prominent role. There is a careful account of the relationship to the work of Armstrong–Sirakov–Smart given in [1]. Historical Reﬂections In 1982 the authors showed that for each calibration on a riemannian manifold there is an associated family of minimal subvarieties, forming a calibrated geometry [13]. More recently [16] it was discovered that the cali- bration also determines a potential theory of functions whose restrictions to each of the distinguished submanifolds are subharmonic. Although there is an analoguein this setting of the i∂∂operator from complex geometry, that operator does not play a critical role in the development of the potential theory [14]. In fact, somewhat surprisingly, a corresponding potential theory can be established for any collection of submanifolds determined by requiring their tangent spaces to be in an arbitrary given closed subset of the grass- mannian. Even more generally one has the potential theory associated to an elliptic (possibly degenerate) nonlinear inequality F(D2u) ⩾0, provided by viscosity subsolutions ([6]). This raises the possibility of cross-fertilization between two well estab- lished and deep ﬁelds, pluripotential theory (in several complex variables) and nonlinear elliptic theory. This paper, although not the ﬁrst, can be viewed as an example of this phenomenon. The authors believe there are many more to come. 2. The Radial Subequations Associated to a Subequation F In this section we ﬁrst describe the ordinary diﬀerential inequality which governs C2 radial (i.e., spherically symmetric) F-subharmonic functions. Our main result ﬁlls an apparent gap in the literature by extending this character- ization to general upper semi-continuous radial F-subharmonics. Somewhat surprisingly this extension requires the attention of Lemma 2.10 below. – 788 – Tangents to subsolutions: existence and uniqueness, Part I Suppose ψ(t) is of class C2 on an interval contained in the positive real numbers. We also consider ψ as the function ψ(\|x\|) of x on the corresponding annular region in Rn. Lemma 2.1. D2 xψ = ψ′(\|x\|) \|x\| P[x]⊥+ ψ′′(\|x\|)P[x] , (2.1) (2.1) where P[x] = x◦x \|x\|2 denotes orthogonal projection onto the line [x] through x ̸= 0 and P[x]⊥= I −P[x] denotes orthogonal projection onto the hyperplane with normal [x]. Proof. — First note that D(\|x\|) = x \|x\|, and therefore D2(\|x\|) = D( x \|x\|) = 1 \|x\|I − x \|x\|2 ◦ x \|x\| = 1 \|x\|(I −P[x]) = 1 \|x\|P[x]⊥. Hence, Dxψ = ψ′(\|x\|) x \|x\| and D2 xψ = ψ′(\|x\|)D x \|x\| + ψ′′(\|x\|) x \|x\| ◦x \|x\| = ψ′(\|x\|) \|x\| P[x]⊥+ ψ′′(\|x\|)P[x] . □ \|x\| D2 xψ = ψ′(\|x\|)D x \|x\| + ψ′′(\|x\|) x \|x\| ◦x \|x\| = ψ′(\|x\|) \|x\| P[x]⊥+ ψ′′(\|x\|)P[x] . □ Corollary 2.2. — The second derivative D2 xψ has eigenvalues ψ′(\|x\|) \|x\| with multiplicity n −1 and ψ′′(\|x\|) with multiplicity 1. Let F ⊂Sym2(Rn) be a pure second-order constant coeﬃcient sub- equation. Then by Lemma 2.1 a radial C2-function u(x) = ψ(\|x\|) is F-subharmonic on an annular region in Rn if and only if D2 xu = ψ′(t) t Pe⊥+ ψ′′(t)Pe ∈F , (2.2) (2.2) for t = \|x\| in the corresponding interval in (0, ∞). We use λ = ψ′(t) and a = ψ′′(t) as one-variable jet coordinates. Then the basic one-variable sube- quation associated with F is deﬁned as follows. for t = \|x\| in the corresponding interval in (0, ∞). We use λ = ψ′(t) and a = ψ′′(t) as one-variable jet coordinates. Then the basic one-variable sube- quation associated with F is deﬁned as follows. Definition 2.3. — The radial subequation associated with F is the reduced variable coeﬃcient subequation RF on (0, ∞) whose ﬁbre at t is (RF )t ≡ (λ, a) ∈R2 : λ t Pe⊥+ aPe ∈F, ∀\|e\| = 1 . F. Reese Harvey and H. Blaine Lawson Theorem 2.4 (Radial Subharmonics). — The function u(x) ≡ψ(\|x\|) is F-subharmonic on an annular region in Rn if and only if ψ(t) is RF - subharmonic on the corresponding open sub-interval of (0, ∞). Theorem 2.4 (Radial Subharmonics). — The function u(x) ≡ψ(\|x\|) is F-subharmonic on an annular region in Rn if and only if ψ(t) is RF - subharmonic on the corresponding open sub-interval of (0, ∞). Remark 2.5. — In all but this section of the paper, the subequations F will be assumed to be cones, unless explicitly stated to the contrary. For such subequations the maximum principle holds, i.e., it holds for each F-subharmonic function u(x) (see Theorem A.2). Consequently, if u(x) = ψ(\|x\|) is a radial F-subharmonic on a ball about 0, then ψ(t) must be increas- ing in t. This motivates focusing on an “increasing” version of Theorem 2.4. We will use the fact, which is elementary to establish, that for an upper semi-continuous function ψ(t), ψ(t) is increasing ⇐⇒ ψ is {λ ⩾0}-subharmonic. (2.4) See [12] for a proof.) ψ(t) is increasing ⇐⇒ ψ is {λ ⩾0}-subharmonic. (2.4) (See [12] for a proof.) (2.4) (See [12] for a proof.) Definition 2.6. — The increasing radial subharmonic equation R↑ F on (0, ∞) is deﬁned by R↑ F = RF ∩{λ ⩾0}. (2.5) (2.5) In light of (2.3), it is obvious that for C2-functions ψ(t): In light of (2.3), it is obvious that for C2-functions ψ(t): ψ(t) is R↑ F -subharmonic ⇐⇒ ψ(\|x\|) is F ∩{x·p ⩾0}-subharmonic (2.6 In light of (2.3), it is obvious that for C functions ψ(t): ψ(t) is R↑ F -subharmonic ⇐⇒ ψ(\|x\|) is F ∩{x·p ⩾0}-subharmonic (2.6) where the variable coeﬃcient ﬁrst-order subequation {x · p ⩾0} is the con- straint x · Dxu ⩾0 on C2-functions. The equivalence (2.6) can be extended as in Theorem 2.4. (2.6) ψ( ) F ψ(\| \|) { p ⩾} ( ) where the variable coeﬃcient ﬁrst-order subequation {x · p ⩾0} is the con- straint x · Dxu ⩾0 on C2-functions. The equivalence (2.6) can be extended as in Theorem 2.4. Theorem 2.7 (Increasing Radial Subharmonics). — The function u(x) ≡ψ(\|x\|) is an increasing, radial F-subharmonic function if and only if ψ(t) is R↑ F -subharmonic. Remark 2.8. — We will sometimes blur the distinction between ψ(t) and u(x) = ψ(\|x\|) by calling ψ(t) a radial (or increasing radial) F-subharmonic. Remark 2.8. 2. The Radial Subequations Associated to a Subequation F Thus for C2-functions we have that Thus for C2-functions we have that u(x) ≡ψ(\|x\|) is F subharmonic ⇐⇒ ψ(t) is RF subharmonic. (2.3) u(x) ≡ψ(\|x\|) is F subharmonic ⇐⇒ ψ(t) is RF subharmonic. (2.3) (2.3) We extend this to the viscosity setting where F-subharmonic functions are just upper semi-continuous (see [5, 6, 14, 17] for deﬁnitions). The proof given below of the implication ⇒is elementary, whereas the proof of ⇐ will require a lemma. Note that the equivalence: u(x) = ψ(\|x\|) is upper semicontinuous ⇐⇒ψ(t) is upper semicontinuous, is obvious. – 789 – F. Reese Harvey and H. Blaine Lawson — We will sometimes blur the distinction between ψ(t) and u(x) = ψ(\|x\|) by calling ψ(t) a radial (or increasing radial) F-subharmonic. Remark 2.9. — The statement and proof of a theorem analogous to 2.7 for decreasing radial subharmonics is left to the reader. Remark 2.9. — The statement and proof of a theorem analogous to 2.7 for decreasing radial subharmonics is left to the reader. Proof of Theorem 2.4. Proof of Theorem 2.4. (⇒): Suppose u(x) ≡ψ(\|x\|) is F-subharmonic. If ϕ(t) is a test function for ψ(t) at t0, then ϕ(\|x\|) is a test function for ψ(\|x\|) at any point on the t0- sphere in Rn. Therefore D2 x0ϕ ∈F. Applying the formula for D2 x0ϕ in terms of ϕ′(t0) and ϕ′′(t0), the equivalence (2.3), and the deﬁnition of (RF )t0, we have J2 t0ϕ ∈RF . This proves that ψ(t) is RF -subharmonic. (⇒): Suppose u(x) ≡ψ(\|x\|) is F-subharmonic. If ϕ(t) is a test function for ψ(t) at t0, then ϕ(\|x\|) is a test function for ψ(\|x\|) at any point on the t0- sphere in Rn. Therefore D2 x0ϕ ∈F. Applying the formula for D2 x0ϕ in terms of ϕ′(t0) and ϕ′′(t0), the equivalence (2.3), and the deﬁnition of (RF )t0, we have J2 t0ϕ ∈RF . This proves that ψ(t) is RF -subharmonic. – 790 – Tangents to subsolutions: existence and uniqueness, Part I (⇐): Suppose that ψ(t) is RF -subharmonic. We must show that u(x) ≡ ψ(\|x\|) is F-subharmonic. That is, given a test function ϕ(x) for u(x) at a point x0, we must show that D2 x0ϕ ∈F. Suppose that there exists a smooth function ψ(t), deﬁned near t0 = \|x0\|, such that ϕ(x) ≡ψ(\|x\|) satisﬁes u(x) ⩽ϕ(x) ⩽ϕ(x) (2.7) (2.7) near x0. Then ψ(t) is a test function for ψ(t) at t0. Hence, the 2-jet of ψ at t0 belongs to RF . By Lemma 2.1 and the discussion above, this implies that D2 x0ϕ ∈F. The inequality ϕ(x) ⩽ϕ(x) (with equality at x0) implies that D2 x0ϕ = D2 x0ϕ + P for some P ⩾0, which proves that D2 x0ϕ ∈F as desired. To complete this argument by ﬁnding ψ(t) there is some ﬂexibility given by Lemma 2.4 in [17] so that not all test functions ϕ(x) need be considered. First we may choose new coordinates z = (t, y) near x0 so that t ≡\|x\|. F. Reese Harvey and H. Blaine Lawson (Thus t = constant deﬁnes the sphere of radius t near x0.) Furthermore, we may assume that ϕ(z) is a polynomial of degree ⩽2 in z = (t, y) and that it is a strict local test function, i.e., u(z) < ϕ(z) for z ̸= z0. Now Lemma 2.10 below ensures the existence of ϕ(x) = ψ(\|x\|) satisfying (2.7). □ Let z = (t, y) denote standard coordinates on Rn = Rk × Rℓ. Fix a point z0 = (t0, y0) and let u(t) be an upper semi-continuous function (of t alone) and ϕ(z) a C2-function, both deﬁned in a neighborhood of z0. Lemma 2.10. — Suppose u(t) < ϕ(z) for z ̸= z0 with equality at z0. If ϕ(z) is a polynomial of degree ⩽2, then there exists a polynomial ϕ(t) of degree ⩽2 with u(t) ⩽ϕ(t) ⩽ϕ(z) near z0 . u(t) ⩽ϕ(t) ⩽ϕ(z) near z0 . (2.8) (2.8) u(t) ⩽ϕ(t) ⩽ϕ(z) near z0 . Proof. — We may assume z0 = 0 and u(0) = ϕ(0) = 0. Then ϕ(z) = ⟨p, t⟩+ ⟨q, y⟩+ ⟨At, t⟩+ 2⟨Bt, y⟩+ ⟨Cy, y⟩. We assume u(t) < ϕ(t, y) for \|t\| ⩽ϵ and \|y\| ⩽δ with (t, y) ̸= (0, 0). Proof. — We may assume z0 = 0 and u(0) = ϕ(0) = 0. Then ϕ(z) = ⟨p, t⟩+ ⟨q, y⟩+ ⟨At, t⟩+ 2⟨Bt, y⟩+ ⟨Cy, y⟩. We assume u(t) < ϕ(t, y) for \|t\| ⩽ϵ and \|y\| ⩽δ with (t, y) ̸= (0, 0). — We may assume z0 = 0 and u(0) = ϕ(0) = 0. Then ϕ(z) = ⟨p, t⟩+ ⟨q, y⟩+ ⟨At, t⟩+ 2⟨Bt, y⟩+ ⟨Cy, y⟩. ϕ(z) = ⟨p, t⟩+ ⟨q, y⟩+ ⟨At, t⟩+ 2⟨Bt, y⟩+ ⟨Cy, y⟩. We assume u(t) < ϕ(t, y) for \|t\| ⩽ϵ and \|y\| ⩽δ with (t, y) ̸= (0, 0). Setting t = 0, we have 0 = u(0) < ⟨q, y⟩+ ⟨Cy, y⟩for y ̸= 0 suﬃciently small. Therefore, q = 0 and C > 0 (positive deﬁnite). Now deﬁne Setting t = 0, we have 0 = u(0) < ⟨q, y⟩+ ⟨Cy, y⟩for y ̸= 0 suﬃciently small. Therefore, q = 0 and C > 0 (positive deﬁnite). Now deﬁne ϕ(t) ≡⟨p, t⟩+ ⟨(A −BtC−1B)t, t⟩. F. Reese Harvey and H. Blaine Lawson F. Reese Harvey and H. Blaine Lawson Proof of Theorem 2.7. — The arguments given for Theorem 2.4 along with the following missing steps provide the proof. If ϕ(t) is a test function for ψ(t) at a point t0, then ϕ(\|x\|) is a test function for ψ(\|x\|) at x0 whenever \|x0\| = t0. Now Dx0ϕ = ϕ′(\|x0\|) x0 \|x0\| and hence x0 · Dx0ϕ = \|x0\|ϕ′(\|x0\|). (2.11) Dx0ϕ = ϕ′(\|x0\|) x0 \|x0\| and hence x0 · Dx0ϕ = \|x0\|ϕ′(\|x0\|). (2.11) (2.11) Hence, if ψ(\|x\|) is {p·x ⩾0}-subharmonic, then ψ(t) is {λ ⩾0}-subharmonic, and thus increasing. Conversely, if ψ(t) is increasing and ϕ(x) is a test func- tion for ψ(\|x\|) at x0, then ϕ(t) ≡ϕ( tx0 \|x0\|) is a test function for ψ(t) at t0 = \|x0\|. Hence, ϕ′(t0) ⩾0. However, ϕ′(t0) = (Dx0ϕ) · x0. □ □ F. Reese Harvey and H. Blaine Lawson (2.9) (2.9) The inequalities in (2.8) follow from the fact that for t suﬃciently small, The inequalities in (2.8) follow from the fact that for t suﬃciently small, ϕ(t) = inf \|y\|⩽δ ϕ(z) = ⟨p, t⟩+ ⟨At, t⟩+ inf \|y\|⩽δ{2⟨Bt, y⟩+ ⟨Cy, y⟩}. (2.10) ϕ(t) = inf \|y\|⩽δ ϕ(z) = ⟨p, t⟩+ ⟨At, t⟩+ inf \|y\|⩽δ{2⟨Bt, y⟩+ ⟨Cy, y⟩}. (2.10) ϕ(t) = inf \|y\|⩽δ ϕ(z) = ⟨p, t⟩+ ⟨At, t⟩+ inf \|y\|⩽δ{2⟨Bt, y⟩+ ⟨Cy, y⟩}. (2.10) To prove (2.10) ﬁx t and consider the function 2⟨Bt, y⟩+ ⟨Cy, y⟩. Since C > 0, it has a unique minimum point at the critical point y = −C−1Bt. The minimum value is −⟨BtC−1Bt, t⟩. If t is suﬃciently small, the critical point y satisﬁes \|y\| < δ, which proves (2.7). □ – 791 – 3. ST-Invariant Cone Subequations: The Riesz Characteristic This section is devoted to investigating the cone subequations which sat- isfy a weak form of invariance which will be referred to as spherical transi- tivity (ST). Two characteristic numbers (p, q) will be associated with each such subequation F. They uniquely determine the radial subequation for F and, as we shall show in this and the following sections, can be easily computed in any example. Moreover, we give a complete description of all possible examples (of ST-invariant subequations with characteristics (p, q)) in the second subsection here. Most readers will prefer to come back to this subsection. Although it adds important perspective to the scope of ST-cone subequations, it is not used in the subsequent results of the paper. Recall from the introduction that a subequation F ⊂Sym2(Rn) is said to be ST-invariant if there exists a subgroup G ⊂O(n) which acts transitively on the sphere Sn−1 ⊂Rn and leaves F invariant (under the induced action of G on Sym2(Rn)). For an ST-invariant cone subequation F, For an ST-invariant cone subequation F, the slices F ∩span{Pe⊥, Pe} for e ∈Sn−1 are all isomorphic. (3.1) Note that span{Pe⊥, Pe} = span{I, Pe} and that the induced action on Sym2(Rn) sends Pe to Pg(e). In particular, the slices F ∩span{Pe⊥, Pe} for e ∈Sn−1 are all isomorphic. (3.1) Note that span{Pe⊥, Pe} = span{I, Pe} and that the induced action on Sym2(Rn) sends Pe to Pg(e). In particular, the slices F ∩span{Pe⊥, Pe} for e ∈Sn−1 are all isomorphic. (3.1) (3.1) the slices F ∩span{Pe⊥, Pe} for e ∈Sn−1 are all isomorphic. (3.1) Note that span{Pe⊥, Pe} = span{I, Pe} and that the induced action on Sym2(Rn) sends Pe to Pg(e). In particular, λPe⊥+ µPe ∈F for one e ∈Sn−1 ⇐⇒ λPe⊥+ µPe ∈F for all e ∈Sn−1. (3.2) (3.2) This weakening of ST-invariance will be referred to as weak invariance. This weakening of ST-invariance will be referred to as weak invariance. – 792 – Tangents to subsolutions: existence and uniqueness, Part I Lemma 3.4. — For ST-invariant cone subequations F (1) pF = 1 ⇐⇒Pe⊥∈∂F ⇐⇒F = P (2) pF = ∞⇐⇒−Pe ∈F ⇐⇒−Pe ∈∂F. (3) pF < ∞⇐⇒−Pe /∈F ⇐⇒Pe ∈Int eF ⇐⇒P−{0} ⊂Int eF. (1) pF = 1 ⇐⇒Pe⊥∈∂F ⇐⇒F = P (2) pF = ∞⇐⇒−Pe ∈F ⇐⇒−Pe ∈∂F. (3) pF < ∞⇐⇒−Pe /∈F ⇐⇒Pe ∈Int eF ⇐⇒P−{0} ⊂Int eF. (1) pF = 1 ⇐⇒Pe⊥∈∂F ⇐⇒F = P (2) pF = ∞⇐⇒−Pe ∈F ⇐⇒−Pe ∈∂F. (3) pF < ∞⇐⇒−Pe /∈F ⇐⇒Pe ∈Int eF ⇐ Actually, as noted above, it is easy to compute the exact value of pF in all the examples. Actually, as noted above, it is easy to compute the exact value of pF in all the examples. Proof of (1). — Note ﬁrst that pF > 1 ⇐⇒ Pe⊥−ϵPe ∈F for all small ϵ > 0. Now if F contains an element A with at least one eigenvalue strictly negative, then by positivity and the cone property there is an element A′ = Pe⊥−ϵPe ∈F. Hence F ̸= P ⇒pF > 1. Proof of (2). — Note ﬁrst that −Pe ∈F ⇒αPe⊥−Pe ∈F ∀α ⩾0 ⇒ Pe⊥−(p −1)Pe ∈F ∀p ⩾1 ⇒pF = ∞. On the other hand −Pe /∈F ⇒ ϵPe⊥−Pe /∈F ∀ϵ ⩾0 small ⇒Pe⊥−(p −1)Pe /∈F ∀p large ⇒pF < ∞. To complete the proof of (2), note that −Pe ∈Int F cannot occur unless F = Sym2(Rn) since −Pe ∈Int F ⇒0 ∈Int F. Proof of (3). — By (DE) above we have ∼(−F) = Int eF, and the ﬁrst part of (3) follows from the ﬁrst part of (2). For the last ⇒note that P is the convex cone hull of the Pe’s. The last ⇐is obvious. □ □ The primary application of the Riesz characteristics (and the reason for choosing the name) is the fact that the solutions of the associated increasing radial equation R↑ p are given by the Riesz kernels. Proposition 3.5. — An ST-invariant cone subequation F has ﬁnite Riesz characteristic p = pF if and only if the increasing radial harmonics for F are: ΘKp(\|x\|) + C (3.5) (3.5) where Θ ⩾0, C ∈R, and Kp(t) is the pth Riesz function deﬁned on 0 < t < ∞by Kp(t) =      t2−p if 1 ⩽p < 2 log t if p = 2 − 1 tp−2 if 2 < p < ∞. (3.6) (3.6) Proof. The Riesz Characteristics Blaine Lawson F. Reese Harvey and H. Blaine Lawson Lemma 3.4. — For ST-invariant cone subequations F The Riesz Characteristics We begin by focusing on the ﬁrst of the two characteristics (p, q). Al- though there is an abundance of interesting ST-invariant cone subequations in dimensions ⩾3, there are not many increasing radial subequations. In fact they are described by a single “characteristic” number p between 1 and ∞, which determines a one-variable subequation as follows. Definition 3.1. — For each p with 1 ⩽p < ∞, the increasing radial subequation R↑ p is deﬁned by R↑ p : a + (p −1) t λ ⩾0 and λ ⩾0, (3.3) (3.3) while for p = ∞, the subequation R↑ ∞is ﬁrst-order and deﬁned by R↑ ∞= {λ ⩾0}. while for p = ∞, the subequation R↑ ∞is ﬁrst-order and deﬁned by R↑ ∞= {λ ⩾0}. Definition 3.2 (The Increasing Riesz Characteristic). — Suppose F is an ST-invariant cone subequation. The increasing characteristic pF of F is deﬁned to be pF ≡sup{p : Pe⊥−(p −1)Pe ∈F}. (3.4a) Equivalently, for ﬁnite Riesz characteristic, pF is the unique number p such that Pe⊥−(p −1)Pe ∈∂F . (3.4b) (3.4b) Proposition 3.3 (Increasing). — Suppose that F is an ST-invariant cone subequation. Then the increasing radial subequation R↑ F equals R↑ p where p = pF is the increasing Riesz characteristic of F. Proof. — Using Deﬁnitions 2.3, 2.6, 3.1 and 3.2, we must show that for λ ⩾0 λ t Pe⊥+ aPe ∈F ⇐⇒ a + p −1 t λ ⩾0. ⩾ λ t Pe⊥+ aPe ∈F ⇐⇒ a + p −1 t λ ⩾0. Set −(p −1) ≡at/λ, so that λ t Pe⊥+ aPe ∈F ⇐⇒Pe⊥−(p −1)Pe ∈F. Then p ⩽p ⇐⇒−at λ ⩽p −1 ⇐⇒a + p−1 t λ ⩾0. □ Note that by Deﬁnition 3.2, the positivity condition for F, and the fact that 0 ∈F, we have that pF ⩾1. Thus 1 ⩽pF ⩽∞. The only equation with pF = 1 is P. At the other extreme we have pF = ∞. Here there is a test which is very simple to apply in all the ST- invariant examples, namely: pF = ∞iﬀ−Pe ∈F. Hence, determining when pF < ∞is also simple, namely: pF < ∞iﬀ−Pe /∈F. We recall the fact that for a subequation F, the dual subequation eF is deﬁned as e eF = −(∼Int F) = ∼(−Int F). eF = −(∼Int F) = ∼(−Int F). (DE) (DE) – 793 – F. Reese Harvey and H. Lemma 3.4. — For ST-invariant cone subequations F — From (3.4b) it is easy to see that u(x) ≡ψ(\|x\|) is F-subhar- monic if and only if ψ(t) is R↑ p-subharmonic. The ordinary diﬀerential equa- tion given by equality in (3.3) is easily solved, and ΘKp(t) + C are the viscosity solutions. One can check directly using Lemma 2.1 that D2Kp(\|x\|) = 1 \|x\|p P[x]⊥−(p −1)P[x] and DKp = x \|x\|p (3.7) (3.7) – 794 – Tangents to subsolutions: existence and uniqueness, Part I Tangents to subsolutions: existence and uniqueness, Part I where Kp has been renormalized to Kp ≡ 1 \|p −2\|Kp if p ̸= 2 and K2 = K2 . (3.8) □ (3.8) □ (3.8) 3.8) □ □ The sign of Kp(t) has been chosen so that Kp(\|x\|) is a increasing or downward-pointing F-harmonic on Rn −{0}. The actual normalization in (3.6) is simpler when the focus is on the function u, while the normaliza- tion in (3.8) is simpler when the focus is on the ﬁrst and second derivatives of u. The second of the two numbers (p, q) can also be deﬁned in several equiv- alent ways. Definition 3.6 (The Decreasing Riesz Characteristic). — For each ST- invariant cone subequation F, this characteristic, denoted qF , is deﬁned by qF = sup {¯q : −Pe⊥+ (¯q −1)Pe /∈F}, (3.9a) (3.9a) r equivalently qF is the unique number q such that or equivalently qF is the unique number q such that −Pe⊥+ (q −1)Pe ∈∂F , (3.9b) (3.9b) or ﬁnally, qF can be deﬁned to be the increasing characteristic of the dual subequation, i.e. qF = pe F . (3.9c) (3.9c) Since ∂eF = −∂F, the equivalence of (3.9c) follows easily from (3.4b). Thus the decreasing characteristic of F might also be called the dual char- acteristic of F. For each 1 ⩽q < ∞set R↓ q : a + q −1 t λ ⩾0 and λ ⩽0 (3.10) (3.10) while for q = ∞the subequation R↓ q is ﬁrst-order and deﬁned by R↓ ∞= {λ ⩽0}. Then the decreasing versions of Propositions 3.3, Lemma 3.4(3) and Proposition 3.5 state the following. Proposition 3.7 (Decreasing). R↓ F = R↓ q with q ≡qF . (3.11a) (Decreasing). R↓ F = R↓ q with q ≡qF . (3.11a) R↓ F = R↓ q with q ≡qF . Lemma 3.4. — For ST-invariant cone subequations F (3.11a) F has ﬁnite decreasing characteristic qF ⇐⇒ Pe ∈Int F , (3.11b) which in turn holds if and only if the decreasing radial F-harmonics are F has ﬁnite decreasing characteristic qF ⇐⇒ Pe ∈Int F , (3.11b) which in turn holds if and only if the decreasing radial F-harmonics are which in turn holds if and only if the decreasing radial F-harmonics are −ΘKq(\|x\|) + C where Θ ⩾0 and C ∈R, and q = qF . (3.11c) −ΘKq(\|x\|) + C – 795 – F. Reese Harvey and H. Blaine Lawson Remark. — In summary we have that: (1) For some p ﬁnite, Kp(\|x\|) is an increasing (or downward-pointing) F-harmonic on Rn −{0} ⇐⇒ −Pe /∈F ⇐⇒ F has ﬁnite increasing characteristic. (2) For some q ﬁnite, −Kq(\|x\|) is an decreasing (or upward-pointing) F-harmonic on Rn −{0} ⇐⇒ Pe ∈Int F ⇐⇒ F has ﬁnite decreasing characteristic. (3) Both Kp(\|x\|) and −Kq(\|x\|) are F-harmonic on Rn −{0} ⇐⇒F has both characteristics (p, q) ﬁnite ⇐⇒−Pe /∈F and Pe ∈Int F. These criteria hold for a signiﬁcant number of degenerate (non uniformly elliptic) subequations. (See the next section and Appendix A in Part II.) However, in case (3) if either F or eF is convex, then both are uniformly elliptic. Conversely, uniform ellipticity always implies that (p, q) are both ﬁnite even in the non-convex case. Tangents to subsolutions: existence and uniqueness, Part I Tangents to subsolutions: existence and uniqueness, Part I Theorem 3.11 (Universal Solvability of the Dirichlet Problem). — Sup- pose that F is an ST-invariant cone subequation for which both Riesz char- acterstics pF and qF are ﬁnite (or equivalently for which the simple con- dition Pe ∈Int F and −Pe /∈F holds). Then for every domain Ω⊂⊂Rn with smooth boundary ∂Ω, and for every ϕ ∈C(∂Ω), there exists a unique h ∈C(Ω) such that (1) h is F-harmonic on Ω, and (2) h ∂Ω= ϕ. (1) h is F-harmonic on Ω, and (2) h ∂Ω= ϕ. (1) h is F-harmonic on Ω, and (2) h ∂Ω= ϕ. Remark 3.12. — In fact Theorem 3.11 holds for any constant coeﬃcient second-order subequation F if and only if its asymptotic cone subequation −→ F satisﬁes Pe ∈Int F and −Pe /∈F for all \|e\| = 1. Finally, combining both characteristics we have Finally, combining both characteristics we have Proposition 3.8. — If F has characteristics (p, q), then the radial sube- quation for F is RF = R↑ p ∪R↓ q . (3.12) (3.12) Remark 3.9 (Boundary Convexity and the Riesz Characteristic). — The ﬁniteness of the two characteristics of F, which is so easy to ascertain, is equivalent to automatic boundary convexity for all domains. Proposition 3.10. — The boundary ∂Ωof every smoothly bounded do- main Ω⊂⊂Rn is (1) strictly F-convex ⇐⇒ pe F = qF < ∞ ⇐⇒ Pe ∈Int F, (2) strictly eF-convex ⇐⇒ pF = qe F < ∞ ⇐⇒ −Pe /∈F, (3) both strictly F- and eF-convex ⇐⇒ (pF , qF ) is ﬁnite ⇐⇒ Pe ∈Int F and −Pe /∈F. (2) strictly eF-convex ⇐⇒ Proof. — We ﬁrst prove (2). By Lemma 5.3(ii′) in [14], ∂Ωis strictly eF-convex at x ∈∂Ωfor all domains Ωif and only if ∀B ∈Sym2(W), B + tPe ∈Int eF for all t ⩾some t0 . (3.13) where \|e\| = 1 and W = e⊥. Now (3.13) ⇒Pe ∈Int eF ⇒1 t B + Pe ∈Int eF for all t ⩾some t0 ⇒(3.13). Thus (3.13) is equivalent to pF < ∞by Lemma 3.4(3). The proof of (1) follows by duality, and (1) and (2) together imply (3). □ ∀B ∈Sym2(W), B + tPe ∈Int eF for all t ⩾some t0 . (3.13) where \|e\| = 1 and W = e⊥. Now (3.13) ⇒Pe ∈Int eF ⇒1 t B + Pe ∈Int eF for all t ⩾some t0 ⇒(3.13). Thus (3.13) is equivalent to pF < ∞by Lemma 3.4(3). The proof of (1) follows by duality, and (1) and (2) together imply (3). □ (3.13) Results in [14] immediately imply the following. Results in [14] immediately imply the following. – 796 – Proof. — L Pmin/2 p ⇒A(p that A(p) ∈∂ Each A ∈ using the ord λ1Pe1 + λnPe If A ∈Pmi p and F have th B0 ∈F. Howe For the ot Now A ⩽B1 given by (3.1 A ∈Pmin/max p This impo Corollar Proof. — I quation, then istic p, we ha satisﬁes q −1 Remark. — teristics (p, q) by using Prop It is just a note that the F. Reese Harvey and H. Blaine Lawson A Description of all ST-Invariant Cone Subequations Although it is always easy to compute the characteristics (p, q) of a given F, it is still enlightening to give a description (or construction) of all the possible ST-invariant cone subequations with characteristics (p, q). The following speciﬁc examples are instrumental in this description. For A ∈Sym2(Rn) let λ1(A) ⩽· · · ⩽λn(A) denote the ordered eigenvalues of A, and set λmin(A) ≡λ1(A) and λmax(A) ≡λn(A). We then deﬁne Pmin/max p ≡{A : λmin(A) + (p −1)λmax(A) ⩾0} (3.14) Pmin/2 p ≡{A : λmin(A) + (p −1)λ2(A) ⩾0} (3.15) (3.15) It is clear that both of these are O(n)-invariant cone subequations. Both A ≡Pe⊥−(p −1)Pe and B ≡−Pe⊥+ 1 p−1Pe have the property that λmin + (p −1)λmax = 0, which shows that A, B ∈∂Pmin/max p and hence Pmin/max p has characteristics (p, q) where q satisﬁes (p−1)(q−1) = 1. Similarly, Pmin/2 p has characteristics (p, ∞) if n ⩾3. It is clear that both of these are O(n)-invariant cone subequations. Both A ≡Pe⊥−(p −1)Pe and B ≡−Pe⊥+ 1 p−1Pe have the property that λmin + (p −1)λmax = 0, which shows that A, B ∈∂Pmin/max p and hence Pmin/max p has characteristics (p, q) where q satisﬁes (p−1)(q−1) = 1. Similarly, Pmin/2 p has characteristics (p, ∞) if n ⩾3. Our general discussion is a characterization in terms of these two exam- ples and their duals. Proposition 3.13. — Suppose that F is an ST-invariant (not necessar- ily convex) cone subequation. Then F has a ﬁnite (increasing) Riesz charac- teristic p if and only if Pmin/2 p ⊂F ⊂Pmin/max p . (3.16) (3.16) Equivalently, Kp(\|x\|) is an increasing (or downward-pointing) radial F-har- monic. In particular, both the “smallest” and the “largest” subequations, Pmin/2 p and Pmin/max p , have Riesz characteristic p. – 797 – F. Reese Harvey and H. Blaine Lawson (3.20) (3.20) – 798 – Tangents to subsolutions: existence and uniqueness, Part I Tangents to subsolutions: existence and uniqueness, Part I Proof. — Pmin/2 q ⊂eF ⊂Pmin/max q ⇐⇒ ePmin/max q ⊂F ⊂ePmin/2 q . □ □ Finally, it is possible to describe all the ST-invariant cone subequations with both characteristics ﬁnite. Proposition 3.16. — Suppose that F is an ST-invariant cone subequa- tion. Then F has both Riesz characteristics (p, q) ﬁnite if and only if Pmin/2 p ∪ePmin/max q ⊂F ⊂Pmin/max p ∩ePmin/2 q . (3.21) ations exist if and only if (3.21) Such subequations exist if and only if (p −1)(q −1) ⩾1, (3.22) (3.22) and so in particular if this constraint holds for (p, q), then both and so in particular if this constraint holds for (p, q), then both Pmin/2 p ∪ePmin/max q and Pmin/max p ∩ePmin/2 q have characteristics (p, q). (3.23) Pmin/2 p ∪ePmin/max q and Pmin/max p ∩ePmin/2 q have characteristics (p, q). (3.23) Pmin/2 p ∪ePmin/max q and Pmin/max p ∩ePmin/2 q have characteristics (p, q). (3.23) Proof. — Note that (3.21) holds if and only if both (3.16) and (3.20) hold. Thus by Propositions 3.13 and 3.15, F has ﬁnite Riesz characteristics (p, q) if and only if (3.21) holds. Corollary 3.14 states that if F has characteristics (p, q), then (3.22) must hold. Now suppose that (3.22) holds. Then ePmin/max q ⊂Pmin/max p and Pmin/2 p ⊂ePmin/2 q (3.24) because λmax +(q−1)λmin ⩾0 ⇒λmin +(p−1)λmax ⩾0 if p−1 ⩾1/(q−1); and λmin+(p−1)λ2 ⩾0 ⇒λn−1+(p−1)λmax ⩾0 ⇒λmax+(q−1)λn−1 ⩾0 if q−1 ⩾1/(p−1). Finally, (3.24) implies that Pmin/2 p ∪ePmin/max q ⊂Pmin/max p ∩ e i /2 ePmin/max q ⊂Pmin/max p and Pmin/2 p ⊂ePmin/2 q (3.24) because λmax +(q−1)λmin ⩾0 ⇒λmin +(p−1)λmax ⩾0 if p−1 ⩾1/(q−1); and λmin+(p−1)λ2 ⩾0 ⇒λn−1+(p−1)λmax ⩾0 ⇒λmax+(q−1)λn−1 ⩾0 if q−1 ⩾1/(p−1). Finally, (3.24) implies that Pmin/2 p ∪ePmin/max q ⊂Pmin/max p ∩ e i /2 (3.24) ePmin/2 q so that both of these subequations have characteristics (p, q). □ 4. Some Illustrative Examples For the basic subequations the Riesz characteristic is quite easy to com- pute. We shall illustrate this with a selection of examples of diﬀering types. A detailed discussion of subequations of characteristic p, and further results, are given in Appendix A of Part II. For A ∈Sym2(Rn) we let λ1(A) ⩽λ2(A) ⩽· · · ⩽λn(A) (4.1) denote the ordered eigenvalues of A. For A ∈Sym2(Rn) we let For A ∈Sym2(Rn) we let ) λ1(A) ⩽λ2(A) ⩽· · · ⩽λn(A) (4.1) genvalues of A. λ1(A) ⩽λ2(A) ⩽· · · ⩽λn(A) (4. denote the ordered eigenvalues of A. (4.1) F. Reese Harvey and H. Blaine Lawson Proof. — Let A(p) ≡Pe⊥−(p −1)Pe. If F satisﬁes (3.16), then A(p) ∈ Pmin/2 p ⇒A(p) ∈F, and A(p) /∈Int Pmin/max p ⇒A(p) /∈Int F, which proves that A(p) ∈∂F, and hence F has characteristic p. Each A ∈Sym2(Rn) can be written as a sum A = λ1Pe1 + · · · + λnPen using the ordered eigenvalues of A. Set B0 ≡λ1Pe1 + λ2Pe⊥ 1 , and B1 ≡ λ1Pe1 + λnPe⊥ 1 , and note that B0 ⩽A ⩽B1. If A ∈Pmin/2 p , then λ1 + (p −1)λ2 ⩾0. Thus, B0 ∈Pmin/2 p . Since Pmin/2 p and F have the same proﬁle given by (3.1) (and λ2 ⩾0), we conclude that B0 ∈F. However, B0 ⩽A proving that A ∈F. For the other inclusion, pick A ∈F. Since F ⊂eP, we have λmax ⩾0. Now A ⩽B1 implies B1 ∈F. Again F and Pmin/max p have the same proﬁle given by (3.1). Therefore, B1 ∈Pmin/max p . This implies by deﬁnition that A ∈Pmin/max p . □ This imposes a constraint on the decreasing characteristic q of F. Corollary 3.14. — The characteristics of F satisfy (p −1)(q −1) ⩾1. (3.17) (3.17) Proof. — It follows from Deﬁnition 3.6(3.9a) that if one shrinks a sube- quation, then its decreasing characteristic goes up. Thus if F has character- istic p, we have Pmin/max p ⊃F and so the decreasing characteristic q of F satisﬁes q −1 ⩾qPmin/max p −1 = 1/(p −1). □ □ Remark. — The only ST-invariant cone subequation with given charac- teristics (p, q) satisfying equality in (3.17) is Pmin/max p . This can be proved by using Proposition 3.15 below, but details are omitted here. It is just as easy to describe all examples with dual characteristic q. First note that the duals of the two subequations in (3.16) are given by ePmin/2 p : λmax(A) + (p −1)λn−1(A) ⩾0, (3.18) ePmin/max p : λmax(A) + (p −1)λmin(A) ⩾0. (3.19) (3.18) (3.19) Note that the increasing characteristics of these two subequations are both ∞, and the decreasing characteristics are p by (3.9c). Applying Proposition 3.13 to eF now yields the following result. Proposition 3.15. — Suppose that F is an ST-invariant (not necessar- ily convex) cone subequation. Then F has a ﬁnite (decreasing) Riesz char- acteristic q if and only if ePmin/max q ⊂F ⊂ePmin/2 q . F. Reese Harvey and H. Blaine Lawson F. Reese Harvey and H. Blaine Lawson F. Reese Harvey and H. Blaine Lawson For p an integer the Pp-subharmonic functions are characterized by the fact that their restrictions to minimal submanifolds of dimension p are intrinsi- cally subharmonic. For this and a discussion of the geometry associated with this equation, see [22]. (Results for integer p go back to H. Wu [37, 45].) Note, by the way, that P1 = P is the homogeneous Monge–Ampère subequation and Pn = ∆is the standard Laplacian. There are complex and quaternionic analogues PC p and PH p deﬁned by (4.2) but using the eigenvalues of the complex (respectively quaternionic) hermitian symmetric part of A = D2u. When p = 1 this yields the homo- geneous complex and quaternionic Monge–Ampère subequations. The PC p - subharmonic functions are characterized by the fact that their restrictions to complex p-dimensional submanifolds are ∆-subharmonic. The Riesz char- acteristics of PC p and PH p are 2p and 4p respectively. See Lemma 4.8 below. Example 4.2 (The Elementary Symmetric or Hessian Equations). — For each integer k, 1 ⩽k ⩽n, let σk(A) denote the kth elementary symmetric function of the eigenvalues of A ∈Sym2(Rn). The convex cone subequation Σk = {A : σ1(A) ⩾0, σ2(A) ⩾0, . . . , σk(A) ⩾0} (4.3) (4.3) has Riesz characteristic has Riesz characteristic pΣk ≡n k . (4.4) pΣk ≡n k . (4.4) These subequations, often called hessian equations, have been the focus of much study (e.g., [33, 34, 35, 42, 43, 44]). There are again complex and quaternionic analogues ΣC k and ΣH k with Riesz characteristics 2n/k and 4n/k respectively. Example 4.3 (The δ-Uniformly Elliptic Equations). — The δ-uniformly elliptic regularization of the basic subequation P ≡{A ⩾0} (cf. Exam- ple 4.10) is P (δ) ≡ A : A + δ ntr(A)I ⩾0 . (4.5) (4.5) n These are convex cone subequations with Riesz characteristic p = n(1 + δ)/ (n + δ). Given p with 1 ⩽p ⩽n and setting δ = n(p −1) n −p . (4.6) (4.6) Lemma A.2 of Part II states that P(δ) is the largest O(n)-invariant convex cone subequation with Riesz characteristic p. There are again complex and quaternionic analogues described in Example 4.7 below. Example 4.4 (Geometrically Deﬁned Subequations). — These important examples account for our choice of normalization in deﬁning the Riesz charac- teristic. denote the ordered eigenvalues of A. Example 4.1 (The p-Convexity Equation). — For each real number p with 1 ⩽p ⩽n, the smallest (see Lemma A.2 in Part II) convex cone subequation with characteristic p is also one of the most basic: Pp ≡{A : λ1(A) + · · · + λ[p](A) + (p −[p])λ[p]+1(A) ⩾0}. (4.2) Pp ≡{A : λ1(A) + · · · + λ[p](A) + (p −[p])λ[p]+1(A) ⩾0}. (4.2) (4.2) – 799 – F. Reese Harvey and H. Blaine Lawson Fix a compact subset G ⊂G(p, Rn) in the Grassmannian of p-planes in Rn, and deﬁne F(G) ≡{A : trW (A) ⩾0 for all W ∈G} (4.7) (4.7) – 800 – Tangents to subsolutions: existence and uniqueness, Part I where trW (A) denotes the trace of A W . Assuming the ST-invariance of G, the Riesz characteristic is easily seen to be (4.8) pF (G) = p. (4.8) pF (G) = p. Many interesting subequations arise this way. When G = G(p, Rn), GC(p, Cn) and GH(p, Hn) we retrieve the integer cases in Example 4.1 above. There are many other interesting examples. One such is LAG ⊂GR(n, Cn), the set of Lagrangian n-planes in Cn. Closely related are the sets of isotropic p-planes, and p-planes satisfying certain CR (Cauchy–Riemann) conditions. Also of in- terest is SLAG ⊂LAG, the special Lagrangian planes (cf. [13]). This latter is an example of a subequation associated to a calibration [16]. Other par- ticularly interesting examples come from the associative and coassociative calibrations in R7 and the Cayley calibration in R8. All the speciﬁc subequa- tions in this paragraph have the property that they are ST-invariant, i.e., invariant under a subgroup G ⊂O(n) which acts transitively on the sphere Sn−1 ⊂Rn. These geometrically deﬁned subequations will be the sole focus of Part II of this paper. These geometrically deﬁned subequations will be the sole focus of Part II of this paper. Example 4.5 (Branches of Gårding Operators). — In many of the cases above, one can associate a homogeneous polynomial operator Φ(D2u). When the polynomial Φ is Gårding hyperbolic with respect to the identity I (which is typically the case), the equation has many branches [10, 15, 21]. The simplest case is P = P1 where the operator is Φ(A) = detR(A). Here the branches are given by {λk(A) ⩾0} (see (4.1)). Unfortunately, in this case the branches for k > 1 have inﬁnite characteristic. For the general Gårding polynomial Φ(A) of degree m, there are ordered eigenvalues, Λ1(A) ⩽Λ2(A) ⩽· · · ⩽Λm(A), and Φ(A) = Λ1(A) · · · Λm(A). (4.1 Λ1(A) ⩽Λ2(A) ⩽· · · ⩽Λm(A), and Φ(A) = Λ1(A) · · · Λm(A). (4.1′) (4.1′) Just as with detR(A), the kth branch is deﬁned by {Λk(A) ⩾0} for k = 1, . . . , m. F. Reese Harvey and H. Blaine Lawson The Riesz characteristics p1 ⩽· · · ⩽pm of these respective branches are determined by the eigenvalues of Pe (assuming ST-invariance). They are exactly the numbers 1/Λj(Pe), j = 1, . . . , m arranged in increasing order (see Proposition A.10 in Part II). Therefore the number of branches with ﬁnite Riesz characteristic equals the number of non-zero eigenvalues of Pe. Only the ﬁrst and smallest branch is convex, and it is uniformly elliptic ⇐⇒all branches are uniformly elliptic ⇐⇒Φ(Pe) > 0. Gårding operators are plentiful. For instance, in each of our ﬁrst three examples there is an associated Gårding operator, and hence each comes equipped with branches. To illustrate, for the case where p is an integer in – 801 – F. Reese Harvey and H. Blaine Lawson Example 4.1, we have Φ(A) = Y i1<···<ip (λi1(A)+· · ·+λip(A)) = det (DA : ΛpRn −→ΛpRn) . (4.9) (4.9) Said diﬀerently, ΛI(A) = λi1(A) + · · · + λip(A) are the eigenvalues. Here DA is the extension of A as a derivation. The kth branch is given by requiring that the kth ordered p-fold sum of the λi’s be ⩾0. One easily computes that the ﬁrst n−1 p−1 branches have Riesz characteristic p and the remaining branches have inﬁnite characteristic. In Example 4.2 the Gårding operator is Φ(A) = σk(A). Although the eigenvalues Λj(A) of Φ do not have an explicit formula in terms of the standard eigenvalues of A, the eigenvalues of A = Pe are all zero except for one which equals k/n. Hence, Σk has characteristic n/k and all other branches have characteristic ∞. In Example 4.3 the eigenvalues are Λk(A) = λk(A) + δ n(1 + δ)tr(A), k = 1, . . . , n. Hence, each of the kth branches {Λk(A) ⩾0}, for k ⩾2, has the same Riesz characteristic p = n(1 + 1 δ ), which is ﬁnite but larger than n, while as noted above, the ﬁrst branch P(δ) has characteristic n(1 + δ)/(n + δ). Hence, each of the kth branches {Λk(A) ⩾0}, for k ⩾2, has the same Riesz characteristic p = n(1 + 1 δ ), which is ﬁnite but larger than n, while as noted above, the ﬁrst branch P(δ) has characteristic n(1 + δ)/(n + δ). Example 4.6 (Trace Powers of the Hessian). F. Reese Harvey and H. Blaine Lawson — Consider the non-convex cone subequation F ≡{A : tr (Aq) ⩾0} where q > 1 is an odd integer. More generally one could deﬁne Aq for any q > 0 by using the function tq for t ⩾0 and −\|t\|q for t < 0. In all cases one computes that the Riesz characteristic is pF = 1 + (n −1) 1 q . More generally, for k ∈[1, n] and q > 0 real numbers, there is the subequation F ≡{A : λq 1(A) + · · · + λq [k](A) + (k −[k])λq [k]+1 ⩾0} y, ∈[ , ] q > 0 , F ≡{A : λq 1(A) + · · · + λq [k](A) + (k −[k])λq [k]+1 ⩾0} with tq deﬁned as above. Here the Riesz characteristic is with tq deﬁned as above. Here the Riesz characteristic is pF = 1 + (k −1) 1 q . Example 4.7 (Complex and Quaternionic Analogues). — Suppose F ⊂Sym2(Rn) is an O(n)-invariant subequation. Then F can be deﬁned by the constraint set E ⊂Rn imposed by F on the eigenvalues λ(A) = (λ1(A), . . . , λn(A)). Thus A ∈F ⇐⇒λ(A) ∈E. The equation F has com- plex and quaternionic analogues F C and F H, deﬁned on Cn = (R2n, J) and – 802 – Tangents to subsolutions: existence and uniqueness, Part I Hn = (R4n, I, J, K) respectively, as follows. For A ∈Sym2(R2n) consider the hermitian symmetric part AC ≡1 2(A −JAJ) whose eigenspaces are complex lines with ordered eigenvalues λ1(AC) ⩽· · · ⩽ λn(AC). One now deﬁnes F C by applying the eigenvalue constraints E of F to these eigenvalues of AC. The story in the quaternionic case is parallel and uses the quaternionic hermitian symmetric part AH ≡1 4(A−IAI −JAJ −KAK) and eigenvalues λk(AH). whose eigenspaces are complex lines with ordered eigenvalues λ1(AC) ⩽· · · ⩽ λn(AC). One now deﬁnes F C by applying the eigenvalue constraints E of F to these eigenvalues of AC. The story in the quaternionic case is parallel and uses the quaternionic hermitian symmetric part AH ≡1 4(A−IAI −JAJ −KAK) and eigenvalues λk(AH). Lemma 4.8. — If F is an O(n)-invariant cone subequation with Riesz characteristic p, then the Riesz characteristics of F C and F H are pF C = 2p and pF H = 4p. 5. Kp-Convexity and Monotonicity In this section we give a fairly complete discussion of the classical one- variable results that underlie this paper. They concern the properties of subsolutions to the one-variable subequation Rp introduced below. In the following section we will prove that associated to each F-sub- harmonic function u there are three functions of r (denoted M(r), S(r) and V (r)), which are subsolutions of Rp, and which capture much of the asymptotic behavior of u. By Lemma 5.1(3) below this will imply the key double monotonicity result, Theorem 6.4, which is needed for deﬁning the notion of density and for proving our main theorems. Fix a real number p with 1 ⩽p < ∞, and for r > 0 consider the one- variable Riesz kernel Kp(r) ≡ 1 (2 −p)r2−p if p ̸= 2 and K2(r) = log r. (5.1) (5.1) With this normalization K′ p(r) = 1 rp−1 for all 1 ⩽p < ∞. Note that Kp(r) is a strictly increasing solution to the subequation Rp : ψ′′(r) + p −1 r ψ′(r) ⩾0 on (0, ∞). (5.2a) (5.2a) Alternatively, Rp : d dr rp−1ψ′(r) = d dr ψ′(r) K′p(r) ⩾0 on (0, ∞). (5.2b) All solutions of Rp are of the form All solutions of Rp are of the form h(r) ≡CKp(r) + k with C, k ∈R. (Riesz Harmonics) (5.3) Note that h(r) is increasing if and only if C ⩾0. h(r) ≡CKp(r) + k with C, k ∈R. (Riesz Harmonics) (5.3) Note that h(r) is increasing if and only if C ⩾0. (5.3) The change of variables s = Kp(r) along with its inverse r = K−1 p (s) (5.4) s = Kp(r) along with its inverse r = K−1 p (s) (5.4) (5.4) p play an important role. The inverse r(s) = K−1 p (s) is deﬁned on the range of Kp which is the interval (0, ∞) when 1 ⩽p < 2, all of R when p = 2, and (−∞, 0) for 2 < p < ∞. play an important role. The inverse r(s) = K−1 p (s) is deﬁned on the range of Kp which is the interval (0, ∞) when 1 ⩽p < 2, all of R when p = 2, and (−∞, 0) for 2 < p < ∞. Lemma 5.1 (The Equivalences). F. Reese Harvey and H. Blaine Lawson pF C = 2p and pF H = 4p. Proof. — We consider the complex case. If A = Pe⊥−(p −1)Pe ∈ Sym2(R2n), then one computes that AC = PCe⊥− p 2 −1 PCe and AH = PHe⊥− p 4 −1 PHe (4.10) which displays the eigenvalues of AC and AH. □ AC = PCe⊥− p 2 −1 PCe and AH = PHe⊥− p 4 −1 PHe (4.10) which displays the eigenvalues of AC and AH. □ □ which displays the eigenvalues of AC and AH. which displays the eigenvalues of AC and AH. Example 4.9 (The Subequation Determined by a Gårding Operator and a Universal Eigenvalue Constraint). — The procedures above can be greatly generalized. Note, to begin, that given an O(m)-invariant subequation F, the eigenvalue set E ≡λ(F) is closed, invariant under permutation of coordi- nates and Rm +-monotone. Conversely, any such eigenvalue set E determines an O(m)-invariant subequation F = λ−1(E). Each such E is a universal eigenvalue subequation in the sense that, for each degree-m Gårding oper- ator Φ on Sym2(Rn), the set F ≡λ−1 Φ (E) is a subequation on Rn, where λΦ : Sym2(Rn) →Rm is the eigenvalue map associated to Φ. See Proposi- tion A.8 in [23] for the details and further discussion. Example 4.10 (The δ-Uniformly Elliptic Regularization of a Subequation). Given a cone subequation F ⊂Sym2(Rn) and δ > 0, deﬁne F(δ) ≡ A : A + δ ntr(A)I ∈F . (4.11) (4.11) This equation satisﬁes the uniformly elliptic condition: This equation satisﬁes the uniformly elliptic condition: F(δ) + P(δ) ⊂F(δ). (4.12) (4.12) One computes that One computes that F has Riesz characteristic p ⇐⇒F(δ) has Riesz characteristic pn(1 + δ) n + δp . F has Riesz characteristic p ⇐⇒F(δ) has Riesz characteristic pn(1 + δ) n + δp . F has Riesz characteristic p ⇐⇒F(δ) has Riesz characteristic pn(1 + δ) n + δp . – 803 – F. Reese Harvey and H. Blaine Lawson F. Reese Harvey and H. Blaine Lawson T (2) (Kp- of va of s. (3) (Kp-M (4) (Rp-C the in Proof. — is smooth. Th ψ′(r) = f For general ψ ϕ(r(s)) is a te smooth case. convexity (see Now (3) is f(s) ≡ψ(r(s tion (4) is ju comparison w Corollar ma 5.1. Then (1) the fu (2) the le Proof. — ψ(r(s)) with function f. Densities The rema function ψ(r) the Hypothe 5 1 Then (1) the function ψ(r) is locally Lipschitz continuous, (2) the left and right hand derivatives ψ′ ±(r) exist. (1) the function ψ(r) is locally Lipschitz continuous, (2) the left and right hand derivatives ψ′ ±(r) exist. Proof. — The corresponding statements for the function f(s) ≡ ψ(r(s)) with r(s) = K−1(s) are standard classical facts about the convex function f. □ T (2) (Kp- of va of s. (3) (Kp-M (4) (Rp-C the in Proof. — is smooth. Th ψ′(r) = f For general ψ ϕ(r(s)) is a te smooth case. convexity (see Now (3) is f(s) ≡ψ(r(s tion (4) is ju comparison w Corollar ma 5.1. Then (1) the fu (2) the le Proof. — ψ(r(s)) with function f. Densities The rema function ψ(r) the Hypothe 5 1 Tangents to subsolutions: existence and uniqueness, Part I (2) (Kp-Convexity) ψ(r) is Kp-convex, meaning that under the change of variables (5.4)), the function f(s) ≡ψ(r(s)) is a convex function of s. f (3) (Kp-Monotonicity) ψ(r)−ψ(t) Kp(r)−Kp(t) is non-decreasing in r and t (r ̸= t). (3) (Kp-Monotonicity) ψ(r)−ψ(t) Kp(r)−Kp(t) is non-decreasing in r and t (r ̸= t). (4) (R C i ) If ψ( ) ⩽CK ( ) + k f d t th (3) (Kp-Monotonicity) ψ( ) ψ( ) Kp(r)−Kp(t) is non-decreasing in r and t (r ̸= t). (4) (Rp-Comparison) If ψ(r) ⩽CKp(r) + k for r = s and r = t, then the inequality holds for all r between s and t. p( ) p( ) (4) (Rp-Comparison) If ψ(r) ⩽CKp(r) + k for r = s and r = t, then the inequality holds for all r between s and t. p p (4) (Rp-Comparison) If ψ(r) ⩽CKp(r) + k for r = s and r = t, then the inequality holds for all r between s and t. Proof. — Now f(s) ≡ψ(r(s)) implies ψ(r) = f(Kp(r)). First, assume ψ is smooth. Then ψ′(r) = f ′(s)K′ p(r) and hence d dr ψ′(r) K′p(r) = f ′′(s)K′ p(r). (5.5) (5.5) For general ψ, the fact that: ϕ(r) is a test function for ψ at r0 if and only if ϕ(r(s)) is a test function for ψ(r(s)) at s0 ≡s(r0), reduces the proof to the smooth case. Since viscosity convexity f ′′(s) ⩾0 is equivalent to classical convexity (see for example, [14, Prop. 2.6]), this proves that (1) ⇐⇒(2). For general ψ, the fact that: ϕ(r) is a test function for ψ at r0 if and only if ϕ(r(s)) is a test function for ψ(r(s)) at s0 ≡s(r0), reduces the proof to the smooth case. Since viscosity convexity f ′′(s) ⩾0 is equivalent to classical convexity (see for example, [14, Prop. 2.6]), this proves that (1) ⇐⇒(2). Now (3) is just monotonicity of the slopes of secant lines to the function f(s) ≡ψ(r(s)), and hence it is equivalent to the convexity of f(s). Asser- tion (4) is just the statement that f(s) is convex if and only if f satisﬁes comparison with aﬃne functions Cs + k. □ Corollary 5.2. — Let ψ(r) satisfy the equivalent conditions in Lem- ma 5.1. 5. Kp-Convexity and Monotonicity — The following conditions on an up- per semi-continuous function ψ(r), deﬁned on a subinterval of (0, ∞), are equivalent. (1) (Rp-Subharmonic) ψ(r) satisﬁes the subequation Rp deﬁned by (5.2). – 804 – Densities The remainder of this section is devoted to describing properties of a function ψ(r), deﬁned on an interval (0, r0) (with r0 = ∞possible), under the Hypothesis 5.3. — ψ satisﬁes the equivalent conditions (1)–(4) of Lem- ma 5.1. The Properties (1) and/or (3) enable us to introduce the following. – 805 – F. Reese Harvey and H. Blaine Lawson F. Reese Harvey and H. Blaine Lawson F. Reese Harvey and H. Blaine Lawson Corollary 5.4 (Existence of Densities). — The decreasing limits 5.4 (Existence of Densities). — The decreasing limits Corollary 5.4 (Existence of Densities). — The decreasing limits Θψ = lim r,t→0 t>r>0 ψ(t) −ψ(r) K(t) −K(r) = lim r→0 ψ′ ±(r) K′(r) (5.6) (5.6) exist and deﬁne the density Θψ. Moreover, if ψ is increasing, then 0 ⩽Θψ<∞. Proof. — To see that the two decreasing limits in (5.6) agree divide the numerator and denominator of ψ(r+δ)−ψ(r) K(r+δ)−K(r) by δ and let δ →0. □ In this one-variable context, rather than in its later applications, it might be better to call this “the derivative of ψ(r(s)) at r = 0”. Note that the monotonicity quotient in (3) remains unchanged if ψ is replaced by a translate ψ −c with c ∈R. In particular, the densities of u and u −c are the same. This point is critical in establishing the following. Lemma 5.5. — If ψ is increasing, then there exists c ∈R and r0 such that ψ(r) −c K(r) decreases to Θψ as 0 < r < r0 decreases down to 0. ψ(r) −c K(r) decreases to Θψ as 0 < r < r0 decreases down to 0. Moreover, ψ(r) −ψ(0) K(r) decreases to Θψ if 1 ⩽p < 2, and (5.7a) decreases to Θψ if 1 ⩽p < 2, and (5.7a) lim r→0 ψ(r) K(r) = Θψ if 2 ⩽p < ∞. (5.7b) (5.7b) (Note: if we set ψ(0) = 0 when 1 ⩽p < 2, then we have in all cases (1 ⩽p < ∞) that limr→0 ψ(r)/K(r) = Θψ.) (Note: if we set ψ(0) = 0 when 1 ⩽p < 2, then we have in all cases (1 ⩽p < ∞) that limr→0 ψ(r)/K(r) = Θψ.) Proof. — For any value of p, 1 ⩽p < ∞, there is exactly one point in[0, ∞] where K vanishes. Densities Since the graph of a(s) is a supporting line for the epigraph of f over (−∞, s0), this extension f is convex and increasing on (−∞, 0]. Observe now that by translating our original ψ by a suitable additive constant, we can insure that f < 0 on (−∞, 0]. Now set ψ(t) ≡f(K(t)), where 0 < t ⩽1 if p = 2 and 0 < t ⩽∞if 2 < p. Finally, by (5.7b′) where ψ(1) is ﬁnite, and (5.7c′) where ψ(∞) is ﬁnite, the fact that K(0) = −∞ implies that limr→0 ψ(r)/K(r) = Θψ. □ Remark 5.6. — The subequation Rp : ψ′′ + p−1 r ψ′(r) ⩾0 is linear and could have been interpreted in the distributional sense as well as the viscosity sense. Densities However there are three cases: K(0) = 0 if 1 ⩽p < 2, K(1) = 0 if p = 2, and K(∞) = 0 if 2 < p. First let us suppose that the function ψ is deﬁned and ﬁnite on an interval containing the point where K vanishes. Then one can take r in (5.6) to be that point, and the Proposition follows immediately from the double monotonicity in (3) of Lemma 5.1. Speciﬁcally, in the three cases we obtain that: (1⩽p < 2) ψ(t)−ψ(0) K(r) decreases to Θψ as 0 < t < r0 decreases to 0, (5.7a′) (p = 2) ψ(t)−ψ(1) K(t) decreases to Θψ as 0 < t < r0 decreases to 0, (5.7b′) (2 < p) ψ(t)−ψ(∞) K(t) decreases to Θψ as 0 < t < r0 decreases to 0. (5.7c′) (1⩽p < 2) ψ(t)−ψ(0) K(r) decreases to Θψ as 0 < t < r0 decreases to 0, (5.7a′) (p = 2) ψ(t)−ψ(1) K(t) decreases to Θψ as 0 < t < r0 decreases to 0, (5.7b′) (2 < p) ψ(t)−ψ(∞) K(t) decreases to Θψ as 0 < t < r0 decreases to 0. (5.7c′) – 806 – Tangents to subsolutions: existence and uniqueness, Part I This leaves us with an extension problem in the last two cases. Namely we must prove that there exists an r0 > 0 such that the restriction of the given ψ to (0, r0) (p = 2) has an extension ψ to (0, 1] satisfying Lemma 5.1(3), and (5.8b) (2 < p) has an extension ψ to (0, ∞) satisfying Lemma 5.1(3) with ψ(∞) < ∞. (5.8c) (5.8c) Suppose that ψ has domain containing (0, r0] (and if p = 2 that r0 < 1 since if r0 ⩾1 in this case we are ﬁnished.) Make the change of variables s0 ≡K−1(r0) as in Lemma 5.1(2). Since 2 ⩽p < ∞, we have s0 < 0. The convex increasing function f(s) on (−∞, s0) can be extended to a convex increasing function f on (−∞, 0] by deﬁning f to be the aﬃne function a(s) ≡f ′ (s0)(s −s0) + f(s0) (5 9) a(s) ≡f ′ −(s0)(s −s0) + f(s0) (5.9) (5.9) on s0 ⩽s ⩽0. Since the graph of a(s) is a supporting line for the epigraph of f over (−∞, s0), this extension f is convex and increasing on (−∞, 0]. on s0 ⩽s ⩽0. 6. Monotonicity and Stability of Averages for F-Subharmonic Functions In this section we discuss three of the basic ways of taking an average of an F-subharmonic function, and show that each average produces a radial F-subharmonic. Since the radial F-subharmonics are just one-variable Rp- subharmonics (Proposition 3.3), they are well understood and enjoy all the properties of Lemma 5.1. In particular, they satisfy the double monotonicity described in Theorem 6.4 below, which provides the vehicle for deﬁning the densities explored in the next section. Finally, the stability of these averages under the tangential ﬂow is established in Lemma 6.5. We assume as always that the subequation F is an ST-invariant cone with invariance group G ⊂O(n). We further assume that the Riesz charac- teristic p of F is ﬁnite. This is because when p = ∞, the increasing radial – 807 – F. Reese Harvey and H. Blaine Lawson subequation R↑ F is simply g′(t) ⩾0 (Proposition 3.3 and Deﬁnition 3.1). Thus, when p = ∞, all increasing functions g(t) determine increasing radial subharmonics g(\|x\|), and no sensible notion of density is possible. subequation R↑ F is simply g′(t) ⩾0 (Proposition 3.3 and Deﬁnition 3.1). Thus, when p = ∞, all increasing functions g(t) determine increasing radial subharmonics g(\|x\|), and no sensible notion of density is possible. To begin we set some notation. Let Br(x0) = {x : \|x −x0\| ⩽r} denote the ball of radius r about x0, and set Sr(x0) ≡∂Br(x0). Let A(a, b; x0) ≡ {x : a < \|x −x0\| < b} denote an annular region centered at x0. Here and elsewhere, when x0 = 0, reference to it will be dropped from the notation. Thus, Br = Br(0) and Sr = ∂Br. Similarly we set B = B1 and S = ∂B. The ﬁrst average only requires that F be an ST-invariant cone (not nec- essarily convex). We denote the (spherical) maximum for an F-subharmonic function u deﬁned on a region containing Sr(x0) by M(u, x0; r) ≡sup S u(x0 + rx), (6.1a) (6.1a) Note that if u is F-subharmonic on BR(x0), then by the maximum principle M(u, x0; r) = sup B u(x0 + rx) = sup Br(x0) u (6.1b) M(u, x0; r) = sup B u(x0 + rx) = sup Br(x0) u and hence is increasing for 0 ⩽r ⩽R. (6.1b) (6.1b) By the ST-invariance of F By the ST-invariance of F M(u, x0; \|x\|) ≡sup g∈G u(x0 + gx). (6.2) (6.2) We now simplify by setting x0 = 0 and using the abbreviated notation M(r) ≡M(u; r) = M(u, 0; r) when the meaning is obvious. Lemma 6.1. — If u is F-subharmonic on an annular region A(a, b), then M(\|x\|) is a radial F-subharmonic function on A(a, b). If u is F-subharmonic on BR, then M(r) is also increasing in r. Proof. — Let ug(x) ≡u(gx) with g ∈G. Then M(\|x\|) = supg∈G ug(x). Since F is G-invariant, each ug is F-subharmonic. Therefore, by the standard “families locally bounded above” property for F, it suﬃces to prove that M(t) is upper semi-continuous. This is done as follows. For each δ > 0, Nδ ≡{x : u(x) < M(t)+δ} is an open set containing ∂Bt. Hence the annulus A(t −ϵ, t + ϵ) is contained in Nδ for ϵ > 0 small. Thus M(r) < M(t) + δ if t −ϵ ⩽r ⩽t + ϵ, proving that M(t) is upper semi-continuous, and hence M(\|x\|) is F-subharmonic. □ For the other averages we make the further standing assumption that F is convex. In this case we note the following. F is an ST-invariant convex cone ⇒ F ⊂∆≡{tr(A) ⩾0}. (6.3) (6.3) – 808 – Tangents to subsolutions: existence and uniqueness, Part I Tangents to subsolutions: existence and uniqueness, Part I Proof. — If F ∩{tr(A) = c < 0} is non-empty, then invariance plus convexity implies that −c nI ∈F. Now by the cone property, −λI ∈F for all λ > 0. This along with positivity implies that F = Sym2(Rn). Since tr(Pe⊥−(p −1)Pe) = n −p, the condition F ⊂{tr A ⩾0} implies pF ⩽n. Therefore, F is an ST-invariant convex cone ⇒ 1 ⩽pF ⩽n. (6.4) F is an ST-invariant convex cone ⇒ 1 ⩽pF ⩽n. (6.4) (6.4) We now deﬁne the spherical and volume averages of u at x0 by S(u, x0; r) ≡1 \|S\| ˆ σ∈S u(x0 + rσ) dσ ≡ S u(x0 + rσ) dσ, (6.5a) V (u, x0; r) ≡ 1 \|B\| ˆ x∈B u(x0 + rx) dx ≡ B u(x0 + rx) dx. By the ST-invariance of F (6.5b) (6.5b) Note that for any upper semi-continuous function u, each of these func- tions is jointly upper semi-continuous in (x0, r) since u(x0 + rx) is the inﬁ- mum of ϕ(x0 + rx) taken over continuous functions ϕ ⩾u. □ Lemma 6.2. — Suppose that u is F-subharmonic on the annulus A(a, b). Then S(u; \|x\|) is a radial F-subharmonic on A(a, b). If u is F-subharmonic on the ball BR, then both S(u; \|x\|) and V (u; \|x\|) are increasing radial F- subharmonic functions on BR (with limiting values S(u, 0) = V (u, 0) = u(0) at x = 0). Proof. — As noted above S(u; r) and V (u; r) are upper semi-continuous in r, and hence so are the functions S(u; \|x\|) and V (u; \|x\|) of x deﬁned on BR. The statement about their limiting values at x = 0 is a standard fact about ∆-subharmonic functions. It remains to show that S(u; \|x\|) and V (u; \|x\|) are F-subharmonic on BR. Note that S(\|x\|) = ˆ G u(gx) dg (6.6) (6.6) for a suitably normalized invariant measure dg on G, and that V (\|x\|) = n ˆ 1 0 S(ρ\|x\|)ρn−1 dρ since \|B\| = 1 n\|S\|. (6.7) (6.7) To prove (6.7), set \|x\| = r and compute V (r) = 1 \|B\| ´ B u(ry) dy using polar coordinates. Now since F is a convex cone subequation, averages such as (6.6) and (6.7) preserve F-subharmonicity. This is explained further by Theorem 9.5 and Remark 9.6. □ y □ Remark 6.3. — By Theorem 2.4 and Theorem 2.7, the lemmas above could have been restated by concluding that the functions M(r), S(r) and V (r) are R↑ F -subharmonic on (0, R), or Rp-subharmonic on (a, b) in the annular cases. – 809 – F. Reese Harvey and H. Blaine Lawson The properties of upper semi-continuous functions ψ(r) satisfying Rp have been presented in detail in Section 5. We make full use of those results by applying them to the three functions M(u, x0, r), S(u, x0, r) and V (u, x0, r), where u is an F-subharmonic function. This includes the Kp-convexity, the Kp-monotonicity and the Rp-comparison properties of Lemma 5.1. In particular, the Kp-monotonicity, part (3) of Lemma 5.1, gives the following basic result. Theorem 6.4 (Double Monotonicity). — Let u be F-subharmonic in an annular region about the origin in Rn. Then M(u, r) −M(u, s) K(r) −K(s) is increasing in r and s. (1.5) (1.5) for all 0 < s < r where M is deﬁned. for all 0 < s < r where M is deﬁned. for all 0 < s < r where M is deﬁned. Furthermore, if F is convex, the same statement holds with M(u, r) re- placed by S(u, r); or by V (u, r) provided that u is F-subharmonic on a ball about the origin. It is an important fact that each of these averages is stable under limits in L1. This basic classical fact can be found in [26, §III.3.2]. We state it here in slightly diﬀerent form needed later for tangents. Lemma 6.5 (Stability of Averages). — Suppose uj is a sequence of ∆- subharmonic functions on BR converging in L1(BR) to a ∆-subharmonic function U. Then for 0 < r < R, (1) M(U, r) = limj→∞M(uj, r), (2) S(U, r) = limj→∞S(uj, r), (2) S(U, r) = limj→∞S(uj, r), (3) V (U, r) = limj→∞V (uj, r), (3) V (U, r) = limj→∞V (uj, r), Proof. — Taking K ≡Br in (3.2.7) of Theorem 3.2.1 in [26] gives us that lim sup j→∞ M(uj, r) ⩽M(u, r). Proof. — Taking K ≡Br in (3.2.7) of Theorem 3.2.1 in [26] gives us that lim sup j→∞ M(uj, r) ⩽M(u, r). Proof. — Taking K ≡Br in (3.2.7) of Theorem 3.2.1 in [26] gives us that lim sup j→∞ M(uj, r) ⩽M(u, r). Suppose there exists C < M(u, r) such that M(uj, r) ⩽C for all j suﬃ- ciently large. Then in the L1-limit we would have u −C ⩽0 a.e. on Br. However, for ∆-subharmonic functions, this implies that u −C ⩽0 ev- erywhere on Br, contrary to the deﬁnition of M(u, r). We conclude that lim supj→∞M(uj, r) = M(u, r). The fact that this is also true for all subse- quences proves (1). As discussed in the paragraph prior to Proposition 3.2.14 in [26], the Theorem 3.2.13 can be applied to spherical measure σr on ∂Br. Thus ujσr – 810 – Tangents to subsolutions: existence and uniqueness, Part I converges to Uσr in the weak topology of measures, yielding (2). Finally, (3) is implied directly by the hypothesis of L1(Br)-convergence. □ converges to Uσr in the weak topology of measures, yielding (2). Finally, (3) is implied directly by the hypothesis of L1(Br)-convergence. □ 7. Densities for F-Subharmonic Functions: Upper Semi-Continuity From the results of the last section and Corollary 5.4 we have three densities, ΘM(u, x), ΘS(u, x) and ΘV (u, x) ΘM(u, x), ΘS(u, x) and ΘV (u, x) associated to an F-subharmonic function u deﬁned in a neighborhood of the origin. For the second two densities, we must assume that F is convex. Under this convexity assumption there exists a fourth , even more classical density. The Mass Density Note that by (6.3) u is classically ∆-subharmonic. Thus ∆u is a measure µ ⩾0, which means ∆u has a “mass density”. Given a measure µ ⩾0 deﬁned in a neighborhood of a point x0 ∈Rn, and 0 < k ⩽n, the limit Θk(µ, x0) ≡lim r↓0 µ (Br(x0)) α(k)rk , (7.1) (7.1) if it exists, is called the k-dimensional mass density of µ at x0. (See, for example, [9, 2.10.19] for discussion and deﬁnition of the constants α(k).) When k is an integer, α(k) = \|Bk\|, the volume of the unit ball in Rk. Suppose Θk(µ, x) exists everywhere or replace lim by lim sup in (7.1). Fix an open set X, a constant c > 0, and deﬁne Ec ≡{x ∈X : Θk(µ, x) ⩾c}. Then the Hausdorﬀk-measure satisﬁes (cf. [39, p. 11]) cHk(Ec) ⩽µ(X). cHk(Ec) ⩽µ(X). Comparing Densities The next proposition states that: All densities but ΘM “agree”, where “agree” means “are equal up to universal factors”. Proposition 7.1. — Suppose that u is F-subharmonic near x0 where F is convex with characteristic p, and set µ = ∆u. Then when p ̸= 2, ΘS(u, x0) = n −p + 2 n ΘV (u, x0) = α(n −p) n\|p −2\|α(n)Θn−p(µ, x0), (7.2) (7.2) – 811 – F. Reese Harvey and H. Blaine Lawson d when p = 2 we have that ΘS(u, x0) = ΘV (u, x0) = α(n −2) nα(n) Θn−2(µ, x0). (7.3) and when p = 2 we have that ΘS(u, x0) = ΘV (u, x0) = α(n −2) nα(n) Θn−2(µ, x0). (7.3) (7.3) The discussion of all densities is completed by showing that the maximum density and the spherical density are in general “comparable”, and in fact equal when p = 2. Proposition 7.2. — Suppose that u is F-subharmonic near x0 where F is convex and of characteristic p. Then there exists a constant C = C(p, n) >1 such that ΘM(u, x0) ⩽ΘS(u, x0) ⩽CΘM(u, x0) if 2 < p < ∞, and (7.4) ΘS(u, x0) ⩽ΘM(u, x0) ⩽CΘS(u, x0) if 1 < p < 2, while (7.5) ΘM(u, x0) = ΘS(u, x0) if p = 2. (7.6) ΘM(u, x0) ⩽ΘS(u, x0) ⩽CΘM(u, x0) if 2 < p < ∞, and (7.4) ΘS(u, x0) ⩽ΘM(u, x0) ⩽CΘS(u, x0) if 1 < p < 2, while (7.5) ΘM(u, x0) = ΘS(u, x0) if p = 2. (7.6) (7.6) Remark 7.3. — Kiselman proved the equality in (7.6) in the plurisubhar- monic case where F = PC on Cn (see [29, p. 161, line 2ﬀ.]) by using Harnack’s Inequality for ∆-subharmonic functions. The same proof works for any con- vex F of characteristic p = 2. Note that for p = 1 the left inequality in (7.5) holds but the right inequality fails, even for linear functions. Proof of Proposition 7.1. — We give the proof of the ﬁrst equality for all p using (6.7). Taking x0 = 0 and dropping u and x0 from the notation, it says that V (r) = n ˆ 1 0 S(rt)tn−1 dt. (7.7) (7.7) Hence, we have V (r) K(r) = n ˆ 1 0 S(rt) K(rt) K(rt) K(r) tn−1 dt. Comparing Densities When p ̸= 2, K(rt)/K(r) = 1/tp−2, so that letting r ↓0 and integrating yields the ﬁrst equality in (7.2). When p = 2, When p ̸= 2, K(rt)/K(r) = 1/tp−2, so that letting r ↓0 and integrating yields the ﬁrst equality in (7.2). When p = 2, K(rt) K(r) = 1 + log t log r , K(rt) K(r) = 1 + log t log r , K(rt) K(r) = 1 + log t log r , so letting r ↓0 and integrating yields the ﬁrst equality ΘV (u) = ΘS(u) in (7.3). so letting r ↓0 and integrating yields the ﬁrst equality ΘV (u) = ΘS(u) in (7.3). For the proof of the second equalities we show that the mass density Θn−p(µ) (µ = ∆u) is the desired multiple of the spherical density ΘS(u). Recall the classical fact that µ(Br) = (n −2)\|S\| S′ −(r) K′n(r) . (7.8) (7.8) – 812 – Tangents to subsolutions: existence and uniqueness, Part I (See in [26, Thm. 3.2.16, (3.2.13)′] for a proof.) Since n −2 K′n(r) = rn−1 = \|p −2\|rn−p K′p(r) when p ̸= 2, we have rp−nµ(Br) = \|p −2\|\|S\| S′ −(r) K′n(r) when p ̸= 2. (7.8′) (7.8′) If p = 2, this holds with \|p −2\| replaced by 1. Finally, letting r ↓0 and using (5.6) completes the proof. □ Proof of Proposition (7.2). — For simplicity let x0 = 0. Note that for all p and r we have S(u, r) ⩽M(u, r). On the other hand, K(r) < 0 when p ⩾2 and K(r) > 0 when p < 2. Dividing by K(r) and letting r ↓0 then gives the inequalities on the left as well as the inequality ΘM(u) ⩽ΘS(u) when p = 2 (since u and u + c have the same density, we can assume that u(0) = 0 when p < 2.) The remainder of the proof is a consequence of Harnack’s inequality. The standard form of this inequality is for a function v ⩽0 which is ∆- subharmonic on Bρ. It says, with ϕ deﬁned by ϕ(λ) ≡ 1 −λ (1 + λ)n−1 for 0 < λ < 1, that M(v, λr) ⩽ϕ(λ)S(v, r) for all 0 < r ⩽ρ. (7.9) M(v, λr) ⩽ϕ(λ)S(v, r) for all 0 < r ⩽ρ. (7.9) (7.9) (See, for example [7, Prop. Comparing Densities Using the fact that K(λr) = λ2−pK(r) and letting r ↓0 gives Using the fact that K(λr) = λ2−pK(r) and letting r ↓0 gives ψ(λ)ΘM(u, 0) ⩽ΘS(u, 0) with ψ(λ) = 1 + (1 + λ)n−1 1 −λ (λ2−p −1). Now direct calculation shows that limλ↓0 ψ′(λ) = ∞, and so c ≡ sup0<λ<1 ψ(λ) > 0. This gives the desired result with C = 1/c. Now direct calculation shows that limλ↓0 ψ′(λ) = ∞, and so c ≡ sup0<λ<1 ψ(λ) > 0. This gives the desired result with C = 1/c. It remains to prove that ΘS(u) ⩽ΘM(u) when p = 2. Set λ = 1/e in (7.11) and note the fact that K(r) = log r = log r e + 1 = K(λr) + 1 = K(λr)(1 + o(r)). Then taking the limit as r →0 in (7.11) yields 0 = ΘM(u) −ΘM(u) ⩽ΘS(u) −ΘM(u) by Corollary 5.4. This completes the proof of Proposition 7.2. □ Comparing Densities 4.2.2].) For an arbitrary ∆-subharmonic function v, the function v −M(v, r) is ⩽0 on Br. Hence, (7.9) gives the following more general form of Harnack’s inequality M(v, λr) −M(v, r) ⩽ϕ(λ) S(v, r) −M(v, r) for all 0 < r ⩽ρ. (7.10 for functions not necessarily ⩽0. M(v, λr) −M(v, r) ⩽ϕ(λ) S(v, r) −M(v, r) for all 0 < r ⩽ρ. (7.10) for functions not necessarily ⩽0. M(v, λr) −M(v, r) ⩽ϕ(λ) S(v, r) −M(v, r) for all 0 < r ⩽ρ. (7.10) for functions not necessarily ⩽0. Suppose ﬁrst that p > 2. We may assume u(0) = −∞since otherwise the assertion is trivial. Then u is negative near 0, and we can apply the standard form (7.9)of Harnack’s inequality to obtain M(u, λr) K(λr) ⩾λp−2ϕ(λ)S(u, r) K(r) . Letting r ↓0 gives ΘM(u, 0) ⩾cΘS(u, 0) where c = λp−2ϕ(λ) > 0. This gives (7.4) with C = 1/c. (Note that c ≡supλ λp−2ϕ(λ) provides the best constant C.) Letting r ↓0 gives ΘM(u, 0) ⩾cΘS(u, 0) where c = λp−2ϕ(λ) > 0. This gives (7.4) with C = 1/c. (Note that c ≡supλ λp−2ϕ(λ) provides the best constant C.) Suppose now that 1 < p < 2. Replace u by u(x) −u(0) so that u(0) = 0. Since densities are unchanged by adding a constant, we have ΘM(u, 0) = limr↓0 M(u, r)/K(r) and ΘS(u, 0) = limr↓0 S(u, r)/K(r) by Corollary 5.4. – 813 – F. Reese Harvey and H. Blaine Lawson Since u may not be ⩽0, we must use the general form (7.10) of Harnack. Dividing by K(r) gives Since u may not be ⩽0, we must use the general form (7.10) of Harnack. Dividing by K(r) gives (1 + λ)n−1 1 −λ M(u, λr) K(r) −M(u, r) K(r) ⩽S(u, r) K(r) −M(u, r) K(r) . (7.11) Using the fact that K(λr) = λ2−pK(r) and letting r ↓0 gives (7.11) (1 + λ)n 1 1 −λ M(u, λr) K(r) −M(u, r) K(r) ⩽S(u, r) K(r) −M(u, r) K(r) . Using the fact that K(λr) = λ2−pK(r) and letting r ↓0 gives 1 −λ K(r) K(r) ⩽K(r) K(r) . The Upper Semi-Continuity of Density Theorem 7.4. — Each of the densities ΘM(u, x), ΘS(u, x), and ΘV (u, x) considered above is an upper semi-continuous function of x. Proof. — Because of Proposition 7.1 there are only two cases to consider. We must show that lim sup x→x0 x̸=x0 Θ(u, x) ⩽Θ(u, x0). (7.12) (7.12) Set x0 = 0. Assume 0 < \|x\| < r < t. Then Θψ(u, x) ⩽ψ(u, x, t) −ψ(u, x, r) K(t) −K(r) . (7.13) Set x0 = 0. Assume 0 < \|x\| < r < t. Then Θψ(u, x) ⩽ψ(u, x, t) −ψ(u, x, r) K(t) −K(r) . (7.13) (7.13) Case 1. ψ = M. — By using the facts that Bt(x) ⊂Bt+\|x\|(0) and Br−\|x\|(0) ⊂Br(x), we see that the last quantity above is ⩽ supBt+\|x\|(0) u −supBr−\|x\|(0) u K(t) −K(r) . The function M(u, 0, r) ≡supBr(0) u is continuous (see Corollary 5.2(1)) and increasing. Therefore, lim sup x→0 x̸=0 ΘM(u, x) ⩽ supBt(0) u −supBr(0) u K(t) −K(r) , 0 < r < t. Finally, the limit of the RHS as r, t →0 equals ΘM(u, 0). This proves the ﬁrst case. Finally, the limit of the RHS as r, t →0 equals ΘM(u, 0). This proves the ﬁrst case. – 814 – Tangents to subsolutions: existence and uniqueness, Part I Case 2. ψ = V . — It suﬃces to note that limx→0 V (u, x, t) = V (u, 0, t), which follows since V (u, x, t) = ﬄ B u(x + ty) dy and u converges in L1(B) to u(ty) as x →0. □ Case 2. ψ = V . — It suﬃces to note that limx→0 V (u, x, t) = V (u, 0, t), which follows since V (u, x, t) = ﬄ B u(x + ty) dy and u converges in L1(B) to u(ty) as x →0. □ Remark. — By using Theorem 3.2.13 in [26], one can show that u(x + tσ) dσ converges weakly in measure to u(tσ) dσ as x →0. This gives a direct proof that S(u, x, t) is continuous in x at x = 0 without using Proposition 7.1 Corollary 7.5. — For all c > 0, the set Ec ≡{x : Θ(u, x) ⩾c} is closed. Corollary 7.5. — For all c > 0, the set Ec ≡{x : Θ(u, x) ⩾c} is closed. Remark. The Upper Semi-Continuity of Density — When p = 1 the set where Θ(u) = 0 is just the set of diﬀer- entiability points of u (see (5.5) in Part II). 8. Maximality of Subharmonics with Harmonic Averages In this section we extend the standard notion of maximality in pluripo- tential theory to each F-potential theory. This notion extends the notion of being F-harmonic, but is still very close to it. In fact, a maximal function is harmonic if and only if it is continuous. Our main result, Theorem 8.2, is key for the study of tangents. It provides a new criterion for an F-subharmonic function to be F-maximal. An excellent reference for pluripotential theory is [31]. Definition 8.1. — An F-subharmonic function u on an open set X ⊂ Rn is said to be F-maximal on X if for each F-subharmonic function v on X and each compact subset K ⊂X, v ⩽u on X −K ⇒ v ⩽u on X. (8.1) v ⩽u on X −K ⇒ v ⩽u on X. (8.1) Note that by replacing v with max{u, v}, condition (8.1) is equivalent to v ⩾u on X and v = u on X −K ⇒ v = u on X. (8.1′) v ⩽u on X −K ⇒ v ⩽u on X. (8.1) Note that by replacing v with max{u, v}, condition (8.1) is equivalent to v ⩾u on X and v = u on X −K ⇒ v = u on X. (8.1′) (8.1) ote that by replacing v with max{u, v}, condition (8.1) is equivalent to v ⩾u on X and v = u on X −K ⇒ v = u on X. (8.1′) (8.1′) Most of the previous results come together in the proof of the next result. Most of the previous results come together in the proof of the next result. Theorem 8.2 (The Maximality Criterion). — Suppose that F is an ST- invariant convex cone subequation, and U is an F-subharmonic function on the annulus A = {x : a < \|x\| < b}. If the spherical average S(U, t) ≡ S U(tσ) dσ determines an (8.2) S(U, t) ≡ S U(tσ) dσ determines an increasing F harmonic S(U, \|x\|) on A(a, b), (8.2) (8.2) increasing F harmonic S(U, \|x\|) on A(a, b), ( ) increasing F harmonic S(U, \|x\|) on A(a, b), then the function U is F-maximal on A. (8.3) (8.3) – 815 – F. Reese Harvey and H. Blaine Lawson Proof. — The hypothesis on U can be restated as the condition S(U, t)is R↑ F harmonic on (a, b), (8.2′) Proof. 8. Maximality of Subharmonics with Harmonic Averages — The hypothesis on U can be restated as the condition S(U, t)is R↑ F harmonic on (a, b), (8. (8.2′) y Theorem 2.7. By Proposition 3.3, R↑ F = R↑ p, so by Proposition 3.5 this roves that (8.2′) is equivalent to by Theorem 2.7. By Proposition 3.3, R↑ F = R↑ p, so by Proposition 3.5 this proves that (8.2′) is equivalent to by Theorem 2.7. By Proposition 3.3, R↑ F = R↑ p, so by Proposition 3.5 this proves that (8.2′) is equivalent to S(U, t) = ΘK(t) + c on (a, b) (8.2′′) (8.2′′) for constants Θ ⩾0 and c ∈R. Now by the homogeneity of S and K, this is equivalent to for constants Θ ⩾0 and c ∈R. Now by the homogeneity of S and K, this is equivalent to S(U, t) −S(U, r) K(t) −K(r) = Θ ⩾0 for all r < t in (a, b) (8.2′′′) nstant Θ ⩾0. S(U, t) −S(U, r) K(t) −K(r) = Θ ⩾0 for all r < t in (a, b) (8.2′′′) (8.2′′′) for some constant Θ ⩾0. for some constant Θ ⩾0. As in (8.1′) assume that v is F-subharmonic on A with v ⩾U and that outside a compact subset K ⊂A we have v = U. By the fundamental double monotonicity Theorem 6.4 we have that for a < r < t < b, S(v, t) −S(v, r) K(t) −K(r) is increasing in r and t. (8.4) (8.4) Since v = U outside K, this quotient equals Θ if both r and t are suﬃciently close to a or suﬃciently close to b. Hence, this quotient equals Θ for all r < t in (a, b). That is, S(v, t) satisﬁes (8.2′′′). It follows that S(v, t), in addition to S(U, t), satisﬁes (8.2′′). Therefore, S(v, t) = S(U, t) + c ∀t ∈(a, b). (8.5) (8.5) Taking t close to a shows that c = 0. Now the fact that S(v, t) = S(U, t) for all t ∈(a, b) combined with the inequality U ⩽v implies that U = v on A, thus proving that U is F-maximal on A. □ Taking t close to a shows that c = 0. Tangents to subsolutions: existence and uniqueness, Part I X0 ⊂C, when considered as a function u(z) ≡u(z1) on X = X0 × Cn−1 with n ⩾2, is PC-maximal. (If v(z) ⩽u(z1) on X −K, then by applying the maximum principle to v on slices z1 = constant, we get v(z) ⩽u(z) on X.) Now u(z) ≡u(z1) is PC-harmonic if and only if u is continuous, however, u is not necessarily continuous even if it is bounded. X0 ⊂C, when considered as a function u(z) ≡u(z1) on X = X0 × Cn−1 with n ⩾2, is PC-maximal. (If v(z) ⩽u(z1) on X −K, then by applying the maximum principle to v on slices z1 = constant, we get v(z) ⩽u(z) on X.) Now u(z) ≡u(z1) is PC-harmonic if and only if u is continuous, however, u is not necessarily continuous even if it is bounded. Proposition 8.5. — If u is F-maximal and continuous on X, then u is F-harmonic on X. Proposition 8.5. — If u is F-maximal and continuous on X, then u is F-harmonic on X. Proof. — This is the standard “bump-function” argument which occurs for example as far back as [2] or in [28]. It goes as follows. Suppose u is not F-harmonic but is F-maximal, and therefore F-subharmonic. Then v ≡−u is not eF-subharmonic. Therefore, by Lemma 2.4 in [17], there exist x ∈X, ϵ > 0 and a quadratic polynomial Q(y) such that v(y) < Q(y) −ϵ\|y −x\|2 on Br(x)−{x} with equality at y = x, but D2 xQ /∈eF, i.e., −D2 xQ ∈Int F. Thus, w ≡−Q + δ is strictly F-subharmonic at x, and hence in a neighborhood Br(x). Pick δ > 0 suﬃciently small that v(y) < Q(y)−δ = −w(y) on ∂Br(x). Then w(y) < u(y) on ∂Br(x), but w(x) = u(x) + δ. This proves that u is not maximal. □ □ F-harmonic functions may not be closed under decreasing limits. For in- stance in Example 8.4 each u(z1) which is ∆-subharmonic is the decreasing limit of functions uj(z1) which are smooth and ∆-subharmonic. The exten- sions uj →u to Cn give an example for the case F = PC. This defect is corrected by enlarging the space of F-harmonic functions to the space of F-maximal functions. (This is the smallest such enlargement by Theorem 8.7 below.) Proposition 8.6. — If u is the decreasing limit of a sequence of F- maximal functions, then u is F-maximal. 8. Maximality of Subharmonics with Harmonic Averages Now the fact that S(v, t) = S(U, t) for all t ∈(a, b) combined with the inequality U ⩽v implies that U = v on A, thus proving that U is F-maximal on A. □ The following additional facts about F-maximal functions are standard in pluripotential theory, where F = PC. The proofs easily adapt to the more general subequation F, but since these results are not part of the viscosity literature, we inlcude them for the convenience of the reader. Throughout the remainder of this section F is an arbitrary subequation, i.e., a closed set F ⊂Sym2(Rn) which satisﬁes F + P ⊂F. Proposition 8.3. — If u is F-harmonic on X, then u is F-maximal on X. This is immediate since comparison holds for F (cf. [19, Thm. 6.4]). The only thing standing in the way of the converse is the continuity of u. Example 8.4. — The subequation F = PC of pluripotential theory has many functions, such as log \|z1\| on C2, which are maximal but not F- harmonic. In fact any function u(z1), which is ∆-subharmonic on a domain – 816 – Tangents to subsolutions: existence and uniqueness, Part I is F-subharmonic on X. Proof. — Sup-convolution provides a decreasing sequence uϵ ↓u of con- tinuous F-subharmonic functions which are deﬁned on subdomains which contain Ωand increase to X. Set vϵ δ ≡ ( max{uϵ + δ, v} on Ω uϵ + δ on X −Ω. Since {v < uϵ+δ} is a relatively open subset of Ωcontaining ∂Ω, the function vϵ δ is F-subharmonic on domains containing Ωwhich increase to X as ϵ ↓0. Finally, vϵ δ ↓v as ϵ, δ ↓0, proving that v is F-subharmonic on X. □ Using this Lemma 8.8, the deﬁnitions (8.1) and (8.1′) of F-maximality on X can be further reﬁned as follows: For each domain Ω⊂⊂X and v ∈USC(Ω) which is F-subharmonic on Ω, v ⩽u on ∂Ω ⇒ v ⩽u on Ω. (8.1′′) (8.1′′) Using this deﬁnition of F-maximality together with the fact that on balls B ⊂Rn the Dirichlet problem is uniquely solvable by the Perron function, it is easy to prove Theorem 8.7. Using this deﬁnition of F-maximality together with the fact that on balls B ⊂Rn the Dirichlet problem is uniquely solvable by the Perron function, it is easy to prove Theorem 8.7. Proof of Theorem 8.7. — Suppose u is maximal on X and B ⊂X is a closed ball. Choose ϕj ∈C(∂B) such that ϕj ↓u ∂Ω. Let uj ∈C(Ω) denote the solution to the Dirichlet Problem on B with uj ∂Ω= ϕj and uj F-harmonic on B. Since uj is the Perron function for boundary values ϕj, we have u ⩽uj for all j and uj ↓v ≡limj uj is decreasing. Thus u ⩽v. Also v ∂B = lim uj ∂B = lim ϕj = u ∂B, and v is F-subharmonic on B. Thus, by (8.1′′) above, v ⩽u on B. Hence, u = v = lim uj. □ F. Reese Harvey and H. Blaine Lawson is F-subharmonic on X. Tangents to subsolutions: existence and uniqueness, Part I Proposition 8.6. — If u is the decreasing limit of a sequence of F- maximal functions, then u is F-maximal. Proof. — Suppose {uj} are F-maximal and uj ↓u on an open set X. Fix a compact set K ⊂X. Then v ⩽u on X −K ⇒v ⩽uj on X −K ⇒v ⩽uj on X ⇒v ⩽u on X. □ This fact has a strong converse. Theorem 8.7. — If u is locally F-maximal, then u is locally the de- creasing limit u = limj→∞uj of F-harmonic functions uj. The proof of this fact requires a lemma. The proof of this fact requires a lemma. Lemma 8.8. — Suppose u is F-subharmonic on X, Ωopen ⊂⊂X, and v ∈USC(Ω) is F-subharmonic on Ω. If v ⩽u on ∂Ω, then v ≡ ( max{u, v} on Ω u on X −Ω – 817 – – 817 – F. Reese Harvey and H. Blaine Lawson 9. Tangents to Subharmonics Now we come to the main topic of the paper: introducing the notion of tangents to F-subharmonics. In this section the ST-invariant cone subequa- tion F on Rn is assumed to be convex. We shall work at a ﬁxed point, which for simplicity is assumed to be the origin. That is, given an F-subharmonic function u deﬁned in a neighborhood of 0, we deﬁne the notion of tangent functions to u at 0. A required clariﬁcation is given by Proposition 9.4. The basic properties of a tangent U to u at 0 are then established in Theo- rems 10.4 and 11.2. – 818 – Tangents to subsolutions: existence and uniqueness, Part I Tangents to subsolutions: existence and uniqueness, Part I Definition 9.1. — Suppose that u is F-subharmonic on the ball about the origin of radius ρ. The tangential p-ﬂow (or p-homothety) determined by the Riesz characteristic p = pF of F is deﬁned as follows. (1) ur(x) = rp−2u(rx) if p > 2, (2) ur(x) = 1 r2−p [u(rx) −u(0)] if 1 ⩽p < 2, and (3) ur(x) = u(rx) −M(u, r) if p = 2. (1) ur(x) = rp−2u(rx) (1) ur(x) = rp−2u(rx) Remark 9.2. — Suppose 1 ⩽p < 2. Since u(0) = M(u, 0) is ﬁnite, some readers may prefer to assume once and for all in part (2) that u(0) = 0 so that the p-ﬂows for all p ̸= 2 are the same, namely that ur(x) = rp−2u(rx) if p ̸= 2. (9.1) (9.1) Others may wish to make this assumption in the proofs. Note that the functions ur are F-subharmonic on Bρ/r, and as r →0, these balls expand to Rn. An upper semi-continuous function U(x) on Rn taking values in [−∞, ∞) is invariant under this ﬂow if and only if there exists an u.s.c. function g on the unit sphere S such that U(x) = \|x\|p−2g x \|x\| in the cases where p ̸= 2, while in the case where p = 2, we leave it to the reader to prove that U(x) = Θ log \|x\| + g x \|x\| with sup Sn−1 g = 0 and Θ ⩾0 a constant. U(x) = Θ log \|x\| + g x \|x\| with sup Sn−1 g = 0 and Θ ⩾0 a constant. Functions of this form will be said to have Riesz homogeneity p. Our ﬁrst result clariﬁes this Deﬁnition. Our ﬁrst result clariﬁes this Deﬁnition. Proposition 9.4. — Each tangent U to u at 0 is an entire F-subharm- onic function on Rn. Moreover, U belongs to the L1 loc-class U ∈L1 loc(Rn) and is the unique F-subharmonic function in this L1 loc-class. Proposition 9.4. — Each tangent U to u at 0 is an entire F-subharm- onic function on Rn. Moreover, U belongs to the L1 loc-class U ∈L1 loc(Rn) and is the unique F-subharmonic function in this L1 loc-class. To prove Proposition 9.4 we use the following result established in [18, Cor. 5.4] (see [20] for generalizations). We say that a subequation F can be deﬁned using fewer of the variables in Rn if there exist an (n−1)-dimensional subspace W ⊂Rn and a subequation F ′ ⊂Sym2(W) which determines F by: A ∈F ⇐⇒A W ∈F ′. An important point in the following result is that the same representative u of the L1 loc-class u (given by (9.4)) is the correct representative, no matter which subequation F is being considered. Theorem 9.5 (Distributional versus Viscosity Subharmonics). — Sup- pose F is a convex cone subequation which cannot be deﬁned using fewer of the variables in Rn. (1) If u is F-subharmonic in the viscosity sense, then u is L1 loc and F-subharmonic in the distributional sense. (2) If u is an F-subharmonic distribution, then u ∈L1 loc and the limit u(x) = lim r→0 ess sup Br(x) u exists at each point (9.4) (9.4) and deﬁnes an upper semi-continuous function u in the L1 loc-class u which is F-subharmonic in the viscosity sense. Moreover, u is the unique such representative of u. and deﬁnes an upper semi-continuous function u in the L1 loc-class u which is F-subharmonic in the viscosity sense. Moreover, u is the unique such representative of u. Remark 9.6. — We refer the reader to Sections 3, 4 and 5 of [18] for a fuller discussion of this result and the deﬁnition of an F-subharmonic distribution (Deﬁnition 4.1 and Proposition 4.3). However, the terminology used in [18] is somewhat diﬀerent. Here we use the terminology employed in [20]. In [18] a convex cone subequation F is called a “positive cone” and denoted P+. The polar cone is denoted by P+. A convex cone subequation which cannot be deﬁned using fewer of the variables in Rn is called an “elliptic cone”. 9. Tangents to Subharmonics Functions of this form will be said to have Riesz homogeneity p Under our assumptions on F each F-subharmonic function u is L1 loc since it is ∆-subharmonic by (6.3). Definition 9.3 (Tangents). — Suppose that u is an F-subharmonic function deﬁned in a neighborhood of the origin. For each sequence rj ↘0 such that U ≡lim j→∞urj converges in L1 loc(Rn), (9.2) (9.2) the point-wise deﬁned function U(x) ≡lim r→0 ess sup Br(x) U (9.3) (9.3) is called a tangent to U at 0. We let T0(u) denote the set of all such tan- gents U. (We will refer to U, satisfying (9.2), as an L1 loc-tangent when the distinction between the function U and the equivalence class of functions U is important.) – 819 – F. Reese Harvey and H. Blaine Lawson Our ﬁrst result clariﬁes this Deﬁnition. From the distributional point of view it is straightforward to see that averages, or more generally convolution, of an F-subharmonic function u with any non-negative measure is again F-subharmonic. – 820 – Tangents to subsolutions: existence and uniqueness, Part I Proof of Proposition 9.4. — We use these facts about the ST-invariant convex cone subequation F: (1) F ⊂∆; (2) 1 ⩽pF ⩽n; (3) F cannot be deﬁned using fewer of the variables in Rn. Properties (1) and (2) have already been noted in (6.3) and (6.4). For Property (3) note that the ST-invariance of F rules out the possibility that F could be deﬁned using fewer of the variables in Rn. Because of (3) one can apply Theorem 9.5. Suppose U = limj→∞urj in L1 loc(Rn) is an L1 loc tangent to u at 0. Since F is a cone, each ur is viscosity F-subharmonic, and hence in L1 loc and distri- butionally F-subharmonic by Part (1) of Theorem 9.5. Hence, in the limit, U is distributionally F-subharmonic. Now apply Part (2) of Theorem 9.5 to U to complete the proof. □ In light of Proposition 9.4 we frequently drop the distinction between U and U. 10. Uniqueness of Averages of Both Tangents and of Flows Most of the properties of tangents can be deduced from the following result, which proves that averages of tangents are always unique by showing that they are radial harmonics. Theorem 10.1 (Averages of Tangents). — Suppose that u is an F- subharmonic function deﬁned in a neighborhood of the origin in Rn. Let p = pF be the Riesz characteristic of F. If p ̸= 2, then each tangent U to u at 0 has averages n each tangent U to u at 0 has averages M(r) ≡sup S U(rσ) = ΘM(u)K(r), S(r) ≡ S U(rσ) dσ = ΘS(u)K(r), and V (r) ≡ B U(rx) dx = ΘV (u)K(r). (10.1) (10.1) In particular, ΘΨ(U) = ΘΨ(u) for Ψ = M, S, or V. (10.2) (10.2) – 821 – F. Reese Harvey and H. Blaine Lawson When p = 2, all the densities of u and any tangent U to u at 0, agree, and will be simply denoted by Θ = Θ(u). Speciﬁcally, we have Θ(u) = ΘM(U) = ΘS(U) = ΘV (U) = ΘM(u) = ΘS(u) = ΘV (u). (10.3) Moreover, the averages of a tangent U to u are given by M(r) = Θ log r, S(r) = Θ log r + S U, and V (r) = Θ log r + B U , (10.4) with with −CΘ ⩽ S U ⩽0 and −(C +1)Θ ⩽ B U, where C = 1 ϕ 1 e > 1, (10.5) and where ϕ(λ) = (1 −λ)/(1 + λ)n−1. When p ̸= 2, these formulas show that any two tangents have the same maxima M(r) and the same spherical averages S(r) and volume averages V (r), all being the appropriate density times K(r). When p = 2, M(r), S(r) and V (r) all agree with Θ log r modulo an additive constant, but the constant depends on the tangent U, not just on u. In all cases, for each tangent U, the function S(U, \|x\|) is F-harmonic on Rn −{0} since ΘK(\|x\|) + C is F-harmonic there (Proposition 3.5). Combining Theorem 8.2 and Theorem 10.1 is one of the main ingredients of the paper and has the following immediate consequence. Theorem 10.2. — Every tangent to an F-subharmonic function is F- maximal. Theorem 10.2. — Every tangent to an F-subharmonic function is F- maximal. Applying Proposition 8.5 yields the following. Applying Proposition 8.5 yields the following. Corollary 10.3. 10. Uniqueness of Averages of Both Tangents and of Flows — Every continuous tangent to an F-subharmonic function is F-harmonic. Theorem 10.1, the uniqueness of averages of tangents, follows from the stability of averages (Lemma 6.5) and the uniqueness of the averages of a ﬂow. Its proof is given at the end of this section. We may assume that u(0) = 0 if 1 ⩽p < 2 (Remark 9.2), and that u(0) = −∞if 2 ⩽p < ∞. Theorem 10.4 (Averages of Flows). — For p ̸= 2 and Ψ = M, S or V , (10.6) lim s↓0 Ψ(us, r) = ΘΨ(u)K(r). (10.6) – 822 – Tangents to subsolutions: existence and uniqueness, Part I Tangents to subsolutions: existence and uniqueness, Part I Tangents to subsolutions: existence and uniqueness, Part I For p = 2, if Ψ = M we also have lim s↓0 M(us, r) = ΘM(u)K(r) = ΘM(u) log r. (10.6a) (10.6a) In this case the limit is decreasing and uniform in r ⩽R. For Ψ = S or V we have In this case the limit is decreasing and uniform in r ⩽R. For Ψ = S or V we have lim inf s↓0 S(us, r) ⩾ΘM(u)(log r −C), (10.6b) and lim inf s↓0 V (us, r) ⩾ΘM(u)(log r −C −1) (10.6c) (10.6b) with C as in (10.5). Direct calculations from the deﬁnitions of the ﬂow and the averages es- tablish the next result. Lemma 10.5. — For Ψ = M, S, or V : Ψ(us, r) = sp−2Ψ(u, sr) = Ψ(u, sr) K(sr) K(r) if p ̸= 2, (10.7) Ψ(us, r) = Ψ(u, sr) −M(u, s) = Ψ(u, sr) −M(u, s) K(sr) −K(s) K(r) if p = 2. (10.8) Lemma 10.5. — For Ψ = M, S, or V : Ψ(us, r) = sp−2Ψ(u, sr) = Ψ(u, sr) K(sr) K(r) if p ̸= 2, (10. if p ̸= 2, (10.7) Ψ(us, r) = Ψ(u, sr) −M(u, s) = Ψ(u, sr) −M(u, s) K(sr) −K(s) K(r) if p = 2. (10. Proof. — For example, when Ψ is the volume average V and p ̸= 2, we have Proof. — For example, when Ψ is the volume average V and p ̸= 2, we have V (us, r) = 1 \|B\| ˆ B us(rx) dx = sp−2 \|B\| ˆ B u(rsx) dx = sp−2V (u, rs). □ The remaining calculations are left to the reader. Proof of Theorem 10.4. 10. Uniqueness of Averages of Both Tangents and of Flows Since V (U, et) and S(U, et) are entire convex functions of t, the linear inequalities Θ(t −C) ⩽S(U, et) ⩽Θt and Θ(t −C −1) ⩽V (U, et) ⩽Θt imply that S(U, et) = Θ(t + k) and V (U, et) = Θ(t + k′ −1) where k and k′ satisfy −C ⩽k, k′ ⩽0. □ Θ(t −C) ⩽S(U, et) ⩽Θt and Θ(t −C −1) ⩽V (U, et) ⩽Θt imply that S(U, et) = Θ(t + k) and V (U, et) = Θ(t + k′ −1) where k and k′ satisfy −C ⩽k, k′ ⩽0. □ 10. Uniqueness of Averages of Both Tangents and of Flows — By Lemma 5.5, the identity (10.7) implies (10.6) for p ̸= 2. In the case where p = 2 the limit (10.6a) for the max- imum follows from (10.8) by the double monotonicity Theorem 6.4. The limit (10.6c) for V follows from the limit (10.6c) for S since V (us, r) = n ´ 1 0 S(us, t) tn−1 dt by (6.7), and n ´ 1 0 (log rt −C) tn−1 dt = log r −C −1. It remains to prove (10.6b). Harnack’s inequality in the form (7.10) with v = us and λ = 1/e states that C M us, r e −M(us, r) + M(us, r) ⩽S(us, r). We know the limit of the terms involving M as s ↓0. This gives CΘM(u) log r e −log r + ΘM(u) log r ⩽lim inf s↓0 S(us, r) We know the limit of the terms involving M as s ↓0. This gives CΘM(u) log r e −log r + ΘM(u) log r ⩽lim inf s↓0 S(us, r) □ as desired. Proof of Theorem 10.1. — The density statements for u are contained in Propositions 7.1 and 7.2. The density statements for U follow from the formulas in Theorem 10.1 and the density statements for u. The formulas – 823 – F. Reese Harvey and H. Blaine Lawson in Theorem 10.1 follow immediately from the formulas in Theorem 10.4 for the averages of ﬂows and the stability of averages (Lemma 6.5), with the exception of (10.4) for S and V , and the estimates in (10.5). in Theorem 10.1 follow immediately from the formulas in Theorem 10.4 for the averages of ﬂows and the stability of averages (Lemma 6.5), with the exception of (10.4) for S and V , and the estimates in (10.5). The estimates (10.6b) and (10.6c) and the Stability Lemma 6.5 show that for any tangent U to u at 0, ΘM(u) (log r −C) ⩽S(U, r) and ΘM(u) (log r −C −1) ⩽V (U, r) for all 0 < r < ∞. Also we have that V (U, r) ⩽S(U, r) ⩽M(U, r) = ΘM(u) log r. Tangents to subsolutions: existence and uniqueness, Part I Theorem 11.2. — Suppose that u is an F-subharmonic function deﬁned in a neighborhood of the origin in Rn. Then the tangent set T0(u) to u at 0 satisﬁes: (1) T0(u) is non-empty. (2) T0(u) is a compact subset of L1 loc(Rn). (3) T0(u) is invariant under the homothety U →Ur. 1 (4) T0(u) is a connected subset of L1 loc(Rn). Proof. — Parts (1) and (2) are immediate from Theorem 11.1. The argu- ments for parts (3) and (4) are given in [38, Prop. 1.1.1]. We include them here for completeness. To prove (3) note that U(x) = limrj↓0 urj(x) implies Ur(x) = limsj↓0 usj(x) with sj = rrj. To prove (4) suppose urj →U0 and utj →U1 with U0 and U1 elements of disjoint open sets N0 and N1 which cover T0(u). We can assume rj < tj for all j and choose sj between rj and tj with usj /∈N0 ∪N1. (Note that s 7→us is a continuous map into L1 loc.) By Theorem 11.1 the sequence usj has a convergent subsequence, and its limit is in neither N0 nor N1, a contradiction. □ □ 11. Existence of Tangents We now address the basic existence question. Again F is assumed here to be convex. However, in the case where 1 ⩽p < 2 much stronger results are true even if F is just a cone and not necessarily convex. These stronger results are established in Section 15. Theorem 11.1 (Existence of Tangents). — Suppose that u is F-sub- harmonic on a ball Bρ. For each R > 0 there exists δ > 0 such that the family {ur}0<r⩽δ is unformly bounded above and bounded in norm in L1(BR). In particular, the set {ur}0<r⩽δ is precompact in L1(BR). Theorem 11.1 (Existence of Tangents). — Suppose that u is F-sub- harmonic on a ball Bρ. For each R > 0 there exists δ > 0 such that the family {ur}0<r⩽δ is unformly bounded above and bounded in norm in L1(BR). In particular, the set {ur}0<r⩽δ is precompact in L1(BR). Proof. — An upper bound for u can be chosen to be any number greater than ΘM(u)K(R) by (10.6) if p ̸= 2 and by (10.6a) if p = 2. Consequently the boundedness in L1(BR) is equivalent to a lower bound for V (us, R) which is uniform in s. This lower bound can be chosen to be any number less than ΘV (u)K(R) if p ̸= 2, or ΘM(u)(log R−C −1) if p = 2, by (10.6) and (10.6c) respectively in Theorem 10.4. □ The basic properties of the tangent set T0(u) are contained in the fol- lowing theorem. Again see Section 15 for the stronger versions of parts (2) and (4) using the Hölder topology instead of the L1 loc-topology when 1 ⩽ p < 2. – 824 – Tangents to subsolutions: existence and uniqueness, Part I 12. Uniqueness of Tangents In this section we discuss some basic situations where tangents are unique. Our main uniqueness results are are stated and proved in subsequent sec- tions. As in Sections 9–11 we assume that F is convex with ﬁnite Riesz characteristic p. Definition 12.1. — Suppose u is an F-subharmonic function deﬁned in a neighborhood of the origin. (1) If T0(u) = {U} is a singleton, then we say that uniqueness of tan- gents holds for u. If uniqueness of tangents holds for all such u, we say the that uniqueness of tangents holds for F. (2) If T0(u) = {ΘK(\|x\|)} with Θ ⩾0 a constant, then we say that strong uniqueness of tangents holds for u. If strong uniqueness of tangents holds for all such u, then we say that strong uniqueness of tangents holds for F. (3) If every tangent U to u satisﬁes Ur = U ∀r, then we say that homo- geneity of tangents holds for u. If homogeneity of tangents holds for all such u, then we say that homogeneity of tangents holds for F. (3) If every tangent U to u satisﬁes Ur = U ∀r, then we say that homo- geneity of tangents holds for u. If homogeneity of tangents holds for all such u, then we say that homogeneity of tangents holds for F. Now (2) ⇒(1) ⇒(3). The ﬁrst implication is obvious. For the second, note that (1) can be rephrased since Now (2) ⇒(1) ⇒(3). The ﬁrst implication is obvious. For the second, note that (1) can be rephrased since T0(u) = {U} ⇐⇒ lim r→0 ur exists in L1 loc(Rn) and equals U. (12.1) T0(u) = {U} ⇐⇒ lim r→0 ur exists in L1 loc(Rn) and equals U. (12.1) (12.1) – 825 – F. Reese Harvey and H. Blaine Lawson Thus by (1), urj and urrj have the same limit U, but urrj has limit Ur, which proves (3). Thus by (1), urj and urrj have the same limit U, but urrj has limit Ur, which proves (3). In general, S(u, r) ⩽M(u, r). Therefore, For 2 ⩽p ⩽n, ΘM(u) ⩽ΘS(u), In general, S(u, r) ⩽M(u, r). Therefore, For 2 ⩽p ⩽n, ΘM(u) ⩽ΘS(u), and for 1 ⩽p < 2, ΘS(u) ⩽ΘM(u) (12.2) (12.2) by (5.7) since K > 0 in the ﬁrst case and K < 0 in the second case. Tangents to subsolutions: existence and uniqueness, Part I Now one checks that: for n ⩾3, (K ∗ν)r = K ∗(( 1 r)∗ν) and for n = 2, (K ∗ν)(rx) = K ∗(( 1 r)∗ν)(x) + ν(1) log r, so that M(K ∗ν, r) = M(K ∗ ( 1 r)∗ν, 1) + ν(1) log r. Now limr→0( 1 r)∗ν always exists weakly in the space of measures and equals Θ[0], where Θ = limr→0 ν(Br) is the zero-dimensional density of ν at 0. Since K ∈L1 loc(Rn), the limit of (K ∗ν)r exists in L1 loc(Rn) and equals K ∗(Θ[0]) = ΘK. (Note that for n = 2, M(K ∗( 1 r)∗ν, 1) has limit M(Θ log \|x\|, 1) = 0.) In the n = 2 case there is a diﬀerent proof following Kiselman [29]. Note that by (10.4) we have M(U, r) = Θ log r for any tangent U to u at 0. In particular, U(x) −Θ log \|x\| is ⩽0 on R2 and ∆-subharmonic on R2 −{0}. Hence, it can be extended to R2 as a subharmonic function, and then by Liouville’s Theorem it must be constant. Since M(ur, 1) = 0 for all r small, M(U, r) = 0, proving that the constant is zero. □ 12. Uniqueness of Tangents (In the n = 2 case ur and vr + hr diﬀer by M(v, r) + M(h, r) −M(u, r), but this error has limit zero.) – 826 – Tangents to subsolutions: existence and uniqueness, Part I Tangents to subsolutions: existence and uniqueness, Part I 12. Uniqueness of Tangents How- ever, if strong uniqueness holds for u, then all densities “agree” because of Proposition 7.1 and the following. by (5.7) since K > 0 in the ﬁrst case and K < 0 in the second case. How- ever, if strong uniqueness holds for u, then all densities “agree” because of Proposition 7.1 and the following. If for some Θ ⩾0, T0(u) = {ΘK}, then ΘM(u) = ΘS(u) = Θ. (12.3) This follows from (10.2) and the fact that ΘM(K) = ΘS(K) = 1. (12.3) There are two classical cases where strong uniqueness holds, that will prove useful later. For the sake of completeness we include proofs. There are two classical cases where strong uniqueness holds, that will prove useful later. For the sake of completeness we include proofs. Proposition 12.2 (Radial Subharmonics). — Suppose that u(x)=f(\|x\|) is a radial F-subharmonic function deﬁned on a neighborhood of 0. Then lim r→0 ur = Θ(u)Kp(\|x\|) in L1 loc(Rn) and uniformly on compact subsets in Rn −{0}. Thus, T0(u) = {ΘKp}. in L1 loc(Rn) and uniformly on compact subsets in Rn −{0}. Thus, T0(u) = {ΘKp}. Proof. — Since u is radial, we have that ur(x) = M(ur, \|x\|), but by Theorem 10.4 we know that limr↓0 M(ur, \|x\|) = ΘKp(\|x\|) uniformly in 0 < \|x\| ⩽R. □ Remark 12.3. — The conclusion of convergence in C(Rn −{0}) only re- quires F to be an ST-invariant cone subequation with ﬁnite characteristic. It does not require convexity. Proposition 12.4 (Newtonian Case). — Suppose u is a ∆-subharmonic function deﬁned on a neighborhood of 0. Then lim r→0 ur(x) = −Θ(u) \|x\|n−2 in L1 loc(Rn) when n ⩾3, and lim r→0 ur(x) = Θ(u) log \|x\| in L1 loc(Rn) when n = 2. in L1 loc(Rn) when n = 2. in L1 loc(Rn) when n = 2. Proof. — Each such u is of the form u = v + h where v = K ∗v is a Newtonian potential and h is harmonic near the origin. (Take the measure ν to be a cut-oﬀof the measure µ = ∆u in a small ball about the origin.) This reduces the proof to the case v ≡K ∗ν. Proposition 12.4 can be partly generalized. Proposition 12.4 can be partly generalized. Proposition 12.4′ (Riesz Potentials, p > 2). — Suppose u = Kp ∗ν where ν ⩾0 is a compactly supported measure. Then lim r→0 ur = −Θ(ν) \|x\|p−2 in L1 loc(Rn) where, up to a universal constant, Θ(ν) = limr→0 ν(Br). where, up to a universal constant, Θ(ν) = limr→0 ν(Br). Proof. — Ignoring constants, we have (cf. [36]) Proof. — Ignoring constants, we have (cf. [36]) ∆u = (∆Kp) ∗ν = Kp+2 ∗ν ≡µ. ∆u = (∆Kp) ∗ν = Kp+2 ∗ν ≡µ. Note that Kn ∗µ = Kn ∗Kp+2 ∗ν = Kp ∗ν = u. We compute that ur(x) = rp−2u(rx) = rp−2(Kp ∗ν)(rx) is equal to Kp ∗ 1 r ∗ν , and observe that limr↓0 1 r ∗ν = Θ(ν)[0]. ur(x) = rp−2u(rx) = rp−2(Kp ∗ν)(rx) is equal to Kp ∗ 1 r ∗ν , and observe that limr↓0 1 r ∗ν = Θ(ν)[0]. □ □ We complete this section with a ﬁnal case where strong uniqueness holds. Proposition 12.5 (Zero Density). — Suppose that u is F-subharmonic in a neighborhood of the origin and F is convex with p > 1. If any of the densities of u is zero at 0, then all the densities of u vanish at 0, and in this case lim r→0 ur = 0 in L1 loc(Rn). (12.4) (12.4) If F is not convex but 1 ⩽p < 2, then ΘM(u, 0) = 0 implies that (12.5) lim r→0 ur = 0 locally in α Holder norm, α = 2 −p. (12.5) lim r→0 ur = 0 locally in α Holder norm, α = 2 −p. (12.5) – 827 – F. Reese Harvey and H. Blaine Lawson Proof. — The equality of zero densities is a direct consequence of Propo- sitions 7.1 and 7.2, while (12.4) follows from Theorem 10.4. The proof of the ﬁnal assertion of Proposition 12.5 is postponed as it follows immediately from (15.11). □ 13. The Strong Uniqueness Theorem I In this section we give two proofs of one of our two main results concern- ing strong uniqueness. Recall that every O(n)-invariant subequation F has complex and quaternionic analogues F C and F H, which are invariant under U(n) and Sp(n) respectively (see Example 4.7). Theorem 13.1. — Suppose that F is O(n)-invariant and convex with ﬁnite Riesz characteristic p. Then, except for the case F = P, strong unique- ness of tangents holds for F. Furthermore, except for the cases PC and PH, strong uniqueness of tangents also holds for the complex and quaternionic analogues F C and F H of F. Remark 13.2. — For the subequations P, PC and PH, strong uniqueness fails dramatically. Nonetheless, tangents are classiﬁed in these cases. This is discussed in Part II of this paper. Proof. — Let u be F-subharmonic in a neighborhood of the origin and choose U ∈T0(u). Then U(x) = lim j→∞urj(x) for a sequence rj ↓0, where the ﬂow urj(x), given in Deﬁnition 9.1, depends on p. for a sequence rj ↓0, where the ﬂow urj(x), given in Deﬁnition 9.1, depends on p. Theorem 10.2 states that orem 10.2 states that U ∈T0(u) ⇒ U is F-maximal on Rn −{0}, (13.1) U ∈T0(u) ⇒ U is F-maximal on Rn −{0}, (13.1) (13.1) and and U ∈T0(u) and U ∈C(Rn −{0}) ⇒U is F-harmonic on Rn −{0}. (13.2) We ﬁrst prove the theorem under the additional assumption that F is uni- formly elliptic. (Note, however, from Section 4 that there many examples of subequations F which are not uniformly elliptic, but for which the theorem still applies.) Proposition 13.3. — If, in addition to the hypotheses of Theorem 13.1, F is uniformly elliptic, then strong uniqueness of tangents holds for F. – 828 – Tangents to subsolutions: existence and uniqueness, Part I Tangents to subsolutions: existence and uniqueness, Part I Proof of Proposition 13.3. — Two regularity results are needed for F. They can be stated as follows. Fact 13.4. — A sequence {uj} of F-harmonics on Xopen ⊂Rn, which is bounded in L∞(K) for each compact K ⊂X, is precompact in C(X). Fact 13.5. — Each F-harmonic function is C1. The reader is referred to [4] and [41] for these results. 13. The Strong Uniqueness Theorem I Also, for Fact 13.4, one can use the Krylov–Safanov Hölder Estimate 4 in [8] which holds with f = 0 because of the First Linearization on p. 107. Recall that F is assumed to be invariant under a subgroup G ⊆O(n) which acts transitively on Sn. Lemma 13.6. (1) Suppose U ∈T0(u). Then g∗U ∈T0(g∗u) for each g ∈G, and the densities ΘS(g∗U) = ΘS(U) = ΘS(u) = ΘS(g∗u) are all equal. (2) If U ∈T0(u) and V ∈T0(v), then max{U, V } ∈T0(max{u, v}). (3) If U ∈T0(u) and g ∈G, then max{U, g∗U} ∈T0(max{u, g∗u}). F. Reese Harvey and H. Blaine Lawson Fact 13.8. — Let f : R →R be a 2π-periodic function with the property that for all a ∈R, the function Fa(x) ≡max{f(x), f(x+a)} is diﬀerentiable. Then f ≡constant. Fact 13.8. — Let f : R →R be a 2π-periodic function with the property that for all a ∈R, the function Fa(x) ≡max{f(x), f(x+a)} is diﬀerentiable. Then f ≡constant. We see this as follows. If f is not constant, there exists a point x with f ′(x) > 0. Since it is periodic, there must also exist a point y with f ′(y) < 0. Set a = y −x. Then the left hand derivative of Fa at x is < 0 (if it exists), and the right hand one is > 0. This completes the argument for G = O(n). Consider now the general case of a closed subgroup G ⊂O(n). Fix x ∈ Sn−1 and decompose the Lie algebra as g = k ⊕h (orthogonal with respect to the Killing form of so(n)), where k = g∩so(n−1) is the Lie algebra of the subgroup K ≡{g ∈G : g(x) = x}. Now the diﬀerential of the G-action at x gives an isomorphism g ∼= Tx(Sn−1), and for every 1-parameter subgroup ϕt ⊂G generated by an element of g, the orbit is a great circle. The argument made above for O(n) now applies, and Lemma 13.7 is proved. □ □ Taken together, these two lemmas prove that the punctured harmonic U(x) is radial (constant on spheres about the origin). Therefore, by Propo- sition 3.5, U = ΘK + C, and by (10.1), C = 0. This completes the proof of Proposition 13.3 if U ∈C(Rn −{0}). For the next step we establish the following strengthening of Propo- sition 8.5 which reduces the case U ∈L∞ loc(Rn −{0}) to the case U ∈ C(Rn −{0}). Proposition 13.9. — Suppose F is uniformly elliptic. Then each locally bounded F-maximal function is F-harmonic. Proposition 13.9. — Suppose F is uniformly elliptic. Then each locally bounded F-maximal function is F-harmonic. Proof of Proposition 13.9. — Suppose u is an F-maximal L∞ loc-function on a domain X ⊂Rn. By Theorem 8.7 for any compact set K ⊂X, u is the decreasing limit of a sequence {uj}j of F-harmonic functions on a neighborhood of K. By Fact 13.4, the limit u is continuous, and hence F- harmonic by Proposition 8.5. The straightforward proofs are omitted. The straightforward proofs are omitted. The proof of Proposition 13.3 will progress in three stages. First we es- tablish strong uniqueness for continuous tangents, then for tangents which are locally bounded, and ﬁnally for general tangents. The proof that U = ΘKp for U ∈C(Rn −{0}) is as follows. Note that for g ∈G, max{U, g∗U} ∈C(Rn −{0}), and therefore by Lemma 13.6 and (13.2), max{U, g∗U} is F-harmonic on Rn −{0} for each g ∈G. (13.3) max{U, g∗U} is F-harmonic on Rn −{0} for each g ∈G. (13.3 max{U, g∗U} is F-harmonic on Rn −{0} for each g ∈G. (13.3) By the C1-regularity result Fact 13.5 we have that max{U, g∗U} is C1 on Rn −{0} for each g ∈G. (13.4) (13.3) (13.4) Although the maximum of two F-subharmonics is always subharmonic, it is unusual for the maximum of two distinct F-harmonics to be F-harmonic. In fact we have the following. Lemma 13.7. — Let f be a function on the unit sphere in S ⊂Rn with the property that max{f, g∗f} ∈C1(S) for all g ∈G. Then f = constant. Proof of Lemma 13.7. — We begin with the case G = O(n). If we can prove constancy on every great circle in Sn−1, we are done. So we are im- mediately reduced to the case n = 2. Lifting to the covering R →S1 we are then reduced to the following elementary fact: – 829 – F. Reese Harvey and H. Blaine Lawson F. Reese Harvey and H. Blaine Lawson □ Proof of Proposition 13.9. — Suppose u is an F-maximal L∞ loc-function on a domain X ⊂Rn. By Theorem 8.7 for any compact set K ⊂X, u is the decreasing limit of a sequence {uj}j of F-harmonic functions on a neighborhood of K. By Fact 13.4, the limit u is continuous, and hence F- harmonic by Proposition 8.5. □ This completes the second stage of the proof of Proposition 13.3 where U ∈L∞ loc(Rn −{0}). It remains to prove the last stage where U is a general tangent. By Lemma 13.6(2), for each N > 0 we have U N ≡max{U, NKp} ∈ T0(max{u, NKp}). Since U N ∈L∞ loc(Rn −{0}), U N is a multiple of Kp. We now observe that U N decreases down to U as N →∞. Hence, if each U N is a multiple of the Riesz kernel, then so is U. This completes the proof of Proposition 13.3 □ □ The last result needed for the proof of Theorem 13.1 in the O(n)-invariant case is the following proposition, which reduces the case of our general F – 830 – Tangents to subsolutions: existence and uniqueness, Part I of characteristic p, to a speciﬁc maximal such equation, which is uniformly elliptic. Proposition 13.10. — The subequation Plargest p def = A : A + p −1 n −p(trA)I ⩾0 contains all the O(n)-invariant convex cone subequations F of Riesz char- acteristic p, and has Riesz characteristic p itself. Since contains all the O(n)-invariant convex cone subequations F of Riesz char- acteristic p, and has Riesz characteristic p itself. Since Plargest p = P(δ) with δ = (p −1)n n −p (see Example 4.3), the subequation Plargest p is uniformly elliptic when p > 1. Proof. — Suppose A = λ1Pe1 + · · · + λnPen is in diagonal form with (see Example 4.3), the subequation Plargest p is uniformly elliptic when p > 1. Proof. — Suppose A = λ1Pe1 + · · · + λnPen is in diagonal form with λ1 ⩽· · · ⩽λn. Then by deﬁnition (4.5) we know that Proof. — Suppose A = λ1Pe1 + · · · + λnPen is in diagonal form with λ1 ⩽· · · ⩽λn. F. Reese Harvey and H. Blaine Lawson Then by deﬁnition (4.5) we know that A /∈P(δ) ⇐⇒ ⟨A, Pe1 + δ nI⟩= λ1 + δ n (λ1 + · · · + λn) < 0. A /∈P(δ) ⇐⇒ ⟨A, Pe1 + δ nI⟩= λ1 + δ n (λ1 + · · · + λn) < 0. If µ′ = π(λ′) is a permutation of λ′ = (λ2, . . . , λn), then Aπ ≡λ1Pe1 + µ2Pe2+· · ·+µnPen also belongs to the open half-space H deﬁned by ⟨A, Pe1+ δ nI⟩< 0, and H is disjoint from P(δ). Averaging A over these permutations yields B ≡λ1Pe1 + Σ n−1pe⊥ 1 where Σ ≡λ2 + · · · + λn. Since B ∈H we have B /∈P(δ). Hence setting e ≡e1 and using the fact that P(δ) is a cone, we can rescale to obtain B′ ≡Pe⊥−(p′ −1)Pe /∈P(δ). Since the characteristic of P(δ) is equal to p, this proves that p′ > p. Now if A ∈F, then since F is O(n)-invariant and convex, the average B ∈F. Finally since F is a cone, B′ ∈F. Since p′ > p, this proves that F has Riesz characteristic > p, contrary to assumption. □ □ Proposition 13.10 says that if U is a tangent to an F-subharmonic func- tion, where F satisﬁes the hypotheses, then U is Plargest p -tangent. Since the subequation Plargest p is uniformly elliptic, Proposition 13.3 applies, which completes the proof of Theorem 13.1 in the orthogonally invariant case. Remark 13.11. — Some (in fact, many) readers may be uncomfortable with the assertion that P(δ)-harmonics have the regularity of viscosity solu- tions to equations which are convex and uniformly elliptic in the conventional sense. A discussion of this point is given in Appendix B. Consider now the complex analogue F C of F on Cn. Then we have F C ⊂ PC(δ), the complex analogue of the subequation deﬁned in Proposition 13.10. Now for any A ∈Sym2 R(Cn) one has that tr(A) = 2trC(AC) and λ1(A) ⩽ λC 1 (AC). Hence, P( δ 2) ⊂PC(δ) as subsets of Sym2(R2n) = Sym2 R(Cn). It follows that PC(δ) is uniformly elliptic (for p > 1). The arguments given above now go through to establish the theorem in this case. – 831 – F. Reese Harvey and H. Blaine Lawson – 832 – Tangents to subsolutions: existence and uniqueness, Part I We now ﬁx g0 ∈G and deﬁne We now ﬁx g0 ∈G and deﬁne V ϵ(x) ≡U ϵ(g0x) = lim j→∞rp−2 j uϵ(rjg0x) where the second equality comes from Lemma 13.12. Clearly V ϵ is a tangent, and it satisﬁes S(V ϵ, r) = S(U ϵ, r) = ΘSK(r). In particular, V ϵ is also maximal. Furthermore, note that where the second equality comes from Lemma 13.12. Clearly V ϵ is a tangent, and it satisﬁes S(V ϵ, r) = S(U ϵ, r) = ΘSK(r). In particular, V ϵ is also maximal. Furthermore, note that max{U ϵ(x), V ϵ(x)} ≡lim j→∞rp−2 j max{uϵ(rjx), uϵ(rjg0x)} is also a tangent and hence maximal. We have proved the following. is also a tangent and hence maximal. We have proved the following. Proposition 13.14. — For all g ∈G and all ϵ > 0 the function max{U ϵ, g∗U ϵ} is F-harmonic. Proposition 13.14. — For all g ∈G and all ϵ > 0 the function max{U ϵ, g∗U ϵ} is F-harmonic. As in the ﬁrst proof we now apply elliptic regularity and Lemma 13.7 to conclude that each function max{U ϵ, g∗U ϵ} is C1, and therefore that U ϵ is constant on each sphere. Then by Corollary 10.3 U ϵ is an increasing radial harmonic and therefore a multiple of the Riesz kernel. Since U ϵ →U in L1 loc, we conclude that U = ΘS(u)K(\|x\|). This completes our second proof in the orthogonally invariant case. Arguments for the complex and quaternionic analogous proceed as above. □ Proof of Lemma 13.12. — Let Uj(x) ≡rp−2 j u(rjx), so that Uj →U in L1 loc(Rn −{0}). Set A = {r ⩽\|x\| ⩽R}. Then U ϵ j −U ϵ L1(A) = ˆ A ˆ G {Uj(gx)χ(g) −U(gx)χ(g)} dg dx ⩽ ˆ G ˆ A \|Uj(gx) −U(gx)\| dx χ(g) dg = ˆ G ∥g∗Uj −g∗U∥L1(A) χ(g) dg = ˆ G ∥Uj −U∥L1(A) χ(g) dg = ∥Uj −U∥L1(A) . Thus limj→∞U ϵ j = {limj→∞Uj}ϵ as claimed. Thus limj→∞U ϵ j = {limj→∞Uj}ϵ as claimed. Thus limj→∞U ϵ j = {limj→∞Uj}ϵ as claimed. Thus limj→∞U ϵ j = {limj→∞Uj}ϵ as claimed. □ □ Proof of Lemma 13.13. — It is standard that the restriction of U ϵ to each sphere {\|x\| = r} is continuous (in fact, smooth). We see this as follows. F. Reese Harvey and H. Blaine Lawson F. Reese Harvey and H. Blaine Lawson The case of the quaternionic analogue F H is proved in exactly the same way. This completes the proof of Theorem 13.1. □ For the interested reader we present a second argument for Theorem 13.1 where the passage from maximal to harmonic is based on regularization via the group G, a technique which is discussed, for example, in [27]. A Slightly Diﬀerent Proof of Theorem 13.1. — Let u be F-subharmonic in a neighborhood of the origin and choose U ∈T0(u). For clarity of exposi- tion we work in the case p > 2. Then U(x) = lim j→∞rp−2 j u(rjx) for a sequence rj ↓0. Let χ = χϵ : G →[0, ∞) be a family of smooth functions converging to the δ-function at the identity in G, and for any function f which is L1 loc in Rn −{0} and in L1(Sn−1(r)) for all r, deﬁne for a sequence rj ↓0. Let χ = χϵ : G →[0, ∞) be a family of smooth functions converging to the δ-function at the identity in G, and for any function f which is L1 loc in Rn −{0} and in L1(Sn−1(r)) for all r, deﬁne f ϵ(x) ≡ ˆ G f(gx)χ(g) dg where dg is Haar measure with unit volume on G. The following lemma is proved below. where dg is Haar measure with unit volume on G. The following lemma is proved below. Lemma 13.12. U ϵ(x) = lim j→∞rp−2 j uϵ(rjx). Now by the Fubini Theorem, U ϵ satisﬁes Now by the Fubini Theorem, U ϵ satisﬁes S(U ϵ, r) = ˆ \|x\|=1 U ϵ(rx) dx = ˆ \|x\|=1 ˆ G U(grx)χ(g) dg dx = ˆ G (ˆ \|x\|=1 U(rgx) dx ) χ(g) dg = ˆ G S(U, r) χ(g) dg = S(U, r) = ΘSK(r). From this we conclude that U ϵ is maximal by Theorem 8.2. The next lemma is also proved below. From this we conclude that U ϵ is maximal by Theorem 8.2. The next lemma is also proved below. Lemma 13.13. — U ϵ is continuous and converges to U in L1 loc(Rn−{0}) as ϵ →0. Note that the continuity of U ϵ implies that it is F-harmonic (Proposi- tion 8.5). T Now by th However, we a ˆ Sn−1 eV since the last ΘK(t0) = S U ϵ(t0x) = Vt0 ous for all ϵ. Now it is a convergence is that C∞ 0 is de Lemma 13.13 Example 1 rem 13.1, the equations of fails. To see th P (See Appendi the one above the following z = (x, y), and where Kp is g eigenvalues from which it Pmin/max p -harm therefore its o ness fails for P Straightfor nels” are not istic p given in Tangents to subsolutions: existence and uniqueness, Part I Now by the upper semi-continuity of U ϵ we have Now by the upper semi-continuity of U ϵ we have he upper semi-continuity of U ϵ we have eV (x) = lim j→∞Vtj(x) = lim j→∞U ϵ(tjx) ⩽U ϵ(t0x). (13.5) eV (x) = lim j→∞Vtj(x) = lim j→∞U ϵ(tjx) ⩽U ϵ(t0x). (13.5) (13.5) However, we also have that However, we also have that Sn−1 eV (x) dx = lim j→∞ ˆ Sn−1 Vtj(x) dx = lim j→∞ ˆ Sn−1 U ϵ(tjx) dx = ˆ Sn−1 U ϵ(t0x) dx. = ˆ Sn−1 U ϵ(t0x) dx. since the last two terms are just the averages S(U ϵ, tj) = ΘK(tj) → ΘK(t0) = S(U ϵ, t0). By the inequality (13.5) we conclude that eV (x) = U ϵ(t0x) = Vt0(x) for all x ∈Sn−1. Thus we have shown that U ϵ is continu- ous for all ϵ. Now it is a general fact that f ϵ →f in L1 loc. The proof is easy and the convergence is uniform when f ∈C∞ 0 . The general case follows from the fact that C∞ 0 is dense in L1 on compact domains. This completes the proof of Lemma 13.13. □ Example 13.15. — If one drops the convexity hypothesis in Theo- rem 13.1, then in dimensions n ⩾3 there are orthogonally invariant sub- equations of every ﬁnite Riesz characteristic for which strong uniqueness fails. To see this we consider the largest such subequation of characteristic p: Pmin/max p ≡{A : λmin(A) + (p −1)λmax(A) ⩾0} . (See Appendix A in Part II for a proof that there exists a largest and it is the one above.) To see that strong uniqueness fails for Pmin/max p we consider the following functions. Write Rn = Rm × Rn−m, m < n with coordinates z = (x, y), and consider the function Pmin/max p ≡{A : λmin(A) + (p −1)λmax(A) ⩾0} . u(x, y) ≡Kp(\|x\|) where Kp is given by (3.8). Then D2 zu = 1 \|x\|p (Px⊥−(p −1)Px) has ordered eigenvalues where Kp is given by (3.8). Then D2 zu = 1 \|x\|p (Px⊥−(p −1)Px) has ordered eigenvalues ( 1) 1 1 −(p −1) \|x\|p , 0, . . . , 0, 1 \|x\|p , . . . F. Reese Harvey and H. Blaine Lawson Suppose xj →x in {\|x\| = r}. By transitivity we can write xj = gjx where – 833 – F. Reese Harvey and H. Blaine Lawson gj \|U ϵ(xj) −U ϵ(x)\| = ˆ G U(gxj)χ(g) dg − ˆ G U(gx)χ(g) dg = ˆ G U(ggjx)χ(g) dg − ˆ G U(gx)χ(g) dg = ˆ G U(hx)χ(hg−1 j ) dh − ˆ G U(gx)χ(g) dg = ˆ G U(gx) χ(gg−1 j ) −χ(g) dg ⩽ ˆ G \|U(gx)\| χ(gg−1 j ) −χ(g) dg ⩽ (ˆ {\|x\|=r} \|U(x)\| dg ) sup g∈G χ(gg−1 j ) −χ(g) → We also know that U ϵ is maximal, and in particular upper semi-continuous with S(U ϵ, t) ≡ΘK(t) for all t. Now for \|x\| = r, g0 ∈G, and any r1 < r < r2, the calculation above also shows that \|U ϵ(g0x) −U ϵ(x)\| ⩽ sup r1⩽t⩽r2 (ˆ {\|x\|=t} \|U(x)\| dg ) sup g∈G χ(gg−1 0 ) −χ(g) . Now every y with \|y\| = t and \|y −x\| < δ can be written as y = g0x with d(g0, 1) < ϵ(δ) where ϵ(δ) →0 as δ →0. Thus we have Now every y with \|y\| = t and \|y −x\| < δ can be written as y = g0x with d(g0, 1) < ϵ(δ) where ϵ(δ) →0 as δ →0. Thus we have \|U ϵ(y) −U ϵ(x)\| ⩽ sup r1⩽t⩽r2 (ˆ {\|x\|=t} \|U(x)\| dg ) sup d(g0,1)<ϵ(δ) sup g∈G χ(gg−1 0 ) −χ(g) ⩽Cϕ(δ) \|U ϵ(y) −U ϵ(x)\| ⩽ sup r1⩽t⩽r2 (ˆ {\|x\|=t} \|U(x)\| dg ) sup d(g0,1)<ϵ(δ) sup g∈G χ(gg−1 0 ) −χ(g) ⩽Cϕ(δ) for all \|x\| = t, \|y\| = t, \|y −x\| < δ and r1 ⩽t ⩽r2. This shows that the family of functions for all \|x\| = t, \|y\| = t, \|y −x\| < δ and r1 ⩽t ⩽r2. This shows that the family of functions Vt ≡U ϵ(tx)is uniformly equicontinuous on the sphere Sn−1 = {\|x\| = 1}. Claim. Claim. lim t→t0 sup Sn−1 \|Vt −Vt0\| = 0. Proof. — Let tj →t0 be any sequence. Then by the equicontinuity above, there is a subsequence such that Vtj converges uniformly to a limit eV on Sn−1. We are done if we show that eV = Vt0. – 834 – 14. The Structure of the Sets Ec where the Density is ⩾c In this section we assume the subequation F on Rn is convex with ﬁnite Riesz characteristic p ⩾2. Fix u ∈F(X) where X is an open subset in Rn. Let Θ = ΘV : X →R be the density function (for the volume function). For c > 0 deﬁne Ec(u) ≡{x ∈X : Θ(x) ⩾c}. For classical plurisubharmonic functions in Cn (where F = PC), these sets have been of central importance. A deep theorem, due to L. Hörmander, E. Bombieri and in its ﬁnal form by Siu ([25, 3, 40]), states that in this case Ec is a complex analytic subvariety. One straightforwardly deduces from this result that for the subequation P2 in R2n the set Ec is discrete, since PC(J) ⊂P2 for all parallel complex structures J on R2n. This strong corollary has a quite general extension. Theorem 14.1. — Suppose strong uniqueness of tangents holds for F (e.g., F = Pp). Then for any F-subharmonic function u the set Ec(u) is discrete. This result is essentially sharp. See Remark 14.2 below. This result is essentially sharp. See Remark 14.2 below. We will prove Theorem 14.1 in the following equivalent form. Consider an F-subharmonic function u where F has Riesz characteristic p with 2 < p < ∞. Theorem 14.1′. — Suppose strong uniqueness of tangents holds for u at a point x0, that is, suppose that the p-ﬂow of u has limit lim r↓0 ur(x0; x) = ΘK(\|x −x0\|) in L1 loc(Rn), for some Θ ⩾0. (14.1) lim r↓0 ur(x0; x) = ΘK(\|x −x0\|) in L1 loc(Rn), for some Θ ⩾0. (14.1) (14.1) Then lim x→x0 x̸=x0 Θ(u, x) = 0. lim x→x0 ̸ Θ(u, x) = 0. lim x→x0 x̸=x0 Θ(u, x) = 0. Proof. — Suppose the conclusion fails. Then there exists a sequence xj → x0 with Θ(u, xj) ⩾c > 0 for all j. Assume x0 = 0, and set xj = rjσj with rj = \|xj\|. Then rj →0, and passing to a subsequence we can assume that σj →σ ∈Sn−1. The idea now is to apply the sequence of rj-homotheties to u. This will give a sequence urj of F-subharmonics with Θ(urj, σj) ⩾c. With appropriate estimates from monotonicity, this will contradict (14.1). T Now by th However, we a ˆ Sn−1 eV since the last ΘK(t0) = S U ϵ(t0x) = Vt0 ous for all ϵ. Now it is a convergence is that C∞ 0 is de Lemma 13.13 Example 1 rem 13.1, the equations of fails. To see th P (See Appendi the one above the following z = (x, y), and where Kp is g eigenvalues from which it Pmin/max p -harm therefore its o ness fails for P Straightfor nels” are not istic p given in , 1 \|x\|p , from which it is clear that u is Pmin/max p -subharmonic on Rn and, in fact, Pmin/max p -harmonic for x ̸= 0. Note that u has Riesz homogeneity p and is therefore its own tangent at points of the form (0, y). Hence strong unique- ness fails for Pmin/max p . from which it is clear that u is Pmin/max p -subharmonic on Rn and, in fact, Pmin/max p -harmonic for x ̸= 0. Note that u has Riesz homogeneity p and is therefore its own tangent at points of the form (0, y). Hence strong unique- ness fails for Pmin/max p . Straightforward calculation shows, however, that these “partial Riesz ker- nels” are not subharmonic for the largest convex subequation of character- istic p given in Proposition 13.10 above. – 835 – F. Reese Harvey and H. Blaine Lawson 14. The Structure of the Sets Ec where the Density is ⩾c To begin pick ρ > 0 small, and note that To begin pick ρ > 0 small, and note that V urj, σj, ρ K(ρ) = V (u, xj, rjρ) K(rjρ) (14.2) (14.2) – 836 – Tangents to subsolutions: existence and uniqueness, Part I Tangents to subsolutions: existence and uniqueness, Part I since since since V urj, σj, ρ = B urj (σj + ρx) dx = rp−2 j B u (xj + rjρx) dx and rp−2 j K(ρ) = 1 K(rjρ) . V urj, σj, ρ = B urj (σj + ρx) dx = rp−2 j B u (xj + rjρx) dx and rp−2 j K(ρ) = 1 K(rjρ) . Next we show that for all j V (u, xj, rjρ) K(rjρ) ⩾c 2 . (14.3) (14.3) In fact, this uniform bound from below, on the convergence of V (u,xj,t) K(t) to Θ(u, xj), independent of xj, is obtained from the monotonicity property (Theorem 6.4) as follows. Set α ≡2 1 p−2 . Fix xj and abbreviate notation by setting t = rjρ and V (t) = V (u, xj, t) = V (u, xj, rjρ). We now apply the identity V (t) K(t) = V (αt) −V (t) K(αt) −K(t) 1 −K(αt) K(t) 1 −V (αt) V (t) , (14.4) (14.4) with the constant α > 0 chosen so that K(αt) K(t) = α−(p−2) = 1 2. We assume u and hence V (t) is ⩽0 which can be obtained by subtracting a constant, or noting that limx→0 u(x) = −∞since Θ(u, 0) ⩾c by Theorem 7.4. with the constant α > 0 chosen so that K(αt) K(t) = α−(p−2) = 1 2. We assume u and hence V (t) is ⩽0 which can be obtained by subtracting a constant, or noting that limx→0 u(x) = −∞since Θ(u, 0) ⩾c by Theorem 7.4. Then V (t) ⩽V (αt) ⩽0 since V (t) is increasing in t, which implies that the reciprocal of 1 −V (αt) V (t) is ⩾1. By Theorem 6.4 this proves that, as desired, By Theorem 6.4 this proves that, as desired, By Theorem 6.4 this proves that, as desired, V (t) K(t) ⩾c 2 . (14.3′) (14.3′) Combining (14.2) and (14.3) we have Combining (14.2) and (14.3) we have Combining (14.2) and (14.3) we have V urj, σj, ρ K(ρ) ⩾c 2 . this implies that c = 0, a contradiction. this implies that c = 0, a contradiction. Remark 14.2. — For F as above, any ﬁnite set can occur as the set Ec for an F-subharmonic function. In fact, more is true. In a separate paper [24] we construct F-subharmonics with prescribed asymptotics at a ﬁnite set of points and prescribed boundary values. Theorem 14.3 ([24]). — Let Ω⊂Rn be a domain with smooth boundary ∂Ωwhich is strictly convex (or more generally strictly F-convex, cf. [14]). Let E = {xj}N j=1 ⊂Ωbe a ﬁnite subset, and {Θj}N j=1 any set of positive real numbers. Then given any ϕ ∈C(∂Ω), there exists a unique u ∈USC(Ω) such that: (1) u is F-harmonic in Ω−E, (2) u ∂Ω= ϕ, and (3) Θ(u, xj) = Θj for j = 1, . . . , N. (1) u is F-harmonic in Ω−E, (2) u ∂Ω= ϕ, and 15. Subequations with Riesz characteristic 1 ⩽p < 2 When the Riesz characteristic satisﬁes 1 ⩽p < 2, the behavior and study of F-subharmonics diﬀers greatly from the case p ⩾2. 14. The Structure of the Sets Ec where the Density is ⩾c (14.5) (14.5) By the hypothesis (14.1) we have By the hypothesis (14.1) we have By the hypothesis (14.1) we have By the hypothesis (14.1) we have lim rj↓0 V urj, σj, ρ = lim rj↓0 Bρ(σj) urj = Θ Bρ(σ) K(\|y\|) dy. Therefore, by (14.5) −ρp−2Θ Bρ(σ) K(\|y\|) dy ⩾c 2 . – 837 – lim rj↓0 V urj, σj, ρ = lim rj↓0 Bρ(σj) urj = Θ Bρ(σ) K(\|y\|) dy. Therefore, by (14.5) −ρp−2Θ Bρ(σ) K(\|y\|) dy ⩾c 2 . – 837 – F. Reese Harvey and H. Blaine Lawson Since lim ρ→0 Bρ(σ) K(\|y\|) dy = K(1) = −1, □ C0,α Regularity of Subharmonics To begin, all F-subharmonics (not just the F-harmonics) are regular. To be completely clear we formulate two hypotheses on a function u. Hypothesis A. — u ∈F(X) where F is a (not necessarily convex) ST-invariant cone subequation with characteristic 1 ⩽p < 2. Hypothesis B. — u ∈USC(X) satisﬁes the Maximum Principle (or (MP) for short) and Kp double monotonicity, that is, for all y ∈X M(u, y, t) −M(u, y, s) Kp(t) −Kp(s) is non decreasing in s and t (15.1) (15.1) for all 0 ⩽s < t < dist(y, ∂X). for all 0 ⩽s < t < dist(y, ∂X). – 838 – Tangents to subsolutions: existence and uniqueness, Part I By Theorem 2.7 and Theorem 6.4 Theorem 15.1 holds under Theorem 15.1 holds under Hypothesis A′ (0 < α ⩽1). — The function u satisﬁes the subequation λmin(D2u) + (1 −α)λmax(D2u) ⩾0 on X Hypothesis A′ (0 < α ⩽1). — The function u satisﬁes the subequation λmin(D2u) + (1 −α)λmax(D2u) ⩾0 on X in the viscosity sense. Said diﬀerently, Hypothesis A and Hypothesis A′ are the same. Remark 15.5. — The subequations Pmin/max p are never convex unless p = 1. In addition we have 1. In addition we have Pmin/max p ⊂∆ ⇐⇒ p ⩽1 + 1 n −1 ⇐⇒ n −2 n −1 ⩽α ⩽1. To see this, note that λ1 + (p −1)λn ⩾0 ⇒λ1 + · · · + λn ⩾0 if and only if p −1 ⩽ 1 n−1 since λ1 + · · · + λn ⩾(n −1)λ1 + λn = (n −1)(λ1 + 1 n−1λn). n −2 n −1 ⩽α ⩽1. By Theorem 2.7 and Theorem 6.4 Hypothesis A ⇒ Hypothesis B. (15.2) (15.2) Note that under Hypothesis B the density Θ(u, y) exists with 0 ⩽Θ(u, y) < ∞for each point y ∈X. For an arbitrary function u, we abbreviate the Hölder norm on a compact set K (allowing the value +∞) by ∥u∥α(K) ≡∥u∥C0,α(K). (15.3) (15.3) Theorem 15.1. — Assume Hypothesis B. Then u is locally Hölder con- tinuous on X with exponent α ≡2 −p. More speciﬁcally, if B3ρ(x0) ⊂X, then (Bρ(x0)) ⩽ Rα (R −ρ)α −ρα M(u, x0, R) −u(x0) Rα (15.4) ∥u∥α (Bρ(x0)) ⩽ Rα (R −ρ)α −ρα M(u, x0, R) −u(x0) Rα (15.4) for all 0 < 3ρ ⩽R < dist(x0, ∂X). (In particular, u(x0) > −∞, i.e., u is ﬁnite-valued at each point x0 ∈X.) ∥u∥α (Bρ(x0)) ⩽ Rα (R −ρ)α −ρα M(u, x0, R) −u(x0) Rα (15.4) for all 0 < 3ρ ⩽R < dist(x0, ∂X). (In particular, u(x0) > −∞, i.e., u is ﬁnite-valued at each point x0 ∈X.) (15.4) Proof. — Assume x, y ∈Bρ(x0). Note that x ∈∂B\|x−y\|(y). Hence, u(x) −u(y) \|x −y\|α ⩽M(u, y, \|x −y\|) −u(y) \|x −y\|α . Proof. — Assume x, y ∈Bρ(x0). Note that x ∈∂B\|x−y\|(y). Hence, u(x) −u(y) \|x −y\|α ⩽M(u, y, \|x −y\|) −u(y) \|x −y\|α . Choose R ⩾3ρ. Since x, y ∈Bρ(x0), we have \|x −y\| ⩽2ρ and hence R ⩾ \|x −y\| + ρ, or R −ρ ⩾\|x −y\|. Therefore, by the monotonicity Hypothesis B M(u, y, \|x −y\|) −u(y) \|x −y\|α ⩽M(u, y, R −ρ) −M(u, y, ρ) (R −ρ)α −ρα . (15.5) (15.5) Now BR−ρ(y) ⊂BR(x0) since y ∈Bρ(x0). This proves that M(u, y, R −ρ) ⩽M(u, x0, R). (15.6) M(u, y, R −ρ) ⩽M(u, x0, R). (15.6) Also x0 ∈Bρ(y) and hence u(x0) ⩽M(u, y, ρ), or equivalently −M(u, y, ρ) ⩽−u(x0). (15.7) (15.7) Now (15.6) and (15.7) imply that M(u, y, R−ρ)−M(u, y, ρ) ⩽M(u, x0, R)− u(x0) and (15.4) follows from (15.5). □ Deﬁne the inﬁnitesimal Hölder norm of u at x0 to be ∥u∥α(x0) ≡lim ρ→∞∥u∥α (Bρ(x0)) . (15.8) (15.8) Proposition 15.2. — Under Hypothesis B, ∥u∥α(x0) ⩽M(u, x0, R) −u(x0) Rα ⩽∥u∥α (BR(x0)) . (15.9) ∥u∥α(x0) ⩽M(u, x0, R) −u(x0) Rα ⩽∥u∥α (BR(x0)) . (15.9) for all 0 < R < dist(x0, ∂X). for all 0 < R < dist(x0, ∂X). – 839 – F. Reese Harvey and H. Blaine Lawson Proof. By Theorem 2.7 and Theorem 6.4 — For the ﬁrst inequality, let ρ →0 on both sides of the inequal- ity (15.4) in Theorem 15.1. By the (MP) there exists y ∈∂BR(x0) such that M(u, x0, R) = u(y), and hence M(u, x0, R) −u(x0) Rα = u(y) −u(x0) \|y −x\|α ⩽∥u∥α (BR(x0)) . Now it is easy to prove that the inﬁnitesimal Hölder norm and the density are the same thing. Corollary 15.3. ∥u∥α(x0) = Θ(u, x0). Proof. — Take the limit as R →0 in (15.9) and apply the deﬁnition of the density. □ □ Remark 15.4 (Hypothesis A). — Lemma A.1 in Part II states that Pmin/max p ≡{A : λmin(A)+(p−1)λmax(A) ⩾0} is the maximal subequation of characteristic p, i.e. it contains every other subequation F of characteristic p. Thus the relevance of Theorem 15.1 for pure second-order subequations can be stated as follows. Existence of Tangents In the range 1 ⩽p < 2 the arguments for the existence and structure of tangents have a diﬀerent ﬂavor from the case p ⩾2. Recall that in this range the tangent ﬂow ur(x) = 1 rα (u(rx) −u(0)) where α = 2 −p, – 840 – Tangents to subsolutions: existence and uniqueness, Part I is deﬁned in Deﬁnition 9.1(2). Tangents to subharmonics have only been deﬁned when F is convex (see Deﬁnition 9.3). However, because of the Hölder continuity when 1 ⩽p < 2, the deﬁnition can be extended to the more general cone case in Hypothe- sis A. In fact, Hypothesis B is enough. Give C(Rn) the topology of uniform convergence on compact subsets. Definition 15.6 (Tangents). — Suppose that u satisﬁes Hypothesis B in a neighborhood of the origin in Rn. For each sequence rj ↘0 such that U ≡lim j→∞urj converges in C(Rn), (15.10) (15.10) the limit function U is called a tangent to u at 0, and T0(u) denotes the space of all such tangents. the limit function U is called a tangent to u at 0, and T0(u) denotes the space of all such tangents. The version of Theorem 11.1 for 1 ⩽p < 2 is given as follows. Theorem 15.7 (Existence of Tangents). — Suppose u satisﬁes Hypoth- esis B on a ball about the origin. Then for each ρ > 0 there exists a δ > 0 such that the family {ur}0<r⩽δ is bounded in norm in C0,α(Bρ). In fact, lim sup r↓0 ∥ur∥α (Bρ) ⩽ΘM(u, 0) ∀ρ > 0. (15.11) (15.11) In particular, the set {ur}0<r⩽δ is precompact in C(Rn). Proof. — Note that ur(0) = 0 so that Theorem 15.1 states that the α-Hölder norm of ur on Bρ satisﬁes ∥ur∥α (Bρ) ⩽ Rα (R −ρ)α −ρα M(ur, 0, R) Rα if rR is small and 0 < 3ρ ⩽R. Now by the deﬁnition of ur M(ur, 0, R) = M(u, 0, rR) −u(0) rα , and therefore ∥ur∥α (Bρ) ⩽ Rα (R −ρ)α −ρα M(u, 0, rR) −u(0) (rR)α . ∥ur∥α (Bρ) ⩽ Rα (R −ρ)α −ρα M(u, 0, rR) −u(0) (rR)α . Taking the lim sup as r ↓0 yields lim sup r↓0 ∥ur∥α (Bρ) ⩽ Rα (R −ρ)α −ρα ΘM(u, 0). Finally we can let R →∞, proving (15.11). Taking the lim sup as r ↓0 yields lim sup r↓0 ∥ur∥α (Bρ) ⩽ Rα (R −ρ)α −ρα ΘM(u, 0). l ( ) lim sup r↓0 ∥ur∥α (Bρ) ⩽ Rα (R −ρ)α −ρα ΘM(u, 0). Finally we can let R →∞, proving (15.11). Finally we can let R →∞, proving (15.11). The proof is similar to that of Theorem 11.2 and is omitted. As a consequence of Theorem 15.8 the Hölder norm of a tangent is ﬁnite on all of Rn. Corollary 15.9. — If U ∈T0(u), then ∥U∥α(Rn) = Θ(u, 0) = ∥u∥α(x0). where C0,β(Rn) = C(Rn) when β = 0. □ where C0,β(Rn) = C(Rn) when β = 0. Remark. — If F is convex, then our previous L1 loc Deﬁnition 9.3 of a tangent U to u at 0 is also applicable. It agrees with Deﬁnition 15.6 because of the precompactness. Remark. — If F is convex, then our previous L1 loc Deﬁnition 9.3 of a tangent U to u at 0 is also applicable. It agrees with Deﬁnition 15.6 because of the precompactness. The analogue of Theorem 11.2 is the same except that L1 loc(Rn) is re- placed by C(Rn). The analogue of Theorem 11.2 is the same except that L1 loc(Rn) is re- placed by C(Rn). Theorem 15.8. — The tangent set T0(u) to an F-subharmonic function u satisﬁes: Theorem 15.8. — The tangent set T0(u) to an F-subharmonic function u satisﬁes: (1) T0(u) is non-empty. (1) T0(u) is non-empty. (2) T0(u) is a compact subset of C(Rn). (3) T0(u) is invariant under the tangent ﬂow U →Ur. (4) T0(u) is a connected subset of C(Rn). The proof is similar to that of Theorem 11.2 and is omitted. is deﬁned in Deﬁnition 9.1(2). By the standard compact embedding theorem this proves that (taking the topology of Hölder norms on compact subsets) {ur}0<r⩽δ is precompact in C0,β(Rn) for each 0 ⩽β < α, (15.12) (15.12) – 841 – F. Reese Harvey and H. Blaine Lawson Uniqueness, Strong Uniqueness, and Homogeneity of Tangents The three concepts are deﬁned exactly as in Deﬁnition 12.1. For instance, uniqueness of tangents holds for u at 0 if T0(u) = {U} is a singleton, or equivalently (cf. (12.1)) lim r→0 ur exists in C(Rn) and equals U . (15.13) (15.13) Strong uniqueness holds for u at 0 if this limit U = ΘKp where Θ = ΘM(u, 0). In this setting strong uniqueness for u is equivalent to the no- tion of asymptotic equivalence u ∼Θ\|y\|α deﬁned by (15.3) below. Lemma 15.10. — Strong uniqueness of tangents for u at 0 holds, i.e., lim r→0 ur = ΘKp = Θ\|x\|α in C(Rn) with Θ ⩾0 (15.14) (15.14) if and only if u(y) ∼Θ\|y\|α, i.e., lim y→0 u(y) −u(0) \|y\|α = Θ ⩾0. (15.15) (15.15) – 842 – Tangents to subsolutions: existence and uniqueness, Part I Proof. — Actually, the equivalence of (15.14) and (15.15) is an elemen- tary fact which holds for any continuous function deﬁned in a neighborhood of the origin. We can assume u(0) = 0. We ﬁrst show that (15.15) ⇒(15.14). The inequality ( ) u(y) \|y\|α −Θ ⩽ϵ \|y\| can be rewritten, with y = rx, as \|ur(x) −Θ\|x\|α\| ⩽ϵ\|x\|α. If the ﬁrst holds for \|y\| ⩽δ, then the second holds for \|x\| ⩽R and r ⩽δ/R. Thus we have \|ur(x) −Θ\|x\|α\| ⩽ϵRα, for all \|x\| ⩽R and r ⩽δ/R, which is enough to prove (15.13). For the converse we need only assume that ur →ΘK uniformly on some sphere ∂BR. The inequality \|ur(x) −Θ\|x\|α\| ⩽ϵ \|ur(x) −Θ\|x\|α\| ⩽ϵ can be rewritten, with y = rx, as u(y) \|y\|α −Θ ⩽ ϵ \|x\|α . \| r( ) can be rewritten, with y = rx, as u(y) \|y\|α −Θ ⩽ ϵ \|x\|α . If the ﬁrst holds for all \|x\| = R and r ⩽δ, then the second holds for all \|y\| ⩽δR with the right-hand side replaced by ϵ/Rα. This is enough to prove that limy→0 u(y)/\|y\|α = 0. □ Remark 15.11. — We say that strong uniqueness holds for a subequation F if it holds for all F-subharmonics at 0. Recall that by Theorem 13.1 Strong Uniqueness of Tangents to subharmonics holds for every convex O(n)- invariant subequation F with ﬁnite Riesz characteristic except F = P. This section is only concerned with the cases 1 ⩽p < 2, or 1 < p < 2 when P is excluded. Uniqueness, Strong Uniqueness, and Homogeneity of Tangents This includes the subequations: Pp (1 < p < 2), Σk (p ≡n k < 2), P(δ) (δ < n n−2), and others. Theorem 15.12. — Let F be as above. Then for u F-subharmonic in a neighborhood of 0, every tangent U ∈T0(u) is F-harmonic in Rn −{0}. Harmonicity of Tangents when F is convex If F is a convex cone ST-invariant subequation with ﬁnite characteristic, then by Theorem 10.2 every tangent to a subharmonic is maximal, and by Proposition 8.5, every continuous maximal function is F-harmonic. Thus the regularity result Theorem 15.1 implies the following for 1 ⩽p < 2. Theorem 15.12. — Let F be as above. Then for u F-subharmonic in a neighborhood of 0, every tangent U ∈T0(u) is F-harmonic in Rn −{0}. – 843 – F. Reese Harvey and H. Blaine Lawson H is F-harmonic across x0 Proof. — Assume that x0 = 0. By Proposition A.5 in [24], the strong uniqueness hypothesis can be restated as an asymptotic equivalence limx→0 (H(x)−H(0)) \|x\|α = Θ ⩾0, which was denoted there as H(x) ∼Θ\|x\|α, at x0 = 0. Suppose Θ = 0. Then for all ϵ > 0, ∃δ > 0 such that H(x)−H(0) ⩽ϵ\|x\|α if \|x\| ⩽δ. Set Vϵ(x) ≡−(H(x) −H(0)) + 2ϵ\|x\|α. Then ϵ\|x\|α ⩽Vϵ(x) on \|x\| ⩽δ, which implies that Vϵ has no test functions at 0. Since eF + Pp ⊂eF, the Addition Theorem (cf. [19]) implies that Vϵ is eF-subharmonic on Bδ−{0}. Thus Vϵ is eF-subharmonic on Bδ. Since Vϵ decreases to −H(x) + H(0) as ϵ →0, this proves that −H is eF-subharmonic on Bδ, and hence H is F- harmonic. Suppose Θ > 0. Then for 0 < ϵ < Θ there exists 0 < δ < 1 with ϵ\|x\|α ⩽H(x) −H(0) on Bδ. Therefore, −(H(x) −H(0)) ⩽−ϵ\|x\|α ⩽−ϵ\|x\|2 if \|x\| ⩽δ, which proves that −ϵ\|x\|2 is a test function for −H(x) at 0, and hence −H is not subaﬃne. Finally, 0 ∈F ⇒P ⊂F ⇒eF ⊂eP, which proves that −H is not eF-subharmonic. □ Removable Point Singularities The next result should be compared with Theorem 1.9 (the case α∗< 0) in [ASS], where F is assumed to be uniformly elliptic. Theorem 15.13. — Suppose that F is a cone subequation with a Riesz characteristic p and 1 < p < 2. Suppose Strong Uniqueness of Tangents holds for F and F + Pp ⊂F (i.e., F is Pp-monotone). For each function H which is F-harmonic in a punctured neighborhood of x0 and F-subharmonic across x0, one has that Appendix A. Subaﬃne Functions and a Dichotomy For punctured radial subharmonics, i.e., a radial F-subharmonic function deﬁned on a ball, there is a useful dichotomy between those which are in- creasing and those which are decreasing, which we now discuss. The subaﬃne equation eP = {λmax ⩾0} is an important special case, since it contains every subequation F (including itself) for which the maximum principle holds. It is also a special case in that the radial subequation Re P on (0, ∞) is constant coeﬃcient. Using the jet variables (λ, a), we have Re P = ^ R+ × R+ ≡{(λ, a) : either λ ⩾0 or a ⩾0}. (A.1) (A.1) – 844 – Tangents to subsolutions: existence and uniqueness, Part I It is important to note that the maximum principle holds for this one-variable subequation. This dual subequation ^ R+ × R+ is more restrictive than one might guess. The next result shows that near the left endpoint of (a, b) there is a di- chotomy for a subharmonic. It is either increasing or it is convex and de- creasing. Lemma A.1 (Increasing/Decreasing). — Suppose that ψ is a general upper semi-continuous ^ R+ × R+-subharmonic function on an open interval (a, b). Then either Lemma A.1 (Increasing/Decreasing). — Suppose that ψ is a general upper semi-continuous ^ R+ × R+-subharmonic function on an open interval (a, b). Then either (1) ψ is increasing on (a, b), or (1) ψ is increasing on (a, b), or (2) ψ is decreasing and convex on (a, b), or (2) ψ is decreasing and convex on (a, b), or (3) ∃c ∈(a, b) such that ψ is decreasing and convex on (a, c) and in- creasing on (c, b). (3) ∃c ∈(a, b) such that ψ is decreasing and convex on (a, c) and in- creasing on (c, b). Proof. — Suppose that ψ is not increasing on all of (a, b), that is, ψ(r) > ψ(s) for some a < r < s < b. We claim that ψ is decreasing on (a, r). If not, there exist r1, r2 with a < r1 < r2 < r and ψ(r1) < ψ(r2). If ψ(r2) < ψ(r), then since ψ(r) > ψ(s), ψ has a strict maximum on (r2, s). Thus ψ(r2) ⩾ ψ(r) > ψ(s), and since ψ(r1) < ψ(r2), we must have a strict maximum on (r1, s). (3) 0 /∈Int F. ^ (1) The maximum principle holds for F. e e F. Reese Harvey and H. Blaine Lawson Proof. — Parts (1)–(3) were proved in [14, Lem. 2.2 and Prop. 4.8]. For part (4) note that F ⊂eP ⇒(RF )t ⊂(Re P)t = ^ R+ × R+. If F is not contained in eP ≡{A : λmax(A) ⩾0}, then there exists B < 0 with B ∈F. By positivity −ϵI ∈F for some ϵ > 0, which implies that (Re P)t is not contained in ^ R+ × R+. □ These two results can be combined as follows. Corollary A.3. — If the (MP) holds for F, then the conclusions (1), (2) and (3) of the Increasing/Decreasing Lemma A.1 hold for any radial F- subharmonic function u(x) = ψ(\|x\|) deﬁned on an annulus. (In particular, if u is F-subharmonic on a ball, then ψ(t) must be increasing.) Proof. — By Theorem 2.4 and Theorem A.2, ψ is ^ R+ × R+-subharmonic, and hence Lemma A.1 applies to ψ. □ Appendix A. Subaﬃne Functions and a Dichotomy Suppose further that ψ is not decreasing on all of (a, b), that is, ψ(s) < ψ(t) for some r < s < t < b. The argument above shows that there exists a maximal c ∈(s, t) so that ψ is decreasing on (a, c). Now ψ must be increasing on (c, b) for if not, it would have a strict interior maximum on that interval. When ψ is decreasing on (a, c), it must be convex there. To see this let ϕ be a test function for ψ at t0 ∈(a, c). Then 0 ⩽ψ(t)−ψ(t0) ⩽ϕ(t)−ϕ(t0) for t < t0. This implies that ϕ′(t0) ⩽0. If ϕ′(t0) = 0, then the same inequality implies that ϕ′′(t0) ⩾0. On the other hand, if ϕ′(t0) < 0, then ϕ′′(t0) ⩾0 because ψ is ^ R+ × R+-subharmonic. □ We say that the maximum principle (MP) holds for a subequation F if it holds for all F-subharmonic functions. Theorem A.2. — The following conditions on a subequation F ⊂ Sym2(Rn) are equivalent. Theorem A.2. — The following conditions on a subequation F ⊂ Sym2(Rn) are equivalent. (1) The maximum principle holds for F. e e (2) F ⊂eP (i.e., the subequation eP is universal for (MP)). ( ) / (2) F ⊂eP (i.e., the subequation eP is universal for (MP)). (3) 0 / I t F ( ) / (4) RF ⊆ ^ R+ × R+. – 845 – F. Reese Harvey and H. Blaine Lawson Bibliography [1] S. N. Armstrong, C. K. Smart & B. Sirakov, “Fundamental solutions of homo- geneous fully nonlinear elliptic equations”, Commun. Pure Appl. Math. 64 (2011), no. 6, p. 737-777. [2] E. Bedford & B. A. Taylor, “The Dirichlet problem for a complex Monge-Ampère equation”, Invent. Math. 37 (1976), p. 1-44. [3] E. Bombieri, “Algebraic values of meromorphic maps”, Invent. Math. 10 (1970), p. 267-287, addendum in ibid. 11 (1970), p. 163-166. [4] L. A. Caffarelli & X. Cabré, Fully Nonlinear Elliptic Equations, Colloquium Publications, vol. 43, American Mathematical Society, 1995, v+104 pages. [5] M. G. Crandall, “Viscosity solutions: a primer”, in Viscosity solutions and appli- cations, Lecture Notes in Mathematics, vol. 1660, Springer, 1997, p. 1-43. [6] M. G. Crandall, H. Ishii & P.-L. Lions, “User’s guide to viscosity solutions of second order partial diﬀerential equations”, Bull. Am. Math. Soc. 27 (1992), no. 1, p. 1-67. [7] J.-P. Demailly, “Complex analytic and diﬀerential geometry”, http: //www-fourier.ujf-grenoble.fr/~demailly/documents.html. [8] L. C. Evans, “Regularity for fully nonlinear elliptic equations and motion by mean curvature”, in Viscosity solutions and applications, Lecture Notes in Mathematics, vol. 1660, Springer, 1997, p. 97-133. g [9] H. Federer, Geometric measure theory, Die Grundlehren der mathematischen Wis- senschaften, vol. 153, Springer, 1969, xiv+676 pages. [10] L. Gårding, “An inequality for hyperbolic polynomials”, J. Math. Mech. 8 (1959), no. 2, p. 957-965. [11] F. R. Harvey, “Removable singularities and structure theorems for positive cur- rents”, in Partial diﬀerential equations, Proc. Sympos. Pure Math., vol. 23, American Mathematical Society, 1973, p. 123-133. 12] F. R. Harvey & H. B. Lawson, “Subequation characterizations of various classical functions”, in preparation. [13] ——— , “Calibrated geometries”, Acta Math. 148 (1982), p. 47-157. [14] ——— , “Dirichlet duality and the nonlinear Dirichlet problem”, Commun. Pure Appl. Math. 62 (2009), no. 3, p. 396-443. [15] ——— , “Hyperbolic polynomials and the Dirichlet problem”, https://arxiv.org/ abs/0912.5220, 2009. [16] ——— , “An introduction to potential theory in calibrated geometry”, Am. J. Math. 131 (2009), no. 4, p. 893-944. [17] ——— , “Dirichlet duality and the nonlinear Dirichlet problem on Riemannian man- ifolds”, J. Diﬀer. Geom. 88 (2011), no. 3, p. 395-482. [18] ——— , “Plurisubharmonicity in a general geometric context”, in Geometry and anal- ysis, No. 1, Advanced Lectures in Mathematics, vol. 17, International Press; Higher Education Press, 2011, p. 363-401. [19] ——— , “The AE Theorem and addition theorems for quasi-convex functions”, https://arxiv.org/abs/1309.1770, 2013. Appendix B. Uniform Ellipticity and P(δ) The point of this section is to make clear that viscosity harmonics for the subequation P(δ′) = A ∈Sym2(Rn) : A + δ tr(A) ⩾0 , δ = δ′ n , are solutions to a uniformly elliptic equation F(D2u) = 0 as deﬁned in [4], [6], [41], etc. We deﬁne the operator are solutions to a uniformly elliptic equation F(D2u) = 0 as deﬁned in [4], [6], [41], etc. We deﬁne the operator F : Sym2(Rn) −→R by F(A) ≡λmin(A) + δtr(A). It is straightforward to verify that for all P ⩾0 one has It is straightforward to verify that for all P ⩾0 one has δ tr(P) ⩽F(A + P) −F(A) ⩽(1 + δ) tr(P). which is one of the standard equivalent versions of uniform ellipticity for the operator F appearing in the sources above. which is one of the standard equivalent versions of uniform ellipticity for the operator F appearing in the sources above. Now since P(δ′) = {A : F(A) ⩾0} and Int P(δ′) = {A : F(A) > 0} it is completely straightforward to verify that a continuous function u is a viscosity solution of F(D2u) = 0 if and only if (in our terminology) u is P(δ′)-harmonic. – 846 – Tangents to subsolutions: existence and uniqueness, Part I F. Reese Harvey and H. Blaine Lawson [23] ——— , “Tangents to subsolutions – existence and uniqueness, Part II”, https: arxiv.org/abs/1408.5851, 2014. [24] ——— , “The Dirichlet Problem with Prescribed Interior Singularities”, Adv. Mat 303 (2016), p. 1319-1357. [25] L. Hörmander, An Introduction to Complex Analysis in Several Variables, 3rd ed., North-Holland Mathematical Library, vol. 7, North-Holland, 1990, xii+254 pages. [26] ——— , Notions of Convexity, Progress in Mathematics, vol. 127, Birkhäuser, 1994, viii+414 pages. [27] L. Hörmander & R. Sigurdsson, “Limit sets of plurisubharmonic functions”, Math. Scand. 65 (1989), no. 2, p. 308-320. [28] H. Ishii, “On uniqueness and existence of viscosity solutions of fully nonlinear second- order elliptic PDE’s”, Commun. Pure Appl. Math. 42 (1989), no. 1, p. 15-45. [29] C. O. Kiselman, “Tangents of plurisubharmonic functions”, in International Sympo- sium in Memory of Hua Loo Keng, Vol. II (Beijing, 1988), Springer, 1991, p. 157-167. [30] ——— , “Plurisubharmonic functions and potential theory in several complex va ables”, in Development of mathematics 1950-2000, Birkhäuser, 2000, p. 655-714. [31] M. Klimek, Pluripotential theory, London Mathematical Society Monographs, New Series, vol. 6, Clarendon Press, 1991, xiv+266 pages. [32] N. V. Krylov, “On the general notion of fully nonlinear second-order elliptic equa- tions”, Trans. Am. Math. Soc. 347 (1995), no. 3, p. 857-895. [33] D. A. Labutin, “Isolated singularities for fully nonlinear elliptic equations”, J. Diﬀer. Equations 177 (2001), no. 1, p. 49-76. [34] ——— , “Potential estimates for a class of fully nonlinear elliptic equations”, Duke Math. J. 111 (2002), no. 1, p. 1-49. [35] ——— , “Singularities of viscosity solutions of fully nonlinear elliptic equations”, in Viscosity Solutions of Diﬀerential Equations and Related Topics, RIMS Kôkyûroku, vol. 1287, Kyoto University, 2002, p. 45-57. [36] N. S. Landkof, Foundations of Modern Potential Theory, Die Grundlehren der mathematischen Wissenschaften, vol. 180, Springer, 1972, x+424 pages. [37] J. Sha, “p-convex riemannian manifolds”, Invent. Math. 83 (1986), p. 437-447. [38] R. Sigurdsson, “Growth properties of analytic and plurisubharmonic functions of ﬁnite order”, Math. Scand. 59 (1986), no. 2, p. 235-304. [39] L. Simon, Lectures on Geometric Measure Theory, Proceedings of the Centre for Mathematical Analysis, Australian National University, vol. 3, Australian National University, 1983, vii+272 pages. [40] Y.-T. Siu, “Analyticity of sets associated to Lelong numbers and the extension o closed positive currents”, Invent. Math. 27 (1974), p. 53-156. 41] N. S. Trudinger, “Hölder gradient estimates for fully nonlinear equations”, Proc. R. Soc. Edinb., Sect. A 108 (1988), no. Bibliography [20] ——— , “The equivalence of viscosity and distributional subsolutions for convex sube- quations – the strong Bellman principle”, Bull. Braz. Math. Soc. 44 (2013), no. 4, p. 621-652. [21] ——— , “Gårding’s theory of hyperbolic polynomials”, Commun. Pure Appl. Math. 66 (2013), no. 7, p. 1102-1128. [22] ——— , “p-convexity, p-plurisubharmonicity and the Levi problem”, Indiana Univ. Math. J. 62 (2013), no. 1, p. 149-169. – 847 – F. Reese Harvey and H. Blaine Lawson 1-2, p. 57-65. 42] N. S. Trudinger & X.-J. Wang, “Hessian measures. I”, Topol. Methods Nonlinear Anal. 10 (1997), no. 2, p. 225-239. [43] ——— , “Hessian measures. II”, Ann. Math. 150 (1999), no. 2, p. 579-604. [44] ——— , “Hessian measures. III”, J. Funct. Anal. 193 (2002), no. 1, p. 1-23. [45] H.-H. Wu, “Manifolds of partially positive curvature”, Indiana Univ. Math. J. 36 (1987), no. 3, p. 523-548. – 848 –
https://openalex.org/W4293858655	https://bcpublication.org/index.php/SSH/article/download/1596/1598	English	null	Analysis of the Japanese printing and dyeing techniques: Yuzen dyeing	BCP social sciences & humanities	2,022	cc-by	4,106	1. Introduction The application of painting in dyeing technique is a major feature of Yuzen Dyeing, and the combination of painting and printing pattern technology make the form of pattern expression more free and flexible than traditional printing and dyeing technology. The humanistic thought and national characteristics contained in Yuzen patterns are the main reasons for their enduring popularity, as well as the national spirit in it. Analysis of the Japanese printing and dyeing techniques: Yuzen dyeing Jiayi Zhang Jiayi Zhang College of Art and Design, Wuhan Textile University, Wuhan 430073, China Abstract. Yuzen dyeing is one of the traditional Japanese printing and dyeing techniques. Its production process is complex and exquisite, colorful, using Yuzen patterns dyed by Yuzen dyeing technique. In Japan, according to the region, it can be divided into Kyouyuzen, Kagayuzen and Tokyoyuzen. The cloth dyed by Yuzen is usually made into clothing, bags and other products. Yuzen patterns bear the creativity and spiritual culture of the Japanese nation. This paper will mainly analyze the background origin and artistic characteristics of Yuzen patterns, hoping to provide help for traditional printing and dyeing techniques and patterns. Keywords: Yuzen; Pattern; Traditional craft. Keywords: Yuzen; Pattern; Traditional craft. BCP Social Sciences & Humanities Volume 19 (2022) BCP Social Sciences & Humanities Volume 19 (2022) ECSS 2022 3. The origin of Yuzen dyeing Yuzen dyeing first appeared in the sector drawing process. The term “yuzen” originated in the book The Tale of Genji, published in Jogan fourth year in Japan. In this book, Yuzen dyeing first appeared in the sector drawing process and then used in clothing production. In modern Japan, yuzen textile productions include some bags, scarves and other decorations. According to The Tale of Genji, yuzen had already gained popularity in 1681 (the first year of Japan's Tenwa era). In the following year, the Japanese novelist, Ihara Saikaku, published his first erotic novel A Lustful Man(Koshoku ichidaiotoko), in which he mentioned that Yuzen fans appeared as the belongings of rich dudes at that time. In addition to this book, published in 1682, the popularity of Yuzen fan in Japanese people's culture was more or less mentioned in successive books published in Japan in the following years, indicating that Yuzen fan was very popular at that time. g y p p The use of Yuzen craft in clothing production was mainly due to a ban on kimono in the second year of Tenwa era (1682), which restricted the use of some expensive materials and elaborate production techniques used in clothing. During the Edo Period, Japan's commodity economy, handicraft industry and culture were all well developed. At this time, the peasant class, who already had economic strength, began to create a civilian culture that reflected their self-consciousness. As the economic development at that time brought powerful strength to the townspeople, it also strengthened their artistic creativity gradually. In the middle of Edo period, there was a trend of enjoyment in the society. The wealthy began to add precious materials and more elaborate manufacturing techniques to their costumes, which led to the prevalence of social comparison. As a result, the Shogunate issued a series of laws banning clothing from being made by embroidery, mesh cloth and other expensive materials and complicated and elaborate techniques. In response to these bans, the peasant class began to take corresponding reactions, which did not violate the regulations, but also met the public's demand for clothing decoration. Japanese fan painting master Miyazaki Tomozen applied unique yuzen fan painting techniques into costumes, satisfying the peasant class’s desire for clothing decoration. And it was more flexible and exquisite compared with traditional printing and dyeing, so yuzen dyeing started developing in clothing. 2. The background of Yuzen dyeing The Japanese Yuzen pattern originated in the Edo period. At the beginning of the Edo period, the political system established by Tokugawa Ieyasu was very strict, and the two rule over the shogunate by two generations of generals, Tokugawa Hideyoshi and Tokugawa lemitsu, gradually stabilized. As the biggest feudal Lord in the Edo era, the general directly managed a quarter of Japan's regions and major cities, while the rest of the country was divided into more than 200 vassals of different sizes. The lords of vassals were called vassals, who directly took orders from the general. In the Tokugawa era, the Japanese people were strictly divided into four classes: samurai, farmers, craftsmen and merchants, which resulted in the differences of customs and cultures between different regions showed by Yuzen patterns. Among different classes, there were a series of prohibitions against the use of certain crafts or patterns in order to ensure the authority of shogunate and samurai. Such class barriers also cause differences in patterns among different classes. With the premise of political stability, the economy also develops. During the Tokugawa Tsunayoshi era, the economy developed well, so that the status of businessmen was greatly improved. Genroku culture flourished among merchants and farmers, resulting in a new style with bright colors and abstract patterns.This is a kind of artistic style based on the life of urban businessmen, reflecting the growth of urban citizens' class and the new awareness of citizens' class, with the tendency of anti- feudal naturalism and realism. In terms of arts and crafts, Hishikawa Moronobu, a painter who came from a family of embroidery workers, created Ukiyo-e, namely popular genre paintings, mainly focus on women, actors and sumo wrestlers, which gained popularity among people after being printed by woodblock printing. Also, colorful printed silks created by Miyazaki Tomozen in Kyoto, all of which are arts and crafts developed during this period. In the first half of the 18th century, the emergence of capitalism and the new mode of production fundamentally shook the foundation of Tokugawa era. After the Meiji Restoration, the Yuzen dyeing also started a new development with the trend of the times. 142 BCP Social Sciences & Humanities Volume 19 (2022) ECSS 2022 ECSS 2022 4. The craft of yuzen dyeing Yuzen dyeing is a dye-proof process.At the early stage, Yuzen Dyeing used plant dyes, and used glutinous rice flour and glutinous rice bran powder to blend into paste as dye-proof material, while at the later stage, modern industrial dyes were used more. While Chinese blue dyeing craft uses a wide range of anti-dyeing materials. Taking batik as an example, beeswax and paraffin wax are often used as anti-dyeing paste, while paraffin wax, due to its material properties, will produce cracks in the production process and produce beautiful crack-like patterns on the cloth, while glutinous rice anti- dyeing paste can't achieve this effect, so this pattern can’t be found in Yuzen pattern. In the production process, firstly, the outline of the pattern is sketched with anti-dye paste, so as to prevent the dye of adjacent patterns from being cross-colored. After the color blocks in the picture are drawn in layers, the painted part is covered with paste to prevent the dye from seeping in when it is put back into the dye bath. In terms of the production process, yuzen is mainly divided into hand-painted yuzen and type yuzen. Hand-painted yuzen, as its name implies, means that the whole pattern picture is made by hand painting. First, draw the pattern on silk cloth, and then draw the dye-proof paste on the cloth surface with a cone-shaped tool to prevent cross-color between different color block areas.After the whole pattern is colored, a layer of anti-dyeing paste will be spread on the picture to prevent the background color from affecting the pattern that has been drawn when dyeing. Hand-painted yuzen is much time- consuming with exquisite process, so the price is high. The emergence of type yuzen dated back to Meiji restoration, when a large number of brightly colored chemical dyes entered Japan after the western industrial revolution, and the type yuzen was developed by hand-painted yuzen dyer Hirose 143 BCP Social Sciences & Humanities Volume 19 (2022) ECSS 2022 ECSS 2022 Jisuke. The making method of type yuzen is mainly made of hollow paper. When making patterns, each color will use a unique piece of paper. According to the difficulty of patterns and different shades of colors, hundreds of pieces of paper will sometimes be used to make a Yuzen pattern. 4. The craft of yuzen dyeing In mass production, in order to ensure the accuracy of the connection, there is no trace at the connection of each paper pattern, and the skill of the craftsmen is highly required. Although the production of type yuzen is not simple, it is much simpler than the hand-painted yuzen, and the paper used can be reused, which also provides great convenience for the future mass production. After the Meiji Restoration, the Japanese royal family moved from Kyoto to Edo, and Edo changed its name to Tokyo. During the Meiji Restoration, Japan began to implement industrialization and mass production, and introduced many advanced technologies and materials from the West, which also promoted the change of Yuzen Dyeing to a certain extent, and reduced the production cost by using new materials and technologies. In addition to material changes, the Meiji Restoration also liberated people's minds, and the government began to gradually abolish the unequal provisions on the social class system, including the restrictions on the use of some patterns, which also promoted the industrial production of Yuzen dyeing. After World War II, Japan's Yuzen dyeing attracted the attention of experts and scholars all over the world, and therefore Yuzen dyeing gained better publicity worldwide. The Japanese government also began to protect its traditional handicrafts and issued a series of protection policies. However, due to the economic development, the power of businessmen gradually grew, and environmental pollution, chemical dyes and machine production appeared in yuzen dyeing's production, which to some extent also led to the shoddy yuzen dyeing process. Moreover, yuzen dyeing has higher requirements for dyes and water sources, and in modern times, yuzen dyeing has used shallow high-quality water sources in Kyoto. But due to environment pollution, it’s difficult to get high-quality water from deeper bottom, which has a great impact on the dyeing and weaving industry. In Kyoto, however, with the spirit of craftsman, a group of designers with unique understanding of the new era have emerged. They kept high demands on yuzen dyeing and added their own understanding of the spirit of the times, endowing yuzen dyeing with new inspirations for its development in modern times. 5. Philosophy contained in yuzen dyeing Some aesthetic thoughts about "feeling the truth" and "emptiness" are reflected in the patterns of Japan's Yuzen, which may come from Japan's geographical location. Because of typhoons and frequent earthquakes all the year round, they have a higher reverence for life, and they have different feelings for the rotation of the four seasons and the replacement of vegetation. Therefore, plant patterns are used to express their praise for life and desire to be one with heaven and man. In the yuzen dyeing patterns, plants often appear as the main characters in the picture. There are many kinds of Japanese traditional plant patterns, among which there are many, such as vortex pattern, vertical surge pattern and grass roll pattern. As we all know, Japanese culture has absorbed and developed a large number of excellent foreign cultures, including China. After absorbing the patterns of other nationalities, many other plant patterns have been added to the traditional plant patterns of Japan, such as chrysanthemum pattern, peony pattern, pine pattern, lily pattern, etc. Some have been deformed and adjusted to form patterns with their own characteristics, such as crab peony, which is a new pattern formed by combining peony patterns with crab patterns. Heian Period is the development period of Japanese plant patterns, when plant patterns with Japanese traditional national style were formed and the style of foreign patterns was not completely abandoned, and the color and pattern design became richer and more exquisite. In Japanese culture, many patterns are endowed with unique meanings. When the seasons change, Japanese people will hold some flower admiring activities to express their cherish of life and gratitude to nature. Cherry blossoms, or sakura symbolizes diligence, courage and wisdom in Japan. Although Japanese cherry blossoms are the most famous and well-known, in fact, cherry blossoms are not native flowers in Japan, but were brought to Japan by envoys of Tang Dynasty in China and loved by 144 BCP Social Sciences & Humanities Volume 19 (2022) ECSS 2022 ECSS 2022 Volume 19 (2022) the people. Although cherry blossoms are the mainstream of flower viewing in Japan now, in Nara era, plum blossoms that can bloom even in a cold and harsh environment were the main attraction of flower viewing. The resolute spirit embodied by plum blossoms has always been appreciated by the Japanese nation, and plum blossom patterns have a long history in Japan. 5. Philosophy contained in yuzen dyeing Besides, bamboo has long been regarded as a sacred plant by the Japanese because it is described in traditional Japanese novel The Tale of the Bamboo Cutter that it gave birth to Kaguya (the goddess of the moon). In Japanese patterns, pine, bamboo and plum patterns are often combined to form a new pattern, which is called "Three Friends", and its connotation is basically the same as that of Chinese "Three Friends of Winter". Pomegranate patterns were also introduced into Japan from China, which symbolize reproduction and vitality, and are often combined with bergamot to express longevity and good luck. The tung pattern is used by the Japanese royal family and has a long history. The culture of tung pattern originated from China. According to legend, the phoenix perches on the phoenix tree, symbolizing the auspicious sign of the birth of the holy son of heaven. Therefore, tung pattern is a very noble pattern in Japan and represents power. In addition to the above plant patterns, the common plant patterns in Yuzen dyeing include autumn grass, vines, peach patterns, grapes, maple, and so on, all of which are endowed with many meanings, reflecting the ancient Japanese people's eulogy and blessing for life 5. Philosophy contained in yuzen dyeing Born in the Heian period and widely used in the Edo period, pine wood grain is considered a symbol of good luck and longevity in Japan because the pine tree is always green. Bamboo is also one of the common plant patterns in yuzen dyeing, and it grows straight and upward, which contains the spirit of self-improvement. Besides, bamboo has long been regarded as a sacred plant by the Japanese because it is described in traditional Japanese novel The Tale of the Bamboo Cutter that it gave birth to Kaguya (the goddess of the moon). In Japanese patterns, pine, bamboo and plum patterns are often combined to form a new pattern, which is called "Three Friends", and its connotation is basically the same as that of Chinese "Three Friends of Winter". Pomegranate patterns were also introduced into Japan from China, which symbolize reproduction and vitality, and are often combined with bergamot to express longevity and good luck. The tung pattern is used by the Japanese royal family and has a long history. The culture of tung pattern originated from China. According to legend, the phoenix perches on the phoenix tree, symbolizing the auspicious sign of the birth of the holy son of heaven. Therefore, tung pattern is a very noble pattern in Japan and represents power. In addition to the above plant patterns, the common plant patterns in Yuzen dyeing include autumn grass, vines, peach patterns, grapes, maple, and so on, all of which are endowed with many meanings, reflecting the ancient Japanese people's eulogy and blessing for life the people. Although cherry blossoms are the mainstream of flower viewing in Japan now, in Nara era, plum blossoms that can bloom even in a cold and harsh environment were the main attraction of flower viewing. The resolute spirit embodied by plum blossoms has always been appreciated by the Japanese nation, and plum blossom patterns have a long history in Japan. Born in the Heian period and widely used in the Edo period, pine wood grain is considered a symbol of good luck and longevity in Japan because the pine tree is always green. Bamboo is also one of the common plant patterns in yuzen dyeing, and it grows straight and upward, which contains the spirit of self-improvement. BCP Social Sciences & Humanities Volume 19 (2022) BCP Social Sciences & Humanities Volume 19 (2022) BCP Social Sciences & Humanities Volume 19 (2022) ECSS 2022 ECSS 2022 Besides natural plants, the theme of Tokyoyuzen also likes to draw wavy patterns, with compact pictures. It also likes to take people's lives as objects, drawing all kinds of people's lives, and the pictures are full of life and fun. The theme of Tokyoyuzen is very close to the public, so this is also a major reason why Tokyoyuzen can be widely spread in the folk j y y y y p The reasons for this factional difference may be the following: First of all, it is the difference of political regions. In 794 AD, Emperor Kanmu moved its capital to Heiankyo, and it was the capital of Japan until 1868 AD, when Tokyo established its capital. The historical and cultural accumulation made Kyoto rich in cultural heritage, and it was one of the important towns of Japanese traditional culture. At the same time, Kyoto Prefecture had a large population, which was not only the spiritual hometown of Japanese, but also the origin of Japanese culture, representing Japanese culture. The Kaga is different. Before the Meiji Restoration, there were many vassal states in Japan. Although they claimed to be ruled by the Tokugawa shogunate, they were actually independent. When the U. S. army attacked Japan and opened the Japanese door, some vassal states, such as Tosa Domain, Choshu Domain, Satsuma, and Aizu, all tried to pull the tide at the end of the Shogunate, which could be called strong vassals. However, the Kaga was the rich vassal state in Japan’s history that could be compared with the Tokugawa Shogunate, so that the style of yuzen patterns presented in Kaga is relatively bright and colorful is reasonable for some political regional reasons. Edo Castle has been one of the major cities of Japan since the Tokugawa Shogunate. After being renamed Tokyo during the Meiji Restoration, it developed into the center of Japan's politics, economy, culture, transportation and other fields. BCP Social Sciences & Humanities Volume 19 (2022) In the process of development, Edo Castle has formed two different regional types: one is private area, namely the center gathered by businessmen, vendors and craftsmen, and they are mainly engaged in water transportation, goods distribution and various commercial activities; and the other is the area where the upper class act, includes the daimyo and Chimoto residential areas, which are the political centers of Edo Castle. The political and economic development has also brought cultural prosperity. The emergence of civilian culture also symbolizes the heyday of cultural development in Edo Castle. After World War II, Tokyo not only became the center of world commerce, finance, popular culture and fashion, but also one of the most developed and wealthy cities in the world. Therefore, Tokyoyuzen also produced different effects under the cultural influence of different classes and the capital relocation. Secondly, the natural environment is different, and the requirements of dyeing and weaving industry for water resources are relatively strict. Kyoto, located in the west of Japan, is an inland city, located in the northern half of the Kyoto Basin (mountain city) and the eastern mountainous area of the Tamba Plateau, so Kyoto provides high-quality superficial water for the dyeing of Kyouyuzen. Located in the southwest of Ishikawa County, Kaga is blessed with abundant natural resources such as the sea, mountains and rivers. Since ancient times, Kaga has also been known as the hometown of hot springs. In addition, there are two rivers running in Kaga, namely the Rhinokawa and Asano River. The rich water resources give Kaga a great advantage in yuzen dyeing. Tokyo has subtropical monsoon climate, four distinct seasons, abundant rainfall and Edogawa, but the development of modern industry has affected the quality of water resources. In addition to water resources, natural dyes produced in different regions are bound to be different. For example, the Kaga region is rich in minerals, which can also be of great help in dyes. 6. Yuzen dyeing in different regions In the Edo era, the technique of yuzen dyeing was used in clothing from the sector by Kyoto master Miyazaki Tomozen, and it was called “Kyouyuzen”. Then it was introduced into Kanazawa in kaga, known as “Kagayuzen”. After the Meiji Restoration, the Japanese royal family moved to Tokyo, and the yuzen dyeing developed in Tokyo was also called “Tokyoyuzen”. Since then, the traditional yuzen dyeing were divided into three factions, and Kyouyuzen, Kagayuzen and Tokyoyuzen own their unique characteristics. The specific difference between them lies in the difference of color and pattern content. p p In terms of color and tone, Kyouyuzen uses rich and magnificent colors, with red and gold predominating and embroidery as the main feature, while Kagayuzen’s colors are soft and dominated by brighter tones, and it adopts the classic "Kaga Colorful", namely "blue, purple, rouge, khaki and grass green" five colors, which are more vivid than those used by Kyouyuzen. One of the major characteristics of Tokyoyuzen is freshness, and it often uses blue and other cold colors, and the color is more stable and simple. In terms of pattern contents, Kyouyuzen patterns are mainly plant and flower patterns and ancient style patterns, and prefer to more concrete patterns. Elegant curve lines are detailed and smooth, with the traditional ancient style patterns, such as professional patterns and Linpai patterns, and professional patterns are decorative patterns on ancient official clothing, supplies and carriage equipment, Linpai patterns are gold and silver foil as the background, the theme is mostly flowers, trees, birds and beasts or character stories. In the Kyouyuzen pattern, the inner color of flowers is often aggravated, and the outer color is lighter, which makes it a major feature, and it can be called the best product. Kagayuzen is somewhat similar to Kyouyuzen in themes, but compared with it, the pictures of Kagayuzen are more calm and elegant. In plant painting, Kagayuzen will emphasize the external color and lighten the internal color, which is different from Kyouyuzen patterns, and contrast colors are used in some natural patterns. In addition, Kagayuzen also uses a painting technique called "insect- eating" in the description of fabric patterns, adding dark spots in the part of the leaves in the pattern, forming the visual effect of the insect bite. 145 7. Conclusion Yuzen dyeing is not only a traditional printing and dyeing technique in Japan, but also a treasure of the world printing and dyeing culture. The good wishes for life contained in its patterns and the excellent national spirit inherited by craftsmen are the main factors that make Yuzen dyeing last for a long time, which makes Yuzen dyeing still shine brilliantly all over the world after hundreds of years. 146 BCP Social Sciences & Humanities Volume 19 (2022) ECSS 2022 ECSS 2022 Volume 19 (2022) Volume 19 (2022) [3] Zhang Hong. A study on the influence of Chinese Zen aesthetic thought on the origin and development of You Zen patterns [J]. Beauty and the Times (Upper Journal),2010(11):50-51. [2] BALANCE. Kaga Tomochan:Cherry real dreams[J]. Fairview,2018,11(11):12-15. [4] Zhang Fuya, Li Xinyang. A study on the composition of kimono pattern by Kunihiko Moriguchi [J]. Art and Design: Theory Edition,2020(12):131-134. ] Nakagawa Seiji. Kaga Tomozen[J]. Textile Consumer Science,2020,61(11 TN.672):14-17. ] Liang Shuang. An analysis of Japanese You Zen dyeing plant patterns[J]. Peony, 2021 (08): 1 References [1] Liang Shuang. An analysis of Japanese You Zen dyeing plant patterns[J]. Peony, 2021 (08): 176-177. [2] BALANCE. Kaga Tomochan:Cherry real dreams[J]. Fairview,2018,11(11):12-15. [3] Zhang Hong. A study on the influence of Chinese Zen aesthetic thought on the origin and development of You Zen patterns [J]. Beauty and the Times (Upper Journal),2010(11):50-51. [4] Zhang Fuya, Li Xinyang. A study on the composition of kimono pattern by Kunihiko Moriguchi [J]. Art and Design: Theory Edition,2020(12):131-134. [5] Nakagawa Seiji. Kaga Tomozen[J]. Textile Consumer Science,2020,61(11 TN.672):14-17. [2] BALANCE. Kaga Tomochan:Cherry real dreams[J]. Fairview,2018,11(11):12 15. [3] Zhang Hong. A study on the influence of Chinese Zen aesthetic thought on the origin and development of You Zen patterns [J]. Beauty and the Times (Upper Journal),2010(11):50-51. [4] Zhang Fuya, Li Xinyang. A study on the composition of kimono pattern by Kunihiko Moriguchi [J]. Art and Design: Theory Edition,2020(12):131-134. 147
https://openalex.org/W2900867346	https://europepmc.org/articles/pmc6256130?pdf=render	English	null	Review of the subgenus Plumiger of Myrmecophyes, with description of a new species (Heteroptera, Miridae, Halticini)	ZooKeys	2,018	cc-by	10,105	Keywords Al d Alpine meadows, Caucasus, diagnosis, female genitalia, key to species, male genitalia, systematics http://zoobank.org/CC96882F-7123-4A2D-8345-7C5E36F817C1 Citation: Konstantinov FV, Simov N (2018) Review of the subgenus Plumiger of Myrmecophyes, with description of a new species (Heteroptera, Miridae, Halticini). In: Wheeler Jr AG (Ed.) A Festschrift Recognizing Thomas J. Henry for a Lifetime of Contributions to Heteropteran Systematics. ZooKeys 796: 215–239. https://doi.org/10.3897/zookeys.796.21877 Abstracth The Caucasian subgenus Plumiger Horváth, 1927 of the halticine genus Myrmecophyes Fieber, 1870 is revised. A key, updated diagnoses, and data on distribution are given for the subgenus and its four species, including M. tomi sp. n. (Georgia and Dagestan), and the previously unknown male of M. armeniacus Drapolyuk, 1989. Illustrations of the male and female genitalia, photographs of the dorsal habitus, and SEM micrographs of selected structures are provided for all species of the subgenus. f a new species... 215 Launched to accelerate biodiversity research A peer-reviewed open-access journal f a new species... 215 Launched to accelerate biodiversity research A peer-reviewed open-access journal Review of the subge ZooKeys 796: 215–239 (2018) doi: 10.3897/zookeys.796.21877 http://zookeys.pensoft.net Review of the subge ZooKeys 796: 215–239 (2018) doi: 10.3897/zookeys.796.21877 http://zookeys.pensoft.net of Myrmecophyes, w RESEARCH ARTICLE Fedor V. Konstantinov1,2, Nikolay Simov3 Fedor V. Konstantinov1,2, Nikolay Simov3 1 Department of Entomology, Faculty of Biology, St. Petersburg State University, Universitetskaya nab. 7/9, St. Petersburg 199034, Russia 2 Zoological Institute, Russian Academy of Sciences, Universitetskaya nab. 1, St. Petersburg 199034, Russia 3 National Museum of Natural History, Bulgarian Academy of Sciences, 1 Tsar Osvoboditel Blvd., 1000 Sofia, Bulgaria Corresponding author: Fedor V. Konstantinov (f.konstantinov@spbu.ru) cademic editor: A. Wheeler \| Received 26 October 2017 \| Accepted 19 April 2018 \| Published 15 November Copyright Fedor V. Konstantinov, Nikolay Simov. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Introduction Myrmecophyes Fieber, 1870 is a distinctive and strikingly myrmecomorphic halticine genus comprising 30 currently recognized species (Schuh 2002–2013, Tatarnic and Cassis 2012, Konstantinov et al. 2013). All Myrmecophyes spp. are brachypterous, re stricted to relatively small distributional areas and inhabit montane grasslands, except M. alboornatus, which spans a large area in Europe and Siberia, reaching Kazakhstan, 216 Fedor V. Konstantinov & Nikolay Simov / ZooKeys 796: 215–239 (2018) Mongolia, and northern China in the south. The genus is most speciose in the Central Asian and Caucasian mountains. Species found elsewhere are M. gallicus Wagner, 1976 in the Pyrenees; M. latus Wagner, 1975 and M. montenegrinus Wagner, 1976 described from Western Balkan Mountains; and M. oregonensis Schuh & Lattin, 1980, the only North American species, found in Oregon at altitudes from 1500 to 1700 m. Horváth (1927) described the subgenus Plumiger within Myrmecophyes to ac commodate M. heterocerus Horváth, 1927. His diagnosis of the monotypic subgenus was based on the modified first two antennal segments in males. Drapolyuk (1989) published a review of the Caucasian Myrmecophyes and described two more species of the subgenus. g Recent collecting by the authors from the Armenian Highlands produced a sub stantial number of Myrmecophyes spp., including long series of Myrmecophyes (Plu miger) armeniacus Drapolyuk, 1989, originally described from two females. Subse quent examination of holdings from the Zoological Institute, Russian Academy of Sciences (ZISP), led to the discovery of a new species belonging to the same subgenus. Thus we recognize four species within Plumiger, all restricted to the Caucasus. A phy logenetic analysis of the genus is needed to clarify the status of the subgenus. Pending such a study, which might reveal interesting biogeographic patterns, we are certain that the group is sufficiently distinct to warrant recognition at the subgeneric level. The monophyly of Plumiger is corroborated by characters presented in our diagnosis that are not shared by other species of Myrmecophyes.h The present paper provides the description of a new species, a key to males and females, a revised diagnosis, and a redescription of the subgenus. A diagnosis, descrip tion, measurements, distributional information, a dorsal habitus photograph, and il lustrations of male and female genitalia are given for each species of the subgenus.h We are delighted to dedicate this paper to our eminent colleague Dr. Thomas J. Henry on the occasion of his 70th birthday. Introduction During a long and distinguished career at the USDA, c/o Smithsonian National Museum of Natural History, Tom Henry has made singular contributions to our knowledge of the Heteroptera worldwide. Despite his many duties, Tom always manages to find time to help his colleagues in their stud ies and to provide an energetic and supportive environment to all researchers working with the USNM Heteroptera collection. Materials and methods Slightly more than 600 specimens were examined for this study. All specimens are retained in the Zoological Institute, St. Petersburg, Russia (ZISP) and the National Museum of Natural History, Sofia, Bulgaria (SOFM). The holotype of Myrmecophyes tomi sp. n. is deposited in the Heteroptera collection of the Zoological Institute in St. Petersburg. All ZISP specimens were associated with bar code labels (“unique specimen identifiers” or “USIs”), which were printed as a matrix code label that also provides an alphanumeric string, e.g., AMNH_PBI 00343231. USI numbers explicitly identify Review of the subgenus Plumiger of Myrmecophyes, with description of a new species... 217 particular specimens and are listed for each species in the “Material examined” section. Additional specimen information for a selected species, including additional figures not included in this paper, can be obtained from the Heteroptera Species Pages (http:// research.amnh.org/pbi/heteropteraspeciespage/), which assemble available data from a specimen database. Geo-reference data for each locality were obtained from gazetteers, atlases, handheld GPS and other sources. The distributional maps were created using QGIS 2.18.4 software. Unless otherwise stated, all measurements are in millimeters. Measurements shown in Table 1 include body length, clypeus to apex of wing pad length, head and pronotum length and width, interocular distance, length of hind tibia, and antennal segments I and II. Observations, measurements, and digital dorsal color images were made with a Nikon SMZ 1500 stereomicroscope equipped with Nikon D700 digital SLR cam era. Images of the genitalic structures were taken with a Leica DM2500 microscope equipped with Leica DFC 450 digital camera. Partially focused images of each specimen or structure were stacked using the Helicon Focus 6.2.2 software. The terminology used for genitalia follows Konstantinov (2003) for males and Schwartz (2011) for females. Taxonomy Genus Myrmecophyes Fieber, 1870 Subgenus Plumiger Horváth, 1927 Plumiger Horváth, 1927: 189 (new subgenus). Type species by monotypy: Myrmeco phyes heterocerus Horváth, 1927. Plumiger Horváth, 1927: 189 (new subgenus). Type species by monotypy: Myrmeco phyes heterocerus Horváth, 1927. Plumiger Horváth, 1927: 189 (new subgenus). Type species by monotypy: Myrmeco phyes heterocerus Horváth, 1927. Diagnosis. Antenna sexually dimorphic (Figs 5–13), female filiform, male with first two segments modified and clothed ventrally with dense spatulate whitish scales (Figs 20, 21), segment I distinctly swollen, segment II curved and flattened apically; antennal segment III distinctly longer than other segments in both sexes; endosoma of aedeagus without sclerites, composed of several minutely dentate lobes, entirely mem branous (Figs 37, 39) or slightly sclerotized (Figs 47, 49).f Remarks. All species of the subgenus Myrmecophyes differ from Plumiger in having filiform (rather than sexually dimorphic) antennae and two or three variously shaped and typically large sclerites of the endosoma (Bykov 1971: figs 48–96, Schuh and Lat tin 1980: fig. 10, Drapolyuk 1989: figs 7–10, Drapolyuk and Kerzhner 2000: figs 14, 22, Konstantinov et al. 2013: fig. 3). Antennal segments in males and females of Myr mecophyes spp. are straight, rod-shaped, and thin, with the second segment the longest and only slightly thinner than the first. i Redescription. Male. Total body length 2.8–3.8, brachypterous and distinctly antlike. Coloration (Figs 5–7): Dorsum, thoracic pleura, and venter uniformly dark i Redescription. Male. Total body length 2.8–3.8, brachypterous and distinctly antlike. Coloration (Figs 5–7): Dorsum, thoracic pleura, and venter uniformly dark Fedor V. Konstantinov & Nikolay Simov / ZooKeys 796: 215–239 (2018) 218 Table 1. Measurements (mm). Abbreviations: Clyp-Wing – distance between apex of clypeus and apex of wing pad in dorsal view, Head Length – distance between apex of clypeus and the highest point of vertex, AntSeg1 – AntSeg2 – length of antennal segments I and II, InterOcDi – width of vertex between inner margins of eyes in dorsal view. Subgenus Plumiger Horváth, 1927 Species Length Width Body Clyp-Wing Head Pronotum Tibia3 AntSeg1 AntSeg2 Head InterOcDi Pronotum Myrmecophyes armeniacus ♂♂ (n = 5) Mean 3.46 2.07 0.55 0.65 3.27 0.48 0.67 1.15 0.56 0.85 SD 0.11 0.07 0.03 0.02 0.11 0.02 0.03 0.02 0.02 0.03 Range 0.25 0.16 0.09 0.04 0.28 0.04 0.07 0.04 0.04 0.07 Min 3.40 2.02 0.50 0.64 3.08 0.46 0.64 1.13 0.53 0.81 Max 3.65 2.18 0.58 0.67 3.36 0.50 0.71 1.17 0.57 0.88 ♀♀ (n = 5) Mean 3.99 2.17 0.52 0.65 3.02 0.51 1.00 1.20 0.60 0.89 SD 0.11 0.11 0.05 0.02 0.11 0.02 0.02 0.04 0.03 0.03 Range 0.25 0.28 0.11 0.04 0.28 0.05 0.04 0.07 0.07 0.07 Min 3.86 2.05 0.46 0.64 2.90 0.48 0.99 1.17 0.57 0.85 Max 4.11 2.34 0.57 0.67 3.19 0.53 1.03 1.24 0.64 0.92 Myrmecophyes heterocerus ♂♂ (n = 5) Mean 2.97 1.71 0.48 0.52 1.88 0.42 0.53 0.99 0.46 0.71 SD 0.16 0.12 0.06 0.02 1.07 0.00 0.03 0.02 0.01 0.03 Range 0.42 0.32 0.14 0.04 2.58 0.00 0.07 0.05 0.04 0.07 Min 2.76 1.52 0.42 0.50 0.00 0.42 0.50 0.97 0.44 0.67 Max 3.19 1.84 0.57 0.53 2.58 0.42 0.57 1.03 0.48 0.74 ♀♀ (n =5) Mean 3.37 1.90 0.53 0.57 2.39 0.48 0.88 1.11 0.56 0.84 SD 0.38 0.10 0.07 0.03 0.10 0.03 0.05 0.04 0.03 0.03 Range 0.92 0.21 0.18 0.07 0.25 0.07 0.11 0.09 0.05 0.07 Min 2.90 1.81 0.46 0.53 2.30 0.46 0.81 1.08 0.53 0.81 Max 3.82 2.02 0.64 0.60 2.55 0.53 0.92 1.17 0.58 0.88 Myrmecophyes nasutus ♂♂ (n = 2) Min 3.60 2.05 0.46 0.60 3.36 0.85 1.56 1.24 0.64 0.85 Max 3.75 2.30 0.57 0.64 3.54 0.85 1.59 1.26 0.65 0.88 ♀♀ (n =5) Mean 4.30 2.19 0.59 0.67 3.25 0.64 1.48 1.30 0.68 0.99 SD 0.11 0.05 0.03 0.01 0.08 0.02 0.07 0.03 0.03 0.03 Range 0.28 0.11 0.07 0.02 0.21 0.04 0.18 0.09 0.07 0.07 Min 4.18 2.12 0.57 0.65 3.12 0.64 1.42 1.26 0.64 0.96 Max 4.46 2.23 0.64 0.67 3.33 0.67 1.59 1.35 0.71 1.03 Myrmecophyes tomi sp. n. Subgenus Plumiger Horváth, 1927 ♂♂ (n = 1) 3.40 1.88 0.46 0.64 3.19 0.57 0.99 1.13 0.57 0.85 ♀♀ (n =5) Mean 3.33 1.84 0.52 0.57 2.48 0.46 0.86 1.10 0.56 0.81 SD 0.16 0.10 0.05 0.03 0.20 0.04 0.04 0.04 0.02 0.04 Range 0.42 0.25 0.11 0.07 0.46 0.07 0.11 0.11 0.05 0.11 Min 3.12 1.70 0.46 0.53 2.30 0.42 0.81 1.03 0.53 0.74 Max 3.54 1.95 0.57 0.60 2.76 0.50 0.92 1.13 0.58 0.85 brown to black with narrow contrastingly whitish stripe along apex of wing pad brown to black, with narrow, contrastingly whitish stripe along apex of wing pad forming transverse band; antenna uniformly black, sometimes with chestnut brown segment I; femora black, sometimes with pale brown apices, rarely almost uniformly Review of the subgenus Plumiger of Myrmecophyes, with description of a new species... 219 Figures 1–4. Habitats and habitus images of live individuals of Myrmecophyes (Plumiger) species. 1, 2 Typical habitats of Armenian Myrmecophyes (Plumiger) species 3, 4 Myrmecophyes armeniacus Drapol yuk, 1989 3 female 4 male. Figures 1–4. Habitats and habitus images of live individuals of Myrmecophyes (Plumiger) species. 1, 2 Typical habitats of Armenian Myrmecophyes (Plumiger) species 3, 4 Myrmecophyes armeniacus Drapol yuk, 1989 3 female 4 male. pale brown, tibiae and tarsi dark brown. Surface and Vestiture: Dorsum and venter rugose, pronotum with fine transverse wrinkles along anterior and posterior margin; abdomen smoother than thoracic dorsum, sometimes shining. Dorsum, venter and legs with very short and thin, reclining, pale simple setae, scarce on head, thorax and femora, more dense on abdomen and tibiae. Head with several spinelike setae on vertex posteriorly and on frons between eye and antennal fossa, apex of frons and base of clypeus with very dense, thin and exceptionally long straight whitish setae; antennal segments I and II with numerous black spinelike setae on dorsal surfaces and with dense spatulate white scales on ventral surfaces except basal one-fourth; seg ments III and IV with mixture of relatively scarce erect spinelike setae shorter than those on previous segments and dense semierect pale simple setae. Pronotum with two black spinelike setae on anterorlateral corners, sometimes with additional setae along anterior and posterior margins; fore coxa with row of black spinelike setae on anterior surface; femora typically with one or two incomplete rows of stiff spinelike setae along dorsal margin and several apical spines dorsally and ventrally; tibia with scattered black spines. Subgenus Plumiger Horváth, 1927 Genital capsule with relatively long, thin, simple setae api pale brown, tibiae and tarsi dark brown. Surface and Vestiture: Dorsum and venter rugose, pronotum with fine transverse wrinkles along anterior and posterior margin; abdomen smoother than thoracic dorsum, sometimes shining. Dorsum, venter and legs with very short and thin, reclining, pale simple setae, scarce on head, thorax and femora, more dense on abdomen and tibiae. Head with several spinelike setae on vertex posteriorly and on frons between eye and antennal fossa, apex of frons and base of clypeus with very dense, thin and exceptionally long straight whitish setae; antennal segments I and II with numerous black spinelike setae on dorsal surfaces and with dense spatulate white scales on ventral surfaces except basal one-fourth; seg ments III and IV with mixture of relatively scarce erect spinelike setae shorter than those on previous segments and dense semierect pale simple setae. Pronotum with two black spinelike setae on anterorlateral corners, sometimes with additional setae along anterior and posterior margins; fore coxa with row of black spinelike setae on anterior surface; femora typically with one or two incomplete rows of stiff spinelike setae along dorsal margin and several apical spines dorsally and ventrally; tibia with scattered black spines. Genital capsule with relatively long, thin, simple setae api Fedor V. Konstantinov & Nikolay Simov / ZooKeys 796: 215–239 (2018) 220 Figures 5–13. Dorsal habitus of Myrmecophyes (Plumiger) species. 5, 8 Myrmecophyes armeniacus Drapolyuk 1989 5 male 8 female 6, 9 Myrmecophyes heterocerus Horváth, 1927 6 male 9 female 7, 10 Myrmecophyes tom sp. n. 7 male 10 female 11–13 Myrmecophyes nasutus Drapolyuk, 1989 11 male 12 pale female 13 dark female Figures 5–13. Dorsal habitus of Myrmecophyes (Plumiger) species. 5, 8 Myrmecophyes armeniacus Drapolyuk, 1989 5 male 8 female 6, 9 Myrmecophyes heterocerus Horváth, 1927 6 male 9 female 7, 10 Myrmecophyes tomi sp. n. 7 male 10 female 11–13 Myrmecophyes nasutus Drapolyuk, 1989 11 male 12 pale female 13 dark female. Review of the subgenus Plumiger of Myrmecophyes, with description of a new species... 221 cally and near parameres. Subgenus Plumiger Horváth, 1927 Structure: Head: Large, distinctly vertical, twice as high as length or height of pronotum (Figure 15); frons flat; vertex broad and almost flat, in frontal view at about same level as dorsal margin of eyes; eyes relatively small, not stylate, projecting well beyond lateral margins of pronotum; antennal fossa below ventral margin of eye; antennal segment I distinctly swollen, barrel-shaped, 2.5–3.5 × as wide as segment II, 1.5–2.4 × as long as wide (Figure 18); segment II gradually curved and somewhat flattened at apex, segments III-IV thin, segment III distinctly or at least slightly longer than any other antennal segment; labium stout, always surpassing hind coxa and reaching basal abdominal segments. Thorax: Pronotum with anterior margin slightly convex, flattened and weakly reflexed, lateral margins strongly convex and smoothly rounded, posterior margin flattened, straight or slight ly concave; calli not delimited; exposed part of mesonotum large, slightly shorter than pronotal length, distinctly convex, gibbous in lateral view; scutellum not de limited from mesonotum; hemelytron reduced to undifferentiated, broadly rounded wing pad reaching extreme base of abdomen, somewhat convex in basal two-thirds, with flat apical edging; veins and claval suture absent; pronotum about 2.5–2.8 × as long as wing commissure; metathoracic spiracle surrounded by distinct microscu lpture; scent gland evaporatory area roughly triangular, with flat peritreme (Figure 17); hind femur enlarged and somewhat flattened, distinctly surpassing apex of ab domen, pretarsus as in Figs 22, 23, with smoothly curved claws and fleshy, apically convergent parempodia, pulvilli absent. Abdomen constricted at base in lateral view and greatly expanded posterior to basal segments. Subgenus Plumiger Horváth, 1927 Genitalia: genital capsule wide, heavily sclerotized, partly retracted into pregenital segments, without distinctive or namentation or processes (Figure 24), ventral wall with narrow cone-shaped exten sion caudally, genital opening wide, oval; parameres of typical halticine shape, left paramere sickle-shaped, gradually curving and terminating with oblique T-shaped (Figs 30, 34) or harpoon-shaped (Figs 41, 44) blade; right paramere larger than left one, with long basal process, apically flattened and concave, flag-shaped, subquadrate in lateral view, with apical denticle on inner margin (Figs 33, 36, 43, 46); phallotheca of aedeagus (Figs 38, 40, 48, 50) voluminous, with dorsal wall entirely sclerotized, upturned apically, ventral wall sclerotized apically, membranous at base, equipped with pair of sclerotized and rounded subapical outgrowths at sides; ductus seminis relatively short, basal half membranous, coiled, with distinct sclerotized rings, api cal half somewhat wider, straight and heavily sclerotized, terminating in horseshoe- shaped, sculptured secondary gonopore; endosoma without sclerites, membranous, folded, with several eversible, finely dentate and sometimes slightly sclerotized lobes (Figs 37, 39, 47, 49). Female. Similar to male in coloration, surface, and main structural details, but differing in structure and vestiture of antennal segments, abdomen strongly ex panded at middle, and body proportions (Figs 8–10, 12, 13). Body larger on aver age, total length 2.9–4.5. Coloration: As in male but antennal segment I usually dirty yellow, rarely dark brown, segments III and IV black, sometimes partly or entirely dirty pale brown; femora entirely dirty yellow to dark brown with paler Fedor V. Konstantinov & Nikolay Simov / ZooKeys 796: 215–239 (2018) 222 Figures 14–19. Scanning electron micrographs. 14, 15 Head and thorax in lateral view 14 Myrme cophyes armeniacus Drapolyuk, 1989, female 15 Myrmecophyes heterocerus Horváth, 1927, male 16, 17 Metathoracic scent gland evaporative area and spiracle 16 M. armeniacus 17 M. heterocerus 18 Head of M. heterocerus male in lateral view 19 Antennal segment I of M. heterocerus male. Figures 14–19. Scanning electron micrographs. 14, 15 Head and thorax in lateral view 14 Myrme cophyes armeniacus Drapolyuk, 1989, female 15 Myrmecophyes heterocerus Horváth, 1927, male 16, 17 Metathoracic scent gland evaporative area and spiracle 16 M. armeniacus 17 M. heterocerus 18 Head of M. heterocerus male in lateral view 19 Antennal segment I of M. heterocerus male. apices. Subgenus Plumiger Horváth, 1927 Surface and vestiture: As in male, but frons and clypeus without long, thin, whitish setae and black spinelike setae; antennal segment I with regularly distrib uted, minute, black, adpressed simple setae and 6–12 spinelike setae on mesial surface; segments II-IV with mixture of relatively scarce erect black setae and dense, Review of the subgenus Plumiger of Myrmecophyes, with description of a new species... 223 semierect, pale and thin simple setae. Structure: Similar to male, but antenna not modified, segment I short, slightly and uniformly swollen, at most twice as wide as second, segment II rod-shaped, straight; labium always reaching and usually sur passing hind coxa; abdomen more expanded at middle than in male, broadly oval in lateral and dorsal view. Genitalia: dorsal labiate plate of bursa copulatrix mem branous, with inconspicuous anterior and posterior margins and medially recurved lateral margins, without additional sclerotization and microtrichia; sclerotized ring broadly oval to subtriangular, more or less strongly curved along longitudinal axis, sometimes with strongly and narrowly elongate apex (Figs 51–54); posterior wall membranous, with almost flat, minutely dentate or coarsely rugose interramal scle rites (Figs 55–58); inner margins of first gonapophyses vestibulum with simple and symmetrical, roughly triangular sclerites encircling vulva (Figure 63); first gonapo physis distinctly sagittate, second gonapophysis saber-shaped, gradually tapering. Distribution. The subgenus is endemic to the Caucasus Mountains including Greater and Lesser Caucasus, and Armenian Highlands (Figure 64). Given the recent records of Caucasian halticines in the Pontic Mountains (Dursun and Kartal 2006), species of Plumiger eventually might be found there.h Discussion. The striking sexual dimorphism in Plumiger species and one of the main diagnostic features of the subgenus, is exhibited by the enlarged antennal seg ments I and II and their peculiar vestiture (Figs 18, 19). Males apparently use their antennae to grasp females during copulation in a manner similar to that of several other plant bug genera with sexually dimorphic antennae, e.g., Harpocera Curtis, 1838 (Kullenberg 1944, Stork 1981) and Spanagonicus Berg, 1883. The second antennal segment II of the latter genus is also equipped with spatulate setae that may have an adhesive function (Menard 2015). Natural history. We swept specimens of Plumiger in large numbers between 1950 and 2450 m a.s.l. in different grasslands and steppe habitats, viz. Ponto–Cau casian hay meadows, grass meadow-steppes, feather-grass steppes, and acid subal pine grasslands (Figs 1–4). Myrmecophyes armeniacus and M. heterocerus studied were associated with Poaceae. Subgenus Plumiger Horváth, 1927 Occasionally adults were found on species of Asteraceae. Although no host records are available for M. nasutus and M. tomi sp. n., the label data indicate that they are confined to highland grasslands. Nearly all species of Myr mecophyes s. str. with known host associations also utilize Poaceae, typically occur in large numbers and are considered pests of pastures across highlands of Central Asia (Bykov 1971). In contrast, M. trispiculus and M. geniculatus feed on Artemisia spp. (Drapolyuk and Kerzhner 2000). Almost nothing is known about the phenology of Plumiger species. Based on the presence of eggs in females, the ratio of males to females, and available collection dates, we speculate that M. armeniacus and M. heterocerus have one and two genera tions, respectively. More interesting are our observations of diurnal activity of Arme nian Myrmecophyes. Despite maximal efforts in suitable habitats, we had little success in collecting at noon. The insects became active and started to move up the culm and leaves of grasses approximately two and a half hours before sunset (just after 18:00 in Fedor V. Konstantinov & Nikolay Simov / ZooKeys 796: 215–239 (2018) 224 Figures 20–25. Scanning electron micrographs. 20, 21 Vestiture of male antennal segments in Myrme cophyes heterocerus Horváth, 1927 20 segment I 21 segment II 22, 23 Pretarsus of Myrmecophyes armenia cus Drapolyuk, 1989 24 Genital segment of M. heterocerus male 25 Abdomen of M. armeniacus female. Figures 20–25. Scanning electron micrographs. 20, 21 Vestiture of male antennal segments in Myrme cophyes heterocerus Horváth, 1927 20 segment I 21 segment II 22, 23 Pretarsus of Myrmecophyes armenia cus Drapolyuk, 1989 24 Genital segment of M. heterocerus male 25 Abdomen of M. armeniacus female. June). During the day, the bugs were close to the surface, hidden among lower parts of the grasses. This corresponds with our previous observations on other brachypter ous halticines, viz. Myrmecophyes s. str., Scirtetellus, and Dimorphocoris spp. in the high Review of the subgenus Plumiger of Myrmecophyes, with description of a new species... 225 mountains of the Balkans and Central Asia. Schuh and Lattin (1980) reported plausi ble midmorning and late-afternoon peaks of activity for M. oregonensis sampled south west of Burns, Oregon. mountains of the Balkans and Central Asia. Schuh and Lattin (1980) reported plausi ble midmorning and late-afternoon peaks of activity for M. oregonensis sampled south west of Burns, Oregon. Key to males Left para mere without outgrowth at base of apical process (Figure 35)............heterocerus 3 Antennal segments longer, segment I 1.3–1.4 × as long as pronotum, seg ment II 1.2–1.3 × as long as head width and 1.8–1.9 × as long as width of pronotum at middle. Left paramere with two subapical recurved denticles (Figs 41, 42)......................................................................................nasutus – Antennal segments shorter, segment I 0.9 × as long as pronotum, segment II 0.9 × as long as head width and 1.2 × as long as width of pronotum at middle. Left paramere with single subapical recurved denticle (Figs 44, 45)........tomi Key to males 1 Clypeus distinctly bulging, rectangular in lateral view (Figs 27, 29). Antennal segment II longer than pronotal width at middle and 1.8–1.9 × as long as segment I. Left paramere harpoon-shaped, with one or two recurved denticles apically (Figs 41, 44). Endosoma of aedeagus with three folded and slightly sclerotized lobes (Figs 47, 49)......................................................................3 – Clypeus not bulging, slightly convex in apical half (Figs 26, 28). Antennal segment II shorter than width of pronotum at middle and 1.2–1.4 × as long as segment I. Left paramere ending with oblique T-shaped blade (Figs 30, 34). Endosoma of aedeagus with entirely membranous lobes (Figs 37, 39)..........2 2 Larger, body length 3.4–3.7. Head and abdomen matt. Left paramere with rounded outgrowth at base of apical process (Figs 30, 31)........... armeniacus – Smaller, body length 2.8–3.2. Head and abdomen smooth and shiny. Left para mere without outgrowth at base of apical process (Figure 35)............heterocerus 3 Antennal segments longer, segment I 1.3–1.4 × as long as pronotum, seg ment II 1.2–1.3 × as long as head width and 1.8–1.9 × as long as width of pronotum at middle. Left paramere with two subapical recurved denticles (Figs 41, 42)......................................................................................nasutus – Antennal segments shorter, segment I 0.9 × as long as pronotum, segment II 0.9 × as long as head width and 1.2 × as long as width of pronotum at middle. Left paramere with single subapical recurved denticle (Figs 44, 45)........tomi 1 Clypeus distinctly bulging, rectangular in lateral view (Figs 27, 29). Antennal segment II longer than pronotal width at middle and 1.8–1.9 × as long as segment I. Left paramere harpoon-shaped, with one or two recurved denticles apically (Figs 41, 44). Endosoma of aedeagus with three folded and slightly sclerotized lobes (Figs 47, 49)......................................................................3 – Clypeus not bulging, slightly convex in apical half (Figs 26, 28). Antennal segment II shorter than width of pronotum at middle and 1.2–1.4 × as long as segment I. Left paramere ending with oblique T-shaped blade (Figs 30, 34). Endosoma of aedeagus with entirely membranous lobes (Figs 37, 39)..........2 2 Larger, body length 3.4–3.7. Head and abdomen matt. Left paramere with rounded outgrowth at base of apical process (Figs 30, 31)........... armeniacus – Smaller, body length 2.8–3.2. Head and abdomen smooth and shiny. Key to females 1 Larger, body length 3.9–4.5. Head and abdomen matt (Figs 8, 12, 13).......2 – Smaller, body length 2.8–3.2. Head and abdomen smooth and shiny (Figs 9, 10)...............................................................................................................3 2 Body length 3.9–4.1. Sclerotized rings of dorsal labiate plate broadly oval (Figs 51, 59)............................................................................... armeniacus – Body length 4.2–4.5. Sclerotized rings of dorsal labiate plate subtriangular, long and narrow, with apices distinctly attenuated (Figs 54, 61)........nasutus 3 Sclerotized rings of dorsal labiate plate subtriangular, long and narrow, with apices distinctly attenuated (Figs 53, 60)......................................heterocerus – Sclerotized rings of dorsal labiate plate broadly oval (Figs 52, 62)...........tomi Fedor V. Konstantinov & Nikolay Simov / ZooKeys 796: 215–239 (2018) 226 Figures 26–29. Male head in lateral view: 26 Myrmecophyes armeniacus Drapolyuk, 1989 27 Myrmeco phyes nasutus Drapolyuk, 1989 28 Myrmecophyes heterocerus Horváth, 1927 29 Myrmecophyes tomi sp. n. Figures 26–29. Male head in lateral view: 26 Myrmecophyes armeniacus Drapolyuk, 1989 27 Myrmeco phyes nasutus Drapolyuk, 1989 28 Myrmecophyes heterocerus Horváth, 1927 29 Myrmecophyes tomi sp. n. Myrmecophyes armeniacus Drapolyuk, 1989 Myrmecophyes armeniacus Drapolyuk, 1989 Figs 5, 8, 14, 16, 22, 23, 25, 26, 30–33, 37,38, 51, 55, 59, 64 Myrmecophyes (Plumiger) armeniacus Dapolyuk, 1989: 125; figs 51, 55. Size large, clypeus straight, not bulging apically in either sex (Figs 14, 26), head and abdomen not shiny (Figs 5, 8), antennal segment II in male shorter than width of pronotum; shape of the left paramere as in Figs 30–32, lobes of aedea gus membranous (Figure 37), and sclerotized rings of dorsal labiate plate broadly oval (Figs 51, 55). Remarks. Males of M. armeniacus are most similar to M. heterocerus in structure of the head and male genitalia, but the latter species can be distinguished by the smaller size, shiny head and abdomen, and structure of the left paramere with the apical pro cess narrowing before the T-shaped blade and equipped with a prominent outgrowth at base (compare Figs 30, 34). Females of M. armeniacus and M. nasutus are roughly similar in size and vestiture, and the surface of the dorsum is not shiny; these species can be separated by their differently shaped sclerotized rings (compare Figs 51, 54). p yf y p g ( p g , ) Redescription. Male. Total length 3.4–3.7. Coloration: (Figure 5): entirely black, with whitish transverse stripe along apex of wing pad; fore and middle femora usually, hind femur rarely with pale brown apices. Surface and vestiture: Dorsum matt, head and abdomen at most moderately shiny; vestiture as in subgeneric description. Structure: Body 3.9–4.2 × as long as width of pronotum at middle. Head: Clypeus not bulging (Figure 26), flat at base and slightly convex in apical part, with apical margin straight; vertex 1.8–2.0 × as wide as eye; antennal segment I strongly swollen, short, 0.8–0.9 × as long as length of pronotum, segment II slightly curved and widened apically, rather short, 1.4–1.5 × as long as I, 0.8 × as long as width of pronotum at middle, and 0.6 × as long as width of head; segment III more than 2.8–2.9 × as long as II and distinctly longer than I and II combined; segment IV about 1.5 × as long as II. Thorax: Pronotum at mid dle 1.3 × as wide as long, 0.7–0.8 × as wide as head; hind tibia 3.6–4.0 × as long as width of pronotum at middle. Myrmecophyes (Plumiger) armeniacus Dapolyuk, 1989: 125; figs 51, 55. Myrmecophyes (Plumiger) armeniacus Dapolyuk, 1989: 125; figs 51, 55. Myrmecophyes (Plumiger) armeniacus Dapolyuk, 1989: 125; figs 51, 55. Material examined. Holotype: TURKEY: Kars: Kars, 40.58333°N, 43.06666°E, 04 Jun 1915, Olsufiev, 1♀ (AMNH_PBI 00261681) (ZISP). Paratypes: ARMENIA: Shirak: Gyumri [Leninakan], 40.78333°N, 43.83333°E, 16 Jul 1940, Esterberg, 1♀ (AMNH_PBI 00261680) (ZISP). Other specimens examined: ARMENIA: Gegharkunik: N coast of Sevan Lake, Ar tanish – Shorzha Rd, 40.50109°N, 45.3282°E, 1962 m, 14 Jun 2017, F. Konstantinov & N. Simov, (Poaceae), 15♂ (AMNH_PBI 00343264-AMNH_PBI 00343277, AMNH_ PBI 00343331), 70♀ (AMNH_PBI 00343249-AMNH_PBI 00343263, AMNH_PBI 00343232-AMNH_PBI 00343248, AMNH_PBI 00343196-AMNH_PBI 00343231, AMNH_PBI 00343330, AMNH_PBI 00343376) (ZISP); 16♂, 49♀ (SOFM). N coast of Sevan Lake, Karmir Pass, 4 km ENE of Aghberk, 40.56328°N, 45.29903°E, 2190 m, 15 Jun 2017, F. Konstantinov & N. Simov, (Poaceae), 2♂ (AMNH_PBI 00343351, AMNH_PBI 00343352), 1♀ (AMNH_PBI 00343327) (ZISP); 6♂, 9♀ (SOFM). W coast of Sevan Lake, 2 km ESE of Semyonovka, 40.65074°N, 44.92336°E, 2123 m, 15 Jun 2017, F. Konstantinov & N. Simov, (Poaceae), 24♂ (AMNH_PBI 00343278- AMNH_PBI 00343285, AMNH_PBI 00343288-AMNH_PBI 00343301, AMNH_ PBI 00343332, AMNH_PBI 00343334), 25♀ (AMNH_PBI 00343302-AMNH_PBI 00343312, AMNH_PBI 00343314-AMNH_PBI 00343325, AMNH_PBI 00343333, AMNH_PBI 00343328) (ZISP); 25♂, 34♀ (SOFM). Review of the subgenus Plumiger of Myrmecophyes, with description of a new species... 227 Figures 30–40. Male genitalia: 30–32, 34–35 Left paramere in lateral (30, 34) and dorsal (31, 32, 35) views 30–32 Myrmecophyes armeniacus Drapolyuk, 1989 34, 35 Myrmecophyes heterocerus Horváth, 1927 33, 36 Right paramere in dorsal view 33 M. armeniacus 36 M. heterocerus 37–40 Aedeagus in lateral view (38, 40) and with phallotheca detached from phallobase and partially expanded endosoma (37, 39) 37, 38 M. armeniacus 39, 40 M. heterocerus. Abbreviations: outgr – rounded outgrowth at base of apical process of the left paramere. Figures 30–40. Male genitalia: 30–32, 34–35 Left paramere in lateral (30, 34) and dorsal (31, 32, 35) views 30–32 Myrmecophyes armeniacus Drapolyuk, 1989 34, 35 Myrmecophyes heterocerus Horváth, 1927 33, 36 Right paramere in dorsal view 33 M. armeniacus 36 M. heterocerus 37–40 Aedeagus in lateral view (38, 40) and with phallotheca detached from phallobase and partially expanded endosoma (37, 39) 37, 38 M. armeniacus 39, 40 M. heterocerus. Abbreviations: outgr – rounded outgrowth at base of apical process of the left paramere. Fedor V Konstantinov & Nik 8 22 228 Fedor V. Konstantinov & Nikolay Simov / ZooKeys 796: 215–239 (2018) Diagnosis. Myrmecophyes (Plumiger) armeniacus Dapolyuk, 1989: 125; figs 51, 55. Genitalia: genital capsule comparatively small, about 0.3 of abdomen; left paramere sickle-shaped, thin, apical process of uniform width along entire length, long, somewhat sinuate, flattened, ending with characteristic oblique T-shaped blade; body of paramere with small but distinct rounded outgrowth at base of apical process with long setae (Figs 30–32); right paramere typically flag-shaped, somewhat larger than in M. heterocerus (Figure 33); endosoma of aedeagus voluminous, folded, entirely membranous, with several small, minutely dentate eversible lobes (Figs 37, 38). g Female. Body large, total length 3.9–4.1. Coloration: As in male, but antennal segment I and femora ranging from uniformly rust brown to totally black (Figure 8). Surface and vestiture: As in subgeneric description; dorsum matt, head and abdomen at most moderately shiny; frons and vertex with few erect spinelike setae; antennal segment I with 6–8 similar setae on mesial surface. Structure: Body 4.4–4.6 × as long as width of pronotum at middle. Head: Vertex 1.9–2.1 × as wide as eye; antennal segment I 0.8 × as long as length of pronotum, segment II 1.9–2.1 × as long as I, 1.1–1.2 × as long as width of pronotum at middle, and 0.8–0.9 × as long as width of head; segment III 1.5–1.6 × as long as II; segment IV equal to II in length. Thorax: Pronotum at middle 1.3–1.4 × as wide as long, 0.7–0.8 × as wide as head; hind tibia 3.3–3.5 × as long as width of pronotum at middle. Genitalia: Sclerotized rings of Review of the subgenus Plumiger of Myrmecophyes, with description of a new species... Review of the subgenus Plumiger of Myrmecophyes, with description of a new species... Review of the subgenus Plumiger of Myrmecophyes, with description of a new species... 229 Review of the subgenus Plumiger of Myrmecophyes, with description of a new species... 229 Figures 41 50 Male genitalia: 41 42 44 45 Left paramere in dorsal (41 44) and lateral (42 45) Figures 41–50. Male genitalia: 41–42, 44–45 Left paramere in dorsal (41, 44) and lateral (42, 45) views 41, 42 Myrmecophyes nasutus Drapolyuk, 1989 44, 45 Myrmecophyes tomi sp. n. 43, 46 Right para mere in dorsal view 43 M. nasutus 46 M. tomi sp. n. 47–50 Aedeagus in lateral view (48, 50) and with phallotheca detached from phallobase and partially expanded endosoma (47, 49) 47, 48 M. nasutus, 49, 50 M. tomi sp. n. Myrmecophyes (Plumiger) armeniacus Dapolyuk, 1989: 125; figs 51, 55. Abbreviations: dent –recurved denticle(s) of the left paramere, lob – slightly sclerotized lobes of endosoma. Figures 41–50. Male genitalia: 41–42, 44–45 Left paramere in dorsal (41, 44) and lateral (42, 45) views 41, 42 Myrmecophyes nasutus Drapolyuk, 1989 44, 45 Myrmecophyes tomi sp. n. 43, 46 Right para mere in dorsal view 43 M. nasutus 46 M. tomi sp. n. 47–50 Aedeagus in lateral view (48, 50) and with phallotheca detached from phallobase and partially expanded endosoma (47, 49) 47, 48 M. nasutus, 49, 50 M. tomi sp. n. Abbreviations: dent –recurved denticle(s) of the left paramere, lob – slightly sclerotized lobes of endosoma. Figures 41–50. Male genitalia: 41–42, 44–45 Left paramere in dorsal (41, 44) and lateral (42, 45) views 41, 42 Myrmecophyes nasutus Drapolyuk, 1989 44, 45 Myrmecophyes tomi sp. n. 43, 46 Right para mere in dorsal view 43 M. nasutus 46 M. tomi sp. n. 47–50 Aedeagus in lateral view (48, 50) and with phallotheca detached from phallobase and partially expanded endosoma (47, 49) 47, 48 M. nasutus, 49, 50 M. tomi sp. n. Abbreviations: dent –recurved denticle(s) of the left paramere, lob – slightly sclerotized lobes of endosoma. 230 Fedor V. Konstantinov & Nikolay Simov / ZooKeys 796: 215–239 (2018) dorsal labiate plate broadly oval (Figs 51, 59), interramal sclerites of posterior wall almost flat, densely covered with minute denticles (Figure 55). Distribution. The current distribution of M. armeniacus spans a distance of less than 200 km from northeastern Turkey in the west to northwestern Armenia in the east (Figure 64). Myrmecophyes heterocerus Horváth, 1927 Figs 6, 9, 15, 17–21, 24, 28, 34–36, 39, 40, 53, 56, 60, 63, 64 Myrmecophyes heterocerus Horváth, 1927: 189 Myrmecophyes (Plumiger) heterocerus Drapolyuk, 1989: 135, figs 46–50, 54 (figs, habi tus, male and female genitalia) Material examined. ARMENIA: Gegharkunik: 7 km S from Nshkhark, 39.95378°N, 45.23438°E, 2418 m, 13 Jun 2017, F. Konstantinov & N. Simov, (Poaceae), 56♀ (AMNH_PBI 00343163-AMNH_PBI 00343195, AMNH_PBI 00343338, AMNH_PBI 00343339, AMNH_PBI 00343156-AMNH_PBI 00343162, AMNH_ PBI 00343149-AMNH_PBI 00343155, AMNH_PBI 00343142-AMNH_PBI 00343148), 70♂ (AMNH_PBI 00343378, AMNH_PBI 00343337, AMNH_PBI 00343329, AMNH_PBI 00343071-AMNH_PBI 00343074, AMNH_PBI 00343069, AMNH_PBI 00343067, AMNH_PBI 00343080-AMNH_PBI 00343136, AMNH_ PBI 00343138-AMNH_PBI 00343141), 2 larvae (AMNH_PBI 00343075, AMNH_ PBI 00343137) (ZISP); 45♂, 49♀ (SOFM). AZERBAIJAN: Kalbajar: Istisu nr Kalbacar [Kel’badzhary], 39.96972°N, 45.95°E, 03 Jul 1968 - 04 Jul 1968, Gidayatov, 3♂ (AMNH_PBI 00271255, AMNH_PBI 00261439, AMNH_PBI 00261440), 4♀ (AMNH_PBI 00261638, AMNH_PBI 00261637) (ZISP). Redescription. Male. Total length 2.8–3.2. Coloration: As in M. armeniacus. Sur face and vestiture: Pronotum and scutellum matt, head and abdomen smooth, distinct Myrmecophyes (Plumiger) armeniacus Dapolyuk, 1989: 125; figs 51, 55. GEORGIA: Mtskheta- Mtianeti: Kobi, Voenno-Gruzinskaya doroga [=Georgean Military Rd], 42.5583°N, 44.5122°E, 2144 m, 14 Jul 1925, Kiritshenko, 3♂ (AMNH_PBI 00261433-AMNH_ PBI 00261435), 14♀ (AMNH_PBI 00261608-AMNH_PBI 00261621) (ZISP); 15 Jul 1925, A. N. Kiritshenko, 14♀ (AMNH_PBI 00271256-AMNH_PBI 00271259, AMNH_PBI 00261622-AMNH_PBI 00261631), 1♂ (AMNH_PBI 00261436) (ZISP). RUSSIAN FEDERATION: North Ossetia Rep.: Pass between Verkhnyaya Kora and Ardon Rivers, 42.82306°N, 43.91583°E, 01 Aug 1925, A. N. Kiritshenko, 6♀ (AMNH_PBI 00271254, AMNH_PBI 00261633-AMNH_PBI 00261636, AMNH_ PBI 00261438), 1♂ (AMNH_PBI 00261437) (ZISP). Verkhniy Tsey, 42.80667°N, 43.94028°E, 04 Aug 1925, A. N. Kiritshenko, 1♀ (AMNH_PBI 00261632) (ZISP). Diagnosis. Distinguished from M. armeniacus by the smaller size, shiny abdomen (Figs 6, 9), shape of left paramere (Figs 34, 35) and narrowly triangular sclerotized rings of dorsal labiate plate (Figs 53, 60). The diagnosis of M. armeniacus provides ad ditional characters. Diagnosis. Distinguished from M. armeniacus by the smaller size, shiny abdomen (Figs 6, 9), shape of left paramere (Figs 34, 35) and narrowly triangular sclerotized rings of dorsal labiate plate (Figs 53, 60). The diagnosis of M. armeniacus provides ad ditional characters. Redescription. Male. Total length 2.8–3.2. Coloration: As in M. armeniacus. Sur face and vestiture: Pronotum and scutellum matt, head and abdomen smooth, distinct Review of the subgenus Plumiger of Myrmecophyes, with description of a new species... 231 f g g f y p y p f p Figures 51–54. Dorsal labiate plate of bursa copulatrix: 51 Myrmecophyes armeniacus Drapolyuk, 1989, 52 Myrmecophyes tomi sp. n. 53 Myrmecophyes heterocerus Horváth, 1927 54 Myrmecophyes nasutus Drapolyuk, 1989. ly shiny (Figure 6); vestiture as in subgeneric description. Structure: Body 3.9–4.5 × as long as width of pronotum at middle. Head: Clypeus not bulging (Figure 28), slightly Figures 51–54. Dorsal labiate plate of bursa copulatrix: 51 Myrmecophyes armeniacus Drapolyuk, 1989, 52 Myrmecophyes tomi sp. n. 53 Myrmecophyes heterocerus Horváth, 1927 54 Myrmecophyes nasutus Drapolyuk, 1989. Figures 51–54. Dorsal labiate plate of bursa copulatrix: 51 Myrmecophyes armeniacus Drapolyuk, 1989, 52 Myrmecophyes tomi sp. n. 53 Myrmecophyes heterocerus Horváth, 1927 54 Myrmecophyes nasutus Drapolyuk, 1989. ly shiny (Figure 6); vestiture as in subgeneric description. Structure: Body 3.9–4.5 × as long as width of pronotum at middle. Head: Clypeus not bulging (Figure 28), slightly convex in apical half, with shallow depression at base and apical margin straight; vertex ly shiny (Figure 6); vestiture as in subgeneric description. Structure: Body 3.9–4.5 × as long as width of pronotum at middle. Myrmecophyes (Plumiger) armeniacus Dapolyuk, 1989: 125; figs 51, 55. Head: Clypeus not bulging (Figure 28), slightly convex in apical half, with shallow depression at base and apical margin straight; vertex Fedor V. Konstantinov & Nikolay Simov / ZooKeys 796: 215–239 (2018) 232 1.6–1.9 × as wide as eye; antennal segment I strongly swollen, short, 0.8–0.9 × as long as length of pronotum, segment II slightly curved and widened apically, short, 1.2–1.3 × as long as I, 0.7–0.8 × as long as width of pronotum at middle, and 0.5–0.6 × as long as width of head; segment III more than 2.5 × as long as II and distinctly longer than I and II combined; segment IV about 1.5 × as long as II. Thorax: Pronotum at middle 1.3–1.4 × as wide as long, 0.7 × as wide as head; hind tibia 3.0–3.5 × as long as width of pronotum at middle. Genitalia: genital capsule comparatively small, about 0.3 of abdomen; left paramere sickle-shaped, thin, apical process straight, narrowed subapi cally, ending with characteristic oblique T-shaped blade; body of paramere without outgrowth at base of apical process (Figs 34, 35); right paramere typically flag-shaped, somewhat smaller than in M. armeniacus (Figure 36); endosoma of aedeagus volu minous, folded, entirely membranous, with several small, minutely dentate eversible lobes (Figs 39, 40). Female. Similar to male but body larger on average, total length 2.9–3.8. Colora tion: As in male, but with pale brown to dirty yellow antennal segment I (Figure 9). Surface and vestiture: As in subgeneric description; pronotum and scutellum matt, head and abdomen distinctly shiny; frons and vertex with few erect spinelike setae; anten nal segment I with 6–8 similar setae on mesial surface. Structure: Body 3.6–4.3 × as long as width of pronotum at middle. Head: Vertex 1.9–2.1 × as wide as eye; antennal segment I 0.8–0.9 × as long as length of pronotum, segment II 1.7–2.0 × as long as I, 1.0–1.1 × as long as width of pronotum at middle, and 0.8–0.9 × as long as width of head; segment III 1.3–1.4 × as long as segment II; segment IV equal to II in length. Thorax: Pronotum at middle 1.4–1.5 × as wide as long, 0.7–0.8 × as wide as head; hind tibia 2.8–3.0 × as long as width of pronotum at middle. Genitalia: Sclerotized rings of dorsal labiate plate narrow, subtriangular, with apices narrowly elongate (Figs 53, 60); interramal sclerites flat, finely dentate at base (Figure 56). Myrmecophyes (Plumiger) armeniacus Dapolyuk, 1989: 125; figs 51, 55. li g Distribution. Myrmecophyes heterocerus appears to be the most wide-ranging spe cies of the subgenus, occurring along the Greater Caucasus range from North Ossetia in the west across Georgia to southeastern Dagestan in the east, and along the Greater Caucasus range to central Armenia and western Azerbaijan in the south (Figure 64). Myrmecophyes nasutus Drapolyuk, 1989 Figs 11–13, 27, 41–43, 47, 48, 54, 57, 61, 64 Myrmecophyes (Plumiger) nasutus Drapolyuk, 1989: 125; figs 40–45, 52, 53. Myrmecophyes nasutus Drapolyuk, 1989 Figs 11–13, 27, 41–43, 47, 48, 54, 57, 61, 64 Myrmecophyes (Plumiger) nasutus Drapolyuk, 1989: 125; figs 40–45, 52, 53. Myrmecophyes (Plumiger) nasutus Drapolyuk, 1989: 125; figs 40–45, 52, 53. Material examined. Holotype: GEORGIA: Racha-Lechkhumi and Kvemo Svaneti: Korel’dash, 42.9183°N, 43.144°E, 26 Jul 1957, Akramovskaya, 1♂ (AMNH_PBI 00261441) (ZISP). Paratypes: GEORGIA: Racha-Lechkhumi and Kvemo Svaneti: Korel’dash, 42.9183°N, 43.144°E, 26 Jul 1957, Akramovskaya, 2♀ (AMNH_PBI 00271200, AMNH_PBI 00271201) (ZISP); 30 Jul 1957, Akramovskaya, 1♂ Review of the subgenus Plumiger of Myrmecophyes, with description of a new species... 233 (AMNH_PBI 00261442), 23♀ (AMNH_PBI 00261639-AMNH_PBI 00261661) (ZISP). Lentekhi, Svanetia, 42.78333°N, 42.7°E, 10 Aug 1957, Akramovskaya, 3♀ (AMNH_PBI 00261674-AMNH_PBI 00261676) (ZISP). Tsena, Svanetia, 42.8789°N, 43.1519°E, 25 Jul 1957, Akramovskaya, 15♀ (AMNH_PBI 00271197- AMNH_PBI 00271199, AMNH_PBI 00261662-AMNH_PBI 00261673) (ZISP). Zarsky Pass, Lentekhi Distr., 42.91666°N, 43.16666°E, 27 Jul 1957, Akramovskaya, 3♀ (AMNH_PBI 00261677-AMNH_PBI 00261679) (ZISP). Mtskheta-Mtian eti: Kazbek Mt., 42.68333°N, 44.46666°E, 17 Jul 1910, Unknown collector, 1♀ (AMNH_PBI 00261685) (ZISP). Diagnosis. Distinguished from other Plumiger spp. by the larger size (Figs 11–13), distinctly bulging clypeus (Figure 27), non-shiny head and abdomen, harpoon-shaped left paramere with two subapical recurved denticles (Figs 41, 42), and long, narrow, distinctly attenuate sclerotized rings of dorsal labiate plate (Figs 54, 57). The diagnosis and remarks for M. tomi include a discussion of the distinctive features. Redescription. Male. Total length 3.8–3.9. Coloration (Figure 11): Black, with pale brown apices of femora and dirty white transverse stripe along apex of wing pad. Surface and vestiture: As in generic diagnosis. Structure: Body 4.2–4.6 × as long as width of pronotum at middle. Myrmecophyes (Plumiger) armeniacus Dapolyuk, 1989: 125; figs 51, 55. Head: Clypeus strongly bulging apically, concave and apically rectangular in lateral view (Figure 27), apically dilated, broadly rounded in frontal view; vertex 2.1–2.2 × as wide as eye; antennal segment I strongly swollen, rela tively long, 1.3–1.4 × as long as length of pronotum, segment II gradually curved along entire length and somewhat flattened apically, long, 1.8–1.9 × as long as I, 1.8–1.9 × as long as width of pronotum at middle, and 1.2–1.3 × as long as width of head. Tho rax: Pronotum at middle 1.3–1.5 × as wide as long, 0.7 × as wide as head; hind tibia 3.8–4.2 × as long as width of pronotum at middle. Genitalia: genital capsule about 0.4 of abdomen; left paramere as in M. tomi but slightly larger, with two subapical re curved denticles (Figs 41, 42); right paramere similar to that of M. tomi but larger and equipped with larger apical tooth on inner margin (Figure 43); endosoma of aedeagus voluminous, membranous, with three large, folded, minutely dentate and slightly scle rotized eversible lobes (Figs 47, 48). Female. Body larger on average, total length 4.2–4.5. Coloration: Similar to male, but antennal segment I ranging from dirty orange to entirely black, coxae usually entire ly or at least apically dirty orange, femora from entirely dirty orange to black, with api ces dirty orange (Figs 12, 13). Surface and vestiture: As in M. tomi sp. n. Structure: Body 4.1–4.6 × as long as width of pronotum at middle. Head: Clypeus convex, not bulging apically; vertex 2.0–2.4 × as wide as eye; antennal segment I slightly and uniformly swollen, short, equal to length of pronotum, segment II thin, straight and rod-shaped, 2.2–2.4 × as long as I, 1.4–1.6 × as long as width of pronotum at middle, and 1.1–1.2 × as long as width of head. Thorax: Pronotum at middle 1.5 × as wide as long, 0.7-.0.8 × as wide as head; hind tibia 3.2–3.3 × as long as width of pronotum at middle. Genitalia: Sclerotized rings of dorsal labiate plate long and narrow, gradually curved, with apices gradually attenuated (Figs 54, 61); interramal sclerites flat, finely striated (Figure 57). Fedor V Konstantinov & Ni 4 234 Fedor V. Konstantinov & Nikolay Simov / ZooKeys 796: 215–239 (2018) Distribution. Known from several localities in northern Georgia along the south ern slopes of the Greater Caucasus Mountain range (Figure 64). Distribution. Myrmecophyes (Plumiger) armeniacus Dapolyuk, 1989: 125; figs 51, 55. Known from several localities in northern Georgia along the south ern slopes of the Greater Caucasus Mountain range (Figure 64). Discussion. Drapolyuk (1989) diagnosed M. nasutus as having the aedeagus with a single spicula, although her figure 45 shows a membranous and finely dentate lobe of the endosoma. We observed that the eversible lobes of the endosoma in M. nasutus and M. tomi sp. n. are faintly sclerotized but effectively devoid of spiculae. Myrmecophyes tomi sp. n. http://zoobank.org/A15DCD84-E454-4AE3-8B26-8021EF144F14 Figs 7, 10, 29, 44–46, 49, 50, 52, 58, 62 Type locality. Georgia, Kakheti, Sakhkhova Mts. Range, 42.36666°N, 45.61 yp y g , , g , , Type material. Holotype: GEORGIA: Kakheti: Sakhkhova Mts. Range, Tushetia, 42.36666°N, 45.61666°E, 28 Aug 1959, I. Zaytseva, 1♂ (AMNH_PBI 00261443) (ZISP). Paratypes: GEORGIA: Kakheti: Sakhkheva Mts. Range, Tushe tia, 42.36666°N, 45.61666°E, 28 Aug 1959, I. Zaytseva, 1♀ (AMNH_PBI 00261682) (ZISP). Shida Kartli (South Ossetia): Tskaro, 22 Jun, Demokidov, 1♀ (AMNH_ PBI 00261684) (ZISP). RUSSIAN FEDERATION: Dagestan Rep.: Khochaldag Mt., 42.73333°N, 46.26666°E, 18 Jul 1909, A. Mlokossiewich, 1♂ (AMNH_PBI 00261444), 3♀ (AMNH_PBI 00261686-AMNH_PBI 00261688) (ZISP); 12 Aug 1910, A. Mlokossiewich, 1♀ (AMNH_PBI 00261683) (ZISP). Diagnosis. Distinguished from congeners by the small size (Figs 7, 10), distinctly bulging clypeus in male (Figure 29), antennal segments I and II in male not exceeding length and width of pronotum respectively, shiny head and abdomen, characteristically harpoon-shaped left paramere (Figs 44, 45), faintly sclerotized lobes of endosoma (Fig ure 49), and broadly oval, weakly attenuate sclerotized rings (Figs 52, 58). Remarks. Myrmecophyes tomi is separated from the morphologically similar M. nasutus by the shiny head and abdomen, the smaller body size, the shorter first two antennal segments in the male, the single subapical, recurved denticle of the left para mere, and the broad sclerotized rings. The last-named character would allow females of M. tomi to be distinguished from those of M. heterocerus, which otherwise are similar in body size and proportions, and in having a shiny abdomen. y p p g y Description. Male. Total body length 3.4. Coloration: Dorsum and venter uni formly black, with contrasting yellowish-white stripe along apical margin of wing pad; antennal segment I chestnut brown, remaining segments dark brown; coxae and femora dirty yellow, tibiae and tarsi dark brown (Figure 7). Myrmecophyes (Plumiger) armeniacus Dapolyuk, 1989: 125; figs 51, 55. Surface and vestiture: Head, pronotum and hemelytron rugose, pronotum with fine transverse wrinkles along anterior and posterior margin; abdomen shiny, distinctly smoother than pro notum and scutellum. Dorsum, venter, and legs with very short and thin, reclining, pale brown simple setae, scarce on head, thorax and femora, more dense on abdomen and tibiae; head with several spinelike setae on vertex posteriorly and on frons be tween eye and antennal fossa, apex of frons and base of clypeus with very dense, thin and exceptionally long, straight whitish setae; antennal segments I and II with nu Review of the subgenus Plumiger of Myrmecophyes, with description of a new species... 235 Figures 55–63. Female genitalia: 55–58 Posterior wall of bursa copulatrix 55 Myrmecophyes armeniacus Drapolyuk, 1989 56 Myrmecophyes heterocerus Horváth, 1927 57 Myrmecophyes nasutus Drapolyuk, 1989 58 Myrmecophyes tomi sp. n. 59–62 Sclerotized rings of dorsal labiate plate 59 M. armeniacus 60 M. het erocerus 61 Myrmecophyes nasutus 62 M. tomi sp. n. 63 Vulva of M. heterocerus. Figures 55–63. Female genitalia: 55–58 Posterior wall of bursa copulatrix 55 Myrmecophyes armeniacus Drapolyuk, 1989 56 Myrmecophyes heterocerus Horváth, 1927 57 Myrmecophyes nasutus Drapolyuk, 1989 58 Myrmecophyes tomi sp. n. 59–62 Sclerotized rings of dorsal labiate plate 59 M. armeniacus 60 M. het erocerus 61 Myrmecophyes nasutus 62 M. tomi sp. n. 63 Vulva of M. heterocerus. merous black spinelike setae on dorsal surfaces and dense, white, spatulate scales on l f b l f h III d IV i h l i l Figures 55–63. Female genitalia: 55–58 Posterior wall of bursa copulatrix 55 Myrmecophyes armeniacus Drapolyuk, 1989 56 Myrmecophyes heterocerus Horváth, 1927 57 Myrmecophyes nasutus Drapolyuk, 1989 58 Myrmecophyes tomi sp. n. 59–62 Sclerotized rings of dorsal labiate plate 59 M. armeniacus 60 M. het erocerus 61 Myrmecophyes nasutus 62 M. tomi sp. n. 63 Vulva of M. heterocerus. Figures 55–63. Female genitalia: 55–58 Posterior wall of bursa copulatrix 55 Myrmecophyes armeniacus Drapolyuk, 1989 56 Myrmecophyes heterocerus Horváth, 1927 57 Myrmecophyes nasutus Drapolyuk, 1989 58 Myrmecophyes tomi sp. n. 59–62 Sclerotized rings of dorsal labiate plate 59 M. armeniacus 60 M. het erocerus 61 Myrmecophyes nasutus 62 M. tomi sp. n. 63 Vulva of M. heterocerus. Myrmecophyes (Plumiger) armeniacus Dapolyuk, 1989: 125; figs 51, 55. merous black spinelike setae on dorsal surfaces and dense, white, spatulate scales on ventral surfaces except basal one-fourth; segments III and IV with relatively scarce, erect, spinelike setae shorter than those on previous segments and dense, semierect, merous black spinelike setae on dorsal surfaces and dense, white, spatulate scales on ventral surfaces except basal one-fourth; segments III and IV with relatively scarce, erect, spinelike setae shorter than those on previous segments and dense, semierect, Fedor V. Konstantinov & Nikolay Simov / ZooKeys 796: 215–239 (2018) 236 pale simple setae; femora with incomplete row of stiff spinelike setae along dorsal margin and several apical spines dorsally and ventrally; tibia with scattered black spines. Structure: Body 4.0 × as long as width of pronotum at middle. Head: Vertical, with slightly concave and apically bulging clypeus, rectangular in lateral view (Figure 29) and with somewhat dilated, broadly rounded apex in frontal view; vertex 2.0 × as wide as eye; antennal segment I distinctly swollen, 0.9 × as long as length of pro notum, segment II gradually curved along entire length, somewhat widened and flat tened apically, relatively long, 1.8 × as long as I, 1.2 × as long as width of pronotum at middle, and 0.9 × as long as width of head; remaining segments thin, segment III twice as long as segments II and IV; labium stout, surpassing hind coxa and reaching basal abdominal segments. Thorax: Pronotum at middle 1.3 × as wide as long, 0.8 × as wide as head, metathoracic spiracle and scent efferent system as in M. heterocerus (Figure 17); hind tibia 3.8 × as long as width of pronotum at middle; tarsal segments II and III subequal in length, about 1.5 × as long as segment I, pretarsus as in M. armeniacus (Figs 22, 23). Genitalia: genital capsule about 0.4 of abdomen; left para mere sickle-shaped, comparatively robust, apical process harpoon-shaped, slightly curved, roughly triangular in cross section, tapering towards apex, with subapical recurved denticle; body of paramere with distinct rounded outgrowth at base of api cal process with long setae (Figs 44, 45); right paramere typically flag-shaped, with minute apical tooth on inner margin (Figure 46); endosoma of aedeagus membra nous, similar to that of M. nasutus but somewhat smaller, with three folded, minutely dentate and slightly sclerotized eversible lobes (Figs 49, 50). g y g Female. Myrmecophyes (Plumiger) armeniacus Dapolyuk, 1989: 125; figs 51, 55. Larger than male on average, total body length 3.1–3.5, with antenna unmodified and abdomen broadly oval, strongly expanded at middle. Coloration: Similar to male, but antennal segment I dirty orange, coxae and femora black, with apices dirty orange (Figure 10). Surface and vestiture: Similar to male, but frons with out dark erect spinelike setae and long whitish thin setae; antennal segments I and II without scales and long spinelike setae, segment I with only 8–12 relatively small spinelike erect setae on mesial surface. Structure: Body 3.8–4.5 × as long as width of pronotum at middle. Head: Clypeus convex, not bulging apically; vertex 1.9–2.1 × as wide as eye; antennal segment I slightly and uniformly swollen, short, 0.7–0.9 × as long as length of pronotum, segment II straight and thin, rod-shaped, 1.7–2.0 × as long as I, 1.0–1.1 × as long as width of pronotum at middle, and 0.7–0.8 × as long as width of head; remaining segments filiform, segment III about 1.5 × as long as seg ments II and IV. Thorax: Pronotum at middle 1.4–1.5 × as wide as long, 0.7–0.8 × as wide as head; labium always reaching and usually surpassing hind coxa; hind tibia 2.8–3.3 × as long as width of pronotum at middle. Genitalia: Sclerotized rings of dor sal labiate plate elongate-oval (Figs 52, 62); interramal sclerites convex and covered with dense minute denticles (Figure 58). Etymology. The species is named for Thomas J. Henry in recognition of his unpar alleled contributions to heteropterology. Review of the subgenus Plumiger of Myrmecophyes, with description of a new species... 237 Figure 64. Distribution map of Myrmecophyes (Plumiger) species. Key: ▲ Myrmecophyes armeniacus Drapolyuk, 1989, ● Myrmecophyes heterocerus Horváth, 1927, ■ Myrmecophyes nasutus Drapolyuk, 1989,  Myrmecophyes tomi sp. n. Figure 64. Distribution map of Myrmecophyes (Plumiger) species. Key: ▲ Myrmecophyes armeniacus Drapolyuk, 1989, ● Myrmecophyes heterocerus Horváth, 1927, ■ Myrmecophyes nasutus Drapolyuk, 1989,  Myrmecophyes tomi sp. n. Distribution. Known from northeastern Georgia in the Transcaucasia to south western Dagestan in the North Caucasus (Figure 64).h Distribution. Known from northeastern Georgia in the Transcaucasia to south western Dagestan in the North Caucasus (Figure 64).h Discussion. The new species is described from the specimens originally included in the paratype series of M. nasutus by Drapolyuk (1989), who discussed distinctions in the structure of antennal segment I in male and figured the dorsal labiate plate (Drapolyuk 1989: fig. Myrmecophyes (Plumiger) armeniacus Dapolyuk, 1989: 125; figs 51, 55. 53), but refrained from describing a new taxon. Acknowledgments Funding for this study was provided by the Russian Foundation for Basic Research, project 16–04–01682. The Heteroptera collection of the Zoological Institute, Russian Academy of Sciences, is financially supported by the state research project АААА-А17-117030310210-3. We appreciate the assistance of Alexey Mirolyubov and Svetlana Janson (microscopy and microanalysis facility center, SPSU) for technical support in the operation of the SEM. Michael D. Schwartz (CNC) and Alfred G. Wheeler Jr. (Clemson University) provided valuable comments on an earlier version of the manuscript. Fedor V. Konstantinov & Nikolay Simov / ZooKeys 796: 215–239 (2018) 238 References Bykov AA (1971) Plant bugs of the genus Myrmecophyes Fieb. (Heteroptera, Miridae) in the fauna of the Tien-Shan and the Pamir-Alai. Entomologicheskoe Obozrenie 59: 870–881. [In Russian] Drapolyuk IS (1989) Species of the genus Myrmecophyes Fieb. (Heteroptera, Miridae) fr Drapolyuk IS (1989) Species of the genus Myrmecophyes Fieb. (Heteroptera, Miridae) from the Caucasus. Entomologicheskoe Obozrenie 68: 125–135. [In Russian; English translation: Entomological Review 68: 90–100] Drapolyuk IS, Kerzhner IM (2000) New species of Orthocephalus and Myrmecophyes from Ka zakhstan, Uzbekistan and Turkmenistan (Heteroptera: Miridae). Zoosystematica Rossica 8(2)[1999]: 301–305. Dursun A, Kartal V (2006) Scirtetellus vittatus Kiritshenko, 1951 (Heteroptera: Miridae) üzer ine bir araştırma. Turkish Journal of Entomology 30(3): 235–240. Horváth G (1927) Species Miridarum generis Myrmecophyes Fieber. Annales Historico-Natu rales Musei Nationalis Hungarici 23[1926]: 187–197. Konstantinov FV (2003) Male genitalia in Miridae (Heteroptera) and their significance for su prageneric classification of the family. Part I: general review, Isometopinae and Psallopinae. Belgian Journal of Entomology 5: 3–36. Konstantinov FV, Luo Z, Vinokurov NN (2013) Two new species, new synonymies, and new records of plant bugs (Hemiptera: Heteroptera: Miridae) from Northwestern China. Zootaxa 3666(2): 203–220. https://doi.org/10.11646/zootaxa.3666.2.6 Kullenberg B (1944) Studien über die Biologie der Capsiden. Zoologiska Bidrag från Uppsala 23: 1–522. Menard KL (2015) A review of the genus Spanagonicus Berg (Hemiptera: Miridae: Phylinae: Nasocorini) with the description of novel antennal characters, the description of a new spe cies from Central America, and a key to currently known taxa. Zootaxa 3973(1): 139–158. https://doi.org/10.11646/zootaxa.3973.1.5 Schuh RT (2012–2013) On-line Systematic Catalog of Plant Bugs (Insecta: Heteroptera: Miri dae) http://research.amnh.org/pbi/catalog/ (Accessed October 18, 2017). Schuh RT, Lattin JD (1980) Myrmecophyes oregonensis, a new species of Halticini (Hemiptera, Miridae) from the western United States. American Museum Novitates 2697: 1–11. Schwartz MD (2011) Revision and phylogenetic analysis of the North American genus Slat erocoris Wagner with new synonymy, the description of five new species and a new genus from Mexico, and a review of the genus Scalponotatus Kelton (Heteroptera: Miridae: Or thotylinae). Bulletin of the American Museum of Natural History 354: 1–290. https://doi. org/10.1206/354.1 Stork NE (1981) The structure and function of the adhesive organs on the antennae of male Harpocera thoracica (Fallén) (Miridae; Hemiptera). Journal of Natural History 15(4): 639– 644. https://doi.org/10.1080/00222938100770451 Tatarnic NJ, Cassis G (2012) The Halticini of the world (Insecta: Heteroptera: Miridae: Orthotylinae): generic reclassification, phylogeny, and host plant associations. Zoological Journal of the Linnean Society 164: 558–658. References https://doi.org/10.1111/j.1096- 3642.2011.00770.x Review of the subgenus Plumiger of Myrmecophyes, with description of a new species... 239 Appendix 1 USI numbers of figured specimens. USI numbers of figured specimens. Figure Species Sex USI number 5 Myrmecophyes armeniacus male AMNH_PBI 00343298 6 Myrmecophyes heterocerus male AMNH_PBI 00343084 7 Myrmecophyes tomi male AMNH_PBI 00261443 8 Myrmecophyes armeniacus female AMNH_PBI 00343318 9 Myrmecophyes heterocerus female AMNH_PBI 00343142 10 Myrmecophyes tomi female AMNH_PBI 00261684 11 Myrmecophyes nasutus male AMNH_PBI 00261441 12 Myrmecophyes nasutus female AMNH_PBI 00261674 13 Myrmecophyes nasutus female AMNH_PBI 00261640 14, 16, 22, 23, 25 Myrmecophyes armeniacus female AMNH_PBI 00343376 15, 17–21, 24 Myrmecophyes heterocerus male AMNH_PBI 00343378 26 Myrmecophyes armeniacus male AMNH_PBI 00343291 27 Myrmecophyes nasutus male AMNH_PBI 00261441 28 Myrmecophyes heterocerus male AMNH_PBI 00343083 29 Myrmecophyes tomi male AMNH_PBI 00261443 30–31 Myrmecophyes armeniacus male AMNH_PBI 00343331 32–33 Myrmecophyes armeniacus male AMNH_PBI 00343334 34 Myrmecophyes heterocerus male AMNH_PBI 00343092 35–36 Myrmecophyes heterocerus male AMNH_PBI 00343129 37–38 Myrmecophyes armeniacus male AMNH_PBI 00343268 39 Myrmecophyes heterocerus male AMNH_PBI 00343329 40 Myrmecophyes heterocerus male AMNH_PBI 00343092 41–43 Myrmecophyes nasutus male AMNH_PBI 00261441 44–46 Myrmecophyes tomi male AMNH_PBI 00261443 47–48 Myrmecophyes nasutus male AMNH_PBI 00261441 49–50 Myrmecophyes tomi male AMNH_PBI 00261443 51 Myrmecophyes armeniacus female AMNH_PBI 00343333 52 Myrmecophyes tomi female AMNH_PBI 00261684 53 Myrmecophyes heterocerus female AMNH_PBI 00261637 54 Myrmecophyes nasutus female AMNH_PBI 00261646 55 Myrmecophyes armeniacus female AMNH_PBI 00343333 56 Myrmecophyes heterocerus female AMNH_PBI 00343339 57 Myrmecophyes nasutus female AMNH_PBI 00261646 58 Myrmecophyes tomi female AMNH_PBI 00261444 59 Myrmecophyes armeniacus female AMNH_PBI 00343333 60 Myrmecophyes heterocerus female AMNH_PBI 00261637 61 Myrmecophyes nasutus female AMNH_PBI 00261646 62 Myrmecophyes tomi female AMNH_PBI 00261684 63 Myrmecophyes heterocerus female AMNH_PBI 00261637 AMNH_PBI 00343129 AMNH_PBI 00343268 AMNH_PBI 00343329 AMNH_PBI 00343092 AMNH_PBI 00261441 AMNH_PBI 00261443 AMNH_PBI 00261441
https://openalex.org/W4322734096	https://iris.uniroma1.it/bitstream/11573/1673359/1/Renzi_other-side_2023.pdf	English	null	The other side of COVID-19: A cross-sectional study on mental health in a sample of Italian nurses during the second wave	Frontiers in psychiatry	2,023	cc-by	7,916	TYPE Original Research PUBLISHED 01 March 2023 DOI 10.3389/fpsyt.2023.1083693 TYPE Original Research PUBLISHED 01 March 2023 DOI 10.3389/fpsyt.2023.1083693 TYPE Original Research PUBLISHED 01 March 2023 DOI 10.3389/fpsyt.2023.1083693 KEYWORDS nursing, mental health, COVID-19, post-traumatic stress disorder, generalized anxiety disorder The other side of COVID-19: A cross-sectional study on mental health in a sample of Italian nurses during the second wave OPEN ACCESS EDITED BY Stephen X. Zhang, University of Adelaide, Australia REVIEWED BY Jelena Stojanov, University of Niš, Serbia Teresa Gavaruzzi, University of Padua, Italy CORRESPONDENCE Erika Renzi erika.renzi@uniroma1.it SPECIALTY SECTION This article was submitted to Public Mental Health, a section of the journal Frontiers in Psychiatry RECEIVED 29 October 2022 ACCEPTED 03 February 2023 PUBLISHED 01 March 2023 Erika Renzi 1, Valentin Imeshtari 1, Dima Masud 2, Valentina Baccolini 1, Giuseppe Migliara 1, Giulia Gasperini 3,4, Corrado De Vito 1, Carolina Marzuillo 1, Paolo Villari 1 and Azzurra Massimi 1 1 Department of Public Health and Infectious Diseases, Sapienza University of Rome, Rome, Italy, 2 Emergency Department, Sandro Pertini Hospital, Rome, Italy, 3 Department of Translational and Precision Medicine, Umberto I Teaching Hospital, Rome, Italy, 4 Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy Introduction: The COVID-19 pandemic has led to a drastic increase in the workload of healthcare professionals, particularly nurses, with serious consequences for their psychological well-being. Our study aimed to identify demographic and work-related factors, as well as clinical predictors of post- traumatic stress disorder (PTSD) and generalized anxiety disorder (GAD), in nurses employed during the COVID-19 pandemic. Renzi E, Imeshtari V, Masud D, Baccolini V, Migliara G, Gasperini G, De Vito C, Marzuillo C, Villari P and Massimi A (2023) The other side of COVID-19: A cross-sectional study on mental health in a sample of Italian nurses during the second wave. Methods: We carried out a cross-sectional study between December 2020 and April 2021 on nurses employed during the COVID-19 second wave (October - December 2020). We evaluated PTSD and GAD using two validated questionnaires: i) the Impact of Event Scale – Revised (IES-R); and ii) General Anxiety Disorder –7 (GAD-7). Front. Psychiatry 14:1083693. doi: 10.3389/fpsyt.2023.1083693 2.2. Questionnaire Psychological sequelae secondary to the outbreak of an epidemic have already been reported for Middle East respiratory syndrome (MERS) in 2012 and severe acute respiratory syndrome (SARS) in 2003, in which symptoms of post-traumatic stress disorder (PTSD), sleep disorders, social isolation, work-related stress, burnout, and generalized anxiety disorder (GAD) were reported in HCWs (4–7). In the case of the COVID-19 pandemic, the scientific literature shows that HCWs, especially nurses and women who worked in the front line and in emergency areas, experienced more severe mental health symptoms than others (3). According to a meta-review of systematic reviews, GAD and PTSD were the most prevalent COVID-19 pandemic-related mental health conditions affecting HCWs, especially nurses (8). Several more detailed studies, conducted after the first wave of the pandemic in China, reported that HCWs suffered from anxiety and stress-related symptoms, with prevalence ranging from 28.5 to 36.1% and 24 to 73.4%, respectively (9–11). In addition, some studies have reported high levels of anxiety and acute stress disorders in nurses more than a year after the start of the pandemic (12, 13). We developed a 41-item survey based on the literature. The questionnaire comprised three sections. The first section included 12 items that aimed to collect socio-demographic and occupational data: gender, age, nationality, marital status, employment information (department, area, employment in COVID-19 area during the first wave, and geographical context), and COVID-19 personal history (previous SARS-CoV-2 infection and experience). The second section aimed to investigate anxiety disorders: in this case, Generalized Anxiety Disorder-7 (GAD-7), a questionnaire for GAD screening, was used in its validated Italian version. The scale assigns a score from 0 (not at all) to 3 (almost every day) based on the frequency of reported symptoms over the previous 14 days. The total GAD-7 score for the seven items ranges from 0 to 21. Based on the total score, it is possible to stratify the presence of symptoms of GAD (0–4: minimal anxiety; 5–9: mild anxiety; 10–14: moderate anxiety; 15–21: severe anxiety) (22). The third section assessed acute stress reactions and the increased likelihood of having PTSD using a validated questionnaire, Impact of Event Scale-Revised (IES-R), which has demonstrated good internal consistency in the Italian version (23). IES-R is a 22-item scale that is rated from 0 (not at all) to 4 (extremely) with respect to how distressing each item was during the last 7 days. Front. Psychiatry 14:1083693. doi: 10.3389/fpsyt.2023.1083693 Results: Overall, 400 nurses, whose mean age was 34.3 years (SD ± 11.7), were included in the study. Most were female (78.5%), unmarried (58.5%) and employed in the central (61.5%) regions of Italy. A total of 56.8% of all participants had clinical predictors of PTSD, recording a median IES-R score (IQR) of 37.0 (22.0, 51.0) (range 1-84; cut-off >33 for PTSD). Furthermore, 50% of respondents reported moderate-to-severe symptoms consistent with GAD, recording a median GAD- 7 score (IQR) of 9.5 (6.0,14.0) (range 0-21; cut-off >10 for GAD). Multivariable analysis showed that moderate-to-severe GAD (aOR = 4.54, 95% CI: 2.93 - 7.05), being employed in the critical care area (aOR = 1.74, 95% CI: 1.01 - 3.00) and being female (aOR= 1.88, 95% CI: 1.09 - 3.22) were significantly associated with the presence of clinical predictors of PTSD. COPYRIGHT © 2023 Renzi, Imeshtari, Masud, Baccolini, Migliara, Gasperini, De Vito, Marzuillo, Villari and Massimi. 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. Discussion: The levels of PTSD symptoms and anxiety among nurses were high during the pandemic. PTSD and GAD represent a public health problem that should be addressed in the post-pandemic period. Healthcare organizations need to activate specific support and rehabilitation networks and programs for healthcare professionals employed during the COVID-19 pandemic. nursing, mental health, COVID-19, post-traumatic stress disorder, generalized anxiety disorder 01 01 Frontiers in Psychiatry frontiersin.org Renzi et al. 10.3389/fpsyt.2023.1083693 2.2. Questionnaire For the purposes of our study, we modified the IES-R instructions by contextualizing the questionnaire to the COVID-19 experience of the 7 days prior. The total IES-R score for the 22 items ranges from 0 to 88. The scale scores are determined from three subscales, which reflect intrusion (8 items), avoidance (8 items), and hyperarousal (6 items; see Table 1 for definitions). Focusing attention on the psychological impact of COVID-19 on HCWs, particularly on the Italian nurses who were among the first in Europe to deal with the pandemic and to assist COVID-19 patients (14), is crucial to investigate if such HCWs are to be offered the right tools to face this extraordinary scenario and to preserve their mental health, both as individuals and as actors in the National Health Service. In the Italian context, relatively few studies have investigated the prevalence of GAD and PTSD in HCWs, and most of these were conducted exclusively during the first wave of COVID-19 (15, 16), focusing on local contexts (12, 17, 18). Given this, we conducted a study on a national sample of nurses employed during the second wave of the COVID-19 pandemic to investigate the long-term impact on nurses’ mental health, with the specific aims of (i) determining the prevalence of symptoms of potential GAD and PTSD; (ii) identifying possible predictors of PTSD. 1. Introduction Telegram, Instagram), as well as specific social-media groups for nurses. Data collection was performed between December 2020 and April 2021. Nurses were invited to take part voluntarily in an online survey accessible via smartphone through a Google Form link. The study was performed in accordance with the World Medical Association Declaration of Helsinki. Participants were asked for their consent and were guaranteed anonymity in the information collected. The institutional ethics board of the Umberto I Teaching Hospital/ Sapienza University of Rome approved this study (protocol 0489/2021). Telegram, Instagram), as well as specific social-media groups for nurses. Data collection was performed between December 2020 and April 2021. Nurses were invited to take part voluntarily in an online survey accessible via smartphone through a Google Form link. The study was performed in accordance with the World Medical Association Declaration of Helsinki. Participants were asked for their consent and were guaranteed anonymity in the information collected. The institutional ethics board of the Umberto I Teaching Hospital/ Sapienza University of Rome approved this study (protocol 0489/2021). Since the beginning of the COVID-19 pandemic, healthcare workers (HCWs) were in the front line, assisting patients with COVID-19; they faced an unexpected and sudden increase in healthcare demands and were exposed to a high risk of contracting the disease (1). Furthermore, because of the fear of contagion and the social-distance measures put in place to contain the pandemic, HCWs often could not or chose not to see their families and friends for long periods, so they found themselves alone and with no emotional support. Such conditions are known to have negative effects on the psychological health of HCWs (2, 3). 2.3. Statistical analysis Descriptive statistics were obtained using the median and interquartile range, or mean and standard deviation, for continuous variables, and proportions for dichotomous and categorical variables. The COVID-19 employment area was categorized into five categories: critical care (i.e., emergency departments, intensive care units), medical wards (also including nursing homes), surgical wards, primary care (i.e., COVID-19 vaccination services, COVID-19 testing programs, contact tracing units, and home-care services for COVID-19 patients), and other (i.e., mental-health units, rehabilitation units). The age of the nurses was classified according to the average age (35 years). The presence or absence of symptoms of PTSD and GAD were assessed using validated questionnaires and were classified as either being present or absent (two variables). To Frontiers in Psychiatry 2.1. Setting and participants The final model consisted of the following variables: sex (female vs. male), age (≤ 35 and > 35 years), marital status (married/engaged vs. single/unmarried/separated/divorced), region of employment (northern Italy vs. center, south Italy, and Islands), nurses employed in COVID-19 wards during the first and second waves, COVID-19 employment wards/area (primary care, critical care, medical wards, surgical wards and other), COVID-19 previous infection, symptomatic COVID-19 positive member of family/friends and presence of moderate-to-severe GAD (GAD-7 score ≥ 10). Results were expressed as adjusted odds ratios (aOR), their 95% confidence intervals (CI), and p-value. A p-value <0.05 was considered statistically significant. All analyses were performed using Stata (StataCorp LLC, 4905 Lakeway Drive, College Station, TX, United States), version 17.0. ranged from 0 to 43 years (mean = 16 years, SD= ± 11.9). Most respondents worked in COVID-19 units during both the first and second waves of the pandemic (195; 51.3%); the main areas of work were critical (37.0%) and primary (35.3%) care. Finally, 19% of the sample reported a previous SARS-CoV-2 infection (Table 2). assess GAD-7, we classified a score > 10 as “presence of moderate/ severe symptoms of GAD”. PTSD was classified into two levels: IES-R scores ≥33 were classified as “presence of symptoms of PTSD” and scores <33 as “absence of symptoms of PTSD”. For the univariable analysis, Pearson’s chi-squared test was used for dichotomous and categorical variables, while the Mann–Whitney U Test was used to compare continuous variables between females and males. A multivariable logistic-regression model was built to identify predictors of PTSD. According to the Hosmer and Lemeshow logistic-regression model building strategy, the variables examined by univariate analysis using the appropriate statistical test were included in the model when the p-value was less than 0.25 (see Supplementary Material S2). We also included variables described by opinion of experts and literature to provide a complete control of confounding. All variables initially tested were retained in the final model because their exclusion altered the aORs of the other variables. The only exception was the variable “years of working” which was removed due to collinearity with the variable “age”. The final model consisted of the following variables: sex (female vs. male), age (≤ 35 and > 35 years), marital status (married/engaged vs. single/unmarried/separated/divorced), region of employment (northern Italy vs. 3.2. Generalized anxiety disorder The median GAD-7 score (IQR) was 9.5 (6.0,14.0; range: 0–21). Of the four severity categories, only 63 nurses (15.8%) reported as being in a normal state, while the remaining 337 (84.2%) experienced symptoms of anxiety, from mild (n = 137, 34.2%) to moderate (n = 106, 26.5%), and up to severe GAD (n = 94, 23.5%). Based on the cut-off value of 10, the prevalence of symptoms of GAD was 50% (n = 200). There was a gender difference in score distribution, with females having a higher median than males: 10.0 (6.0,14.0) vs. 8.0 (6.0,14.0; Table 3). 2.1. Setting and participants center, south Italy, and Islands), nurses employed in COVID-19 wards during the first and second waves, COVID-19 employment wards/area (primary care, critical care, medical wards, surgical wards and other), COVID-19 previous infection, symptomatic COVID-19 positive member of family/friends and presence of moderate-to-severe GAD (GAD-7 score ≥ 10). Results were expressed as adjusted odds ratios (aOR), their 95% confidence intervals (CI), and p-value. A p-value <0.05 was considered statistically significant. All analyses were performed using Stata (StataCorp LLC, 4905 Lakeway Drive, College Station, TX, United States), version 17.0. 3.3. Post-traumatic stress disorder Participants recorded IES-R scores ranging from 0 to 84, with a median score (IQR) of 37.0 (22.0, 51.0). According to the predefined cut-off for assessing the presence of PTSD symptoms (IES-R scores ≥33), of the 227 participants (56.8%) who scored above the cut-off, 195 (85.9%) attained scores higher than 39 (Table 4). The median IES-R score (IQR) of female nurses was higher than that of male nurses: 38.0 (24.0, 52.0) vs. 30.0 (22.0, 45.0; Table 2). Similar gender differences were found for the median subscale scores (IQR): female nurses had a higher median score (IQR) for the hyperarousal subscale [8.0 (4.0, 12.0) vs. 6.0 (3.0, 11.0), p-value = 0.034] and intrusion subscale [16.0 (9.0, 21.0) vs. 13.0 (6.0, 9.0), p-value = 0.007]. The items with the highest median score were those in the intrusion subscale (Table 5). Frontiers in Psychiatry 2.1. Setting and participants We conducted a cross-sectional survey, using a convenience sample of Italian nurses who worked on the front line during the second wave of the COVID-19 pandemic (October–December 2020) (19, 20). The survey followed the Checklist for Reporting Results of Internet E-Surveys (CHERRIES) (21) (Supplementary Material S1) and was disseminated through social-media platforms (Facebook, 02 frontiersin.org Renzi et al. 10.3389/fpsyt.2023.1083693 TABLE 1 Impact of Event Scale-Revised (IES-R) subscale definition. TABLE 1 Impact of Event Scale-Revised (IES-R) subscale definition. IES-R subscale (PTSD symptoms) Definition Avoidance The practice or an instance of keeping away from particular situations, environments, individuals, or things because of either (a) the anticipated negative consequence of such an encounter or (b) anxious or painful feelings associated with them (APA Dictionary). For example: avoiding reminders of trauma; losing interest in activities Hyperarousal One of three sets of criteria used to diagnose post-traumatic stress disorder and acute stress disorder. Symptoms of hyperarousal include exaggerated startle response, disturbed sleep, difficulty in concentrating or remembering, and excessive vigilance (APA Dictionary). For example: difficulty sleeping; angry outbursts Intrusion (or intrusive thoughts) Mental events that interrupt the flow of task-related thoughts in spite of efforts to avoid them (APA Dictionary). For example: nightmares; reliving trauma IES-R, impact of Event Scale-Revised; PTSD, post-traumatic stress disorder. IES-R, impact of Event Scale-Revised; PTSD, post-traumatic stress disorder. assess GAD-7, we classified a score > 10 as “presence of moderate/ severe symptoms of GAD”. PTSD was classified into two levels: IES-R scores ≥33 were classified as “presence of symptoms of PTSD” and scores <33 as “absence of symptoms of PTSD”. For the univariable analysis, Pearson’s chi-squared test was used for dichotomous and categorical variables, while the Mann–Whitney U Test was used to compare continuous variables between females and males. A multivariable logistic-regression model was built to identify predictors of PTSD. According to the Hosmer and Lemeshow logistic-regression model building strategy, the variables examined by univariate analysis using the appropriate statistical test were included in the model when the p-value was less than 0.25 (see Supplementary Material S2). We also included variables described by opinion of experts and literature to provide a complete control of confounding. All variables initially tested were retained in the final model because their exclusion altered the aORs of the other variables. The only exception was the variable “years of working” which was removed due to collinearity with the variable “age”. 3.4. Multivariable analysis A total of 400 nurses employed in 19 of the 21 Italian Regions (19 administrative Regions and two autonomous provinces) during the second wave of the COVID-19 pandemic completed the survey. In line with the gender distribution of nurses in Italy [76.45% women (24)], most of the sample was female (78.7%), unmarried (58.5%), and employed in central Italy (61.5%), with a mean age of 34.3 ± 11.8 years. The years of professional experience for the overall sample At the multivariable analysis, higher odds of PTSD symptoms were found for nurses with presence of symptoms for moderate-to- severe GAD (aOR = 4.54, 95% CI: 2.93 to 7.05). Similarly, being female (aOR= 1.88, 95% CI: 1.09 to 3.22) and employment in the critical care area (aOR = 1.74, 95% CI: 1.01 to 3.00) were associated with the 03 frontiersin.org Renzi et al. 10.3389/fpsyt.2023.1083693 TABLE 2 General characteristics of the survey sample. y Variable Frequency (%) Mean (±SD) Age 34.3 ± 11.8 Sex Female 315 (78.7) Male 85 (21.3) Years of working 16.1 ± 11.9 ≤1 year 107 (26.8) 2–5 years 121 (30.2) 5–10 years 45 (11.3) >10 years 125 (31.2) Missing system 2 (0.5) Marital status Single/unmarried 214 (53.5) Married/engaged 166 (41.5) Separated/divorced 20 (5.0) Region of employment North 116 (29.0) Centre 246 (61.5) South and Island 38 (9.5) Employed in COVID-19 wards during first wave (March–May 2020) Yes 195 (51.3) No 205 (48.7) COVID-19 employment wards/area Critical care 148 (37.0) Medical wards 89 (22.2) Surgical wards 6 (1.5) Primary care 141 (35.3) Other 16 (4.0) COVID-19-positive Yes 76 (19.0) No 324 (81.0) COVID-19 cases among family members/friends Yes 83 (20.8) No 317 (79.2) SD, standard deviation. Overall, the results of the present study confirm that the COVID-19 pandemic has had a profoundly negative effect on the mental health of Italian nurses. presence of symptoms of PTSD. In contrast, age, marital status, employment in COVID-19 units continually during both the first and second waves, employment in a COVID-19 area in Northern Italy, and previous SARS-CoV-2 infection did not appear to increase the likelihood of having symptoms of PTSD (Table 6). Generalized anxiety disorder symptoms were reported by half the nursing population sample employed during the second wave of the pandemic. The prevalence of symptoms of anxiety appears in line with the international context. 3.4. Multivariable analysis In fact, during the global pandemic, anxiety was the most prevalent mental disorder in HCWs and prevalence was significantly higher than in the general population, with a similar distribution in the pooled prevalence across all geographic areas: global 42%, (25) Africa 49% (26), South America 35% (27), Eastern Europe 30% (28), and Southeast Asia 23% (9). These results are also consistent with other studies conducted in Italy and other European Frontiers in Psychiatry frontiersin.org 4. Discussion and conclusion This study aimed to assess the prevalence of symptoms of GAD and PTSD among nurses employed during the second wave of COVID-19 in Italy and to evaluate the factors influencing PTSD in this sample. 04 frontiersin.org 10.3389/fpsyt.2023.1083693 Renzi et al. E 3 Distribution of generalized anxiety disorder and post-traumatic stress disorder scores in the study sample and by gender. Severity category (score range) N (%) Female Male p-Value N (%) N (%) GAD-7 0.149 Total sample 400 (100) 315 (100) 85 (100) Normal (0–4) 63 (15.8) 51 (16.2) 12 (14.1) Mild (5–9) 137 (34.2) 99 (31.4) 38 (44.7) Moderate (10–14) 106 (26.5) 87 (27.6) 19 (22.4) Severe (15–21) 94 (23.5) 78 (24.8) 16 (18.8) IES-R Total sample 400 (100) 315 (100) 85 (100) 0.012 Absence of symptoms for PTSD (0–32) 173 (44.2) 126 (40.0) 47 (55.3) Presence of symptoms for PTSD (33–88) 227 (56.8) 189 (60.0) 38 (44.7) GAD-7, generalized anxiety disorder-7 questionnaire; IES-R, impact of Event Scale-Revised; PTSD, post-traumatic stress disorder. ABLE 4 Median of generalized anxiety disorder and post-traumatic stress disorder scores in the study sample and by gender. TABLE 4 Median of generalized anxiety disorder and post-traumatic stress disorder scores in the TABLE 4 Median of generalized anxiety disorder and post traumatic stress disorder scores in the study sample and by gender. Scale Total score, median IQR Female Male p-Value Total score, median IQR Total score, median IQR GAD-7 9.5 6.0–14.0 10.0 6.0–14.0 8.0 6.0–14.0 0.133 IES-R 37.0 22.0–51.0 38.0 24.0–52.0 30.0 22.0–45.0 0.059 IQR, interquartile range; GAD-7, generalized anxiety disorder-7 questionnaire; IES-R, impact of Event Scale-Revised. psychopathology texts: the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) and the U.S. National Center for PTSD define being female as a risk factor for the development of PTSD (42–44). This difference is evident both in the median IES-R score of the sample (female nurses 38.0 vs. male nurses 30.0) and in terms of symptomatology, with a significant difference in the presence of symptoms related to intrusion and hyperarousal. With reference to intrusion symptoms, higher scores were recorded in the female sample, especially for the intrusive emotional experience (reliving negative feelings). Although this characteristic is typical of the period immediately following a traumatic event, its persistence over months seems to be a good predictor of long-term PTSD (45). 4. Discussion and conclusion This suggests that PTSD-related disorders resulting from COVID-19 could be a serious problem that needs addressing during the transition to post-pandemic conditions: interventions will be needed to support nurses to ensure a healthy workforce. countries (25, 29–31). Significant differences were observed in the occurrence and severity of the condition according to gender, with female nurses reporting a higher median GAD-7 score. However, even before the pandemic, anxiety overload in the nursing profession, together with its symptomatic manifestations and their potential effect on patient safety, appeared to be a well-established phenomenon: pre-pandemic studies reported a prevalence of GAD greater than 35% in the nursing population, with a particular impact on female nurses (32–36). Therefore, to rebuild a healthcare system challenged by COVID-19, it will be necessary to increase efforts to screen and diagnose anxiety disorders in healthcare providers. In the case of PTSD, the recorded IES-R scores suggest the presence of PTSD symptoms in almost 60% of the sample. This is markedly higher than the prevalence shown in pre-pandemic studies, which reported PTSD in 7–21% of nurses (37, 38). However, our findings are still consistent with studies carried out during other emergencies/crisis periods when HCWs, particularly nurses and frontline workers, experienced higher levels of psychological distress, anxiety, and PTSD (39). In addition, recent systematic reviews have reported a prevalence of PTSD among HCWs ranging from 21.5% to 73.5% (25, 40); in studies conducted specifically in Italy, during the current pandemic, the reported levels of PTSD symptoms ranged from 37.2% to 52.6% (16, 31). Another finding that emerged from our study was the gender difference in the distribution of mental-health conditions, with a higher prevalence of psychological symptoms of PTSD in females, which is also in line with a recent systematic review of the literature (41). Women are more likely than men to suffer from psychological disorders due to a combination of multiple biological, social, and gender-role factors. Gender differences are extensively described in the literature and in pp y Differences in the prevalence of symptoms of PTSD could be explained by multiple factors, such as the specific temporal and epidemiological context in which the study was conducted, the characteristics of the organization (e.g., the presence of professional support for HCWs), and the context in which nurses are employed (e.g., type of COVID-19 work area) (16). frontiersin.org Frontiers in Psychiatry 4. Discussion and conclusion In our study, nurses employed in the critical care area had a higher likelihood of a diagnosis of PTSD. This result is in line with data from the literature and previous pandemics (46–54). Nurses employed in critical care directly cared for COVID-19 patients and thus experienced patient deaths more frequently and had to make difficult decisions about the allocation of resources and equipment for the people in their care (41, 55–57). They were also exposed to greater health risks as a result of working with 05 frontiersin.org Renzi et al. 10.3389/fpsyt.2023.1083693 TABLE 5 IES-R subscale scores in the study sample and by gender. TABLE 5 IES-R subscale scores in the study sample and by gender. TABLE 5 IES R subscale scores in the study sample and by gender. Impact of Event Scale-Revised (IES-R) Subscale Items Median (IQR) p-Value Total Female Male Avoidance I avoided letting myself get upset when I thought about it or was reminded of it 2.0 (1.0, 3.0) I felt as if it had not happened or wasn’t real 1.0 (0.0, 2.0) I stayed away from reminders of it 1.0 (0.0, 2.0) I tried not to think about it 2.0 (1.0, 3.0) I was aware that I still had a lot of feelings about it, but I did not deal with them 2.0 (1.0, 3.0) My feelings about it were kind of numb 1.0 (0.0, 2.0) I tried to remove it from my memory 1.0 (0.0, 2.0) I tried not to talk about it 1.0 (0.0, 2.0) Median score for Avoidance Subscale 14.0 (9.0, 19.0) 14.0 (9.0, 19.0) 13.0 (8.0, 18.0) 0.426 Hyperarousal I felt irritable and angry 2.0 (1.0, 3.0) I was jumpy and easily startled 2.0 (1.0, 3.0) I had trouble falling asleep. 4. Discussion and conclusion 2.0 (0.0, 3.0) I had trouble concentrating 1.0 (0.0, 2.0) Reminders of it caused me to have physical reactions, such as sweating, trouble breathing, nausea, or a pounding heart 0.0 (0.0, 1.0) I felt watchful and on-guard 2.0 (1.0, 3.0) Median score for Hyperarousal Subscale 8.0 (4.0, 11.00) 8.0 (4.0, 12.0) 6.0 (3.0, 11.00) 0.034 Intrusion Any reminder brought back feelings about it 2.0 (1.0, 3.0) I had trouble staying asleep 2.0 (0.0, 3.0) Other things kept making me think about it 2.0 (1.0, 3.0) I thought about it when I did not mean to 2.0 (1.0, 3.0) Pictures about it popped into my mind 2.0 (1.0, 3.0) I found myself acting or feeling like I was back at that time 2.0 (1.0, 3.0) I had waves of strong feelings about it 1.0 (0.0, 2.0) I had dreams about it 2.0 (1.0, 3.0) Median score for Intrusion Subscale 15.0 (8.0, 20.0) 16.0 (9.0, 21.0) 13.0 (6.0, 19.0) 0.007 *Item response anchors are 0, not at all; 1, a little bit; 2, moderately; 3, quite a bit; 4, extremely; IQR, interquartile range. Some limitations of this study must be addressed. First, the selection of the sample was carried out by a snowball sampling procedure via social media using an online survey link. This has some limitations, as the method does not guarantee that the sample is representative of the larger population, especially due to the possibility that nurses with risk factors were more likely to participate and complete the survey. On the other hand, the selection method is easy to perform and allows the recruitment of a larger number of relevant individuals, and we therefore feel it is suitable for an exploratory analysis. Second, the survey used a self-reported questionnaire, which does not investigate the psychological status of nurses, especially for sensitive topics. However, to avoid bias, we adopted two validated questionnaires with reliable cut-offs that have been widely used in the study population. The above limitations have also been described in some of the pandemic literature and appear to be acceptable and infected patients who required constant nursing care (58). In addition, female nurses, and nurses of both sexes with moderate- to-severe symptoms of GAD, reported more severe clinical predictors of PTSD. Frontiers in Psychiatry frontiersin.org Author contributions ER, PV, and AM contributed to the conception and design of the study. ER, DM, and GG performed data collection. ER, DM, and GG conducted the analyses and contributed to data curation. ER wrote the first draft of the manuscript. VI and AM wrote sections of the manuscript. VB, GM, CD, CM, PV, and AM critically revised the manuscript. All authors contributed to manuscript revision and read and approved the submitted version. 4. Discussion and conclusion The literature supports these findings: in particular, a systematic review with a meta-analysis found a positive correlation between being female, anxiety, and PTSD during the pandemic (40), especially in nurses employed on the front line of care for COVID-19 patients (59). On the other hand, our model did not report associations for some predictors of PTSD described in the pandemic literature. Thus, marital status, being of young age, having worked continuously in COVID-19 units (first and second waves), and having been infected with COVID-19 were not found to be predictors of PTSD for the nurses examined, although they emerged as variables of interest in other studies (39, 60). 06 frontiersin.org 10.3389/fpsyt.2023.1083693 Renzi et al. TABLE 6 Factors associated with post-traumatic stress disorder (IES-R ≥33) in nurses. Post-traumatic stress disorder aOR 95% CI p-value Sex (female) 1.88 1.09–3.22 0.022 Age (≤35 years old) 1.14 0.70–1.85 0.606 Marital status (married) 1.13 0.70–1.84 0.608 Employed in Northern Italy 0.90 0.77–2.10 0.342 Employed in COVID-19 departments during the first and second waves 1.09 0.56–1.45 0.669 COVID-19 employment wards/area Primary care Ref. Critical care 1.74 1.01–3.00 0.046 Medical wards 1.33 0.73–2.43 0.353 Surgical wards 1.12 0.19–6.63 0.901 Other 1.30 0.41–4.13 0.652 COVID-19-positive 1.48 0.82–2.68 0.197 Symptomatic COVID-19-positive family members/friends 0.81 0.51–1.28 0.362 Generalized anxiety disorder (moderate–severe) 4.54 2.93–7.05 0.001 aOR, odds ratio; CI, confidence interval. aOR, odds ratio; CI, confidence interval. strategies that consider the resilience and emotions regulation skills of HCWs as an instrument for growth and psychological well-being. This strategy must be implemented at the organizational level with the purpose of promoting social support (62). understandable given the particularity of the pandemic context. In addition, the authors are aware that multiple other factors, such as a concurrent traumatic event experienced by HCWs, or pre-existing COVID-19 disorders, may have influenced our results. However, this summary allows us to obtain a current view of the emotional and psychological state of the nurses analyzed. Finally, the cross-sectional design of the study is a limitation, especially since it is a single measurement in a changing context (i.e., the pandemic); however, pending longitudinal analyses, these data can provide a useful overview of the health problem in question. Data availability statement The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. In conclusion, levels of PTSD symptoms and anxiety among nurses were high during the pandemic (27, 28) especially in the female sample (10, 61). These findings suggest that, as a primary prevention measure, screening for psychological problems among HCWs should be carefully conducted by healthcare organizations to protect the most vulnerable. Screening for anxiety disorders should be a priority; assessment can be conducted using commonly used validated instruments, such as the GAD-7 questionnaire. Indeed, assessment of anxiety disorders appears to be a reliable predictor for second-level screening of, for example, PTSD in employees with higher-than-average anxiety scores. This would enable organizations to identify the extent of the disorder early and to direct the at-risk population to targeted psychological support interventions. Routine assessment of nurses’ mental well-being is critical considering the results on reported PTDS symptoms, which seem to predict, especially for females, possible long-term sequelae (strong presence of intrusion symptoms). Finally, it seems necessary to implement gender policies in the post-pandemic period to address and modify support services specifically for female nurses, who have emerged as a vulnerable target population. The implementation of an early screening service for GAD and PTSD and the establishment of psychological programs for nurses (especially for those employed in critical care settings) seem to be mandatory actions that are required to promote organizational well-being and improve the quality of care. In addition, the scientific literature highlights that acute COVID-19 stress-related disorders and personal growth are linked and influenced by multiple factors. For this reason, it is necessary to develop support Ethics statement The studies involving human participants were reviewed and approved by the Institutional Ethics Board of the Policlinico Umberto I Teaching Hospital/Sapienza University of Rome (protocol number 0489/2021). The patients/participants provided their written informed consent to participate in this study. Frontiers in Psychiatry Acknowledgments We thank Dr. Federica Patania for reviewing the technical contents concerning the psychiatric area. 07 frontiersin.org Renzi et al. 10.3389/fpsyt.2023.1083693 10.3389/fpsyt.2023.1083693 References 1. Chemali, S, Mari-Sáez, A, Bcheraoui, C, and Weishaar, H. Health care workers’ experiences during the COVID-19 pandemic: a scoping review. Hum Resour Health. (2022). 20:1–17. doi: 10.1186/S12960-022-00724-1/TABLES/2 COVID-19 pandemic in mental healthcare workers. Brain Sci. (2022). 12:408. doi: 10.3390/BRAINSCI12030408 17. Lasalvia, A, Bonetto, C, Porru, S, Carta, A, Tardivo, S, Bovo, C, et al. Psychological impact of COVID-19 pandemic on healthcare workers in a highly burdened area of north-East Italy. Epidemiol Psychiatr Sci. (2020). 30:1158. doi: 10.1017/ S2045796020001158 17. Lasalvia, A, Bonetto, C, Porru, S, Carta, A, Tardivo, S, Bovo, C, et al. Psychological impact of COVID-19 pandemic on healthcare workers in a highly burdened area of north-East Italy. Epidemiol Psychiatr Sci. (2020). 30:1158. doi: 10.1017/ S2045796020001158 2. di Mattei, VE, Perego, G, Milano, F, Mazzetti, M, Taranto, P, di Pierro, R, et al. The “Healthcare workers” wellbeing (Benessere Operatori) “project: a picture of the mental health conditions of Italian healthcare workers during the first wave of the COVID-19 pandemic”. Int J Environ Res Public Health. (2021). 18:5267. doi: 10.3390/ IJERPH18105267 2. di Mattei, VE, Perego, G, Milano, F, Mazzetti, M, Taranto, P, di Pierro, R, et al. The “Healthcare workers” wellbeing (Benessere Operatori) “project: a picture of the mental health conditions of Italian healthcare workers during the first wave of the COVID-19 pandemic”. Int J Environ Res Public Health. (2021). 18:5267. doi: 10.3390/ IJERPH18105267 18. Marcomini, I, Agus, C, Milani, L, Sfogliarini, R, Bona, A, and Castagna, M. COVID-19 and post-traumatic stress disorder among nurses: a descriptive cross- sectional study in a COVID hospital. Med Lav. (2021). 112:241–9. doi: 10.23749/MDL. V112I3.11129 3. Uccella, S, Mongelli, F, Majno-Hurst, P, Pavan, LJ, Uccella, S, Zoia, C, et al. Psychological impact of the very early beginning of the COVID-19 outbreak in healthcare workers: a Bayesian study on the Italian and Swiss perspectives. Front Public Health. (2022). 10:398. doi: 10.3389/FPUBH.2022.768036/BIBTEX 19. Zhang, SX, Arroyo Marioli, F, Gao, R, and Wang, S. A second wave? What do people mean by COVID waves? – a working definition of epidemic waves. Risk Manag Healthc Policy. (2021). 14:3775–82. doi: 10.2147/RMHP.S326051 4. Jung, H, Jung, SY, Lee, MH, and Kim, MS. Assessing the presence of post-traumatic stress and turnover intention among nurses post-Middle East respiratory syndrome outbreak: the importance of supervisor support. Workplace Health Saf. (2020). 68:337–45. doi: 10.1177/2165079919897693 20. Caramello, V, Catalano, A, Macciotta, A, Dansero, L, Sacerdote, C, Costa, G, et al. References Improvements throughout the three waves of COVID-19 pandemic: results from 4 million inhabitants of north-West Italy. J Clin Med. (2022). 11:4304. doi: 10.3390/ JCM11154304/S1 5. Khalid, I, Khalid, TJ, Qabajah, MR, Barnard, AG, and Qushmaq, IA. Healthcare workers emotions, perceived stressors and coping strategies during a MERS-CoV outbreak. Clin Med Res. (2016). 14:7–14. doi: 10.3121/CMR.2016.1303 21. Eysenbach, G. Improving the quality of web surveys: the checklist for reporting results of internet E-surveys (CHERRIES). J Med Internet Res. (2004). 6:E34. doi: 10.2196/JMIR.6.3.E34 6. Maunder, R. The experience of the 2003 SARS outbreak as a traumatic stress among frontline healthcare workers in Toronto: lessons learned. Philos Trans R Soc Lond Ser B Biol Sci. (2004). 359:1117–25. doi: 10.1098/RSTB.2004.1483 22. Spitzer, RL, Kroenke, K, Williams, JBW, and Löwe, B. A brief measure for assessing generalized anxiety disorder: the GAD-7. Arch Intern Med. (2006). 166:1092–7. doi: 10.1001/ARCHINTE.166.10.1092 7. Maunder, RG, Lancee, WJ, Balderson, KE, Bennett, JP, Borgundvaag, B, Evans, S, et al. Long-term psychological and occupational effects of providing hospital healthcare during SARS outbreak. Emerg Infect Dis. (2006). 12:1924–32. doi: 10.3201/ EID1212.060584 23. Weiss, DS, and Marmar, CR. The Impact of Event Scale-Revised In: JP Wilson and TM Keane, editors. Assessing psychological trauma and PTSD, vol. 10. New York: The Guilford Press (1997). 96–107. doi: 10.4236/ME.2019.101007 8. Sahebi, A, Nejati-Zarnaqi, B, Moayedi, S, Yousefi, K, Torres, M, and Golitaleb, M. The prevalence of anxiety and depression among healthcare workers during the COVID-19 pandemic: an umbrella review of meta-analyses. Prog Neuro- Psychopharmacol Biol Psychiatry. (2021). 107:110247. doi: 10.1016/J.PNPBP.2021.110247 24. Federazione Nazionale Ordini Professioni Infermieristiche FNOPI. infermieristica, professione al femminile, ma non per questo sempre “rosa”. Available at: https://www. fnopi.it/2022/03/08/8-marzo-infermiere-2/ [Accessed July 18, 2022] 25. Aymerich, C, Pedruzo, B, Pérez, JL, Laborda, M, Herrero, J, Blanco, J, et al. COVID-19 pandemic effects on health worker’s mental health: systematic review and meta-analysis. Eur Psychiatry. (2022). 65:e10. doi: 10.1192/J.EURPSY.2022.1 9. Pappa, S, Ntella, V, Giannakas, T, Giannakoulis, VG, Papoutsi, E, and Katsaounou, P. Prevalence of depression, anxiety, and insomnia among healthcare workers during the COVID-19 pandemic: a systematic review and meta-analysis. Brain Behav Immun. (2020). 88:901–7. doi: 10.1016/J.BBI.2020.05.026 26. Chen, J, Farah, N, Dong, RK, Chen, RZ, Xu, W, Yin, J, et al. Mental health during the COVID-19 crisis in Africa: a systematic review and meta-analysis. Int J Environ Res Public Health. (2021). 18:10604. doi: 10.3390/IJERPH182010604 10. Publisher’s note The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpsyt.2023.1083693/ 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 Xing, LQ, Xu, ML, Sun, J, Wang, QX, Ge, DD, Jiang, MM, et al. Anxiety and depression in frontline health care workers during the outbreak of Covid-19. Int J Soc Psychiatry. (2021). 67:656–63. doi: 10.1177/0020764020968119 27. Zhang, SX, Batra, K, Xu, W, Liu, T, Dong, RK, Yin, A, et al. Mental disorder symptoms during the COVID-19 pandemic in Latin America – a systematic review and meta-analysis. Epidemiol Psychiatr Sci. (2022). 31:767. doi: 10.1017/S2045796021000767 11. Qi, X, Wang, J, Liu, J, Amporfro, DA, Wang, K, Liu, H, et al. Factors associated with peritraumatic stress symptoms among the frontline healthcare workers during the outbreak of COVID-19 in China. BMJ Open. (2022). 12:e047753. doi: 10.1136/ BMJOPEN-2020-047753 28. Zhang, SX, Miller, SO, Xu, W, Yin, A, Chen, BZ, Delios, A, et al. Meta-analytic evidence of depression and anxiety in Eastern Europe during the COVID-19 pandemic. Eur J Psychotraumatol. (2022). 13:132. doi: 10.1080/20008198.2021.2000132 12. Lasalvia, A, Bodini, L, Amaddeo, F, Porru, S, Carta, A, Poli, R, et al. The sustained psychological impact of the COVID-19 pandemic on health care workers one year after the outbreak-a repeated cross-sectional survey in a tertiary Hospital of North-East Italy. Int J Environ Res Public Health. (2021). 18:3374. doi: 10.3390/IJERPH182413374 29. Conti, C, Fontanesi, L, Lanzara, R, Rosa, I, Porcelli, P, and Fragile heroes, The psychological impact of the COVID-19 pandemic on health-care workers in Italy. PLoS One. (2020). 15:2538. doi: 10.1371/JOURNAL.PONE.0242538 13. Gonzalez Mendez, MJ, Ma, L, Alvarado, R, Ramirez, J, Xu, KP, Xu, HF, et al. A multi-center study on the negative psychological impact and associated factors in Chinese healthcare workers 1 year after the COVID-19 initial outbreak. Int J Public Health. (2022). 67:4979. doi: 10.3389/IJPH.2022.1604979 30. Fattori, A, Cantù, F, Comotti, A, Tombola, V, Colombo, E, Nava, C, et al. Hospital workers mental health during the COVID-19 pandemic: methods of data collection and characteristics of study sample in a university hospital in Milan (Italy). BMC Med Res Methodol. (2021). 21:1–12. doi: 10.1186/S12874-021-01355-1/FIGURES/2 14. D’Arienzo, M, and Coniglio, A. Assessment of the SARS-CoV-2 basic reproduction number, R0, based on the early phase of COVID-19 outbreak in Italy. Biosaf Health. (2020). 2:57–9. doi: 10.1016/J.BSHEAL.2020.03.004 31. Ranieri, J, Guerra, F, Perilli, E, Passafiume, D, Maccarone, D, Ferri, C, et al. Prolonged COVID 19 outbreak and psychological response of nurses in Italian healthcare system: cross-sectional study. Front Psychol. (2021). 12:1003. doi: 10.3389/ FPSYG.2021.608413/BIBTEX 15. Frontiers in Psychiatry References Prevalence and risk factors of depression, anxiety, and stress in a cohort of Australian nurses. Int J Environ Res Public Health. (2018) 16::61. doi: 10.3390/IJERPH16010061 51. Greenberg, N, Weston, D, Hall, C, Caulfield, T, Williamson, V, and Fong, K. Mental health of staff working in intensive care during Covid-19. Occup Med. (2021). 71:62–7. doi: 10.1093/OCCMED/KQAA220 37. Janda, R, and Jandová, E. Symptoms of posttraumatic stress disorder, anxiety and depression among Czech critical care and general surgical and medical ward nurses (2015). 20:298–309. doi: 10.1177/1744987115584211, 52. Heesakkers, H, Zegers, M, van Mol, MMC, and van den Boogaard, M. The impact of the first COVID-19 surge on the mental well-being of ICU nurses: a nationwide survey study. Intensive Crit Care Nurs. (2021). 65:103034. doi: 10.1016/J.ICCN.2021.103034 38. Adriaenssens, J, de Gucht, V, and Maes, S. The impact of traumatic events on emergency room nurses: findings from a questionnaire survey. Int J Nurs Stud. (2012). 49:1411–22. doi: 10.1016/J.IJNURSTU.2012.07.003 53. Hamed, RA, Abd Elaziz, SY, and Ahmed, AS. Prevalence and predictors of burnout syndrome, post-traumatic stress disorder, depression, and anxiety in nursing staff in various departments. Middle East Cur Psychiatry. (2020). 27:1–8. doi: 10.1186/ S43045-020-00044-X/TABLES/3 39. Preti, E, di Mattei, V, Perego, G, Ferrari, F, Mazzetti, M, Taranto, P, et al. The psychological impact of epidemic and pandemic outbreaks on healthcare workers: rapid review of the evidence. Curr Psychiatry Rep. (2020). 22:43. doi: 10.1007/ S11920-020-01166-Z 54. Danella, N, Hamilton, S, and Heinrich, C. Posttraumatic stress disorder in critical care nurses. Nurs Crit Care. (2017). 12:40–6. doi: 10.1097/01.CCN.0000515980.94246.40 55. Wang, Y, Lu, X, Li, Y, Chen, H, Chen, T, Su, N, et al. Clinical course and outcomes of 344 intensive care patients with COVID-19. Am J Respir Crit Care Med. (2020). 201:1430–4. doi: 10.1164/RCCM.202003-0736LE 40. Chutiyami, M, Cheong, AMY, Salihu, D, Bello, UM, Ndwiga, D, Maharaj, R, et al. COVID-19 pandemic and overall mental health of healthcare professionals globally: a meta-review of systematic reviews. Front Psych. (2022). 12:804525. doi: 10.3389/ FPSYT.2021.804525/FULL 56. Murthy, S, Gomersall, CD, and Fowler, RA. Care for Critically ill Patients with COVID-19. JAMA. (2020). 323:1499–500. doi: 10.1001/JAMA.2020.3633 41. D’ettorre, G, Ceccarelli, G, Santinelli, L, Vassalini, P, Pietro, IG, Alessandri, F, et al. Post-traumatic stress symptoms in healthcare workers dealing with the COVID-19 pandemic: a systematic review. Int J Environ Res Public Health. (2021). 18:1–16. doi: 10.3390/IJERPH18020601 57. Ramírez, LPG, Arriaga, RJM, Hernández-Gonzalez, MA, and de la Roca-Chiapas, JM. References Simonetti, V, Durante, A, Ambrosca, R, Arcadi, P, Graziano, G, Pucciarelli, G, et al. Anxiety, sleep disorders and self-efficacy among nurses during COVID-19 pandemic: a large cross-sectional study. J Clin Nurs. (2021). 30:1360–71. doi: 10.1111/JOCN.15685 32. Ramirez-Baena, L, Ortega-Campos, E, Gomez-Urquiza, JL, Cañadas-De la Fuente, GR, de la Fuente-Solana, EI, and Cañadas-De la Fuente, GA. A multicentre study of burnout prevalence and related psychological variables in medical area hospital nurses. J Clin Med. (2019). 8:92. doi: 10.3390/JCM8010092 16. Minelli, A, Silva, RC, Barlati, S, Vezzoli, M, Carletto, S, Isabello, C, et al. The elephant in the room: a cross-sectional study on the stressful psychological effects of the 08 Frontiers in Psychiatry frontiersin.org Renzi et al. 10.3389/fpsyt.2023.1083693 Renzi et al. 48. Crowe, S, Howard, AF, Vanderspank-Wright, B, Gillis, P, McLeod, F, Penner, C, et al. The effect of COVID-19 pandemic on the mental health of Canadian critical care nurses providing patient care during the early phase pandemic: a mixed method study. Intensive Crit Care Nurs. (2021). 63:102999. doi: 10.1016/J.ICCN.2020.102999 33. Tsaras, K, Papathanasiou, IV, Vus, V, Panagiotopoulou, A, Katsou, MA, Kelesi, M, et al. Predicting factors of depression and anxiety in mental health nurses: a quantitative cross-sectional study. Med Arch. (2018). 72:62–7. doi: 10.5455/MEDARH.2017.72.62-67 34. Tran, TTT, Nguyen, NB, Luong, MA, Bui, THA, Phan, TD, Tran, VO, et al. Stress, anxiety and depression in clinical nurses in Vietnam: a cross-sectional survey and cluster analysis. Int J Ment Health Syst. (2019). 13:1–11. doi: 10.1186/S13033-018-0257-4/ TABLES/4 49. Ayotte, BJ, Schierberl Scherr, AE, and Kellogg, MB. PTSD symptoms and functional impairment among nurses treating COVID-19 patients. SAGE Open Nurs. (2022). 8:4651. doi: 10.1177/23779608221074651/ASSET/IMAGES/LARGE/10.1177_23779608221074651- FIG 1.JPEG 49. Ayotte, BJ, Schierberl Scherr, AE, and Kellogg, MB. PTSD symptoms and functional impairment among nurses treating COVID-19 patients. SAGE Open Nurs. (2022). 8:4651. doi: 10.1177/23779608221074651/ASSET/IMAGES/LARGE/10.1177_23779608221074651- FIG 1.JPEG 35. Gao, YQ, Pan, BC, Sun, W, Wu, H, Wang, JN, and Wang, L. Anxiety symptoms among Chinese nurses and the associated factors: a cross sectional study. BMC Psychiatry. (2012). 12:1–9. doi: 10.1186/1471-244X-12-141/TABLES/4 50. Czaja, AS, Moss, M, and Mealer, M. Symptoms of posttraumatic stress disorder among pediatric acute care nurses. J Pediatr Nurs. (2012). 27:357–65. doi: 10.1016/J. PEDN.2011.04.024 50. Czaja, AS, Moss, M, and Mealer, M. Symptoms of posttraumatic stress disorder among pediatric acute care nurses. J Pediatr Nurs. (2012). 27:357–65. doi: 10.1016/J. PEDN.2011.04.024 36. Maharaj, S, Lees, T, and Lal, S. References Psychological distress and signs of post-traumatic stress in response to the COVID-19 health emergency in a Mexican sample. Psychol Res Behav Manag. (2020). 13:589–97. doi: 10.2147/PRBM.S259563 42. How Common is PTSD in Women? - PTSD: National Center for PTSD. Available at: https://www.ptsd.va.gov/understand/common/common_women.asp [Accessed July 18, 2022] 58. Gibson, DM, and Greene, J. Risk for severe COVID-19 illness among health care workers who work directly with patients. J Gen Intern Med. (2020). 35:2804–6. doi: 10.1007/S11606-020-05992-Y 43. Seedat, S, Stein, DJ, and Carey, PD. Post-traumatic stress disorder in women: epidemiological and treatment issues. CNS Drugs. (2005). 19:411–27. doi: 10.2165/00023210-200519050-00004 59. Chen, X, Chen, J, Zhang, M, Dong, RK, Li, J, Dong, Z, et al. Meta-regression on the Heterogenous factors contributing to the prevalence of mental health symptoms during the COVID-19 crisis among healthcare workers. Front Psych. (2022). 13:833865. doi: 10.3389/FPSYT.2022.833865/FULL 44. Post-traumatic stress disorder\|Office on Women’s Health. Available at: https:// www.womenshealth.gov/mental-health/mental-health-conditions/post-traumatic- stress-disorder [Accessed July 18, 2022] 60. Varghese, A, George, G, Kondaguli, SV, Naser, AY, Khakha, DC, and Chatterji, R. Decline in the mental health of nurses across the globe during COVID-19: a systematic review and meta-analysis. J Glob Health. (2021). 11:1–15. doi: 10.7189/JOGH.11.05009 45. Mealer, M, and Jones, J. Posttraumatic stress disorder in the nursing population: a concept analysis. Nurs Forum (Auckl). (2013). 48:279–88. doi: 10.1111/NUF.12045 61. Dragioti, E, Tsartsalis, D, Mentis, M, Mantzoukas, S, and Gouva, M. Impact of the COVID-19 pandemic on the mental health of hospital staff: an umbrella review of 44 meta-analyses. Int J Nurs Stud. (2022). 131:104272. doi: 10.1016/J. IJNURSTU.2022.104272 46. Young, JL, Derr, DM, Cicchillo, VJ, and Bressler, S. Compassion satisfaction, burnout, and secondary traumatic stress in heart and vascular nurses. Crit Care Nurs Q. (2011). 34:227–34. doi: 10.1097/CNQ.0B013E31821C67D5 62. Fino, E, Bonfrate, I, Fino, V, Bocus, P, Russo, PM, and Mazzetti, M. Harnessing distress to boost growth in frontline healthcare workers during COVID-19 pandemic: the protective role of resilience, emotion regulation and social support. Psychol Med. (2021):1–3. doi: 10.1017/S0033291721000519 47. Guttormson, JL, Calkins, K, McAndrew, N, Fitzgerald, J, Losurdo, H, and Loonsfoot, D. Critical care nurse burnout, moral distress, and mental health during the COVID-19 pandemic: a United States survey. Heart Lung. (2022). 55:127–33. doi: 10.1016/J.HRTLNG.2022.04.015 09 Frontiers in Psychiatry frontiersin.org
https://openalex.org/W2051680248	https://www.banglajol.info/index.php/BJP/article/download/17410/12451	English	null	<i>In vitro</i> anti-venom potential of various <i>Jatropha</i> extracts on neutralizing cytotoxic effect induced by phospholipase A2 of crude venom from Indian cobra (<i>Naja naja</i>)	Bangladesh journal of pharmacology	2,014	cc-by	4,783	Abstract In present study various species of Jatropha were evaluated for antidote nature induced by phospholipase A2 (PLA2) cobra venom. Although qualitative phytochemical analysis exhibited less variation across Jatropha species studied, substantial variability in terms of PLA2 inhibition by various solvent extracts across the species and between different parts of same plant was observed. Among all samples methanolic extracts of J. gossypifolia leaf showed highest inhibition of PLA2 toxicity while some aqueous extracts of J. foetida and all aqueous extracts of J. curcas, enhanced PLA2 activity. Present results highlight Jatropha not only as rich source of secondary compounds with antidote property for snake bite but also potent toxic agents as revealed with increased hemolysis by some aqueous extracts of J. curcas and J. foetida. Our findings suggest that methanolic leaf extract of J. gossypifolia contain potent small molecular antagonist(s) to the snake venom PLA2 which will be very useful to design adjuvant therapies in treatments of snake bites. Cite this article: Reddi KVNR, Rajesh SS, Narendra K, Jangala S, Reddy PCO, Satya AK, Sivaraman T, Sekhar AC. In vitro anti- venom potential of various Jatropha extracts on neutralizing cytotoxic effect induced by phospholipase A2 of crude venom from Indian cobra (Naja naja). Bangladesh J Pharmacol. 2014; 9: 22-28. Samejima, 1980), edema (Vishwanath et al., 1987) cardio -toxicity and hemolysis (Condrea et al., 1980, 1981). Samejima, 1980), edema (Vishwanath et al., 1987) cardio -toxicity and hemolysis (Condrea et al., 1980, 1981). In vitro anti-venom potential of various Jatropha extracts on neutralizing cytotoxic effect induced by phospholipase A2 of crud venom from Indian cobra (Naja naja) K. V. N. Rathnakar Reddi1, Sivarathri Siva Rajesh2, Kumara Narendra1, Swathi Jangala1, Puli Chandra Obul Reddy3, Alapati Krishna Satya1, Thirunavukkarasu Sivaraman2 and Akila Chandra Sekhar4 1Departmant of Biotechnology Acharya Nagarjuna University Guntur 522 510 AP India; 2Structural Biology K. V. N. Rathnakar Reddi1, Sivarathri Siva Rajesh2, Kumara Narendra1, Swathi Jangala1, Puli Chandra Obul Reddy3, Alapati Krishna Satya1, Thirunavukkarasu Sivaraman2 and Akila Chandra Sekhar4 1Departmant of Biotechnology, Acharya Nagarjuna University, Guntur 522 510, AP, India; 2Structural Biology Laboratory, Department of Bioinformatics, School of Chemical and Biotechnology, SASTRA University, Thanjavur 613 401, TN, India; 3Plant Molecular Biology Laboratory, Department of Botany, School of Life Sciences, Yogi Vemana University, Kadapa 516 003, AP, India; 4Molecular Genetics and Functional Genomics Laboratory, Department of Biotechnology, School of Life Sciences, Yogi Vemana University, Kadapa 516 003, AP, India. 1Departmant of Biotechnology, Acharya Nagarjuna University, Guntur 522 510, AP, India; 2Structural Biology Laboratory, Department of Bioinformatics, School of Chemical and Biotechnology, SASTRA University, Thanjavur 613 401, TN, India; 3Plant Molecular Biology Laboratory, Department of Botany, School of Life Sciences, Yogi Vemana University, Kadapa 516 003, AP, India; 4Molecular Genetics and Functional Genomics Laboratory, Department of Biotechnology, School of Life Sciences, Yogi Vemana University, Kadapa 516 003, AP, India. 1Departmant of Biotechnology, Acharya Nagarjuna University, Guntur 522 510, AP, India; 2Structural Biology Laboratory, Department of Bioinformatics, School of Chemical and Biotechnology, SASTRA University, Thanjavur 613 401, TN, India; 3Plant Molecular Biology Laboratory, Department of Botany, School of Life Sciences, Yogi Vemana University, Kadapa 516 003, AP, India; 4Molecular Genetics and Functional Genomics Laboratory, Department of Biotechnology, School of Life Sciences, Yogi Vemana University, Kadapa 516 003, AP, India. Article Info Received: 23 December 2013 Accepted: 16 January 2014 Available Online: 20 January 2014 DOI: 10.3329/bjp.v9i1.17410 Article Info Received: 23 December 2013 Accepted: 16 January 2014 Available Online: 20 January 2014 DOI: 10.3329/bjp.v9i1.17410 A Journal of the Bangladesh Pharmacological Society (BDPS); www.bdps.info Bangladesh J Pharmacol 2014; 9: 22-28 Journal homepage: www.banglajol.info Abstracted/indexed in Academic Search Complete, Agroforestry Abstracts, Asia Journals Online, Bangladesh Journals Online, Biological Abstracts, BIOSIS Previews, CAB Abstracts, Current Abstracts, Directory of Open Access Journals, EMBASE/Excerpta Medica, Global Health, Google Scholar, HINARI (WHO), International Pharmaceutical Abstracts, Open J-gate, Science Citation Index Expanded, SCOPUS and Social Sciences Citation Index ISSN: 1991 0088 This work is licensed under a Creative Commons Attribution 3.0 License. You are free to copy, distribute and perform the work. You must attribute the work in the manner specified by the author or licensor. Journal homepage: www.banglajol.info Abstracted/indexed in Academic Search Complete, Agroforestry Abstracts, Asia Journals Online, Bangladesh Journals Online, Biological Abstracts, BIOSIS Previews, CAB Abstracts, Current Abstracts, Directory of Open Access Journals, EMBASE/Excerpta Medica, Global Health, Google Scholar, HINARI (WHO), International Pharmaceutical Abstracts, Open J-gate, Science Citation Index Expanded, SCOPUS and Social Sciences Citation Index ISSN: 1991-0088 Bangladesh Journal of Pharmacology Research Article In vitro anti-venom potential of vari- ous Jatropha extracts on neutralizing cytotoxic effect induced by phos- pholipase A2 of crude venom from Indian cobra (Naja naja) BJP Bangladesh Journal of Pharmacology Research Article In vitro anti-venom potential of vari- ous Jatropha extracts on neutralizing cytotoxic effect induced by phos- pholipase A2 of crude venom from Indian cobra (Naja naja) BJP A Journal of the Bangladesh Pharmacological Society (BDPS); www.bdps.info Bangladesh J Pharmacol 2014; 9: 22-28 Journal homepage: www.banglajol.info Abstracted/indexed in Academic Search Complete, Agroforestry Abstracts, Asia Journals Online, Bangladesh Journals Online, Biological Abstracts, BIOSIS Previews, CAB Abstracts, Current Abstracts, Directory of Open Access Journals, EMBASE/Excerpta Medica, Global Health, Google Scholar, HINARI (WHO), International Pharmaceutical Abstracts, Open J-gate, Science Citation Index Expanded, SCOPUS and Social Sciences Citation Index ISSN: 1991-0088 In vitro anti-venom potential of various Jatropha extracts on neutralizing cytotoxic effect induced by phospholipase A2 of crude venom from Indian cobra (Naja naja) K. V. N. Rathnakar Reddi1, Sivarathri Siva Rajesh2, Kumara Narendra1, Swathi Jangala1, Puli Chandra Obul Reddy3, Alapati Krishna Satya1, Thirunavukkarasu Sivaraman2 and Akila Chandra Sekhar4 1Departmant of Biotechnology, Acharya Nagarjuna University, Guntur 522 510, AP, India; 2Structural Biology Laboratory, Department of Bioinformatics, School of Chemical and Biotechnology, SASTRA University, Thanjavur 613 401, TN, India; 3Plant Molecular Biology Laboratory, Department of Botany, School of Life Sciences, Yogi Vemana University, Kadapa 516 003, AP, India; 4Molecular Genetics and Functional Genomics Laboratory, Department of Biotechnology, School of Life Sciences, Yogi Vemana University, Kadapa 516 003, AP, India. This article was downloaded by you on: Sep 15, 2018 This article was downloaded by you on: Sep 15, 2018 Bangladesh J Pharmacol 2014; 9: 22-28 Journal homepage: www.banglajol.info Abstracted/indexed in Academic Search Complete, Agroforestry Abstracts, Asia Journals Online, Bangladesh Journals Online, Biological Abstracts, BIOSIS Previews, CAB Abstracts, Current Abstracts, Directory of Open Access Journals, EMBASE/Excerpta Medica, Global Health, Google Scholar, HINARI (WHO), International Pharmaceutical Abstracts, Open J-gate, Science Citation Index Expanded, SCOPUS and Social Sciences Citation Index ISSN: 1991-0088 In vitro anti-venom potential of various Jatropha extracts on neutralizing cytotoxic effect induced by phospholipase A2 of crude venom from Indian cobra (Naja naja) Plant selections, collections and material preparations For the present study, three species of Jatropha viz., Jatropha gossypifolia (J.G), Jatropha foetida (J.F) and Jatropha curcas (3 lines) are selected. J. gossypifolia, J. foetida (collected from Seshachalam Hills, Tirupathi), were identified by Dr. A. Madhusudhana Reddy, Assistant Professor, Department of Botany, Yogi Vemana University, Kadapa, Andhra Pradesh, India. Three distinct varieties of J. curcas (these materials were generously provided by Dr. L. Sivarama Prasad, Natu- role Bioenergy Ltd., India, which were originally collected from Sai Petro Chemicals, Maharastra (J.C-1); Aleru, Warangal (J.C-2); and Uganda, Africa (J.C-3); the three varieties of J. curcus are being presently main- tained in Yogi Vemana University, Kadapa) were selected based on genetic diversity shown by molecular genetic studies carried out using RAPD and ISSR profiling (Data not shown). Leaves, stems and roots of each variety were collected separately, thoroughly washed and shade dried. Preparation of RBC cells from blood sample Preparation of RBC cells from blood sample Hemolytic assay Toxicity of the venom and its neutralization by the Jatropha extracts was determined through indirect hemo -lytic assay (Habermann, 1954; Gutierrez, 1988; Paula et al., 2010) by using human erythrocytes and hen’s egg yolk emulsion as substrate. The amount of venom (10 µg/mL) that can produce 70%hemolysis after the incubation was denoted as the Minimum Indirect Hemolytic Dose (MIHD). Experiments were performed by pre-incubating 25 µg of Jatropha extract with a MIH dose of Naja naja venom for 30 min at 37°C prior to evaluating the hemolytic activity. To this, 5 mL of prepared erythrocyte suspension was added and incubated for further thirty min at 37°C. After 30 min, venom activity on the substrate was stopped by adding 3 mL of ice cold PBS buffer to the reaction mixture. The extent of cell lysis/hemolysis caused by the venom on the RBC cells was measured by calculating the amount of hemoglobin released from the cells. The amount of released hemoglobin from the reaction mixture was estimated from the intensity measured at 540 nm using UV spectroscopy. The hemolysis produced by the venom in the absence of Jatropha extracts was taken as 100% and the percentage of inhibition/activation by each Jatropha extract used in the present study against snake venom PLA2s was calculated as shown in the following calculations. All measurements were made in triplicates and results are expressed as mean + standard deviation. Introduction Snakebite is one among vital public health issues of tropical countries including India. Most of the mortality cases come from farmers, hunter gathers of rural areas which lack proper sickbays and antivenin supply (Panfoli et al., 2010). Naja naja commonly called as Indian Elapid is one among the poisonous snakes with toxin sub-fractions like metalloproteases, phospholi- pases, hyaluronidases, neurotoxins, and myotoxins which wreak havoc in victim’s body. Phospholipase A2 is one of well studied fraction of cobra venom with many pharmacological effects like presynaptic neuro- toxicity (Kini and Iwanga, 1986), myotoxicity (Mebs and Till date, antibodies produced in horses are the only source of antivenom, a process of time consuming. Moreover, antibodies need to store in low temperature conditions that lack in rural areas of developing countries. Snake bite victims sensitive to horse products produce hyper-sensitive side reactions when treated with antiserum of equine origin (Nazim et al., 2008). So, identification of alternative medicine with no risk and low temperature storage requirements became impor- tant task. Plant based antidote sources are known from olden ages for snake bites. Jatropha is one among Bangladesh J Pharmacol 2014; 9: 22-28 23 phytochemical estimations and identifications of vari- ous secondary metabolites like, alkaloids, flavonoids, terpenoids, saponins, tannins and phenolics present in the extracts by using standard methods as described elsewhere (Trease and Evans, 1996), Sofowora (Sofo- wora, 1993), and Harborne (Harborne, 1998). The positive results were noted as present (+) and negative results as absent (-). reported to have antidote property and used in some tribal medicines with no scientific evaluation. Phyto- compounds isolated from Jatropha plants are reported to have other therapeutic activities like anti-inflammatory, antitumor, molluscidal, insecticidal and fungicidal activities (Albuquerque, 2006). The present study is aimed on systematic evaluation of antidote potentials of various solvent extracts of Jatro- pha species. Snake venom Lyophilized crude venom of Naja naja (Indian cobra) was purchased from “The Irula Snake-Catchers Indus- trial Cooperative Society (ISCICS), Mamallapuram, Tamil Nadu and the crude venom was stored in airtight containers at 4ºC until used. Plant materials sampling and soxhlet extractions Equal amount of all dried plant materials (viz., leaf, stem and roots separately) were pulverized to fine powder and subjected to soxhlet extraction with chloroform, methanol and water solvents at elevated temperatures of 40, 45 and 75°C respectively. The extract obtained was concentrated through rotavapour at a constant temperature of 38°C for chloroform, methanolic extracts and 46°C for aqueous extracts with the help of water bath. Solid extracts were weighed and stored in dry environment until further use in opaque containers in refrigerator. Materials and Methods Human blood was collected in glass tubes containing sodium citrate as anticoagulant. The blood samples were centrifuged at 800 rpm for 10 min and plasma was carefully removed by sterile Pasteur pipette. The pellet of red blood cells were washed three times by PBS buffer (0.15 M NaCl, 10 mM phosphate buffer, pH 7.0) and then suspended in a fresh PBS buffer at a density of 1.2 x 109 cells/mL monitored by a Neubauer chamber. To this 1% of egg albumin was added (Habermann and Neumann, 1954; Gutierrez, 1988) and the resultant mixture was considered as substrate to snake venom PLA2s in the hemolytic assay described below herein. Plant selections, collections and material preparations % inhibition of PLA2 activity by plant extracts = 100 – 100 x OD of test sample at 540 nm/OD of control sample at 540 nm Results did not cause any membrane lytic effect on red blood cells (results not shown). Of all the aqueous extracts, J. gossypifolia leaf, root and stem showed high hemolytic inhibition of 85.9, 84.5 and 81% respectively (Figure 1). Variation was found in terms of antivenom activity across aqueous extracts of J. foetida plant parts viz., stem, leaf and root. Only leaf extract of J. foetida showed anti-venom property in terms of PLA2 inhibition where- as stem and root aqueous extracts of the plant showed enhanced-hemolytic activity resembling J. curcas extracts when incubated with venom. Selection of Jatropha lines for present phytochemical and antidote analysis (Table I) was based on the phylo- genetic tree constructed from molecular genetic studies of 35 lines using RAPD and ISSR marker profiling (data not shown here) was found to be helpful for initial screening. Our results strengthen the reports of Jatropha’s use as an antidote in folk medicine. For the forty five extracts obtained (3 solvents x 5 plants x 3 parts of each plant–stem, leaf and root) qualitative phytochemical analysis exhibited less variation among the species of Jatropha viz., gossypifolia (J.G- stem, J.G- leaf and J.G- root), foetida (J.F- stem, J.F- leaf and J.F- root), and curcas (J.C- stem, J.C- leaf and J.C- root), and almost no varietal variation was found within the spe- cies (varieties – J.C-1) of curcas and also no significant variation of phytocompounds between different parts of same Jatropha plants is observed (Table I). Methanolic extracts of all three varieties of J. curcas showed good inhibition within a range from 81 to 88.3% and the variation among different parts of a same plant was also negligible (Figure 2). However, extracts of various parts from J. gossypifolia and J. foetida showed high variations in the degree of their inhibition potentials against hemolytic activity of PLA2. Roots of J. foetida (74.5%) and leaf of J. gossypifolia (89.8%) showed high inhibitions. Of all methanolic extracts examined in the present study, leaves extracts of J. gossypifolia showed strongest inhibition of PLA2 (Figure 2). The selection of plants for the present study, based on phylogenetic analysis was found to be worth full as the same kind of variation has been reflected in both phyto- chemical composition and antiophidian property of various Jatropha extracts. Phytochemical screening and analysis % inhibition of PLA2 activity by plant extracts = 100 – 100 x OD of test sample at 540 nm/OD of control sample at 540 nm Equal amount of each extract (45 extracts in total) obtained from Soxhlet method was used for qualitative Bangladesh J Pharmacol 2014; 9: 22-28 24 24 Bangladesh J Pharmacol 2014; 9: 22-28 Table I Phytochemical analysis of Jatropha extracts Phytocompound Alkaloids Flavanoids Terpenoids Saponins Tannins Phenolics Planta Aqueous extract J.G Stem - + + + + - J.G Leaf - + + + + - J.G Root - + + + + - J.F Stem + + + + + - J.F Leaf + + + - + - J.F Root + + + - + - J.C-1 Stem + + + + + + J.C-1 Leaf + + + + + + J.C-1 Root + + + + + + J.C-2 Stem + + + + + + J.C-2 Leaf + + + + + + J.C-2 Root + + + + + + J.C-3 Stem + + + + + + J.C-3 Leaf + + + + + + J.C-3 Root + + + + + + Methanol extract J.G Stem + + - + + + J.G Leaf + + - + + + J.G Root + + - + + + J.F Stem + + + + + + J.F Leaf + + + + + + J.F Root + + + + + + J.C-1 Stem - + + + + + J.C-1 Leaf - + + + + + J.C-1 Root - + + + + + J.C-2 Stem - + + + + + J.C-2 Leaf - + + + + + J.C-2 Root - + + + + + J.C-3 Stem - + + + + + J.C-3 Leaf - + + + + + J.C-3 Root - + + + + + Chloroform extract J.G Stem + + + + + + J.G Leaf + + + + + + J.G Root + + + + + + J.F Stem + - + - + + J.F Leaf + - + - + + J.F Root + - + - + + J.C-1 Stem + + + - + + J.C-1 Leaf + + + - + + J.C-1 Root + + + - + + J.C-2 Stem + + + - + + J.C-2 Leaf + + + - + + J.C-2 Root + + + - + + J.C-3 Stem + + + - + + J.C-3 Leaf + + + - + + J.C-3 Root + + + - + + aJ.G - Jatropha gossypifolia; J.F- Jatropha foetida; Jatropha curcas varieties, J.C-1; J.C-2 and J.C-3 as described in materials Bangladesh J Pharmacol 2014; 9: 22-28 25 Figure 1: Effect of aqueous extracts of stem, leaf and root obtained from various Jatropha herbs used in the present study against hemolysis induced by snake venom PLA2 (Increased hemolysis activity of venom by phytocompound) Figure 1: Effect of aqueous extracts of stem, leaf and root obtained from various Jatropha herbs used in the present study against hemolysis induced by snake venom PLA2 (Increased hemolysis activity of venom by phytocompound) Figure 1: Effect of aqueous extracts of stem, leaf and root obtained from various Jatropha herbs used in th hemolysis induced by snake venom PLA2 (Increased hemolysis activity of venom by phytocompound) Results curcas), and within three selected lines of curcas 26 Bangladesh J Pharmacol 2014; 9: 22-28 Figure 2: Effect of methanolic extracts of stem, leaf and root obtained from various Jatropha herbs used in the present study against hemolysis induced by snake venom PLA2 Figure 3: Effect of chloroform extracts of stem, leaf and root obtained from various Jatropha herbs used in the present study against hemolysis induced by snake venom PLA2 Bangladesh J Pharmacol 2014; 9: 22-28 g J ; 9 Figure 2: Effect of methanolic extracts of stem, leaf and root obtained from various Jatropha herbs used in the present study against hemolysis induced by snake venom PLA2 methanolic extracts of stem, leaf and root obtained from various Jatropha herbs used in the present study against d by snake venom PLA2 Figure 2: Effect of methanolic extracts of stem, leaf and root obtained from various Jatropha herbs used in the present study against hemolysis induced by snake venom PLA2 Figure 2: Effect of methanolic extracts of stem, leaf and root obtained from various Jatropha herbs used in the pre hemolysis induced by snake venom PLA2 acts of stem, leaf and root obtained from various Jatropha herbs used in the present study against m PLA2 Figure 3: Effect of chloroform extracts of stem, leaf and root obtained from various Jatropha herbs used in the present study against hemolysis induced by snake venom PLA2 Figure 3: Effect of chloroform extracts of stem, leaf and root obtained from various Jatropha herbs used in the present study against hemolysis induced by snake venom PLA2 collected from diverse geographical conditions, J. gossypifolia leaf extracts was found to be good choice as antidote for Naja naja venom due to its high inhibition potential against PLA2 activity vis-à-vis that of other extracts as demonstrated above. Among aqueous, methanolic and chloroform extracts of J. curcas, only methanolic extracts were showing high anti-venom properties as inhibition was less in case of extracts from chloroform and negative inhibition could be seen with aqueous extracts from all three varieties of curcas. The high inhibition exerted by the methanol extract of J. collected from diverse geographical conditions, J. gossypifolia leaf extracts was found to be good choice as antidote for Naja naja venom due to its high inhibition potential against PLA2 activity vis-à-vis that of other extracts as demonstrated above. Among aqueous, methanolic and chloroform extracts of J. Results Owing to the records of using Jatropha as antidote for snake bite in folk’s medicine, investigation on different species of Jatropha phytocom- pounds inhibiting PLA2 was carried out. All the aqueous extracts of J. curcas, roots and stem extracts of J. foetida were found to be lethal as the extracts increased hemolysis when compared with positive control which has only venom (MIHD) (Figure 1). Interestingly, it was also found that these extracts alone All chloroform extracts of J. curcas showed very less inhibition of venom hemolysis which were in the range of 2.6 to 22.4%. In J. foetida, leaf showed very less inhibition of 9.1% and root extract showed high inhibition of 81.7%. In J. gossypifolia the leaf showed the highest of 87.5% PLA2 inhibition followed by stem and root (Figure 3). Of all three species of Jatropha (J. foetida, J. gossypifolia and J. Results curcas, only methanolic extracts were showing high anti-venom properties as inhibition was less in case of extracts from chloroform and negative inhibition could be seen with aqueous extracts from all three varieties of curcas. The high inhibition exerted by the methanol extract of J. gossypifolia leaf against the Naja naja venom strongly suggest for presence of phytocompounds possessing therapeutic potentiality with respect to PLA2 inhibitions in the extracts. All chloroform extracts of J. curcas showed very less inhibition of venom hemolysis which were in the range of 2.6 to 22.4%. In J. foetida, leaf showed very less inhibition of 9.1% and root extract showed high inhibition of 81.7%. In J. gossypifolia the leaf showed the highest of 87.5% PLA2 inhibition followed by stem and root (Figure 3). Bangladesh J Pharmacol 2014; 9: 22-28 27 The extracts that were found to be a promising natural antidote, may presumably act by forming complexes with metal ions and proteins which in turn may change the conformation of active site and there by inhibiting the action of phospholipases A2.The use of this kind of natural antidotes may reduce the risk of anaphylactic shocks in patients sensitive to equine products. Also, the natural antidotes have appreciable technical advantages when compared to commercial anti-venoms that need to be stored at very low temperatures (below 273 K) in a highly sophisticated refrigerator which are technically sound and they are presumably cost protective to have at all parts of rural areas of developing, undeveloped and even for developed countries. Work is in progress on identifying potent compounds responsible for anti-venom activities from methonalic leaf extract of J. gossypifolia at molecular/ atomic level resolutions. In these backgrounds, we strongly believe that the outcomes will be very useful to design adjuvant therapies for treatments of snake bites. Discussion The present study is aimed to identify Jatropha species with potent antidote property. Of the three species selected for present study, our results clearly indicate all the species of Jatropha can’t be used as source of antidote for snake venom as indicated from our results based on increased hemolysis activity of snake venom when incubated with aqueous extracts of J. curcas and J. foetida. Though Jatropha is used in local / folk medicine as antidote for snake bite, our observations indicate that all species of Jatropha and all parts of the plant cannot be taken granted as antidote as a few of them are capable of enhancing venom activity in terms of hemolysis and consequently may lead to lethal. Our results highlight that overlooking or adulteration of plant materials with other plant materials at species level or with different parts of the same plant may not only reduce therapeutic efficiency of extracts but may also increase toxic effects as seen in the case of aqueous extracts of J. curcas and J. foetida. Moreover, the above anti-hemolytic studies and phytochemical analysis clari -fied that even the phytocompounds are qualitatively found similar in different parts (stem, leaf and root) of the same plant, their quantities may vary from part to part as extracts of different parts of a plant showed a great degree of variations in their venom inhibition potentials. Hence, choosing of a plant species, part of the plant and extract of the part are of great importance because of great variation in phytochemical distribution of which some are found to have therapeutic value as in present case of J. gossypifolia extracts and some exhibit lethal properties as seen in case of aqueous extracts from all J. curcas varieties and also stem and root extracts of J. foetida. Acknowledgements We would like acknowledge Dr. L. Sivarama Prasad, Naturole, Hyderabad, India for providing J. curcas plant materials from his collections. References Albuquerque UP. Re-examining hypotheses concerning the use and knowledge of medicinal plants: A study in the caatinga vegetation of NE Brazil. J Ethnobiol Ethnomed. 2006; 2: 30. Chiou YL, Shinne R, Wan PH, Long SC. Quercetin modulates activities of Taiwan Naja naja naja phospholipase A2 via its effects on membrane structure and membrane-bound mode of phospholipase A2.. J Biosci. 2012; 37: 277-87. The observations clearly indicate that the aqueous extracts of J. curcas and J. foetida must have micro/ macro compounds or ions that may aid to enhance phospholipase A2 activity of snake venom. There are reports on increased activity of phospholipase A2 when the enzymes combine with some of the divalent ions like Ca2+, Sr2+ and Ba2+ etc., These ions are reported to enhance the activity of phospholipase A2 by inducing conformational changes in the active site and substrate- binding site of the PLA2 and the resultant conformation of the enzyme may either promote or suppress its biological activity (Jiang et al., 1989). The presence of quercetin-like compounds may also be a reason for increased hemolytic activity and they can form a lipid- raft like domains (Chiou et al., 2012). Condrea E, Yang CC, Rosenberg P. Comparison of relatively toxic phospholipase A2 from Naja nigricolis snake venom with that of relatively non toxic Phospholipase A2 from Hemachatus heamachatus snake venom-I. Enzymatic activity on free and membrane bound substrate. Biochem. Pharmacol. 1980; 29: 1555-63. Condrea E, Fletcher JE, Rapuano BE, Yang CC, Rosenberg P. Dissociation of enzymatic activity from lethality and pharmacological properties by cabonylation by lysines in Naja nigrocolis and Naja naja atra snake venom Phospholipase A2 . Toxicon 1981; 19: 705-20. Gutierrez JM, Avila C, Rojas E. An alternative in vitro method for testing the potency of the polyvalent antivenom produced in Costa Rica. Toxicon 1988; 26: 411-13. From a scientific approach carried out in the present study to identifying potent plant-based antidote(s) to crude venom of Naja naja, J. gossypifolia leaf was found to be more effective source for compounds with anti- venom properties to inhibit PLA2 that causes destabilizing the plasma membranes of cells and there by affecting cardiac, nervous and circulatory systems. Habermann E, Neumann W. Egg yolk coagulation method. Physiol Chem. 1954; 297: 174. Harborne JB. Photochemical methods: A guide to modern techniques of plant analysis. London, Chapman A. & Hall, 1998. References Bangladesh J Pharmacol 2014; 9: 22-28 28 hemorrhagic snake venom components: old and new approaches. Toxins 2010; 2: 417-27. Jiang MS, Fletcher JE, Smith LA. Factors influencing the hemolysis of human erythrocytes by cardiotoxins from Naja naja kaouthia and Naja naja atra venoms and a phospholipase A2 with cardiotoxin-like activities from Bungarus fasciatus venom. Toxicon 1989; 27: 247-57. Paula DR, Sanchez EF, Costa TR, Martins CHG, Pereira PS, Lourengo MV, Soares AM, Fuly AL. Antiophidian properties of plant extracts against Lachesis muta venom. J Venom Anim Toxins incl Trop Dis. 2010; 16: 311-23. Kini RM, Iwanga S. Structure-function relationship of phospholipase 1; Prediction of presynaptic neurotoxicity. Toxicon 1986; 24: 527-41. Sofowora A. Medicinal plants and traditional medicines in Africa. New York, John Wiley & Sons, 1993, pp 97-145. Mebs D, Samejima J. Purification from Austalian elapid venoms and properties of phospholipase A which causes myoglobinurea in mice. Toxicon 1980; 18: 443-54. Trease GE, Evans WC. A textbook of pharmacognosy. 14th ed. London, Bailliere Tindall Ltd., 1996. Vishwanath BS, Kini RM, Gowda TV. Characterization of three edema-inducing phospholipase A2 from Vipera russelli venom and its interaction with aristolic acid. Toxicon 1987; 25: 501-15. Nazim MH, Gupta S, Hashmi S, Zuberi J, Wilson A, Roberts L, Karimi K. Retrospective review of snake bite victims. W V Med J. 2008; 104: 30-34. Panfoli I, Calzia D, Ravera S, Morelli A. Inhibition of Author Info Akila Chandra Sekhar (Principal contact) e-mail: acsekhar@yogivemanauniversity.ac.in
https://openalex.org/W3169150470	https://www.mdpi.com/2072-666X/12/6/666/pdf?version=1623048630	English	null	Progress on Self-Powered Wearable and Implantable Systems Driven by Nanogenerators	Micromachines	2,021	cc-by	18,788	Keywords: self-powered systems; nanogenerator; wearable electronics; implantable devices Review Review Citation: Yang, L.; Ma, Z.; Tian, Y.; Meng, B.; Peng, Z. Progress on Self-Powered Wearable and Implantable Systems Driven by Nanogenerators. Micromachines 2021, 12, 666. https://doi.org/10.3390/ mi12060666 Lanxin Yang, Zhihao Ma, Yun Tian, Bo Meng * and Zhengchun Peng Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, College of Physics and Optoelectronic Engineering, Shenzhen University, Shenzhen 518060, China; yanglanxin2020@email.szu.edu.cn (L.Y.); mzh_930930@163.com (Z.M.); 1910454039@email.szu.edu.cn (Y.T.); zcpeng@szu.edu.cn (Z.P.) * Correspondence: bomeng@szu.edu.cn Abstract: With the rapid development of the internet of things (IoT), sustainable self-powered wireless sensory systems and diverse wearable and implantable electronic devices have surged recently. Under such an opportunity, nanogenerators, which can convert continuous mechanical energy into usable electricity, have been regarded as one of the critical technologies for self-powered systems, based on the high sensitivity, ﬂexibility, and biocompatibility of piezoelectric nanogenerators (PENGs) and triboelectric nanogenerators (TENGs). In this review, we have thoroughly analyzed the materials and structures of wearable and implantable PENGs and TENGs, aiming to make clear how to tailor a self-power system into speciﬁc applications. The advantages in TENG and PENG are taken to effectuate wearable and implantable human-oriented applications, such as self- charging power packages, physiological and kinematic monitoring, in vivo and in vitro healing, and electrical stimulation. This review comprehensively elucidates the recent advances and future outlook regarding the human body’s self-powered systems. Citation: Yang, L.; Ma, Z.; Tian, Y.; Meng, B.; Peng, Z. Progress on Self-Powered Wearable and Implantable Systems Driven by Nanogenerators. Micromachines 2021, 12, 666. https://doi.org/10.3390/ mi12060666 Academic Editor: Mengdi Han Received: 27 April 2021 Accepted: 4 June 2021 Published: 7 June 2021 micromachines micromachines Review Progress on Self-Powered Wearable and Implantable Systems Driven by Nanogenerators Lanxin Yang, Zhihao Ma, Yun Tian, Bo Meng * and Zhengchun Peng 1. Introduction g g g Meanwhile, nanogenerators were explored for biological use and quickly played an important role in the development of [17–82] and implantable [83–120] systems. In 2012, Minbaek Lee et al. demonstrated a hybrid nanogenerator composed of ZnO and PVDF, which stimulated the research of wearable PENGs [71]. In 2013, Xiaosheng Zhang et al. proposed a sandwich-shaped TENG and implemented the ﬁrst demonstration of the nanogenerator to directly drive a biomedical microsystem [95]. g Meanwhile, nanogenerators were explored for biological use and quickly played an important role in the development of [17–82] and implantable [83–120] systems. In 2012, Minbaek Lee et al. demonstrated a hybrid nanogenerator composed of ZnO and PVDF, which stimulated the research of wearable PENGs [71]. In 2013, Xiaosheng Zhang et al. proposed a sandwich-shaped TENG and implemented the first demonstration of the nan- ogenerator to directly drive a biomedical microsystem [95]. As brieﬂy illustrated in Figure 1, nanogenerators have been employed to work in multiple parts of the human body. The TENGs and PENGs are integrated into wearable textiles and shoes [32,48] or mounted on human skin [55,60], serving as power supplies or active sensor for motion and vital signs monitoring [32,48,55,60]. In addition, the implanted nanogenerators, which are biocompatible and even biodegradable [85], play an inﬂuential role in biomedical applications to power implanted devices [99,101], to record biological signals, and to stimulate muscles and the nervous system in therapy use [85,98]. g y y [ ] As briefly illustrated in Figure 1, nanogenerators have been employed to work in multiple parts of the human body. The TENGs and PENGs are integrated into wearable textiles and shoes [32,48] or mounted on human skin [55,60], serving as power supplies or active sensor for motion and vital signs monitoring [32,48,55,60]. In addition, the im- planted nanogenerators, which are biocompatible and even biodegradable [85], play an influential role in biomedical applications to power implanted devices [99,101], to record biological signals, and to stimulate muscles and the nervous system in therapy use [85,98]. Figure 1. Overview of self-powered wearable and implantable systems driven by nanogenerators. (a–d) Self-powered wearable systems. (a) A stretchable liquid metal elastomer based TENG patch attached on the knee. Reproduced with permission from Ref. [60]. Copyright © 2020, Wiley-VCH. (b) A self-powered and self-functional cotton sock. Reproduced with permission from Ref. [32]. Copyright © 2019, American Chemical Society. (c) A commercial electric heating sheet powered by PENG. 1. Introduction Wearable electronics and implantable devices have drawn much attention in academic research and industry [1]. However, the traditional wearable and implantable systems were bulky, with high replacement frequency and a short life span [2]. In recent years, with the contemporary increased demands of multiple and ubiquitous wearable and implantable applications, human-oriented self-powered systems have become a hot issue [3,4]. Received: 27 April 2021 Accepted: 4 June 2021 Published: 7 June 2021 pp p y To this end, various technologies have been developed for continuous electricity supply of the wearable and implantable systems. Electrical energy could be transmitted or captured from the ambient environment or the human body itself [3]. Among these approaches, wireless power transmission [5], photovoltaic cells [6], and thermoelectricity [7] all rely too much on external conditions. Due to limited conditions, the utilization rate is not high enough, making them difﬁcult to effectively utilize on a large scale. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. The biomechanical energy produced by human motions and the round-the-clock biological rhythms could be a promising power source to realize self-powered wearable and implantable systems. Diverse mechanisms of energy harvesting have been developed to capture and convert this tiny, ubiquitous, neglected, and wasted biomechanical energy into electricity [3]. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). y In 2006, Wang’s group developed a piezoelectric nanogenerator (PENG) based on ZnO nanowires [8,9], which brought a breakthrough to the miniaturization of energy harvesting. In 2012, Wang’s group invented the triboelectric nanogenerator (TENG) [10], which is a milestone discovery in energy harvesting and self-powered systems. Nanogenerators based on piezoelectric and triboelectric effects have the advantages of low cost [11], high efﬁ- ciency [12], ﬂexibility [13], light weight [14], and strong sustainability [15]. They are widely https://www.mdpi.com/journal/micromachines Micromachines 2021, 12, 666. https://doi.org/10.3390/mi12060666 2 of 18 anogen- ow cost Micromachines 2021, 12, 666 used as a power supplier in portable electronics, the internet of things, and human–machine interfaces, and as active sensors for engineering and environmental monitoring [16]. y y p pp p , g , and human–machine interfaces, and as active sensors for engineering and environmental monitoring [16]. 1. Introduction (e–h) Implantable self-powered systems: (e) A symbiotic cardiac pacemaker. Reproduced with permission from Ref. [101]. Copyright © 2019, Springer Nature. (f) A biodegradable, battery-less electrical stimulator made of piezoelectric nanofibers, serves as a bone scaffold. Reproduced with permission from Ref. [85]. Copyright © 2020, Elsevier. (g) Electrical muscle stimulation directly powered by TENG. Reproduced with permission from Ref. [98]. Cop- yright © 2019, Wiley-VCH. (h) A self-powered treatment to charge implant surface. Reproduced with permission from Ref. [99]. Copyright © 2020, Elsevier. Figure 1. Overview of self-powered wearable and implantable systems driven by nanogenerators. (a–d) Self-powered wearable systems. (a) A stretchable liquid metal elastomer based TENG patch attached on the knee. Reproduced with permission from Ref. [60]. Copyright © 2020, Wiley-VCH. (b) A self-powered and self-functional cotton sock. Reproduced with permission from Ref. [32]. Copyright © 2019, American Chemical Society. (c) A commercial electric heating sheet powered by PENG. Reproduced with permission from Ref. [48] Copyright © 2020, American Chemical Society. (d) A bio- inspired spider-net-coding interface to detect and control multiple directions. Reproduced with permission from Ref. [55]. Copyright © 2019, Wiley-VCH. (e–h) Implantable self-powered systems: (e) A symbiotic cardiac pacemaker. Reproduced with permission from Ref. [101]. Copyright © 2019, Springer Nature. (f) A biodegradable, battery-less electrical stimulator made of piezoelectric nanoﬁbers, serves as a bone scaffold. Reproduced with permission from Ref. [85]. Copyright © 2020, Elsevier. (g) Electrical muscle stimulation directly powered by TENG. Reproduced with permission from Ref. [98]. Copyright © 2019, Wiley-VCH. (h) A self-powered treatment to charge implant surface. Reproduced with permission from Ref. [99]. Copyright © 2020, Elsevier. Figure 1. Overview of self-powered wearable and implantable systems driven by nanogenerators. (a–d) Self-powered wearable systems. (a) A stretchable liquid metal elastomer based TENG patch attached on the knee. Reproduced with permission from Ref. [60]. Copyright © 2020, Wiley-VCH. (b) A self-powered and self-functional cotton sock. Reproduced with permission from Ref. [32]. Copyright © 2019, American Chemical Society. (c) A commercial electric heating sheet powered by PENG. Reproduced with permission from Ref. [48] Copyright © 2020, American Chemical Society. (d) A bio- inspired spider-net-coding interface to detect and control multiple directions. Reproduced with permission from Ref. [55]. Copyright © 2019, Wiley-VCH. (e–h) Implantable self-powered systems: (e) A symbiotic cardiac pacemaker. Reproduced with permission from Ref. [101]. Copyright © 2019, Springer Nature. (f) A biodegradable, battery-less electrical stimulator made of piezoelectric nanofibers, serves as a bone scaffold. 1. Introduction Reproduced with permission from Ref. [48] Copyright © 2020, American Chemical Society. (d) A bio- inspired spider-net-coding interface to detect and control multiple directions. Reproduced with permission from Ref. [55]. Copyright © 2019, Wiley-VCH. (e–h) Implantable self-powered systems: (e) A symbiotic cardiac pacemaker. Reproduced with permission from Ref. [101]. Copyright © 2019, Springer Nature. (f) A biodegradable, battery-less electrical stimulator made of piezoelectric nanofibers, serves as a bone scaffold. Reproduced with permission from Ref. [85]. Copyright © 2020, Elsevier. (g) Electrical muscle stimulation directly powered by TENG. Reproduced with permission from Ref. [98]. Cop- yright © 2019, Wiley-VCH. (h) A self-powered treatment to charge implant surface. Reproduced with permission from Ref. [99]. Copyright © 2020, Elsevier. Figure 1. Overview of self-powered wearable and implantable systems driven by nanogenerators. (a–d) Self-powered wearable systems. (a) A stretchable liquid metal elastomer based TENG patch attached on the knee. Reproduced with permission from Ref. [60]. Copyright © 2020, Wiley-VCH. (b) A self-powered and self-functional cotton sock. Reproduced with permission from Ref. [32]. Copyright © 2019, American Chemical Society. (c) A commercial electric heating sheet powered by PENG. Reproduced with permission from Ref. [48] Copyright © 2020, American Chemical Society. (d) A bio- inspired spider-net-coding interface to detect and control multiple directions. Reproduced with permission from Ref. [55]. Copyright © 2019, Wiley-VCH. (e–h) Implantable self-powered systems: (e) A symbiotic cardiac pacemaker. Reproduced with permission from Ref. [101]. Copyright © 2019, Springer Nature. (f) A biodegradable, battery-less electrical stimulator made of piezoelectric nanoﬁbers, serves as a bone scaffold. Reproduced with permission from Ref. [85]. Copyright © 2020, Elsevier. (g) Electrical muscle stimulation directly powered by TENG. Reproduced with permission from Ref. [98]. Copyright © 2019, Wiley-VCH. (h) A self-powered treatment to charge implant surface. Reproduced with permission from Ref. [99]. Copyright © 2020, Elsevier. Figure 1. Overview of self-powered wearable and implantable systems driven by nanogenerators. (a–d) Self-powered wearable systems. (a) A stretchable liquid metal elastomer based TENG patch attached on the knee. Reproduced with permission from Ref. [60]. Copyright © 2020, Wiley-VCH. (b) A self-powered and self-functional cotton sock. Reproduced with permission from Ref. [32]. Copyright © 2019, American Chemical Society. (c) A commercial electric heating sheet powered by PENG. Reproduced with permission from Ref. [48] Copyright © 2020, American Chemical Society. (d) A bio- inspired spider-net-coding interface to detect and control multiple directions. Reproduced with permission from Ref. [55]. Copyright © 2019, Wiley-VCH. 1. Introduction Reproduced with permission from Ref. [85]. Copyright © 2020, Elsevier. (g) Electrical muscle stimulation directly powered by TENG. Reproduced with permission from Ref. [98]. Cop- yright © 2019, Wiley-VCH. (h) A self-powered treatment to charge implant surface. Reproduced with permission from Ref. [99]. Copyright © 2020, Elsevier. Figure 1. Overview of self-powered wearable and implantable systems driven by nanogenerators. (a–d) Self-powered wearable systems. (a) A stretchable liquid metal elastomer based TENG patch attached on the knee. Reproduced with permission from Ref. [60]. Copyright © 2020, Wiley-VCH. (b) A self-powered and self-functional cotton sock. Reproduced with permission from Ref. [32]. Copyright © 2019, American Chemical Society. (c) A commercial electric heating sheet powered by PENG. Reproduced with permission from Ref. [48] Copyright © 2020, American Chemical Society. (d) A bio- inspired spider-net-coding interface to detect and control multiple directions. Reproduced with permission from Ref. [55]. Copyright © 2019, Wiley-VCH. (e–h) Implantable self-powered systems: (e) A symbiotic cardiac pacemaker. Reproduced with permission from Ref. [101]. Copyright © 2019, Springer Nature. (f) A biodegradable, battery-less electrical stimulator made of piezoelectric nanoﬁbers, serves as a bone scaffold. Reproduced with permission from Ref. [85]. Copyright © 2020, Elsevier. (g) Electrical muscle stimulation directly powered by TENG. Reproduced with permission from Ref. [98]. Copyright © 2019, Wiley-VCH. (h) A self-powered treatment to charge implant surface. Reproduced with permission from Ref. [99]. Copyright © 2020, Elsevier. In this review, as illustrated in Table 1, we focus on a comprehensive overview of recent advances in self-powered wearable and implantable systems that are energized by nanogenerators. Through the development of self-powered systems, we summarize 3 of 18 3 of 18 Micromachines 2021, 12, 666 the optimization of materials and structures in wearable and implantable nanogenerators. Further, we expand on the applications of self-powered wearable and implantable systems. Further, we expand on the applications of self powered wearable and implantable systems. Table 1. Wearable and implantable TENGs and PENGs. 2. Materials and Structural Design of Wearable and Implantable Nanogenerators 2.1. Materials of Wearable and Implantable Nanogenerators 2.1.1. Materials of Wearable and Implantable TENGs f p g 2.1.1. Materials of Wearable and Implantable TENGs Owing to the extensively exiting of triboelectriﬁcation, TENGs have greater selectivity in materials. Surface modiﬁcation on textiles is an efﬁcient approach to obtain excellent and low-cost friction layers for wearable TENG. It has been widely studied [44,59]. Chanho Park et al. put forward a one-step route for developing rapid wet processable surface- conformal nanoporous ﬁlms [59], as shown in Figure 2a, which are made up of a ternary polymer blend of sulfonic-acid-terminated poly(styrene), poly(2-vinylpyridine) and amine- terminated poly(ethylene oxide) in benzene. These mixed materials can result in well- deﬁned nanopores. As shown in Figure 2b, Feng Wen et al. proposed a simple carbon nanotube (CNT)/thermoplastic elastomer coating method to achieve super hydrophobicity of the textile TENG [44]. Biocompatible and biodegradable materials, especially bio- absorbable natural materials such as wood, silk, wheat cotton, and cellulose, provide more opportunities for wearable and implantable applications. Meng Su et al. proposed a CNT-silk mixing layer as the conductive friction material to realize a wholly biodegradable TENG [53]. Qianqian Niu et al. adopted silk nanoribbons with adjustable sizes and stable aqueous conditions and developed an all-silk bio-TENG [61], as shown in Figure 2c. Moreover, biodegradable polymers such as polyvinyl alcohol (PVA) and polycaprolactone (PLC) were widely used. Ruoxing Wang et al. proposed a wearable TENG based on biodegradable PVA [45], as shown in Figure 2d. The fabricated PVA-gelatin composite ﬁlm provides a choice for achieving skin-friendly TENG. Stretchability would be an essential requirement for speciﬁc wearable and implantable TENGs. Conductive 2D materials and liquid metals can help. Md Salauddin et al. presented a conductive fabric-based TENG, which is made up of MXene (Ti3C2Tx) nanosheets and Ecoﬂex composite [56], as shown in Figure 2e. Chengfeng Pan et al. presented an ultra-stretchable TENG based on the sedimented liquid metal elastomer composite [60], as shown in Figure 2f. It possesses excellent conductivity under ultrahigh stretchability. 2.1.2. Materials of Wearable and Implantable PENGs 1. Introduction Nanogenerator Type Wearable TENGs Wearable PENGs Implantable TENGs Implantable PENGs Location of Installation chest [78] knee [80,108]; foot [32,48,77] heart and pericardium [87,101,105] pacemaker lead [97,116] elbow [26,56] chest [29,50]; duodenum [120]; stomach [103] knee [26,30,56] neck [18,19,29,50,52] tumor cells [94] lung [86] waist [21,25,27,45,55,74,81] elbow [49,72,76] the surface of bone [99] heart [83,84,86,106,110,114] eye [23,28] wrist [18,29,50] the subdermal dorsal region [93] blood vessel [29,112,119] ear [20]; foot [42,56,69]; skin [17,78] hand [18,37,76] skin underneath [88,92] skin underneath [104] hand [26,33,38,44,53,65] skin [79] Biomechanical Energy Source walking, running [42,53,56,69,80] walking, running [32,48,77] joint movement [99] motions of leg [104] stretching [21,25,26,33,53,56] stretching [29,72] [76,79] blood pressure [93] blood pressure [112,119] blinking [23,28] joint movement [49,50,81] the peristalsis of duodenum [120] motions of stomach [103] shake and pat [17,38,43,82] breathing [18] heartbeat [87,93,101,105] heartbeat [83,106,110,114,116] motions of ﬁnger [44,65] pulse [19,52] breathing [88,92,93] breathing [29] breathing [21,27]; pulse [45] punching [37] motions of heart lead [84,86,97,115] speaking [20]; touching [55,74,78] Materials PTFE [17,27,42,55] ZnO [48,81] PTFE [87,94,101,105] PVDF [29,83,104,119] Kapton [20,45]; PVDF [33,65] PVDF [29,49,77,79] PLGA [88,93] ZnO [84] Nylon [24,27,74,78] P(VDF-TrFE) [18,72,78] PDMS [87,120] PVDF-TrFE [97,114,116] Mxene [56]; carbon nanotube [44,53] Dopamine [29]; PMN-PT [19,77] PVA [93] PZT [86,103] hydrogel [23,25] balsa wood [50]; PZT [32,37,80] PET [87,92] PMN-PT [106,110] Ecoﬂex [55,56]; liquid metal [38] BaTiO3 [80] Kapton [87,92,101] rubber [21,38]; silicone [25,30] titanium [92,94,99,101] PVA [25,45]; silk [53]; Applications human-machine interface [20,55] motion monitoring [19,29,37,50] anti-bacteria [99] in vivo health monitoring [83,97,119] motion monitoring [21,27,33,56,74] health monitoring [18,49,52] anti-tumor therapy [94] in vivo therapying [29,103,104,114] health monitoring [27,45,78] wound healing [79] in vivo health monitoring [101,105,120] regeneration of tissues [85] eye motion monitoring [23,28] power supply [18,32,72,77] electrical stimulator [98] implanted sensor [112] voice and gesture recognition [20,44,65] power supply [87,88,92,93] power supply [84,106,110] drug delivery [17] power supply [30,38,65,69,81] Table 1. Wearable and implantable TENGs and PENGs. 4 of 18 Micromachines 2021, 12, 666 2. Materials and Structural Design of Wearable and Implantable Nanogenerators 2.1. Materials of Wearable and Implantable Nanogenerators 2.1.1. Materials of Wearable and Implantable TENGs 2.1.2. Materials of Wearable and Implantable PENGs [60]. Copyright © 2020 Wiley VCH Figure 2. Materials of wearable and implantable TENGs. (a) Surface-conformal nanoporous ﬁlms coated on textiles. Reproduced with permission from Ref. [59]. Copyright © 2020, American Chemical Society. (b) A textile TENG with super-hydrophobic coating. Reproduced with permission from Ref. [44]. Copyright © 2020, Wiley-VCH. (c) Natural silk ﬁbers for a wearable TENG. Reproduced with permission from Ref. [61]. Copyright © 2020, Elsevier. (d) TENG built with biodegradable PVA gelatin. Reproduced with permission from Ref. [45]. Copyright © 2020, Wiley-VCH. (e) MXene/Ecoﬂex nanocomposite as a negative friction layer. Reproduced with permission from Ref. [56]. Copyright © 2020, Wiley-VCH. (f) Liquid metal elastomer composite for stretchable TENG. Reproduced with permission from Ref. [60]. Copyright © 2020, Wiley-VCH. 2.1.2. Materials of Wearable and Implantable PENGs presented an ultra-stretchable TENG based on the sedimented liquid metal elastomer composite [60], as shown in Figure 2f. It possesses excellent conductivity under ultrahigh stretchability. Figure 2. Materials of wearable and implantable TENGs. (a) Surface-conformal nanoporous films coated on textiles. Re- produced with permission from Ref. [59]. Copyright © 2020, American Chemical Society. (b) A textile TENG with super- hydrophobic coating. Reproduced with permission from Ref. [44]. Copyright © 2020, Wiley-VCH. (c) Natural silk fibers for a wearable TENG. Reproduced with permission from Ref. [61]. Copyright © 2020, Elsevier. (d) TENG built with bio- degradable PVA gelatin. Reproduced with permission from Ref. [45]. Copyright © 2020, Wiley-VCH. (e) MXene/Ecoflex nanocomposite as a negative friction layer. Reproduced with permission from Ref. [56]. Copyright © 2020, Wiley-VCH. (f) Liquid metal elastomer composite for stretchable TENG. Reproduced with permission from Ref. [60]. Copyright © 2020 Wiley-VCH Figure 2. Materials of wearable and implantable TENGs. (a) Surface-conformal nanoporous ﬁlms coated on textiles. Reproduced with permission from Ref. [59]. Copyright © 2020, American Chemical Society. (b) A textile TENG with super-hydrophobic coating. Reproduced with permission from Ref. [44]. Copyright © 2020, Wiley-VCH. (c) Natural silk ﬁbers for a wearable TENG. Reproduced with permission from Ref. [61]. Copyright © 2020, Elsevier. (d) TENG built with biodegradable PVA gelatin. Reproduced with permission from Ref. [45]. Copyright © 2020, Wiley-VCH. (e) MXene/Ecoﬂex nanocomposite as a negative friction layer. Reproduced with permission from Ref. [56]. Copyright © 2020, Wiley-VCH. (f) Liquid metal elastomer composite for stretchable TENG. Reproduced with permission from Ref. [60]. Copyright © 2020, Wiley-VCH. d i l t bl TENG ( ) S f f l fil t d t til R ble and implantable TENGs. (a) Surface-conformal nanoporous ﬁlms coated on textiles. Figure 2. Materials of wearable and implantable TENGs. (a) Surface-conformal nanoporous films coated on textiles. Re- produced with permission from Ref. [59]. Copyright © 2020, American Chemical Society. (b) A textile TENG with super- hydrophobic coating. Reproduced with permission from Ref. [44]. Copyright © 2020, Wiley-VCH. (c) Natural silk fibers for a wearable TENG. Reproduced with permission from Ref. [61]. Copyright © 2020, Elsevier. (d) TENG built with bio- degradable PVA gelatin. Reproduced with permission from Ref. [45]. Copyright © 2020, Wiley-VCH. (e) MXene/Ecoflex nanocomposite as a negative friction layer. Reproduced with permission from Ref. [56]. Copyright © 2020, Wiley-VCH. (f) Liquid metal elastomer composite for stretchable TENG. Reproduced with permission from Ref. 2.1.2. Materials of Wearable and Implantable PENGs Polyvinylidene ﬂuoride (PVDF) and its copolymers are considered as the most promis- ing candidates for wearable and implantable PENG due to their high ﬂexibility, good biocompatibility and processability [41]. Tong Li et al. designed an all-ﬁber-based PENG using core/shell PVDF/dopamine (DA) nanoﬁbers [29], as shown in Figure 3a. The use of a self-assembly process to form and arrange β-phase PVDF can further enhance the piezo- electric performance while maintaining excellent reliability. Kuntal Maity et al. reported a PENG pressure sensor based on highly aligned PVDF nanoﬁbers arrays and achieved a high sensitivity of 0.8 V/KPa [63]. ZnO nanowire is widely used as well. Congran Jin et al. developed a PENG based on ZnO nanoarrays embedded in a PDMS membrane [84], as shown in Figure 3b. It generates 9.2 V open-circuit voltage and can be stretched to 250%. Typical lead-containing piezoelectric materials are toxic with poor mechanical properties. Well packaged lead-containing PENGs with ﬂexible substrate could also achieve high performance in implantable applications. Geon-Tae Hwang et al. proposed a ﬂexible PENG based on single-crystalline PMN-PT [110], as shown in Figure 3c. This PMN-PT has a piezoelectric charge constant of d33 up to 2500 pC/N. Composites of multiple organic and inorganic piezoelectric materials were studied to develop ﬂexible PENGs with a high charge constant. Xiaoyang Guan et al. proposed a wearable, ﬂexible PENG based on nanocomposite ﬁbers [76], as shown in Figure 3d. This hierarchical micro-structured piezo- electric membrane is fabricated by electrospun P(VDF-TrFE) ﬁbers with polydopamine modiﬁed BATiO3 nanoparticles anchored on the surface. Among the piezoelectric ma- terials for wearable and implantable use, biocompatibility and biodegradability are the kernels to be considered. Eli J. Curry et al. Proposed a biodegradable and biocompati- ble poly(L-lactic acid) (PLLA) nanoﬁber with highly controllable and stable piezoelectric properties [118], as shown in Figure 3e. This PENG has shown good performance for 5 of 18 posite an es- Micromachines 2021, 12, 666 implanted use. Jianguo Sun et al. developed a PENG based on the natural balsa wood [50], as shown in Figure 3f. The piezoelectric wood sponge is fabricated with a simple chemical deligniﬁcation treatment on the natural wood. In addition, it can be decomposed with cellulose-degrading fungi. rials and liquid metals can help. Md Salauddin et al. presented a conductive fabric based TENG, which is made up of MXene (Ti3C2Tx) nanosheets and Ecoflex composite [56], as shown in Figure 2e. Chengfeng Pan et al. 2.1.2. Materials of Wearable and Implantable PENGs Polyvinylidene fluoride (PVDF) and its copolymers a 2.2. Structures of Wearable and Implantable Nanogenerators 2.2.1. Structures of Wearable Nanogenerators proposed a wearable hybrid nanogenerator that shows high performance under low acceleration (≤1 g) and low frequency (≤6 HZ) human motions [64], as shown in Figure 4e. Cheng Yan et al. designed a linear-to-rotary hybrid nanogenerator to achieve high output performance by frequency enhancement [42], as shown in Figure 4f. PENGs with a high charge constant. Xiaoyang Guan et al. proposed a wearable, flexible PENG based on nanocomposite fibers [76], as shown in Figure 3d. This hierarchical micro- structured piezoelectric membrane is fabricated by electrospun P(VDF-TrFE) fibers with polydopamine modified BATiO3 nanoparticles anchored on the surface. Among the pie- zoelectric materials for wearable and implantable use, biocompatibility and biodegrada- bility are the kernels to be considered. Eli J. Curry et al. Proposed a biodegradable and biocompatible poly(L-lactic acid) (PLLA) nanofiber with highly controllable and stable piezoelectric properties [118], as shown in Figure 3e. This PENG has shown good perfor- mance for implanted use. Jianguo Sun et al. developed a PENG based on the natural balsa wood [50], as shown in Figure 3f. The piezoelectric wood sponge is fabricated with a sim- ple chemical delignification treatment on the natural wood. In addition, it can be decom- posed with cellulose-degrading fungi. Figure 3. Materials of wearable and implantable PENGs. (a) A core/shell PVDF/dopamine nanofiber-based PENG. Repro- duced with permission from Ref. [29]. Copyright © 2020, Wiley-VCH. (b) A zinc oxide nanoarrays based PENG. Repro- duced with permission from Ref. [84]. Copyright © 2021, John Wiley and Sons. (c) A PENG based on PMN-PT. Repro- duced with permission from Ref. [110]. Copyright © 2014, Wiley-VCH. (d) A BaTiO3@P(VDF-TrFE) nanocomposite-based PENG. Reproduced with permission from Ref. [76]. Copyright © 2020, Elsevier. (e) A biodegradable PENG based on Figure 3. Materials of wearable and implantable PENGs. (a) A core/shell PVDF/dopamine nanoﬁber-based PENG. Reproduced with permission from Ref. [29]. Copyright © 2020, Wiley-VCH. (b) A zinc oxide nanoarrays based PENG. Reproduced with permission from Ref. [84]. Copyright © 2021, John Wiley and Sons. (c) A PENG based on PMN-PT. Reproduced with permission from Ref. [110]. Copyright © 2014, Wiley-VCH. (d) A BaTiO3@P(VDF-TrFE) nanocomposite- based PENG. Reproduced with permission from Ref. [76]. Copyright © 2020, Elsevier. (e) A biodegradable PENG based on PLLA nanoﬁbers. Reproduced with permission from Ref. [118]. Copyright © 2020, National Academy of Sciences. (f) A PENG based on wood sponge. Reproduced with permission from Ref. [50]. Copyright © 2020, American Chemical Society. 2.1.2. Materials of Wearable and Implantable PENGs Polyvinylidene fluoride (PVDF) and its copolymers a 2.2. Structures of Wearable and Implantable Nanogenerators 2.2.1. Structures of Wearable Nanogenerators Polyvinylidene fluoride (PVDF) and its copolymers are considered as the most prom ising candidates for wearable and implantable PENG due to their high flexibility, good biocompatibility and processability [41]. Tong Li et al. designed an all-fiber-based PENG using core/shell PVDF/dopamine (DA) nanofibers [29], as shown in Figure 3a. The use of The large number of materials enables theoretical models to be transformed into nano- generators with various structures, which not only realizes the functions of the device, but also has the extra advantages of the materials. Beneﬁting from the outstanding ﬂexibility, wearable nanogenerators can be easily designed as simple thin-ﬁlm structures. These nanogenerators are generally attached on the skin. Yang Jiang et al. developed an ultrathin skin-like TENG [74], as shown in Figure 4a. It adopted a single-electrode structure with a stretchable and transparent electrode, and forms a comfortable and conformal device that can attach to the epidermis. Xiao Peng et al. proposed an all-nanoﬁber single-electrode TENG with a hierarchical porous structured friction ﬁlm [58], which is stretchable, breath- able, and biodegradable. Wearable nanogenerators existing as part of textiles, shoes, or other wearable accessories are prevalent as well. Multiple-layered plain structures and 3D textile structures were developed to improve the output performance of nanogenerators. Long Gu et al. proposed a PENG with a three-dimensional intercalation electrode [77], as shown in Figure 4b. It can charge a 1 µF capacitor from 0 V to 8 V in 21 cycles. Seongcheol Ahn et al. proposed a 3D textile structured PENG with pre-strained monoﬁlament [49], as shown in Figure 4c. The 3D structure of the monoﬁlament is employed as a pressure Micromachines 2021, 12, 666 6 of 18 al. pro- gure 3c. 6 of 18 al. pro- gure 3c. transmitter for piezoelectric ampliﬁcation to improve the sensitivity. A direct current fabric TENG with a plain structure was proposed by Chaoyu Chen et al. [81], as shown in Figure 4d. It can produce high DC outputs to harvest the energy from the electrostatic breakdown phenomenon of clothes during human motions. Most of the energy gener- ated by human motions is at low frequency and low acceleration [62]. Inertial structured spring-mass systems have been proved to be an efﬁcient way to achieve high energy har- vesting efﬁciency for wearable nanogenerators. They were usually designed as hybrid nanogenerators. Pukar Maharjan et al. 2.1.2. Materials of Wearable and Implantable PENGs Polyvinylidene fluoride (PVDF) and its copolymers a 2.2. Structures of Wearable and Implantable Nanogenerators 2.2.1. Structures of Wearable Nanogenerators Figure 3. Materials of wearable and implantable PENGs. (a) A core/shell PVDF/dopamine nanofiber-based PENG. Repro- duced with permission from Ref. [29]. Copyright © 2020, Wiley-VCH. (b) A zinc oxide nanoarrays based PENG. Repro- duced with permission from Ref. [84]. Copyright © 2021, John Wiley and Sons. (c) A PENG based on PMN-PT. Repro- duced with permission from Ref. [110]. Copyright © 2014, Wiley-VCH. (d) A BaTiO3@P(VDF-TrFE) nanocomposite-based PENG. Reproduced with permission from Ref. [76]. Copyright © 2020, Elsevier. (e) A biodegradable PENG based on Figure 3. Materials of wearable and implantable PENGs. (a) A core/shell PVDF/dopamine nanoﬁber-based PENG. Reproduced with permission from Ref. [29]. Copyright © 2020, Wiley-VCH. (b) A zinc oxide nanoarrays based PENG. Reproduced with permission from Ref. [84]. Copyright © 2021, John Wiley and Sons. (c) A PENG based on PMN-PT. Reproduced with permission from Ref. [110]. Copyright © 2014, Wiley-VCH. (d) A BaTiO3@P(VDF-TrFE) nanocomposite- based PENG. Reproduced with permission from Ref. [76]. Copyright © 2020, Elsevier. (e) A biodegradable PENG based on PLLA nanoﬁbers. Reproduced with permission from Ref. [118]. Copyright © 2020, National Academy of Sciences. (f) A PENG based on wood sponge. Reproduced with permission from Ref. [50]. Copyright © 2020, American Chemical Society. 7 of 18 hybrid 42], as Micromachines 2021, 12, 666 Figure 4. Structures of wearable nanogenerators. (a) An ultrathin skin-inspired TENG. Reproduced with permission from Ref. [74]. Copyright © 2020, Wiley-VCH. (b) A PENG with a three-dimensional intercalation electrode. Reproduced with permission from Ref. [77]. Copyright © 2020, Springer Nature. (c) A 3D textile structured PENG. Reproduced with permission from Ref. [49]. Copyright © 2020, Elsevier. (d) A textile TENG. Reproduced with permission from Ref. [81]. Copyright © 2020, American Chemical Society. (e) An inertial structured hybrid nanogenerator. Reproduced with permission from Ref. [64]. Copyright © 2020, Wiley-VCH. (f) A linear-to-rotary hybrid wearable nanogenerator. Reproduced with permission from Ref. [42]. Copyright © 2020, Elsevier. Figure 4. Structures of wearable nanogenerators. (a) An ultrathin skin-inspired TENG. Reproduced with permission from Ref. [74]. Copyright © 2020, Wiley-VCH. (b) A PENG with a three-dimensional intercalation electrode. Reproduced with permission from Ref. [77]. Copyright © 2020, Springer Nature. (c) A 3D textile structured PENG. Reproduced with permission from Ref. [49]. Copyright © 2020, Elsevier. (d) A textile TENG. Reproduced with permission from Ref. [81]. Copyright © 2020, American Chemical Society. (e) An inertial structured hybrid nanogenerator. Reproduced with permission from Ref. [64]. 2.1.2. Materials of Wearable and Implantable PENGs Polyvinylidene fluoride (PVDF) and its copolymers a 2.2. Structures of Wearable and Implantable Nanogenerators 2.2.1. Structures of Wearable Nanogenerators Copyright © 2020, Wiley-VCH. (f) A linear-to-rotary hybrid wearable nanogenerator. Reproduced with permission from Ref. [42]. Copyright © 2020, Elsevier. 3.1. Self-Powered Wearable Systems Based on TENG Wearable electronics have brought convenien 3.1. Self-Powered Wearable Systems Based on TENG Wearable electronics have brought conveniences to our daily life. In the face of the increasing demand for self-powered wearable systems, TENGs have played an important role in active sensing and mechanical energy harvesting. Clothing is a necessity in our daily life. Putting the concept of wearable TENG on clothes is bound to be a hot issue pursued by researchers. Textile and fabric-based TENGs have been developed rapidly. Wenjing Fan et al. presented a textile TENG sensor array with high-pressure sensitivity [78]. This device is employed as a noninvasive method to evaluate the signal generated by cardiovascular disease and sleep apnea syndrome. Zhiming Lin et al. reported a smart insole with TENG embedded as an active sensor for real-time gait monitoring [69], as shown in Figure 6a. It has high durability and excellent mechanical robustness to monitor the abnormality of gait for rehabilitation assessment. Liyun Ma et al. proposed an ul- tralight single-electrode textile-based TENG with helical hybridized nano-micro core- shell fiber bundles [65], as shown in Figure 6b. It enables harvesting biomechanical energy and monitoring tiny signals from human motions. TENGs come in a wide range of forms besides fabrics. Yu Song et al. developed a self-powered wearable wireless sweat sensing system based on a TENG [54]. As shown in Figure 6c, Yang Zou et al. designed a bionic stretch TENG by imitating the electric eel’s power generation principle [30]. It has a broad application prospect in underwater motion detection and submarine rescue. With the ad- vent of the intelligent era, objects are connected through the internet, and wearable appli- cations for human–machine interfaces and intelligent systems are also arising. A smart l ith h ti f db k d i d b d TENG t i l h Wearable electronics have brought conveniences to our daily life. In the face of the increasing demand for self-powered wearable systems, TENGs have played an important role in active sensing and mechanical energy harvesting. Clothing is a necessity in our daily life. Putting the concept of wearable TENG on clothes is bound to be a hot issue pursued by researchers. Textile and fabric-based TENGs have been developed rapidly. Wenjing Fan et al. presented a textile TENG sensor array with high-pressure sensitivity [78]. This device is employed as a noninvasive method to evaluate the signal generated by cardiovascular disease and sleep apnea syndrome. Zhiming Lin et al. 2.2.2. Structures of Implantable Nanogenerators For in vivo applications, the implantable nanogenerators usually employ thin-ﬁlm structures and their transformation. Transformative thin-ﬁlm structures are widely used in implantable PENGs. A circular piezoelectric belt is one of the simplest and effective ones [119]. Sophisticated designs of the transformation are implemented to obtain enhanced electrical outputs. Rujie Sun et al. proposed a kirigami stretchable structure of PENG [83], as shown in Figure 5a. It improves the tensile property and ﬂexibility of PENG to implant on the organs and achieves much higher outputs than unstructured design. Lin Dong et al. developed implantable PENGs with a helix structure [116], as shown in Figure 5b and a buckled beam array design [114], as shown in Figure 5c. These PENGs deform through the movement of the pacemaker lead and generate stable electricity. Most of the implantable TENG adapt a contact-separation structure. Owing to the limited separation space in the body, the structures should be well designed to maintain the practical work of TENGs. Bolang Cheng et al. proposed a mechanically asymmetrical TENG [120], as shown in Figure 5d. A 20 µm thick PDMS spacer is used, and the TENG belt can be twisted and rolled up to different shapes. It can monitor the microscopically weak intestinal peristalsis. However, due to the low stiffness of the friction layers, the separation of implantable TENGs will be reduced, which may lead to decreasing output performance. To overcome this, an implantable TENG with a 3D sponge spacer was developed [101], as shown in Figure 5e. A memory alloy ribbon serving as the keel of the friction layer is employed, to obtain a higher long-term stability. Zhao Chaochao et al. ﬁxed two magnets on the back of the friction layers to produce repulsion separation when contact occurs. Thus, the life cycle of the TENG is extended [94]. Well-designed sliding mode TENGs can also work well in vivo. Jun Li et al. reported a stretchable micro-grating structured TENG [115], as 8 of 18 Micromachines 2021, 12, 666 shown in Figure 5f. It was implanted inside a rat’s abdominal cavity to harvest energy from ventral diaphragm movement. R REVIEW 8 of 18 shown in Figure 5f. It was implanted inside a rat’s abdominal cavity to harvest energy from ventral diaphragm movement. R REVIEW 8 of 18 Figure 5. Structures of implantable nanogenerators. (a) A kirigami inspired PENG. Reproduced with permission from Ref. [83]. Copyright © 2019, Wiley-VCH. 2.2.2. Structures of Implantable Nanogenerators (b) A helix structured PENG. Reproduced with permission from Ref. [116]. Copy- right © 2019, Elsevier. (c) A PENG with buckled beam array structure. Reproduced with permission from Ref. [114]. Copyright © 2018, Wiley-VCH. (d) A mechanically asymmetrical TENG. Reproduced with permission from Ref. [120]. Copyright © 2020, Wiley-VCH. (e) An implantable TENG with 3D sponge spacer. Reproduced with permission from Ref. [101]. Copyright © 2019, Springer Nature. (f) A stretchable micro-grating structured TENG. Reproduced with permission from Ref. [115]. Copyright © 2018, American Chemical Society. Figure 5. Structures of implantable nanogenerators. (a) A kirigami inspired PENG. Reproduced with permission from Ref. [83]. Copyright © 2019, Wiley-VCH. (b) A helix structured PENG. Reproduced with permission from Ref. [116]. Copyright © 2019, Elsevier. (c) A PENG with buckled beam array structure. Reproduced with permission from Ref. [114]. Copyright © 2018, Wiley-VCH. (d) A mechanically asymmetrical TENG. Reproduced with permission from Ref. [120]. Copyright © 2020, Wiley-VCH. (e) An implantable TENG with 3D sponge spacer. Reproduced with permission from Ref. [101]. Copyright © 2019, Springer Nature. (f) A stretchable micro-grating structured TENG. Reproduced with permission from Ref. [115]. Copyright © 2018, American Chemical Society. mplantable nanogenerators. (a) A kirigami inspired PENG. Reproduced with permission from Ref. [116] implantable nanogenerators. (a) A kirigami inspired PENG. Reproduced with permission from Figure 5. Structures of implantable nanogenerators. (a) A kirigami inspired PENG. Reproduced with permission from Ref. [83]. Copyright © 2019, Wiley-VCH. (b) A helix structured PENG. Reproduced with permission from Ref. [116]. Copy- right © 2019, Elsevier. (c) A PENG with buckled beam array structure. Reproduced with permission from Ref. [114]. Copyright © 2018, Wiley-VCH. (d) A mechanically asymmetrical TENG. Reproduced with permission from Ref. [120]. Copyright © 2020, Wiley-VCH. (e) An implantable TENG with 3D sponge spacer. Reproduced with permission from Ref. [101]. Copyright © 2019, Springer Nature. (f) A stretchable micro-grating structured TENG. Reproduced with permission from Ref. [115]. Copyright © 2018, American Chemical Society. Figure 5. Structures of implantable nanogenerators. (a) A kirigami inspired PENG. Reproduced with permission from Ref. [83]. Copyright © 2019, Wiley-VCH. (b) A helix structured PENG. Reproduced with permission from Ref. [116]. Copyright © 2019, Elsevier. (c) A PENG with buckled beam array structure. Reproduced with permission from Ref. [114]. Copyright © 2018, Wiley-VCH. (d) A mechanically asymmetrical TENG. Reproduced with permission from Ref. [120]. Copyright © 2020, Wiley-VCH. (e) An implantable TENG with 3D sponge spacer. 2.2.2. Structures of Implantable Nanogenerators Reproduced with permission from Ref. [101]. Copyright © 2019, Springer Nature. (f) A stretchable micro-grating structured TENG. Reproduced with permission from Ref. [115]. Copyright © 2018, American Chemical Society. 3.1. Self-Powered Wearable Systems Based on TENG Wearable electronics have brought convenien 3.1. Self-Powered Wearable Systems Based on TENG The acoustic sensor has ultrahigh sensitivity, which could reach 110 mV/dB. Wearable TENGs are adopted for biomedical applications as well. Zhirong Liu et al. developed a TENG as a stable voltage pulse source to trigger plasma membrane potential and membrane permeability for intracellular drug delivery [67]. The delivery efficiency of this system is 90%, and the cell survival rate is more than 94%. Yonghong Li et al. devised a wearable ionic TENG, which has a stretcha- ble gel composition [75]. The electricity generated by this TENG from biomechanical en- ergy is used in damaged tissues, and it accelerates the wound healing, as shown in Figure 6f. smart glove with a haptic feedback was designed based on TENG to serve as a simple human–computer interaction method [31]. Qiongfeng Shi et al. reported a bio-inspired spider-net-coding interface with great ﬂexibility and scalability [55]. By employing a single-electrode TENG, detection and control of multiple directions are demonstrated, as shown in Figure 6d. Hengyu Guo et al. proposed a self-powered acoustic sensor [20], as shown in Figure 6e. It created a new acoustic system by using TENG. The acoustic sensor has ultrahigh sensitivity, which could reach 110 mV/dB. Wearable TENGs are adopted for biomedical applications as well. Zhirong Liu et al. developed a TENG as a stable voltage pulse source to trigger plasma membrane potential and membrane permeability for intracellular drug delivery [67]. The delivery efﬁciency of this system is 90%, and the cell survival rate is more than 94%. Yonghong Li et al. devised a wearable ionic TENG, which has a stretchable gel composition [75]. The electricity generated by this TENG from biomechanical energy is used in damaged tissues, and it accelerates the wound healing, as shown in Figure 6f. R REVIEW 9 of 18 TENG, detection and control of multiple directions are demonstrated, as shown in Figure 6d. Hengyu Guo et al. proposed a self-powered acoustic sensor [20], as shown in Figure 6e. It created a new acoustic system by using TENG. The acoustic sensor has ultrahigh sensitivity, which could reach 110 mV/dB. Wearable TENGs are adopted for biomedical applications as well. Zhirong Liu et al. developed a TENG as a stable voltage pulse source to trigger plasma membrane potential and membrane permeability for intracellular drug delivery [67]. The delivery efficiency of this system is 90%, and the cell survival rate is more than 94%. Yonghong Li et al. 3.1. Self-Powered Wearable Systems Based on TENG Wearable electronics have brought convenien 3.1. Self-Powered Wearable Systems Based on TENG (f) A wearable ionic TENG patch for wound healing Reproduced with permission from Ref [75] Copyright © 2020, Elsevier Figure 6. Self-powered wearable systems based on TENGs. (a) A TENG-based smart insole. Reproduced with permission from Ref. [69]. Copyright © 2020, Wiley-VCH. (b) An ultralight single-electrode triboelectric yarn with helical hybridized nano-micro core-shell ﬁber bundles. Reproduced with permission from Ref. [65]. Copyright © 2020, American Chemical Society. (c) A bionic stretchable TENG for underwater rescue. Reproduced with permission from Ref. [30]. Copyright © 2020, Springer Nature. (d) A bio-inspired spider-net-coding interface for multiple direction detecting and control. Reproduced with permission from Ref. [55]. Copyright © 2019, Wiley-VCH. (e) A self-powered auditory sensor with ultrahigh sensitivity. Reproduced with permission from Ref. [20]. Copyright © 2020, Elsevier. (f) A wearable ionic TENG patch for wound healing. Reproduced with permission from Ref. [75]. Copyright © 2020, Elsevier. 3.1. Self-Powered Wearable Systems Based on TENG Wearable electronics have brought convenien 3.1. Self-Powered Wearable Systems Based on TENG devised a wearable ionic TENG, which has a stretcha- ble gel composition [75]. The electricity generated by this TENG from biomechanical en- ergy is used in damaged tissues, and it accelerates the wound healing, as shown in Figure 6f. Figure 6. Self-powered wearable systems based on TENGs. (a) A TENG-based smart insole. Reproduced with permission from Ref. [69]. Copyright © 2020, Wiley-VCH. (b) An ultralight single-electrode triboelectric yarn with helical hybridized nano-micro core-shell fiber bundles. Reproduced with permission from Ref. [65]. Copyright © 2020, American Chemical Society. (c) A bionic stretchable TENG for underwater rescue. Reproduced with permission from Ref. [30]. Copyright © 2020, Springer Nature. (d) A bio-inspired spider-net-coding interface for multiple direction detecting and control. Repro- duced with permission from Ref. [55]. Copyright © 2019, Wiley-VCH. (e) A self-powered auditory sensor with ultrahigh sensitivity. Reproduced with permission from Ref. [20]. Copyright © 2020, Elsevier. (f) A wearable ionic TENG patch for wound healing. Reproduced with permission from Ref. [75]. Copyright © 2020, Elsevier. Figure 6. Self-powered wearable systems based on TENGs. (a) A TENG-based smart insole. Reproduced with permission from Ref. [69]. Copyright © 2020, Wiley-VCH. (b) An ultralight single-electrode triboelectric yarn with helical hybridized nano-micro core-shell ﬁber bundles. Reproduced with permission from Ref. [65]. Copyright © 2020, American Chemical Society. (c) A bionic stretchable TENG for underwater rescue. Reproduced with permission from Ref. [30]. Copyright © 2020, Springer Nature. (d) A bio-inspired spider-net-coding interface for multiple direction detecting and control. Reproduced with permission from Ref. [55]. Copyright © 2019, Wiley-VCH. (e) A self-powered auditory sensor with ultrahigh sensitivity. Reproduced with permission from Ref. [20]. Copyright © 2020, Elsevier. (f) A wearable ionic TENG patch for wound healing. Reproduced with permission from Ref. [75]. Copyright © 2020, Elsevier. Figure 6. Self-powered wearable systems based on TENGs. (a) A TENG-based smart insole. Reproduced with permission from Ref. [69]. Copyright © 2020, Wiley-VCH. (b) An ultralight single-electrode triboelectric yarn with helical hybridized nano-micro core-shell fiber bundles. Reproduced with permission from Ref. [65]. Copyright © 2020, American Chemical Society. (c) A bionic stretchable TENG for underwater rescue. Reproduced with permission from Ref. [30]. Copyright © 2020, Springer Nature. (d) A bio-inspired spider-net-coding interface for multiple direction detecting and control. Repro- duced with permission from Ref. [55]. Copyright © 2019, Wiley-VCH. (e) A self-powered auditory sensor with ultrahigh sensitivity. Reproduced with permission from Ref. [20]. Copyright © 2020, Elsevier. 3.1. Self-Powered Wearable Systems Based on TENG Wearable electronics have brought convenien 3.1. Self-Powered Wearable Systems Based on TENG reported a smart insole with TENG embedded as an active sensor for real-time gait monitoring [69], as shown in Figure 6a. It has high durability and excellent mechanical robustness to monitor the abnormality of gait for rehabilitation assessment. Liyun Ma et al. proposed an ultralight single-electrode textile-based TENG with helical hybridized nano-micro core-shell ﬁber bundles [65], as shown in Figure 6b. It enables harvesting biomechanical energy and monitoring tiny signals from human motions. TENGs come in a wide range of forms besides fabrics. Yu Song et al. developed a self-powered wearable wireless sweat sensing system based on a TENG [54]. As shown in Figure 6c, Yang Zou et al. designed a bionic stretch TENG by imitating the electric eel’s power generation principle [30]. It has a broad application prospect in underwater motion detection and submarine rescue. With the advent of the intelligent era, objects are connected through the internet, and wearable applications for human–machine interfaces and intelligent systems are also arising. A 9 of 18 9 of 18 Micromachines 2021, 12, 666 smart glove with a haptic feedback was designed based on TENG to serve as a simple human–computer interaction method [31]. Qiongfeng Shi et al. reported a bio-inspired spider-net-coding interface with great ﬂexibility and scalability [55]. By employing a single-electrode TENG, detection and control of multiple directions are demonstrated, as shown in Figure 6d. Hengyu Guo et al. proposed a self-powered acoustic sensor [20], as shown in Figure 6e. It created a new acoustic system by using TENG. The acoustic sensor has ultrahigh sensitivity, which could reach 110 mV/dB. Wearable TENGs are adopted for biomedical applications as well. Zhirong Liu et al. developed a TENG as a stable voltage pulse source to trigger plasma membrane potential and membrane permeability for intracellular drug delivery [67]. The delivery efﬁciency of this system is 90%, and the cell survival rate is more than 94%. Yonghong Li et al. devised a wearable ionic TENG, which has a stretchable gel composition [75]. The electricity generated by this TENG from biomechanical energy is used in damaged tissues, and it accelerates the wound healing, as shown in Figure 6f. R REVIEW 9 of 18 TENG, detection and control of multiple directions are demonstrated, as shown in Figure 6d. Hengyu Guo et al. proposed a self-powered acoustic sensor [20], as shown in Figure 6e. It created a new acoustic system by using TENG. 3.2. Self-Powered Wearable Systems Based on PENG 3.2. Self-Powered Wearable Systems Based on PENG (a) A PENG arrays integrated on a boxing glove for smart mission from Ref [37] Copyright © 2019 John Wiley and Sons (b) A highly stretchable le systems based on PENG. (a) A PENG arrays integrated on a boxing glove for smart sports. Figure 7. Self-powered wearable systems based on PENG. (a) A PENG arrays integrated on a box sports Reproduced with permission from Ref [37] Copyright © 2019 John Wiley and Sons (b) A Figure 7. Self-powered wearable systems based on PENG. (a) A PENG arrays integrated on a boxing gl sports. Reproduced with permission from Ref. [37]. Copyright © 2019, John Wiley and Sons. (b) A highly stretchable piezoelectric biomechanical sensor. Reproduced with permission from Ref. [80]. Copyright © 2020, American Chemical Society. (c) PENG adapted to drive a commercial electric heating sheet. Reproduced with permission from Ref. [48]. Cop- yright © 2020, American Chemical Society. (d) A self-powered and self-functional sock based on hybrid nanogenerators. Reproduced with permission from Ref. [32]. Copyright © 2019, American Chemical Society. (e) A muscle-fiber-inspired nonwoven piezoelectric textile for health monitoring. Reproduced with permission from Ref. [52]. Copyright © 2020, Wiley-VCH. (f) A highly flexible fabric-based wearable PENG. Reproduced with permission from Ref. [72]. Copyright © 2020, Elsevier. Figure 7. Self powered wearable systems based on PENG. (a) A PENG arrays integrated on a boxing glove for smart sports. Reproduced with permission from Ref. [37]. Copyright © 2019, John Wiley and Sons. (b) A highly stretchable piezoelectric biomechanical sensor. Reproduced with permission from Ref. [80]. Copyright © 2020, American Chemical Society. (c) PENG adapted to drive a commercial electric heating sheet. Reproduced with permission from Ref. [48]. Copyright © 2020, American Chemical Society. (d) A self-powered and self-functional sock based on hybrid nanogenerators. Reproduced with permission from Ref. [32]. Copyright © 2019, American Chemical Society. (e) A muscle-ﬁber-inspired nonwoven piezoelectric textile for health monitoring. Reproduced with permission from Ref. [52]. Copyright © 2020, Wiley-VCH. (f) A highly ﬂexible fabric-based wearable PENG. Reproduced with permission from Ref. [72]. Copyright © 2020, Elsevier. 3.2. Self-Powered Wearable Systems Based on PENG 3.2. Self-Powered Wearable Systems Based on PENG f y Wearable PENGs demonstrate potential applications for power supply, motion mon- itoring and health monitoring in wearable systems as well. Desheng Yao et al. presented a wearable boxing glove based on 3D printed flexible piezoelectric lattice with stretch dominated microarchitectures [37], as shown in Figure 7a. It achieves high electromechan- ical sensitivity and structural functionality. Spatially resolved and time-resolved mapping of reaction punching forces exerted to knuckles of the hand during boxing activities could Wearable PENGs demonstrate potential applications for power supply, motion moni- toring and health monitoring in wearable systems as well. Desheng Yao et al. presented a wearable boxing glove based on 3D printed ﬂexible piezoelectric lattice with stretch domi- nated microarchitectures [37], as shown in Figure 7a. It achieves high electromechanical sensitivity and structural functionality. Spatially resolved and time-resolved mapping of reaction punching forces exerted to knuckles of the hand during boxing activities could Micromachines 2021, 12, 666 10 of 18 be obtained. Iqra Choudhry et al. reported a nanocomposite-based PENG fabricated by dispersing various piezoelectric nanoparticles (BaTiO3, ZnO, and PZT) graphene nano- powder in a silicone matrix [80]. As shown in Figure 7b, it serves as a biomechanical energy harvester and a self-powered motion sensor. Sun Yue et al. proposed a ZnO/PAN nanoﬁber- based PENG integrated with a plate heater for personal thermal management [48], as shown in Figure 7c. Minglu Zhu et al. designed a self-sufﬁcient sock composed of hybrid nanogen- erators [32], as shown in Figure 7d. It shows good ability in energy harvesting and motion sensing. Yuanjie Sun et al. proposed a muscle-ﬁber-inspired nonwoven piezoelectric textile with tunable mechanical properties to mimic the muscle ﬁber, as shown in Figure 7e [52]. It achieves high sensitivity in the monitoring of various physiological signals. Jaegyu Kim et al. developed a highly ﬂexible fabric-based wearable PENG with high efﬁciency and strong integration [72], as shown in Figure 7f. Beyond these, Shu Du et al. developed a bio-inspired hybrid patch with a PENG embedded [79]. The PENG is employed as an electrical stimulator to facilitate skin wound healing. powder in a silicone matrix [80]. As shown in Figure 7b, it serves as a biomechanical en- ergy harvester and a self-powered motion sensor. Sun Yue et al. proposed a ZnO/PAN nanofiber-based PENG integrated with a plate heater for personal thermal management [48], as shown in Figure 7c. Minglu Zhu et al. 3.2. Self-Powered Wearable Systems Based on PENG 3.2. Self-Powered Wearable Systems Based on PENG designed a self-sufficient sock composed of hybrid nanogenerators [32], as shown in Figure 7d. It shows good ability in energy har- vesting and motion sensing. Yuanjie Sun et al. proposed a muscle-fiber-inspired nonwo- ven piezoelectric textile with tunable mechanical properties to mimic the muscle fiber, as shown in Figure 7e [52]. It achieves high sensitivity in the monitoring of various physio- logical signals. Jaegyu Kim et al. developed a highly flexible fabric-based wearable PENG with high efficiency and strong integration [72], as shown in Figure 7f. Beyond these, Shu Du et al. developed a bio-inspired hybrid patch with a PENG embedded [79]. The PENG is employed as an electrical stimulator to facilitate skin wound healing. Figure 7. Self-powered wearable systems based on PENG. (a) A PENG arrays integrated on a boxing glove for smart sports. Reproduced with permission from Ref. [37]. Copyright © 2019, John Wiley and Sons. (b) A highly stretchable piezoelectric biomechanical sensor. Reproduced with permission from Ref. [80]. Copyright © 2020, American Chemical Society. (c) PENG adapted to drive a commercial electric heating sheet. Reproduced with permission from Ref. [48]. Cop- yright © 2020, American Chemical Society. (d) A self-powered and self-functional sock based on hybrid nanogenerators. Reproduced with permission from Ref. [32]. Copyright © 2019, American Chemical Society. (e) A muscle-fiber-inspired nonwoven piezoelectric textile for health monitoring. Reproduced with permission from Ref. [52]. Copyright © 2020, Wiley-VCH. (f) A highly flexible fabric-based wearable PENG. Reproduced with permission from Ref. [72]. Copyright © 2020, Elsevier. Figure 7. Self-powered wearable systems based on PENG. (a) A PENG arrays integrated on a boxing glove for smart sports. Reproduced with permission from Ref. [37]. Copyright © 2019, John Wiley and Sons. (b) A highly stretchable piezoelectric biomechanical sensor. Reproduced with permission from Ref. [80]. Copyright © 2020, American Chemical Society. (c) PENG adapted to drive a commercial electric heating sheet. Reproduced with permission from Ref. [48]. Copyright © 2020, American Chemical Society. (d) A self-powered and self-functional sock based on hybrid nanogenerators. Reproduced with permission from Ref. [32]. Copyright © 2019, American Chemical Society. (e) A muscle-ﬁber-inspired nonwoven piezoelectric textile for health monitoring. Reproduced with permission from Ref. [52]. Copyright © 2020, Wiley-VCH. (f) A highly ﬂexible fabric-based wearable PENG. Reproduced with permission from Ref. [72]. Copyright © 2020, Elsevier. ble systems based on PENG. . Se o e ed I p a tab e Syste s 4.1. Self-Powered Implantable Systems Based on TENG 4.1. Self-Powered Implantable Systems Based on TENG proposed a hybrid energy harvesting system that consisted of a TENG and a glucose fuel cell [92], as shown in Figure 8c. This design strengthened the flexibility of harvesting multiple sources of bioenergies and enhanced electrical outputs. The high-voltage outputs of TENG were adopted to stimulate muscles and nerves as well. Jiahui Wang et al. proposed a self-powered muscle stimulation system based on TENG [98], as shown in Figure 8d. The TENG can directly stimulate the muscle to treat the muscle dysfunction. The multi-channel electrode adheres well to the surface of muscle. Sanghoon Lee et al. proposed a TENG neurostimulator to realize the mechano- neuromodulation of autonomic pelvic nerves [108]. As shown in Figure 8e, the stimulator system consists of a stacked TENG and a flexible neural clip interface. Rui Shi et al. pro- posed a self-powered treatment strategy employing a TENG to charge a titanium implant surface [99], as shown in Figure 8f. The charged titanium implant shows a suitable anti- bacterial property. It can serve as an antibacterial biofilm and helps to promote the osse- ointegration. Figure 8. Self-powered implantable systems based on TENG. (a) A symbiotic cardiac pacemaker powered by TENG. Re- produced with permission from Ref. [101]. Copyright © 2019, Springer Nature. (b) A self-powered endocardial pressure sensor. Reproduced with permission from Ref. [105]. Copyright © 2018, Wiley-VCH. (c) An implanted hybrid energy harvesting system. Reproduced with permission from Ref. [92]. Copyright © 2020, Springer Nature. (d) Electrical muscle stimulation directly powered by TENG. Reproduced with permission from Ref. [98]. Copyright © 2019, Wiley-VCH. (e) A TENG neurostimulator integrated with neural clip interface. Reproduced with permission from Ref. [108]. Copyright © 2019, Elsevier. (f) A self-powered treatment to charge titanium implant surface. Reproduced with permission from Ref. [99]. Copyright © 2020, Elsevier. Figure 8. Self-powered implantable systems based on TENG. (a) A symbiotic cardiac pacemaker powered by TENG. Reproduced with permission from Ref. [101]. Copyright © 2019, Springer Nature. (b) A self-powered endocardial pressure sensor. Reproduced with permission from Ref. [105]. Copyright © 2018, Wiley-VCH. (c) An implanted hybrid energy harvesting system. Reproduced with permission from Ref. [92]. Copyright © 2020, Springer Nature. (d) Electrical muscle stimulation directly powered by TENG. Reproduced with permission from Ref. [98]. Copyright © 2019, Wiley-VCH. (e) A TENG neurostimulator integrated with neural clip interface. Reproduced with permission from Ref. [108]. Copyright © 2019, Elsevier. . Se o e ed I p a tab e Syste s 4.1. Self-Powered Implantable Systems Based on TENG 4.1. Self-Powered Implantable Systems Based on TENG f p y Harvesting energy from the biomechanical energy of heartbeats, blood pressure, and other biological rhythms to power implantable electronic devices has had an upsurge in recent years. Han Ouyang et al. developed an implanted symbiotic cardiac pacemaker based on TENG [101], as shown in Figure 8a. The implanted TENG can obtain 0.495 μJ Harvesting energy from the biomechanical energy of heartbeats, blood pressure, and other biological rhythms to power implantable electronic devices has had an upsurge in recent years. Han Ouyang et al. developed an implanted symbiotic cardiac pacemaker based on TENG [101], as shown in Figure 8a. The implanted TENG can obtain 0.495 µJ 11 of 18 11 of 18 Micromachines 2021, 12, 666 electrical energy in each cardiac cycle. Liu Zhuo et al. reported a self-powered endocardial pressure sensor using TENG [105], as shown in Figure 8b. It can monitor in real time to detect arrhythmias. Owing to its specialty in lightweight and ﬂexibility, TENG can be implanted in subcutaneous tissues. Hu Li et al. proposed a hybrid energy harvesting system that consisted of a TENG and a glucose fuel cell [92], as shown in Figure 8c. This design strengthened the ﬂexibility of harvesting multiple sources of bioenergies and enhanced electrical outputs. The high-voltage outputs of TENG were adopted to stimulate muscles and nerves as well. Jiahui Wang et al. proposed a self-powered muscle stimulation system based on TENG [98], as shown in Figure 8d. The TENG can directly stimulate the muscle to treat the muscle dysfunction. The multi-channel electrode adheres well to the surface of muscle. Sanghoon Lee et al. proposed a TENG neurostimulator to realize the mechano-neuromodulation of autonomic pelvic nerves [108]. As shown in Figure 8e, the stimulator system consists of a stacked TENG and a ﬂexible neural clip interface. Rui Shi et al. proposed a self-powered treatment strategy employing a TENG to charge a titanium implant surface [99], as shown in Figure 8f. The charged titanium implant shows a suitable antibacterial property. It can serve as an antibacterial bioﬁlm and helps to promote the osseointegration. pressure sensor using TENG [105], as shown in Figure 8b. It can monitor in real time to detect arrhythmias. Owing to its specialty in lightweight and flexibility, TENG can be im- planted in subcutaneous tissues. Hu Li et al. . Se o e ed I p a tab e Syste s 4.1. Self-Powered Implantable Systems Based on TENG 4.1. Self-Powered Implantable Systems Based on TENG (f) A self-powered treatment to charge titanium implant surface. Reproduced with permission from Ref. [99]. Copyright © 2020, Elsevier. Figure 8. Self-powered implantable systems based on TENG. (a) A symbiotic cardiac pacemaker powered by TENG. Re- produced with permission from Ref. [101]. Copyright © 2019, Springer Nature. (b) A self-powered endocardial pressure sensor. Reproduced with permission from Ref. [105]. Copyright © 2018, Wiley-VCH. (c) An implanted hybrid energy harvesting system. Reproduced with permission from Ref. [92]. Copyright © 2020, Springer Nature. (d) Electrical muscle stimulation directly powered by TENG. Reproduced with permission from Ref. [98]. Copyright © 2019, Wiley-VCH. (e) A TENG neurostimulator integrated with neural clip interface. Reproduced with permission from Ref. [108]. Copyright © 2019, Elsevier. (f) A self-powered treatment to charge titanium implant surface. Reproduced with permission from Ref. [99]. Copyright © 2020, Elsevier. Figure 8. Self-powered implantable systems based on TENG. (a) A symbiotic cardiac pacemaker powered by TENG. Reproduced with permission from Ref. [101]. Copyright © 2019, Springer Nature. (b) A self-powered endocardial pressure sensor. Reproduced with permission from Ref. [105]. Copyright © 2018, Wiley-VCH. (c) An implanted hybrid energy harvesting system. Reproduced with permission from Ref. [92]. Copyright © 2020, Springer Nature. (d) Electrical muscle stimulation directly powered by TENG. Reproduced with permission from Ref. [98]. Copyright © 2019, Wiley-VCH. (e) A TENG neurostimulator integrated with neural clip interface. Reproduced with permission from Ref. [108]. Copyright © 2019, Elsevier. (f) A self-powered treatment to charge titanium implant surface. Reproduced with permission from Ref. [99]. Copyright © 2020, Elsevier. 4.2. Self-Powered Implantable Systems Based on PENG 4.2. Self-Powered Implantable Systems Based on PENG energy from the lead’s motion caused by heartbeats. Zhiran Yi et al. proposed a self-pow- ered leadless cardiac pacemaker [96]. The PENG used to power the pacemaker obtains a short-circuit current of 30 μA and an open-circuit voltage of 8.1 V. Li Ning et al. proposed an implantable PENG, as shown in Figure 9b. It can directly power a cardiac pacemaker via a rectifier [106]. Xiaoliang cheng et al. presented an implantable self-powered blood pressure monitor based on a PENG [119], as shown in Figure 9c. Good linearity was achieved between the peak output voltage of the PENG and the flow pressure, with a sensitivity of 173 mV/mmHg. As shown in Figure 9d, Qian Yun et al. designed a ZnO based PENG scaffold [100]. It plays a role as an in vivo stimulus to accelerate the speed of tissue healing and nerve conducting. Ritopa Das et al. proposed a new method for bone regeneration [85], as shown in Figure 9e. A biodegradable PENG scaffold driven by ultra- sound is adapted as an electrical stimulator to promote bone regeneration. Liu Zhuo and others designed a PENG to power the photodynamic therapy system for cancer treatment to inhibit the growth of subcutaneous tumor cells in mice [104], as shown in Figure 9f. The inhibition rate reached 87.46%. Figure 9. Self-powered implantable systems based on PENG. (a) A kirigami inspired PENG. Reproduced with permission from Ref. [97]. Copyright © 2021, Wiley-VCH. (b) A self-powered leadless cardiac pacemaker. Reproduced with permis- sion from Ref. [106]. Copyright © 2019, American Chemical Society. (c) PENG for in vivo blood pressure monitoring. Reproduced with permission from Ref. [119]. Copyright © 2016, Elsevier. (d) A PENG scaffold for tissue healing. Repro- duced with permission from Ref. [100]. Copyright © 2020, Wiley-VCH. (e) A battery-less electrical stimulator serving as a bone scaffold. Reproduced with permission from Ref. [85]. Copyright © 2020, Elsevier. (f) A self-powered photodynamic therapy system. Reproduced with permission from Ref. [104]. Copyright © 2020, American Chemical Society. Figure 9. Self-powered implantable systems based on PENG. (a) A kirigami inspired PENG. Reproduced with permission from Ref. [97]. Copyright © 2021, Wiley-VCH. (b) A self-powered leadless cardiac pacemaker. Reproduced with permission from Ref. [106]. Copyright © 2019, American Chemical Society. (c) PENG for in vivo blood pressure monitoring. Reproduced with permission from Ref. [119]. Copyright © 2016, Elsevier. (d) A PENG scaffold for tissue healing. 4.2. Self-Powered Implantable Systems Based on PENG 4.2. Self-Powered Implantable Systems Based on PENG Reproduced with permission from Ref. [100]. Copyright © 2020, Wiley-VCH. (e) A battery-less electrical stimulator serving as a bone scaffold. Reproduced with permission from Ref. [85]. Copyright © 2020, Elsevier. (f) A self-powered photodynamic therapy system. Reproduced with permission from Ref. [104]. Copyright © 2020, American Chemical Society. Figure 9. Self-powered implantable systems based on PENG. (a) A kirigami inspired PENG. Reproduced with permission from Ref. [97]. Copyright © 2021, Wiley-VCH. (b) A self-powered leadless cardiac pacemaker. Reproduced with permis- sion from Ref. [106]. Copyright © 2019, American Chemical Society. (c) PENG for in vivo blood pressure monitoring. Reproduced with permission from Ref. [119]. Copyright © 2016, Elsevier. (d) A PENG scaffold for tissue healing. Repro- duced with permission from Ref. [100]. Copyright © 2020, Wiley-VCH. (e) A battery-less electrical stimulator serving as a bone scaffold. Reproduced with permission from Ref. [85]. Copyright © 2020, Elsevier. (f) A self-powered photodynamic therapy system. Reproduced with permission from Ref. [104]. Copyright © 2020, American Chemical Society. Figure 9. Self-powered implantable systems based on PENG. (a) A kirigami inspired PENG. Reproduced with permission from Ref. [97]. Copyright © 2021, Wiley-VCH. (b) A self-powered leadless cardiac pacemaker. Reproduced with permission from Ref. [106]. Copyright © 2019, American Chemical Society. (c) PENG for in vivo blood pressure monitoring. Reproduced with permission from Ref. [119]. Copyright © 2016, Elsevier. (d) A PENG scaffold for tissue healing. Reproduced with permission from Ref. [100]. Copyright © 2020, Wiley-VCH. (e) A battery-less electrical stimulator serving as a bone scaffold. Reproduced with permission from Ref. [85]. Copyright © 2020, Elsevier. (f) A self-powered photodynamic therapy system. Reproduced with permission from Ref. [104]. Copyright © 2020, American Chemical Society. 4.2. Self-Powered Implantable Systems Based on PENG 4.2. Self-Powered Implantable Systems Based on PENG 4.2. Self-Powered Implantable Systems Based on PENG 4.2. Self-Powered Implantable Systems Based on PENG Implantable PENGs and piezoelectric sensors have shown great potential in the eval- uation and diagnosis of cardiovascular diseases. Great efforts have been made to power cardiac pacemakers by using PENGs. Zhe Xu et al. developed a kirigami inspired PENG Implantable PENGs and piezoelectric sensors have shown great potential in the evaluation and diagnosis of cardiovascular diseases. Great efforts have been made to 12 of 18 12 of 18 Micromachines 2021, 12, 666 power cardiac pacemakers by using PENGs. Zhe Xu et al. developed a kirigami inspired PENG [97], as shown in Figure 9a. The PENG is ﬁxed on the lead of the pacemaker to harvest energy from the lead’s motion caused by heartbeats. Zhiran Yi et al. proposed a self-powered leadless cardiac pacemaker [96]. The PENG used to power the pacemaker obtains a short-circuit current of 30 µA and an open-circuit voltage of 8.1 V. Li Ning et al. proposed an implantable PENG, as shown in Figure 9b. It can directly power a cardiac pacemaker via a rectiﬁer [106]. Xiaoliang cheng et al. presented an implantable self-powered blood pressure monitor based on a PENG [119], as shown in Figure 9c. Good linearity was achieved between the peak output voltage of the PENG and the ﬂow pressure, with a sensitivity of 173 mV/mmHg. As shown in Figure 9d, Qian Yun et al. designed a ZnO based PENG scaffold [100]. It plays a role as an in vivo stimulus to accelerate the speed of tissue healing and nerve conducting. Ritopa Das et al. proposed a new method for bone regeneration [85], as shown in Figure 9e. A biodegradable PENG scaffold driven by ultrasound is adapted as an electrical stimulator to promote bone regeneration. Liu Zhuo and others designed a PENG to power the photodynamic therapy system for cancer treatment to inhibit the growth of subcutaneous tumor cells in mice [104], as shown in Figure 9f. The inhibition rate reached 87.46%. energy from the lead’s motion caused by heartbeats. Zhiran Yi et al. proposed a self-pow- ered leadless cardiac pacemaker [96]. The PENG used to power the pacemaker obtains a short-circuit current of 30 μA and an open-circuit voltage of 8.1 V. Li Ning et al. proposed an implantable PENG, as shown in Figure 9b. It can directly power a cardiac pacemaker via a rectifier [106]. 4.2. Self-Powered Implantable Systems Based on PENG 4.2. Self-Powered Implantable Systems Based on PENG Xiaoliang cheng et al. presented an implantable self-powered blood pressure monitor based on a PENG [119], as shown in Figure 9c. Good linearity was achieved between the peak output voltage of the PENG and the flow pressure, with a sensitivity of 173 mV/mmHg. As shown in Figure 9d, Qian Yun et al. designed a ZnO based PENG scaffold [100]. It plays a role as an in vivo stimulus to accelerate the speed of tissue healing and nerve conducting. Ritopa Das et al. proposed a new method for bone regeneration [85], as shown in Figure 9e. A biodegradable PENG scaffold driven by ultra- sound is adapted as an electrical stimulator to promote bone regeneration. Liu Zhuo and others designed a PENG to power the photodynamic therapy system for cancer treatment to inhibit the growth of subcutaneous tumor cells in mice [104], as shown in Figure 9f. The inhibition rate reached 87.46%. power cardiac pacemakers by using PENGs. Zhe Xu et al. developed a kirigami inspired PENG [97], as shown in Figure 9a. The PENG is ﬁxed on the lead of the pacemaker to harvest energy from the lead’s motion caused by heartbeats. Zhiran Yi et al. proposed a self-powered leadless cardiac pacemaker [96]. The PENG used to power the pacemaker obtains a short-circuit current of 30 µA and an open-circuit voltage of 8.1 V. Li Ning et al. proposed an implantable PENG, as shown in Figure 9b. It can directly power a cardiac pacemaker via a rectiﬁer [106]. Xiaoliang cheng et al. presented an implantable self-powered blood pressure monitor based on a PENG [119], as shown in Figure 9c. Good linearity was achieved between the peak output voltage of the PENG and the ﬂow pressure, with a sensitivity of 173 mV/mmHg. As shown in Figure 9d, Qian Yun et al. designed a ZnO based PENG scaffold [100]. It plays a role as an in vivo stimulus to accelerate the speed of tissue healing and nerve conducting. Ritopa Das et al. proposed a new method for bone regeneration [85], as shown in Figure 9e. A biodegradable PENG scaffold driven by ultrasound is adapted as an electrical stimulator to promote bone regeneration. Liu Zhuo and others designed a PENG to power the photodynamic therapy system for cancer treatment to inhibit the growth of subcutaneous tumor cells in mice [104], as shown in Figure 9f. The inhibition rate reached 87.46%. 5. Conclusions This article h 5. Conclusions This article has reviewed the recent developments of self-powered systems based on TENG and PENG for wearable and implantable applications. The materials and structures for nanogenerators and their wearable and implantable applications are discussed. In This article has reviewed the recent developments of self-powered systems based on TENG and PENG for wearable and implantable applications. The materials and structures Micromachines 2021, 12, 666 13 of 18 for nanogenerators and their wearable and implantable applications are discussed. In terms of materials, biodegradable PVA, PLLA, silk, and so on are introduced to increase the possibility of implantation. Additionally, liquid crystal materials and hydrogel ma- terials are used to increase the tensile strength and afﬁnity. As to the device structures, three-dimensional structures, textile structures, and spring-mass structures of hybrid nano- generators show good performance in wearable applications. In addition, various thin-ﬁlm structures with well-designed transformation or separation are valuable for in vivo energy harvesting as the application of self-powered wearable systems. They can be directly attached to the skin and worn as part of the clothing or accessory for motion monitoring, health monitoring, wound repairing, etc. Implantable systems are supposed to have the characteristics of good biocompatibility and high durability. So far, the applications on the battery-less cardiac pacemaker, in vivo health monitoring, in vivo stimulation, and therapy are promising. Considering the abovementioned progress on wearable and implantable self-powered systems, it is still at its infancy stage of development. Triboelectric and piezoelectric materi- als with high charge density, good biocompatibility and ease of manufacture will be crucial. To realize fully self-powered wearable and implantable systems, power management circuits with high efﬁciency [121,122] would be indispensable for nanogenerators. Author Contributions: Conceptualization, L.Y., Z.M., and B.M.; writing—original draft prepara- tion, L.Y. and Y.T.; writing—review and editing, B.M. and Z.P.; funding acquisition, B.M. and Z.P. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the National Natural Science Foundation of China (Grant No. 61904111), Natural Science Foundation of Guangdong Province (Grant No. 2020A1515011487), and Shenzhen Peacock Team Project (KQTD20170810105439418). Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. Chan, M.; Estève, D.; Fourniols, J.-Y.; Escriba, C.; Campo, E. Smart wearable systems: Current status and future challenges. Artif. Intell. Med. 2012, 56, 137–156. [CrossRef] ; Fourniols, J.-Y.; Escriba, C.; Campo, E. Smart wearable systems: Current status and future challenges. Artif 137–156. [CrossRef] 1. Chan, M.; Estève, D.; Fourniols, J.-Y.; Escriba, C.; Campo, E. Smart wearable systems: Current status an Intell. Med. 2012, 56, 137–156. [CrossRef] 2. Zeng, W.; Shu, L.; Li, Q.; Chen, S.; Wang, F.; Tao, X.-M. Fiber-Based Wearable Electronics: A Review of Materials, Fabrication, Devices, and Applications. Adv. Mater. 2014, 26, 5310–5336. [CrossRef] 3. Gao, M.; Wang, P.; Jiang, L.; Wang, B.; Yao, Y.; Liu, S.; Chu, D.; Cheng, W.; Lu, Y. Power generation for wearable systems. Energy Environ. Sci. 2021, 14, 2114–2157. [CrossRef] 4. Jiang, D.J.; Shi, B.J.; Ouyang, H.; Fan, Y.B.; Wang, Z.L.; Li, Z. Emerging Implantable Energy Harv Implantable Medical Electronics. ACS Nano 2020, 14, 6436–6448. [CrossRef] ; Ouyang, H.; Fan, Y.B.; Wang, Z.L.; Li, Z. Emerging Implantable Energy Harvesters and Self-Powered l Electronics. ACS Nano 2020, 14, 6436–6448. [CrossRef] 5. Xue, R.-F.; Cheng, K.-W.; Je, M. High-Efﬁciency Wireless Power Transfer for Biomedical Implants by Optimal Resonant Load Transformation. IEEE Trans. Circuits Syst. I Regul. Pap. 2012, 60, 867–874. [CrossRef] y g p 6. Kim, B.J.; Kim, D.H.; Lee, Y.-Y.; Shin, H.-W.; Han, G.S.; Hong, J.S.; Mahmood, K.; Ahn, T.K.; Joo, Y.-C.; Hong, K.S.; et al. Highly efﬁcient and bending durable perovskite solar cells: Toward a wearable power source. Energy Environ. Sci. 2015, 8, 916–921. [CrossRef] y g p 6. Kim, B.J.; Kim, D.H.; Lee, Y.-Y.; Shin, H.-W.; Han, G.S.; Hong, J.S.; Mahmood, K.; Ahn, T.K.; Joo, Y.-C.; Hong, K.S.; et al. Highly efﬁcient and bending durable perovskite solar cells: Toward a wearable power source. Energy Environ. Sci. 2015, 8, 916–921. [CrossRef] 7. Leonov, V.; Vullers, R.J.M. Wearable electronics self-powered by using human body heat: The state of the art and the perspective. J. Renew. Sustain. Energy 2009, 1, 062701. [CrossRef] 7. Leonov, V.; Vullers, R.J.M. Wearable electronics self-powered by using human body heat: The state of the art and the perspective. J. Renew. Sustain. Energy 2009, 1, 062701. [CrossRef] 8. Wang, Z.L.; Song, J.H. Piezoelectric nanogenerators based on zinc oxide nanowire arrays. Science 2006, 312, 242–246. [CrossRef] 9. Wang, X.D.; Song, J.H.; Liu, J.; Wang, Z.L. Direct-Current Nanogenerator Driven by Ultrasonic Waves. Science 2007, 316, 102–105. [CrossRef] 8. Wang, Z.L.; Song, J.H. Piezoelectric nanogenerators based on zinc oxide nanowire arrays. Science 2006, 312, 242–246. [CrossRef] 8. Wang, Z.L.; Song, J.H. Piezoelectric nanogenerators based on zinc oxide nanowire arrays. Science 2006, 312, 242–246. ; Fourniols, J.-Y.; Escriba, C.; Campo, E. Smart wearable systems: Current status and future challenges. Artif 137–156. [CrossRef] Eye motion triggered self-powered mechnosensational communication system using triboelectric nanogenerator. Sci. Adv. 2017, 3, e1700694. [CrossRef] [PubMed] 29. Li, T.; Qu, M.H.; Carlos, O.R.; Gu, L.; Jin, F.; Yuan, T.; Wu, X.W.; Xiao, J.J.; Wang, T.; Dong, W.; et al. High-Performance Poly(vinylidene diﬂuoride)/Dopamine Core/Shell Piezoelectric Nanoﬁber and Its Application for Biomedical Sensors. Adv. Mater. 2021, 33, 2006093. [CrossRef] 30. Zou, Y.; Tan, P.C.; Shi, B.J.; Ouyang, H.; Jiang, D.J.; Liu, Z.; Li, H.; Yu, M.; Wang, C.; Qu, X.C.; et al. A bionic stretchable nanogenerator for underwater sensing and energy harvesting. Nat. Commun. 2019, 10, 2695. [CrossRef] g g gy g M.L.; Sun, Z.D.; Zhang, Z.X.; Shi, Q.F.; He, T.Y.Y.; Liu, H.C.; Chen, T.; Lee, C.K. Haptic-feedback smart g g g gy g 31. Zhu, M.L.; Sun, Z.D.; Zhang, Z.X.; Shi, Q.F.; He, T.Y.Y.; Liu, H.C.; Chen, T.; Lee, C.K. Haptic-feedback human-machine interface (HMI) for virtual/augmented reality applications. Sci. Adv. 2020, 6, eaaz869 g g human-machine interface (HMI) for virtual/augmented reality applications. Sci. Adv. 2020, 6, eaaz8693. [CrossRef] [PubMed] 32. Zhu, M.L.; Shi, Q.F.; He, T.Y.Y.; Yi, Z.R.; Ma, Y.M.; Yang, B.; Chen, T.; Lee, C. Self-Powered and Self-Functional Cotton Sock Using Piezoelectric and Triboelectric Hybrid Mechanism for Healthcare and Sports Monitoring. ACS Nano 2019, 13, 1940–1952. [CrossRef] 33. Zhou, Z.H.; Padgett, S.; Cai, Z.X.; Conta, G.; Wu, Y.F.; He, Q.; Zhang, S.L.; Sun, C.C.; Liu, J.; Fan, E.D.; et al. Single-layered ultra-soft washable smart textiles for all-around ballistocardiograph, respiration, and posture monitoring during sleep. Biosens. Bioelectron. 2020, 155, 112064. [CrossRef] [ ] 34. Zhou, T.; Zhang, C.; Han, C.B.; Fan, F.R.; Tang, W.; Wang, Z.L. Woven Structured Triboelectric Nanogenerator for Wearable Devices. ACS Appl. Mater. Interfaces 2014, 6, 14695–14701. [CrossRef] 35. Zhang, W.; Yang, H.M.; Li, L.; Lin, S.Q.; Ji, P.Y.; Hu, C.G.; Zhang, D.Z.; Xi, Y. Flexible piezoelectric nanogenerators based on a CdS nanowall for self-powered sensors. Nanotechnology 2020, 31, 385401. [CrossRef] p 36. Wang, J.; Li, S.; Yi, F.; Zi, Y.; Lin, J.; Wang, X.; Xu, Y.; Wang, Z.L. Sustainably powering wearable electronic energy. Nat. Commun. 2016, 7, 12744. [CrossRef] 37. Yao, D.S.; Cui, H.C.; Hensleigh, R.; Smith, P.; Alford, S.; Bernero, D.; Bush, S.; Mann, K.; Wu, H.F.; Chin-Nieh, M.; et al. Achieving the Upper Bound of Piezoelectric Response in Tunable, Wearable 3D Printed Nanocomposites. Adv. Funct. Mater. 2019, 29, 1903866. [CrossRef] 38. ; Fourniols, J.-Y.; Escriba, C.; Campo, E. Smart wearable systems: Current status and future challenges. Artif 137–156. [CrossRef] [CrossRef] , ; , J ; , J ; g, ; , ; , ; g, Q ; , ; , ; Q , ; g y , self-powered triboelectric auditory sensor for social robotics and hearing aids. Sci. Robot. 2018, 3, eaat2516. [CrossRef] 21. Yi, F.; Lin, L.; Niu, S.M.; Yang, P.K.; Wang, Z.N.; Chen, J.; Zhou, Y.S.; Zi, Y.L.; Wang, J.; Liao, Q.L.; et al. Stretchable-Rubber-Based Triboelectric Nanogenerator and Its Application as Self-Powered Body Motion Sensors. Adv. Funct. Mater. 2015, 25, 3688–3696. [CrossRef] 22. Ning, C.; Dong, K.; Cheng, R.W.; Yi, J.; Ye, C.Y.; Peng, X.; Sheng, F.F.; Jiang, Y.; Wang, Z.L. Flexible and Stretchable Fiber-Shaped Triboelectric Nanogenerators for Biomechanical Monitoring and Human-Interactive Sensing. Adv. Funct. Mater. 2021, 31, 2006679. [CrossRef] 23. Lu, X.; Zheng, L.; Zhang, H.D.; Wang, W.H.; Wang, Z.L.; Sun, C.W. Stretchable, transparent triboelectric nanogenerator as a highly sensitive self-powered sensor for driver fatigue and distraction monitoring. Nano Energy 2020, 78, 105359. [CrossRef] 24. Chen, H.M.; Bai, L.; Li, T.; Zhao, C.; Zhang, J.S.; Zhang, N.; Song, G.F.; Gan, Q.Q.; Xu, Y. Wearable and robust triboelectric sensitive self-powered sensor for driver fatigue and distraction monitoring. Nano Energy 2020, 78, 105359. [CrossRef] 24. Chen, H.M.; Bai, L.; Li, T.; Zhao, C.; Zhang, J.S.; Zhang, N.; Song, G.F.; Gan, Q.Q.; Xu, Y. Wearable and robust triboelectric nanogenerator based on crumpled gold ﬁlms. Nano Energy 2018, 46, 73–80. [CrossRef] 24. Chen, H.M.; Bai, L.; Li, T.; Zhao, C.; Zhang, J.S.; Zhang, N.; Song, G.F.; Gan, Q.Q.; Xu, Y. Wearable and robust triboelectric nanogenerator based on crumpled gold ﬁlms. Nano Energy 2018, 46, 73–80. [CrossRef] g p g gy 25. Guan, Q.B.; Lin, G.H.; Gong, Y.Z.; Wang, J.F.; Tan, W.Y.; Bao, D.Q.; Liu, Y.N.; You, Z.W.; Sun, X.H.; Wen, Z.; et al. Highly efﬁcient self-healable and dual responsive hydrogel-based deformable triboelectric nanogenerators for wearable electronics. J. Mater. Chem. A 2019, 7, 13948–13955. [CrossRef] [ ] 26. Gogurla, N.; Roy, B.; Park, J.-Y.; Kim, S. Skin-contact actuated single-electrode protein triboelectric nanogenerator and strain sensor for biomechanical energy harvesting and motion sensing. Nano Energy 2019, 62, 674–681. [CrossRef] 27. Zhang, H.; Zhang, J.W.; Hu, Z.W.; Quan, L.W.; Shi, L.; Chen, J.K.; Xuan, W.P.; Zhang, Z.C.; Dong, S.R.; Luo, J.K. Waist-wearable wireless respiration sensor based on triboelectric effect. Nano Energy 2019, 59, 75–83. [CrossRef] 28. Pu, X.J.; Guo, H.Y.; Chen, J.; Wang, X.; Xi, Y.; Hu, C.G.; Wang, Z.L. ; Fourniols, J.-Y.; Escriba, C.; Campo, E. Smart wearable systems: Current status and future challenges. Artif 137–156. [CrossRef] [CrossRef] 9 Wang X D ; Song J H ; Liu J ; Wang Z L Direct Current Nanogenerator Driven by Ultrasonic Waves Science 2007 316 102 105 Wang, Z.L.; Song, J.H. Piezoelectric nanogenerators b g g J g y Wang, X.D.; Song, J.H.; Liu, J.; Wang, Z.L. Direct-Current Nanogenerator Driven by Ultrasonic Waves. Scien [CrossRef] [ ] 10. Fan, F.-R.; Tian, Z.-Q.; Wang, Z.L. Flexible triboelectric generator. Nano Energy 2012, 1, 328–334. [CrossRef] Fan, F.-R.; Tian, Z.-Q.; Wang, Z.L. Flexible triboelectric generator. Nano Energy 2012, 1, 328–334. [CrossRef Chen, J.; Yang, J.; Bai, P.; Li, Z.L.; Wang, Z.L. Ultrathin, Rollable, Paper-Based Triboelectric Nanogenerator f 11. Fan, X.; Chen, J.; Yang, J.; Bai, P.; Li, Z.L.; Wang, Z.L. Ultrathin, Rollable, Paper-Based Triboelectric Nanogenerator for Acoustic Energy Harvesting and Self-Powered Sound Recording. ACS Nano 2015, 9, 4236–4243. [CrossRef] gy g g 12. Zhu, G.; Zhou, Y.S.; Bai, P.; Meng, X.S.; Jing, Q.S.; Chen, J.; Wang, Z.L. A Shape-Adaptive Thin-Film-Based Approach for 50% High-Efﬁciency Energy Generation Through Micro-Grating Sliding Electriﬁcation. Adv. Mater. 2014, 26, 3788–3796. [CrossRef] 13 Wu W ; Bai S ; Yuan M ; Qin Y; Wang Z L ; Jing T Lead Zirconate Titanate Nanowire Textile Nanogenerator for Wearable 12. Zhu, G.; Zhou, Y.S.; Bai, P.; Meng, X.S.; Jing, Q.S.; Chen, J.; Wang, Z.L. A Shape-Adaptive Thin-Film-Based Approach for 50% High-Efﬁciency Energy Generation Through Micro-Grating Sliding Electriﬁcation. Adv. Mater. 2014, 26, 3788–3796. [CrossRef] 13. Wu, W.; Bai, S.; Yuan, M.; Qin, Y.; Wang, Z.L.; Jing, T. Lead Zirconate Titanate Nanowire Textile Nanogenerator for Wearable Energy-Harvesting and Self-Powered Devices. ACS Nano 2012, 6, 6231–6235. [CrossRef] gy g 14. Seung, W.; Gupta, M.K.; Lee, K.Y.; Shin, K.-S.; Lee, J.-H.; Kim, T.Y.; Kim, S.; Lin, J.; Kim, J.H.; Kim, S.-W. Nanopatterned Textile-Based Wearable Triboelectric Nanogenerator. ACS Nano 2015, 9, 3501–3509. [CrossRef] 14 of 18 14 of 18 Micromachines 2021, 12, 666 15. Lee, J.-H.; Lee, K.Y.; Gupta, M.K.; Kim, T.Y.; Lee, D.-Y.; Oh, J.; Ryu, C.; Yoo, W.J.; Kang, C.-Y.; Yoon, S.-J.; et al. Highly Stretchable Piezoelectric-Pyroelectric Hybrid Nanogenerator. Adv. Mater. 2014, 26, 765–769. [CrossRef] y y g 16. Fan, F.R.; Tang, W.; Wang, Z.L. Flexible Nanogenerators for Energy Harvesting and Self-Powered Electronics. Adv. Mater. 2016, 28, 4283–4305. [CrossRef] 17. Jiang, Q.; Wu, C.S.; Wang, Z.J.; Wang, A.C.; He, J.H.; Wang, Z.L.; Alshareef, H.N. MXene electrochemical microsupercapacitor integrated with triboelectric nanogenerator as a wearable self-charging power unit. Nano Energy 2018, 45, 266–272. ; Fourniols, J.-Y.; Escriba, C.; Campo, E. Smart wearable systems: Current status and future challenges. Artif 137–156. [CrossRef] [CrossRef] 17. Jiang, Q.; Wu, C.S.; Wang, Z.J.; Wang, A.C.; He, J.H.; Wang, Z.L.; Alshareef, H.N. MXene electrochemical microsupercapacitor integrated with triboelectric nanogenerator as a wearable self-charging power unit. Nano Energy 2018, 45, 266–272. [CrossRef] 18. Wang, A.C.; Hu, M.; Zhou, L.W.; Qiang, X.Y. Self-Powered Wearable Pressure Sensors with Enhanced Piezoelectric Properties of Aligned P(VDF-TrFE)/MWCNT Composites for Monitoring Human Physiological and Muscle Motion Signs. Nanomaterials 2018, J g, Q ; , ; g, J ; g, ; , J ; g, ; , p p integrated with triboelectric nanogenerator as a wearable self-charging power unit. Nano Energy 2018, 45, 266–272. [CrossRef] 18. Wang, A.C.; Hu, M.; Zhou, L.W.; Qiang, X.Y. Self-Powered Wearable Pressure Sensors with Enhanced Piezoelectric Properties of Aligned P(VDF-TrFE)/MWCNT Composites for Monitoring Human Physiological and Muscle Motion Signs. Nanomaterials 2018, 8, 1021. [CrossRef] 18. Wang, A.C.; Hu, M.; Zhou, L.W.; Qiang, X.Y. Self-Powered Wearable Pressure Sensors with Enhanced Piezoelectric Properties of Aligned P(VDF-TrFE)/MWCNT Composites for Monitoring Human Physiological and Muscle Motion Signs. Nanomaterials 2018, 8, 1021. [CrossRef] 19. Jung, Y.H.; Hong, S.K.; Wang, H.S.; Han, J.H.; Pham, T.X.; Park, H.; Kim, J.; Kang, S.; Yoo, C.D.; Lee, K.J. Flexible Piezoelectric Acoustic Sensors and Machine Learning for Speech Processing. Adv. Mater. 2020, 32, 1904020. [CrossRef] Y.H.; Hong, S.K.; Wang, H.S.; Han, J.H.; Pham, T.X.; Park, H.; Kim, J.; Kang, S.; Yoo, C.D.; Lee, K.J. Flexibl Jung, Y.H.; Hong, S.K.; Wang, H.S.; Han, J.H.; Pham, T.X.; Park, H.; Kim, J.; Kang, S.; Yoo, C.D.; Lee, K.J. Acoustic Sensors and Machine Learning for Speech Processing. Adv. Mater. 2020, 32, 1904020. [CrossRef] stic Sensors and Machine Learning for Speech Processing. Adv. Mater. 2020, 32, 1904020. [CrossRef] H.Y.; Pu, X.J.; Chen, J.; Meng, Y.; Yeh, M.-H.; Liu, G.L.; Tang, Q.; Chen, B.D.; Liu, D.; Qi, S.; et al. A hi d t ib l t i dit f i l b ti d h i id S i R b t 2018 3 t2516 [C 20. Guo, H.Y.; Pu, X.J.; Chen, J.; Meng, Y.; Yeh, M.-H.; Liu, G.L.; Tang, Q.; Chen, B.D.; Liu, D.; Qi, S.; et al. A highly sensitive, self-powered triboelectric auditory sensor for social robotics and hearing aids. Sci. Robot. 2018, 3, eaat2516. 38. Yang, Y.Q.; Sun, N.; Wen, Z.; Cheng, P.; Zheng, H.C.; Shao, H.Y.; Xia, Y.J.; Chen, C.; Lan, H.W.; Xie, X.K.; et al. Liquid-Metal-Based Super-Stretchable and Structure-Designable Triboelectric Nanogenerator for Wearable Electronics. ACS Nano 2018, 12, 2027–2034. [CrossRef] ; Fourniols, J.-Y.; Escriba, C.; Campo, E. Smart wearable systems: Current status and future challenges. Artif 137–156. [CrossRef] Yang, Y.Q.; Sun, N.; Wen, Z.; Cheng, P.; Zheng, H.C.; Shao, H.Y.; Xia, Y.J.; Chen, C.; Lan, H.W.; Xie, X.K.; et al. Liquid-Metal-Based Super-Stretchable and Structure-Designable Triboelectric Nanogenerator for Wearable Electronics. ACS Nano 2018, 12, 2027–2034. [CrossRef] 15 of 18 15 of 18 Micromachines 2021, 12, 666 39. Yang, Y.; Pan, H.; Xie, G.Z.; Jiang, Y.D.; Chen, C.X.; Su, Y.J.; Wang, Y.; Tai, H.L. Flexible piezoelectric pressure sensor based on polydopamine-modiﬁed BaTiO3/PVDF composite ﬁlm for human motion monitoring. Sens. Actuators A Phys. 2020, 301, 111789. [CrossRef] [ ] 40. Yang, L.; Zhao, Q.Y.; Chen, K.N.; Ma, Y.Z.; Wu, Y.P.; Ji, H.L.; Qiu, J.H. PVDF-Based Composition-Gradient Multilayered Nanocomposites for Flexible High-Performance Piezoelectric Nanogenerators. ACS Appl. Mater. Interfaces 2020, 12, 11045–11054. [CrossRef] [ ] 41. Yan, J.; Liu, M.; Jeong, Y.G.; Kang, W.M.; Li, L.; Zhao, Y.X.; Deng, N.P.; Cheng, B.W.; Yang, G. Performance enhancements in poly(vinylidene ﬂuoride)-based piezoelectric nanogenerators for efﬁcient energy harvesting. Nano Energy 2019, 56, 662–692. [CrossRef] 42. Yan, C.; Gao, Y.Y.; Zhao, S.L.; Zhang, S.L.; Zhou, Y.H.; Deng, W.L.; Li, Z.W.; Jiang, G.; Jin, L.; Tian, G.; et al. A linear-to-rotary hybrid nanogenerator for high-performance wearable biomechanical energy harvesting. Nano Energy 2020, 67, 104235. [CrossRef] 43 Xi Y Qi X H Qi C H Ti H Y Li Z B Ji Z J A W bl R i ti d P l M it i S t B d 42. Yan, C.; Gao, Y.Y.; Zhao, S.L.; Zhang, S.L.; Zhou, Y.H.; Deng, W.L.; Li, Z.W.; Jiang, G.; Jin, L.; Tian, G.; et al. A linear-to-rotary hybrid nanogenerator for high-performance wearable biomechanical energy harvesting. Nano Energy 2020, 67, 104235. [CrossRef] 43. Xin, Y.; Qi, X.H.; Qian, C.H.; Tian, H.Y.; Ling, Z.B.; Jiang, Z.J. A Wearable Respiration and Pulse Monitoring System Based on PVDF Piezoelectric Film. Integr. Ferroelectr. 2014, 158, 43–51. [CrossRef] 42. Yan, C.; Gao, Y.Y.; Zhao, S.L.; Zhang, S.L.; Zhou, Y.H.; Deng, W.L.; Li, Z.W.; Jiang, G.; Jin, L.; Tian hybrid nanogenerator for high-performance wearable biomechanical energy harvesting. Nano Energy y g g p gy g gy 43. Xin, Y.; Qi, X.H.; Qian, C.H.; Tian, H.Y.; Ling, Z.B.; Jiang, Z.J. A Wearable Respiration and Pulse Monitoring System Based on PVDF Piezoelectric Film. Integr. Ferroelectr. 2014, 158, 43–51. [CrossRef] g 44. Wen, F.; Sun, Z.; He, T.; Shi, Q.; Zhu, M.; Zhang, Z.; Li, L.; Zhang, T.; Lee, C. ; Fourniols, J.-Y.; Escriba, C.; Campo, E. Smart wearable systems: Current status and future challenges. Artif 137–156. [CrossRef] Machine Learning Glove Using Self-Powered Conductive Superhydrophobic Triboelectric Textile for Gesture Recognition in VR/AR Applications. Adv. Sci. 2020, 7, 2000261. [CrossRef] 45. Wang, R.X.; Mu, L.W.; Bao, Y.K.; Lin, H.; Ji, T.; Shi, Y.J.; Zhu, J.H.; Wu, W.Z. Holistically Engineered Polymer–Polymer and Polymer–Ion Interactions in Biocompatible Polyvinyl Alcohol Blends for High-Performance Triboelectric Devices in Self-Powered Wearable Cardiovascular Monitorings. Adv. Mater. 2020, 32, 2002878. [CrossRef] [PubMed] 45. Wang, R.X.; Mu, L.W.; Bao, Y.K.; Lin, H.; Ji, T.; Shi, Y.J.; Zhu, J.H.; Wu, W.Z. Holistically Engine Polymer–Ion Interactions in Biocompatible Polyvinyl Alcohol Blends for High-Performance Triboele g 46. Wang, J.; Li, X.H.; Zi, Y.L.; Wang, S.H.; Li, Z.L.; Zheng, L.; Yi, F.; Li, S.M.; Wang, Z.L. A Flexible Fiber-Based Supercapacitor- Triboelectric-Nanogenerator Power System for Wearable Electronics. Adv. Mater. 2015, 27, 4830–4836. [CrossRef] 47. Viola, G.; Chang, J.K.; Maltby, T.; Steckler, F.; Jomaa, M.; Sun, J.F.; Edusei, J.; Zhang, D.; Vilches, A.; Gao, S.; et al. Bioinspired Multiresonant Acoustic Devices Based on Electrospun Piezoelectric Polymeric Nanoﬁbers. ACS Appl. Mater. Interfaces 2020, 12, 34643–34657. [CrossRef] [PubMed] [ ] [ ] 48. Sun, Y.; Liu, Y.; Zheng, Y.D.; Li, Z.J.; Fan, J.; Wang, L.; Liu, X.Q.; Liu, J.; Shou, W. Enhanced Energy Harvesting Ability of ZnO/PAN Hybrid Piezoelectric Nanogenerators. ACS Appl. Mater. Interfaces 2020, 12, 54936–54945. [CrossRef] [PubMed] 49. Ahn, S.; Cho, Y.; Park, S.; Kim, J.; Sun, J.; Ahn, D.; Lee, M.; Kim, D.; Kim, T.; Shin, H.; et al. Wearable multimode sensors with ampliﬁed piezoelectricity due to the multi local strain using 3D textile structure for detecting human body signals. Nano Energy 2020, 74, 104932. [CrossRef] 50. Sun, J.; Guo, H.Y.; Ribera, J.; Wu, C.S.; Tu, K.K.; Binelli, M.; Panzarasa, G.; Schwarze, F.W.M.R.; Wang, Z.L.; Burgert, I. Sustainable and Biodegradable Wood Sponge Piezoelectric Nanogenerator for Sensing and Energy Harvesting Applications. ACS Nano 2020, 14, 14665–14674. [CrossRef] [PubMed] 51. Su, Y.J.; Wang, J.J.; Wang, B.; Yang, T.N.; Yang, B.X.; Xie, G.Z.; Zhou, Y.H.; Zhang, S.L.; Tai, H.L.; Cai, Z.X.; et al. Alveolus-Inspired Active Membrane Sensors for Self-Powered Wearable Chemical Sensing and Breath Analysis. ACS Nano 2020, 14, 6067–6075. [CrossRef] [PubMed] [ ] [ ] 52. Su, Y.J.; Chen, C.X.; Pan, H.; Yang, Y.; Chen, G.R.; Zhao, X.; Li, W.X.; Gong, Q.C.; Xie, G.Z.; Zhou, Y.H.; et al. Muscle Fibers Inspired High-Performance Piezoelectric Textiles for Wearable Physiological Monitoring. Adv. Funct. Mater. 2021, 31, 2010962. [CrossRef] [ ] 53. Su, M.; Brugger, J.; Kim, B. ; Fourniols, J.-Y.; Escriba, C.; Campo, E. Smart wearable systems: Current status and future challenges. Artif 137–156. [CrossRef] IEEE 2008, 96, 1457–1486. [CrossRef] 63. Maity, K.; Garain, S.; Henkel, K.; Schmeißer, D.; Mandal, D. Self-Powered Human-Health Monitoring through Aligned PVDF Nanoﬁbers Interfaced Skin-Interactive Piezoelectric Sensor ACS Appl Polym Mater 2020 2 862–878 [CrossRef] 63. Maity, K.; Garain, S.; Henkel, K.; Schmeißer, D.; Mandal, D. Self-Powered Human-Health Monitoring through Aligned PVDF 63. Maity, K.; Garain, S.; Henkel, K.; Schmeißer, D.; Mandal, D. Self Powered Human Health Monitoring Nanoﬁbers Interfaced Skin-Interactive Piezoelectric Sensor. ACS Appl. Polym. Mater. 2020, 2, 862–878. y g g ﬁbers Interfaced Skin-Interactive Piezoelectric Sensor. ACS Appl. Polym. Mater. 2020, 2, 862–878. [CrossRef] hatta, T.; Cho, H.; Hui, X.; Park, C.; Yoon, S.; Salauddin, M.; Rahman, M.T.; Rana, S.M.S.; Park, J.Y. A F 64. Maharjan, P.; Bhatta, T.; Cho, H.; Hui, X.; Park, C.; Yoon, S.; Salauddin, M.; Rahman, M.T.; Rana, S.M.S.; Park, J.Y. A Fully Functional Universal Self-Chargeable Power Module for Portable/Wearable Electronics and Self-Powered IoT Applications. Adv. Energy Mater. 2020, 10, 2002782. [CrossRef] gy 65. Ma, L.Y.; Zhou, M.J.; Wu, R.H.; Patil, A.; Gong, H.; Zhu, S.H.; Wang, T.T.; Zhang, Y.F.; Shen, S.; Dong, K.; et al. Continuous and Scalable Manufacture of Hybridized Nano-Micro Triboelectric Yarns for Energy Harvesting and Signal Sensing. ACS Nano 2020, 14, 4716–4726. [CrossRef] [PubMed] 66. Lu, L.J.; Ding, W.Q.; Liu, J.Q.; Yang, B. Flexible PVDF based piezoelectric nanogenerators. Nano Energy 2020, 78, 105251. [CrossRef] 67. Liu, Z.R.; Nie, J.H.; Miao, B.; Li, J.D.; Cui, Y.B.; Wang, S.; Zhang, X.D.; Zhao, G.R.; Deng, Y.B.; Wu, Y.H.; et al. Self-Powered Intracellular Drug Delivery by a Biomechanical Energy-Driven Triboelectric Nanogenerator. Adv. Mater. 2019, 31, 1807795. [CrossRef] [PubMed] 68. Cheng, X.L.; Meng, B.; Zhang, X.S.; Han, M.D.; Su, Z.M.; Zhang, H.X. Wearable electrode-free triboelectric generator for harvesting biomechanical energy. Nano Energy 2015, 12, 19–25. [CrossRef] 69. Lin, Z.M.; Wu, Z.Y.; Zhang, B.B.; Wang, Y.C.; Guo, H.Y.; Liu, G.L.; Chen, C.Y.; Chen, Y.L.; Yang, J.; Wang, Z.L. A Triboelectric Nanogenerator-Based Smart Insole for Multifunctional Gait Monitoring. Adv. Mater. Technol. 2019, 4, 1800360. [CrossRef] 70. Li, T.; Feng, Z.Q.; Qu, M.H.; Yan, K.; Yuan, T.; Gao, B.B.; Wang, T.; Dong, W.; Zheng, J. Core/Shell Piezoelectric Nanoﬁbers with Spatial Self-Orientated Beta-Phase Nanocrystals for Real-Time Micropressure Monitoring of Cardiovascular Walls. ACS Nano 2019, 13, 10062–10073. [CrossRef] [PubMed] 71. Lee, M.; Chen, C.Y.; Wang, S.; Cha, S.N.; Park, Y.J.; Kim, J.M.; Chou, L.J.; Wang, Z.L. A Hybrid Piezoelectric Structure for Wearable Nanogenerators. Adv. Mater. 2012, 24, 1759. [CrossRef] 72. ; Fourniols, J.-Y.; Escriba, C.; Campo, E. Smart wearable systems: Current status and future challenges. Artif 137–156. [CrossRef] Simply Structured Wearable Triboelectric Nanogenerator Based on a Hybrid Composition of Carbon Nanotubes and Polymer Layer. Int. J. Precis. Eng. Manuf. Technol. 2020, 7, 683–698. [CrossRef] 54. Song, Y.; Min, J.H.; Yu, Y.; Wang, H.B.; Yang, Y.R.; Zhang, H.X.; Gao, W. Wireless battery-free wearable sweat sensor powered by human motion. Sci. Adv. 2020, 6, eaay9842. [CrossRef] [PubMed] y 55. Shi, Q.F.; Lee, C.K. Self-Powered Bio-Inspired Spider-Net-Coding Interface Using Single-Electrode Trib Adv. Sci. 2019, 6, 1900617. [CrossRef] Self-Powered Bio-Inspired Spider-Net-Coding Interface Using Single-Electrode Triboelectric Nanogenerator 00617. [CrossRef] 56. Salauddin, M.; Rana, S.M.S.; Sharifuzzaman, M.; Rahman, M.T.; Park, C.; Cho, H.; Maharjan, P.; Bhatta, T.; Park, J.Y. A Novel MXene/Ecoﬂex Nanocomposite-Coated Fabric as a Highly Negative and Stable Friction Layer for High-Output Triboelectric Nanogenerators. Adv. Energy Mater. 2021, 11, 2002832. [CrossRef] g gy 57. Pu, X.; Liu, M.M.; Chen, X.Y.; Sun, J.M.; Du, C.H.; Zhang, Y.; Zhai, J.Y.; Hu, W.G.; Wang, Z.L. Ultrastretchable, transparent triboelectric nanogenerator as electronic skin for biomechanical energy harvesting and tactile sensing. Sci. Adv. 2017, 3, e1700015. [CrossRef] [PubMed] 58. Peng, X.; Dong, K.; Ye, C.Y.; Jiang, Y.; Zhai, S.Y.; Cheng, R.W.; Liu, D.; Gao, X.P.; Wang, J.; Wang, Z.L. A breathable, biodegradable, antibacterial, and self-powered electronic skin based on all-nanoﬁber triboelectric nanogenerators. Sci. Adv. 2020, 6, eaba9624. [CrossRef] [PubMed] [ ] [ ] 59. Park, C.; Koo, M.; Song, G.; Cho, S.M.; Kang, H.S.; Park, T.H.; Kim, E.H.; Park, C. Surface-Conformal Triboelectric Nanopores via Supramolecular Ternary Polymer Assembly ACS Nano 2020 14 755 766 [CrossRef] [PubMed] 59. Park, C.; Koo, M.; Song, G.; Cho, S.M.; Kang, H.S.; Park, T.H.; Kim, E.H.; Park, C. Surface-Conformal Triboelectric Nanopores via Supramolecular Ternary Polymer Assembly. ACS Nano 2020, 14, 755–766. [CrossRef] [PubMed] g g Supramolecular Ternary Polymer Assembly. ACS Nano 2020, 14, 755–766. [CrossRef] [PubMed] 60. Pan, C.F.; Liu, D.Y.; Ford, M.J.; Majidi, C. Ultrastretchable, Wearable Triboelectric Nanogenerator Based on Sedimented Liquid Metal Elastomer Composite. Adv. Mater. Technol. 2020, 5, 2000754. [CrossRef] p 61. Niu, Q.Q.; Huang, L.; Lv, S.S.; Shao, H.L.; Fan, S.N.; Zhang, Y.P. Pulse-driven bio-triboelectric nanogenerator based on silk nanoribbons. Nano Energy 2020, 74, 104837. [CrossRef] 61. Niu, Q.Q.; Huang, L.; Lv, S.S.; Shao, H.L.; Fan, S.N.; Zh nanoribbons. Nano Energy 2020, 74, 104837. [CrossRef] 16 of 18 Micromachines 2021, 12, 666 62. Mitcheson, P.D.; Yeatman, E.M.; Rao, G.K.; Holmes, A.S.; Green, T.C. Energy Harvesting from Human and Machine Motion for Wireless Electronic Devices. Proc. 86. Dagdeviren, C.; Shi, Y.; Joe, P.; Ghaffari, R.; Balooch, G.; Usgaonkar, K.; Gur, O.; Tran, P.L.; Crosby, J.R.; Meyer, M.; et al. Conformal piezoelectric systems for clinical and experimental characterization of soft tissue biomechanics. Nat. Mater. 2015, 14, 728–736. [CrossRef] [PubMed] ; Fourniols, J.-Y.; Escriba, C.; Campo, E. Smart wearable systems: Current status and future challenges. Artif 137–156. [CrossRef] Kim, J.; Byun, S.; Lee, S.; Ryu, J.; Cho, S.; Oh, C.; Kim, H.; No, K.; Ryu, S.; Lee, Y.M.; et al. Cost-effective and strongly integrated fabric-based wearable piezoelectric energy harvester. Nano Energy 2020, 75, 104992. [CrossRef] 73. Khurana, V.; Kisannagar, R.R.; Domala, S.S.; Gupta, D. In Situ Polarized Ultrathin PVDF Film-Based Flexible Piezoelectric Nanogenerators. ACS Appl. Electron. Mater. 2020, 2, 3409–3417. [CrossRef] Dong, K.; Li, X.; An, J.; Wu, D.Q.; Peng, X.; Yi, J.; Ning, C.; Cheng, R.W.; Yu, P.T.; et al. Stretchable, Was 74. Jiang, Y.; Dong, K.; Li, X.; An, J.; Wu, D.Q.; Peng, X.; Yi, J.; Ning, C.; Cheng, R.W.; Yu, P.T.; et al. Stretchable, Washable, and Ultrathin Triboelectric Nanogenerators as Skin-Like Highly Sensitive Self-Powered Haptic Sensors. Adv. Funct. Mater. 2021, 31, 2005584. [CrossRef] 75. Jeong, S.-H.; Lee, Y.; Lee, M.-G.; Song, W.J.; Park, J.-U.; Sun, J.-Y. Accelerated wound healing with an ionic patch assisted by a triboelectric nanogenerator. Nano Energy 2021, 79, 105463. [CrossRef] 76. Guan, X.Y.; Xu, B.G.; Gong, J.L. Hierarchically architected polydopamine modiﬁed BaTiO3@P(VD mats for ﬂexible piezoelectric nanogenerators and self-powered sensors. Nano Energy 2020, 70, 104 p g p 77. Gu, L.; Liu, J.M.; Cui, N.Y.; Xu, Q.; Du, T.; Zhang, L.; Wang, Z.; Long, C.B.; Qin, Y. Enhancing the current density of a piezoelectric nanogenerator using a three-dimensional intercalation electrode. Nat. Commun. 2020, 11, 1030. [CrossRef] [PubMed] g g [ ] [ ] 78. Fan, W.J.; He, Q.; Meng, K.Y.; Tan, X.L.; Zhou, Z.H.; Zhang, G.Q.; Yang, J.; Wang, Z.L. Machine-knitted washable sensor array textile for precise epidermal physiological signal monitoring. Sci. Adv. 2020, 6, eaay2840. [CrossRef] [PubMed] 79. Du, S.; Zhou, N.Y.; Gao, Y.J.; Xie, G.; Du, H.Y.; Jiang, H.; Zhang, L.B.; Tao, J.; Zhu, J.T. Bioinspired hybrid p hydrogel and piezoelectric nanogenerator for promoting skin wound healing. Nano Res. 2020, 13, 2525 79. Du, S.; Zhou, N.Y.; Gao, Y.J.; Xie, G.; Du, H.Y.; Jiang, H.; Zhang, L.B.; Tao, J.; Zhu, J.T. Bioinspired hybrid patches with self-adhesive hydrogel and piezoelectric nanogenerator for promoting skin wound healing. Nano Res. 2020, 13, 2525–2533. [CrossRef] 80. Choudhry, I.; Khalid, H.R.; Lee, H.-K. Flexible Piezoelectric Transducers for Energy Harvesting and Sensing from Human hydrogel and piezoelectric nanogenerator for promoting skin wound healing. Nano Res. 2020, 13, 2525–2533. [CrossRef] 80. Choudhry, I.; Khalid, H.R.; Lee, H.-K. Flexible Piezoelectric Transducers for Energy Harvesting and Sensing from Human Kinematics. ACS Appl. Electron. Mater. 2020, 2, 3346–3357. ; Fourniols, J.-Y.; Escriba, C.; Campo, E. Smart wearable systems: Current status and future challenges. Artif 137–156. [CrossRef] In Vivo Self-Powered Wireless Cardiac Monitoring via Implantable Triboelectric Nanogenerator. ACS Nano 2016, 10, 6510–6518. [CrossRef] [PubMed] g p g [ ] [ ] 88. Li, Z.; Feng, H.Q.; Zheng, Q.; Li, H.; Zhao, C.C.; Ouyang, H.; Noreen, S.; Yu, M.; Su, F.; Liu, R.P.; et al. Photothermally tunable g p g [ ] [ ] 88. Li, Z.; Feng, H.Q.; Zheng, Q.; Li, H.; Zhao, C.C.; Ouyang, H.; Noreen, S.; Yu, M.; Su, F.; Liu, R.P.; et al. Photothermally tunable biodegradation of implantable triboelectric nanogenerators for tissue repairing. Nano Energy 2018, 54, 390–399. [CrossRef] 88. Li, Z.; Feng, H.Q.; Zheng, Q.; Li, H.; Zhao, C.C.; Ouyang, H.; Noreen, S.; Yu, M.; Su, F.; Liu, R.P.; et al. Photothermally tunable bi d d i f i l bl ib l i f i i i N E 2018 54 390 399 [C R f] g p g p g gy 89. Tian, J.J.; Shi, R.; Liu, Z.; Ouyang, H.; Yu, M.; Zhao, C.C.; Zou, Y.; Jiang, D.J.; Zhang, J.S.; Li, Z. Self-powered implantable electrical stimulator for osteoblasts’ proliferation and differentiation. Nano Energy 2019, 59, 705–714. [CrossRef] 89. Tian, J.J.; Shi, R.; Liu, Z.; Ouyang, H.; Yu, M.; Zhao, C.C.; Zou, Y.; Jiang, D.J.; Zhang, J.S.; Li, Z. Self-powe stimulator for osteoblasts’ proliferation and differentiation. Nano Energy 2019, 59, 705–714. [CrossRef] , J J , , y g, , , , J g, J g, J , p p stimulator for osteoblasts’ proliferation and differentiation. Nano Energy 2019, 59, 705–714. [CrossRef] y g J g J g J p p r osteoblasts’ proliferation and differentiation. Nano Energy 2019, 59, 705–714. [CrossRef] p gy 90. Zheng, Q.; Shi, B.J.; Fan, F.R.; Wang, X.X.; Yan, L.; Yuan, W.W.; Wang, S.H.; Liu, H.; Li, Z.; Wang, Z.L. In Vivo Powering of Pacemaker by Breathing-Driven Implanted Triboelectric Nanogenerator. Adv. Mater. 2014, 26, 5851–5856. [CrossRef] g, Q ; , J ; , ; g, ; , ; , ; g, ; , ; , ; g, g Pacemaker by Breathing-Driven Implanted Triboelectric Nanogenerator. Adv. Mater. 2014, 26, 5851–5856. [CrossRef] 91. Yang, J.; Chen, J.; Su, Y.J.; Jing, Q.S.; Li, Z.L.; Yi, F.; Wen, X.N.; Wang, Z.N.; Wang, Z.L. Eardrum-Inspired Active Sensors for Self-Powered Cardiovascular System Characterization and Throat-Attached Anti-Interference Voice Recognition. Adv. Mater. 2015, 27, 1316–1326. [CrossRef] [PubMed] 92. ; Fourniols, J.-Y.; Escriba, C.; Campo, E. Smart wearable systems: Current status and future challenges. Artif 137–156. [CrossRef] Li, H.; Zhang, X.; Zhao, L.M.; Jiang, D.J.; Xu, L.L.; Liu, Z.; Wu, Y.X.; Hu, K.; Zhang, M.R.; Wang, J.X.; et al. A Hybrid Biofuel and Triboelectric Nanogenerator for Bioenergy Harvesting. Nano Micro Lett. 2020, 12, 50. [CrossRef] g gy g 93. Zheng, Q.; Zou, Y.; Zhang, Y.L.; Liu, Z.; Shi, B.J.; Wang, X.X.; Jin, Y.M.; Ouyang, H.; Li, Z.; Wang, Z.L. Biodegradable triboelectric nanogenerator as a life-time designed implantable power source. Sci. Adv. 2016, 2, e1501478. [CrossRef] [PubMed] 93. Zheng, Q.; Zou, Y.; Zhang, Y.L.; Liu, Z.; Shi, B.J.; Wang, X.X.; Jin, Y.M.; Ouyang, H.; Li, Z.; Wang, Z.L. Biodegradable triboelectric nanogenerator as a life-time designed implantable power source. Sci. Adv. 2016, 2, e1501478. [CrossRef] [PubMed] g g p p 94. Zhao, C.C.; Feng, H.Q.; Zhang, L.J.; Li, Z.; Zou, Y.; Tan, P.C.; Ouyang, H.; Jiang, D.J.; Yu, M.; Wang Vivo Cancer Therapy by an Implantable Magnet Triboelectric Nanogenerator. Adv. Funct. Mater. 20 Feng, H.Q.; Zhang, L.J.; Li, Z.; Zou, Y.; Tan, P.C.; Ouyang, H.; Jiang, D.J.; Yu, M.; Wang, C.; et al. Highly E Vivo Cancer Therapy by an Implantable Magnet Triboelectric Nanogenerator. Adv. Funct. Mater. 2019, 29, 1808640. [CrossRef] 95. Zhang, X.-S.; Han, M.-D.; Wang, R.-X.; Zhu, F.-Y.; Li, Z.-H.; Wang, W.; Zhang, H.X. Frequency-Multiplication High-Output Triboelectric Nanogenerator for Sustainably Powering Biomedical Microsystems. Nano Lett. 2013, 13, 1168–1172. [CrossRef] [PubMed] 96. Yi, Z.R.; Xie, F.; Tian, Y.W.; Li, N.; Dong, X.X.; Ma, Y.; Huang, Y.; Hu, Y.L.; Xu, X.B.; Qu, D.; et Heart-Worn Pacemaker Strategy. Adv. Funct. Mater. 2020, 30, 2000477. [CrossRef] 97. Xu, Z.; Jin, C.R.; Cabe, A.; Escobedo, D.; Gruslova, A.; Jenney, S.; Closson, A.B.; Dong, L.; Chen, Z.; Feldman, M.D.; et al. Implantable Cardiac Kirigami-Inspired Lead-Based Energy Harvester Fabricated by Enhanced Piezoelectric Composite Film. Adv. Heal. Mater. 2021, 10, e2002100. [CrossRef] 98. Wang, J.H.; Wang, H.; He, T.Y.Y.; He, B.R.; Thakor, N.V.; Lee, C. Investigation of Low-Current Direct Stimulation for Rehabilitation Treatment Related to Muscle Function Loss Using Self-Powered TENG System. Adv. Sci. 2019, 6, 1900149. [CrossRef] [PubMed] 99. Shi, R.; Zhang, J.S.; Tian, J.J.; Zhao, C.C.; Li, Z.; Zhang, Y.Z.; Li, Y.S.; Wu, C.G.; Tian, W.; Li, Z. An effective self-powered strategy to endow titanium implant surface with associated activity of anti-bioﬁlm and osteogenesis. Nano Energy 2020, 77, 105201. [CrossRef] 98. Wang, J.H.; Wang, H.; He, T.Y.Y.; He, B.R.; Thakor, N.V.; Lee, C. 110. Hwang, G.-T.; Park, H.; Lee, J.-H.; Oh, S.; Park, K.-I.; Byun, M.; Park, H.; Ahn, G.; Jeong, C.K.; No, K.; et al. Self-Powered Cardiac Pacemaker Enabled by Flexible Single Crystalline PMN-PT Piezoelectric Energy Harvester. Adv. Mater. 2014, 26, 4880–4887. [CrossRef] ; Fourniols, J.-Y.; Escriba, C.; Campo, E. Smart wearable systems: Current status and future challenges. Artif 137–156. [CrossRef] [CrossRef] 80. Choudhry, I.; Khalid, H.R.; Lee, H.-K. Flexible Piezoelectric Transducers for Energy Harvestin Kinematics. ACS Appl. Electron. Mater. 2020, 2, 3346–3357. [CrossRef] pp 81. Chen, C.Y.; Guo, H.Y.; Chen, L.J.; Wang, Y.C.; Pu, X.J.; Yu, W.D.; Wang, F.M.; Du, Z.Q.; Wang, Z. Triboelectric Nanogenerator for Biomotion Energy Harvesting. ACS Nano 2020, 14, 4585–4594. [CrossR 82. Cao, R.; Pu, X.J.; Du, X.Y.; Yang, W.; Wang, J.N.; Guo, H.Y.; Zhao, S.Y.; Yuan, Z.Q.; Zhang, C.; Li, C.J.; et al. Screen-Printed Washable Electronic Textiles as Self-Powered Touch/Gesture Tribo-Sensors for Intelligent Human–Machine Interaction. ACS Nano 2018, 12, 5190–5196. [CrossRef] [PubMed] 83. Sun, R.J.; Carreira, S.C.; Chen, Y.; Xiang, C.Q.; Xu, L.L.; Zhang, B.; Chen, M.D.; Farrow, I.; Scarpa, F.; Rossiter, J. Stretchable Piezoelectric Sensing Systems for Self-Powered and Wireless Health Monitoring. Adv. Mater. Technol. 2019, 4, 1900100. [CrossRef] 84. Jin, C.R.; Dong, L.; Xu, Z.; Closson, A.; Cabe, A.; Gruslova, A.; Jenney, S.; Escobedo, D.; Elliott, J.; Zhang, M.; et al. Skin-like Elastomer Embedded Zinc Oxide Nanoarrays for Biomechanical Energy Harvesting. Adv. Mater. Interfaces 2021, 8, 2100094. [CrossRef] 83. Sun, R.J.; Carreira, S.C.; Chen, Y.; Xiang, C.Q.; Xu, L.L.; Zhang, B.; Chen, M.D.; Farrow, I.; Scarpa, F.; Rossiter, J. Stretchable Piezoelectric Sensing Systems for Self-Powered and Wireless Health Monitoring. Adv. Mater. Technol. 2019, 4, 1900100. [CrossRef] 84. Jin, C.R.; Dong, L.; Xu, Z.; Closson, A.; Cabe, A.; Gruslova, A.; Jenney, S.; Escobedo, D.; Elliott, J.; Zhang, M.; et al. Skin-like 84. Jin, C.R.; Dong, L.; Xu, Z.; Closson, A.; Cabe, A.; Gruslova, A.; Jenney, S.; Escobedo, D.; Elliott, J.; Zhang, M.; et al. Skin-like Elastomer Embedded Zinc Oxide Nanoarrays for Biomechanical Energy Harvesting. Adv. Mater. Interfaces 2021, 8, 2100094. [CrossRef] 85. Das, R.; Curry, E.J.; Le, T.T.; Awale, G.; Liu, Y.; Li, S.Y.; Contreras, J.; Bednarz, C.; Millender, J.; Xin, X.; et al. Biodegradable nanoﬁber bone-tissue scaffold as remotely-controlled and self-powering electrical stimulator. Nano Energy 2020, 76, 105028. [CrossRef] 86. Dagdeviren, C.; Shi, Y.; Joe, P.; Ghaffari, R.; Balooch, G.; Usgaonkar, K.; Gur, O.; Tran, P.L.; Crosby, J.R.; Meyer, M.; et al. Conformal piezoelectric systems for clinical and experimental characterization of soft tissue biomechanics. Nat. Mater. 2015, 14, 728–736. [CrossRef] [PubMed] 17 of 18 17 of 18 Micromachines 2021, 12, 666 87. Zheng, Q.; Zhang, H.; Shi, B.J.; Xue, X.; Liu, Z.; Jin, Y.M.; Ma, Y.; Zou, Y.; Wang, X.X.; An, Z.; et al. ; Fourniols, J.-Y.; Escriba, C.; Campo, E. Smart wearable systems: Current status and future challenges. Artif 137–156. [CrossRef] Investigation of Low-Current Direct Stimulation for Rehabilitation Treatment Related to Muscle Function Loss Using Self-Powered TENG System. Adv. Sci. 2019, 6, 1900149. [CrossRef] [PubMed] 99 Shi R Zh J S Ti J J Zh C C Li Z Zh YZ Li YS W C G Ti W Li Z A ff ti lf d t t g y [ ] [ ] 99. Shi, R.; Zhang, J.S.; Tian, J.J.; Zhao, C.C.; Li, Z.; Zhang, Y.Z.; Li, Y.S.; Wu, C.G.; Tian, W.; Li, Z. An effective self-powered strategy to endow titanium implant surface with associated activity of anti-bioﬁlm and osteogenesis. Nano Energy 2020, 77, 105201. [CrossRef] 100. Qian, Y.; Cheng, Y.; Song, J.L.; Xu, Y.; Yuan, W.E.; Fan, C.Y.; Zheng, X.Y. Mechano-Informed Biomimetic Polymer Scaffolds by Incorporating Self-Powered Zinc Oxide Nanogenerators Enhance Motor Recovery and Neural Function. Small 2020, 16, 2000796. [CrossRef] [PubMed] 101. Ouyang, H.; Liu, Z.; Li, N.; Shi, B.J.; Zou, Y.; Xie, F.; Ma, Y.; Li, Z.; Li, H.; Zheng, Q.; et al. Symbiotic cardiac pacemaker. Nat. Commun. 2019, 10, 1821. [CrossRef] 102. Song, P.; Kuang, S.; Panwar, N.; Yang, G.; Tng, D.J.H.; Tjin, S.C.; Ng, W.J.; Majid, M.B.A.; Zhu, G.; Yong, K.-T.; et al. A Self-Powered Implantable Drug-Delivery System Using Biokinetic Energy. Adv. Mater. 2017, 29, 1605668. [CrossRef] p g y y g gy 103. Lu, H.J.; Hong, Y.; Yang, Y.Y.; Yang, Z.B.; Shen, Y.J. Battery-Less Soft Millirobot That Can Mov Remotely by Coupling the Magnetic and Piezoelectric Effects. Adv. Sci. 2020, 7, 2000069. [CrossRef u, H.J.; Hong, Y.; Yang, Y.Y.; Yang, Z.B.; Shen, Y.J. Battery Less Soft Millirobot That Can Move, Sense, a motely by Coupling the Magnetic and Piezoelectric Effects. Adv. Sci. 2020, 7, 2000069. [CrossRef] 104. Liu, Z.; Xu, L.L.; Zheng, Q.; Kang, Y.; Shi, B.J.; Jiang, D.J.; Li, H.; Qu, X.C.; Fan, Y.B.; Wang, Z.L.; et al. Human Motion Driven Self-Powered Photodynamic System for Long-Term Autonomous Cancer Therapy. ACS Nano 2020, 14, 8074–8083. [CrossRef] [PubMed] 105. Liu, Z.; Ma, Y.; Ouyang, H.; Shi, B.J.; Li, N.; Jiang, D.J.; Xie, F.; Qu, D.; Zou, Y.; Huang, Y.; et al. Ultrasensitive Endocardial Pressure Sensor. Adv. Funct. Mater. 2019, 29, 1807560. [CrossRef] Ultrasensitive Endocardial Pressure Sensor. Adv. Funct. Mater. 2019, 29, 1807560. [CrossRef] 106. Li, N.; Yi, Z.R.; Ma, Y.; Xie, F.; Huang, Y.; Tian, Y.W.; Dong, X.X.; Liu, Y.; Shao, X.; Jin, L.; et al. ; Fourniols, J.-Y.; Escriba, C.; Campo, E. Smart wearable systems: Current status and future challenges. Artif 137–156. [CrossRef] Implantable Self-Powered Low-Level Laser Cure System for Mouse Embryonic Osteoblasts’ Proliferation and Differentiation ACS Nano 2015 9 7867 7873 [CrossRef] [PubMed] 114. Dong, L.; Wen, C.S.; Liu, Y.; Xu, Z.; Closson, A.B.; Han, X.M.; Escobar, G.P.; Oglesby, M.; Feldman, M.; Chen, Z.; et al. Piezoelectric Buckled Beam Array on a Pacemaker Lead for Energy Harvesting. Adv. Mater. Technol. 2019, 4, 1800335. [CrossRef] 115. Li, J.; Kang, L.; Long, Y.; Wei, H.; Yu, Y.H.; Wang, Y.H.; Ferreira, C.A.; Yao, G.; Zhang, Z.Y.; Carlos, C.; et al. Implanted Battery-Free Direct-Current Micro-Power Supply from in Vivo Breath Energy Harvesting. ACS Appl. Mater. Interfaces 2018, 10, 42030–42038. [CrossRef] [PubMed] 116. Dong, L.; Closson, A.B.; Oglesby, M.; Escobedo, D.; Han, X.M.; Nie, Y.; Huang, S.C.; Feldman, M.D.; Chen, Z.; Zhang, J.X.J. In vivo cardiac power generation enabled by an integrated helical piezoelectric pacemaker lead. Nano Energy 2019, 66, 104085. [CrossRef] 117. Dagdeviren, C.; Yang, B.D.; Su, Y.W.; Tran, P.L.; Joe, P.; Anderson, E.; Xia, J.; Doraiswamy, V.; Dehdashti, B.; Feng, X.; et al. 116. Dong, L.; Closson, A.B.; Oglesby, M.; Escobedo, D.; Han, X.M.; Nie, Y.; Huang, S.C.; Feldman, M.D.; Chen, Z.; Zhang, J.X.J. In vivo cardiac power generation enabled by an integrated helical piezoelectric pacemaker lead. Nano Energy 2019, 66, 104085. [CrossRef] 117. Dagdeviren, C.; Yang, B.D.; Su, Y.W.; Tran, P.L.; Joe, P.; Anderson, E.; Xia, J.; Doraiswamy, V.; Dehdashti, B.; Feng, X.; et al. Conformal piezoelectric energy harvesting and storage from motions of the heart, lung, and diaphragm. Proc. Natl. Acad. Sci. USA 2014, 111, 1927–1932. [CrossRef] p g y g p p gy , , [ ] 117. Dagdeviren, C.; Yang, B.D.; Su, Y.W.; Tran, P.L.; Joe, P.; Anderson, E.; Xia, J.; Doraiswamy, V.; Dehdashti, B.; Feng, X.; et al. Conformal piezoelectric energy harvesting and storage from motions of the heart, lung, and diaphragm. Proc. Natl. Acad. Sci. USA 2014, 111, 1927–1932. [CrossRef] 118. Curry, E.J.; Le, T.T.; Das, R.; Ke, K.; Santorella, E.M.; Paul, D.; Chorsi, M.T.; Tran, K.T.M.; Baroody, J.; Borges, E.R.; et al. Biodegradable nanoﬁber-based piezoelectric transducer. Proc. Natl. Acad. Sci. USA 2020, 117, 214–220. [CrossRef] 119. Cheng, X.L.; Xue, X.; Ma, Y.; Han, M.D.; Zhang, W.; Xu, Z.Y.; Zhang, H.; Zhang, H.X. Implantable and self-powered blood pressure monitoring based on a piezoelectric thinﬁlm: Simulated, in vitro and in vivo studies. Nano Energy 2016, 22, 453–460. [CrossRef] 120. ; Fourniols, J.-Y.; Escriba, C.; Campo, E. Smart wearable systems: Current status and future challenges. Artif 137–156. [CrossRef] Direct Powering a Real Cardiac Pacemaker by Natural Energy of a Heartbeat. ACS Nano 2019, 13, 2822–2830. [CrossRef] 106. Li, N.; Yi, Z.R.; Ma, Y.; Xie, F.; Huang, Y.; Tian, Y.W.; Dong, X.X.; Liu, Y.; Shao, X.; Jin, L.; et al. Dir Pacemaker by Natural Energy of a Heartbeat. ACS Nano 2019, 13, 2822–2830. [CrossRef] 107. Lee, T.I.; Lee, S.; Lee, E.; Sohn, S.; Lee, Y.; Lee, S.; Moon, G.; Kim, D.; Kim, Y.S.; Myoung, J.M.; et al. High-Power Density Piezoelectric Energy Harvesting Using Radially Strained Ultrathin Trigonal Tellurium Nanowire Assembly. Adv. Mater. 2013, 25, 2920–2925. [CrossRef] 108. Lee, S.; Wang, H.; Peh, W.Y.X.; He, T.Y.Y.; Yen, S.-C.; Thakor, N.V.; Lee, C. Mechano-neuromodulation of autonomic pelvic nerve for underactive bladder: A triboelectric neurostimulator integrated with ﬂexible neural clip interface. Nano Energy 2019, 60, 449–456. [CrossRef] [ ] 109. Kim, D.H.; Shin, H.J.; Lee, H.; Jeong, C.K.; Park, H.; Hwang, G.-T.; Lee, H.-Y.; Joe, D.J.; Han, J.H.; Lee, S.H.; et al. In Vivo Self-Powered Wireless Transmission Using Biocompatible Flexible Energy Harvesters. Adv. Funct. Mater. 2017, 27, 1700341. [CrossRef] 110. Hwang, G.-T.; Park, H.; Lee, J.-H.; Oh, S.; Park, K.-I.; Byun, M.; Park, H.; Ahn, G.; Jeong, C.K.; No, K.; et al. Self-Powered Cardiac Pacemaker Enabled by Flexible Single Crystalline PMN-PT Piezoelectric Energy Harvester. Adv. Mater. 2014, 26, 4880–4887. [CrossRef] 18 of 18 18 of 18 Micromachines 2021, 12, 666 111. Hinchet, R.; Yoon, H.-J.; Ryu, H.; Kim, M.-K.; Choi, E.-K.; Kim, D.-S.; Kim, S.-W. Transcutaneous ultrasound energy harvesting using capacitive triboelectric technology. Science 2019, 365, 491–494. [CrossRef] g p gy 112. Gil, B.; Li, B.; Gao, A.Z.; Yang, G.-Z. Miniaturized Piezo Force Sensor for a Medical Catheter and Implantable Device. ACS Appl. Electron. Mater. 2020, 2, 2669–2677. [CrossRef] [PubMed] Tang, W.; Tian, J.; Zheng, Q.; Yan, L.; Wang, J.; Li, Z.; Wang, Z.L. Implantable Self-Powered Low-Level L Mouse Embryonic Osteoblasts’ Proliferation and Differentiation ACS Nano 2015 9 7867 7873 [CrossRef] 113. Tang, W.; Tian, J.; Zheng, Q.; Yan, L.; Wang, J.; Li, Z.; Wang, Z.L. Implantable Self-Powered Low-Level Laser Cure System for Mouse Embryonic Osteoblasts’ Proliferation and Differentiation. ACS Nano 2015, 9, 7867–7873. [CrossRef] [PubMed] .; Zheng, Q.; Yan, L.; Wang, J.; Li, Z.; Wang, Z.L. Implantable Self-Powered Low-Level Laser Cure System 113. Tang, W.; Tian, J.; Zheng, Q.; Yan, L.; Wang, J.; Li, Z.; Wang, Z.L. ; Fourniols, J.-Y.; Escriba, C.; Campo, E. Smart wearable systems: Current status and future challenges. Artif 137–156. [CrossRef] Cheng, B.L.; Ma, J.X.; Li, G.D.; Bai, S.; Xu, Q.; Cui, X.; Cheng, L.; Qin, Y.; Wang, Z.L. Mechanically Asymmetrical Triboelectric Nanogenerator for Self-Powered Monitoring of In Vivo Microscale Weak Movement. Adv. Energy Mater. 2020, 10, 2000827. [CrossRef] 121. Cheng, X.L.; Miao, L.M.; Song, Y.; Su, Z.M.; Chen, H.T.; Chen, X.X.; Zhang, J.X.; Zhang, H.X. High efﬁciency power management and charge boosting strategy for a triboelectric nanogenerator. Nano Energy 2017, 38, 448–456. [CrossRef] g g gy g gy 122. Brenes, A.; Morel, A.; Juillard, J.; Lefeuvre, E.; Badel, A. Maximum power point of piezoelectric energy harvesters: A review of optimality condition for electrical tuning. Smart Mater. Struct. 2020, 29, 033001. [CrossRef]
https://openalex.org/W2029930110	https://repository.rothamsted.ac.uk/download/2098cb895c281c0a3b4f18b87c3926b9f03cbb57b5efb5e634e95e746846ca0c/930595/1-s2.0-S0038071713002824-main.pdf	English	null	A comparison of two colorimetric assays, based upon Lowry and Bradford techniques, to estimate total protein in soil extracts	Soil biology and biochemistry/Soil biology & biochemistry	2,013	cc-by	7,572	* Corresponding author. Sustainable Soils and Grassland Systems, Rothamsted Research, Harpenden, Herts AL5 2JQ, UK. Tel.: þ44 1582 763133x2417. E-mail addresses: marc.redmile-gordon@othamsted.ac.uk, marc.redmile- gordon@eco888.f9.co.uk (M.A. Redmile-Gordon). 0038-0717/ 2013 Elsevier Ltd. http://dx.doi.org/10.1016/j.soilbio.2013.08.017 Open access under CC BY license. a r t i c l e i n f o Soil extracts usually contain large quantities of dissolved humiﬁed organic material, typically reﬂected by high polyphenolic content. Since polyphenols seriously confound quantiﬁcation of extracted protein, minimising this interference is important to ensure measurements are representative. Although the Bradford colorimetric assay is used routinely in soil science for rapid quantiﬁcation protein in soil- extracts, it has several limitations. We therefore investigated an alternative colorimetric technique based on the Lowry assay (frequently used to measure protein and humic substances as distinct pools in microbial bioﬁlms). The accuracies of both the Bradford assay and a modiﬁed Lowry microplate method were compared in factorial combination. Protein was quantiﬁed in soil-extracts (extracted with citrate), including standard additions of model protein (BSA) and polyphenol (Sigma H1675-2). Using the Lowry microplate assay described, no interfering effects of citrate were detected even with concentrations up to 5 times greater than are typically used to extract soil protein. Moreover, the Bradford assay was found to be highly susceptible to two simultaneous and confounding artefacts: 1) the colour development due to added protein was greatly inhibited by polyphenol concentration, and 2) substantial colour development was caused directly by the polyphenol addition. In contrast, the Lowry method enabled distinction be- tween colour development from protein and non-protein origin, providing a more accurate quantitative analysis. These results suggest that the modiﬁed-Lowry method is a more suitable measure of extract protein (deﬁned by standard equivalents) because it is less confounded by the high polyphenolic content which is so typical of soil extracts. Article history: Received 16 May 2013 Received in revised form 14 August 2013 Accepted 17 August 2013 Available online 3 September 2013 Keywords: Total protein determination Colorimetric protein assay Polyphenol interference Glomalin related soil protein Humic acid GRSP BRSP EPS Soil microbial bioﬁlm Extracellular polymeric substances Article history: Received 16 May 2013 Received in revised form 14 August 2013 Accepted 17 August 2013 Available online 3 September 2013 Keywords: Total protein determination Colorimetric protein assay Polyphenol interference Glomalin related soil protein Humic acid GRSP BRSP EPS Soil microbial bioﬁlm Extracellular polymeric substances 2013 Elsevier Ltd. Open access under CC BY license. Soil Biology & Biochemistry 67 (2013) 166e173 Soil Biology & Biochemistry 67 (2013) 166e173 Contents lists available at ScienceDirect 0038-0717/ 2013 Elsevier Ltd. http://dx.doi.org/10.1016/j.soilbio.2013.08.017 Open access under CC BY license. Table 1 example through secondary metabolism of fungi (Haider et al., 1975), or decreased through termite metabolism (Ji et al., 2000) and microbial priming effects (Kuzyakov et al., 2000). Low molecular weight phenolic fractions have previously been removed from aqueous solutions using polar solid-phases (Ferri et al., 2011) however, this approach is also likely to cause the removal of dissolved protein (Bianchi et al., 1996). Giagnoni et al. (2011) also reported protein losses occur using gel ﬁltration to purify extracts containing humic substances. Removal of humic complexes would also be problematic because polymerisation of extracellular proteins by humic molecules is an inevitable and natural process in most soils (Burns et al., 2013). The quantities of smaller humic-peptides are also highly variable between extracts of different soils (Bonmati et al., 2009). Furthermore, irreversible macromolecular associations are suspected to form in response to extraction (Schmidt et al., 2011). Reducing the magnitude of arte- facts arising from polyphenolic content is therefore a reasonable alternative to attempted removal of the polyphenolic fraction. Lucarini and Kilikian (1999) found that 2 M citrate caused suppression of colour development, leading to an underestimation of about 19% in the Lowry assay and 5% in the Bradford method. The sensitivity of the Lowry assay to citrate might ﬁrst appear prohib- itive. However, extractions of GRSP use citrate concentrations of only 20 mM (pH 7) or 50 mM (pH 8). Furthermore, a modiﬁcation to the Lowry assay employed by Frolund et al. (1995) claimed to enable separation of absorbance due to protein and that from the humic fraction, by inclusion and exclusion of copper sulphate from the Lowry reagent. Although this has been successful for bioﬁlm extracts of waste-water sludges, it has not previously been tested with soil extracts. p p yp The Bradford assay (Bradford,1976) has become the colorimetric method of choice, owing principally to its high sensitivity, perceived linearity, and the speed of analysis (Sapan et al., 1999). The Bradford assay relies on interactions between basic amino acids residues (primarily arginine, lysine and histidine) with the Coo- massie brilliant blue G-250 dye (CBB) in an acidic matrix. The binding of CBB to proteins (or interferands) results in a spectral shift to the blue form of the dye. 2.1. Site description and soil sampling Three soils of contrasting management were obtained from two experimental sites in Southern England with contrasting chemical and physical properties (Table 1). Soil 1 (Field: ‘Long Hoos’) was under wheat cultivation at Rothamsted Research, Hertfordshire, UK (50500 N, 0250 W). Soil 1 is classiﬁed as a ﬂinty clay loam over clay with sandy inclusions (Batcombe series). Soil 2 and Soil 3 were from Woburn Experimental Farm, Bedford, UK (51590 N, 0350 W), and classiﬁed as sandy Cambric Arenosols (FAO). Soils were sampled both from a bare fallow management area (Soil 2) and from permanent grassland (Soil 3). Composite soil samples were collected in April 2010 using a 2.5 cm diameter auger to a depth of 0e23 cm for the arable and bare fallow soils (Soils 1 and 2) and of 0e10 cm for the grassland (Soil 3). Samples were bulked and stored overnight (10 C) before sieving moist (<2 mm) and subsequently air-dried in the dark at 25 C. The citrate extraction technique described by Wright and Upadhyaya (1996) is widely practiced, with protein content commonly referred to as glomalin related soil protein (GRSP). The protein content in these extracts was originally proposed to arise from glomalin producing arbuscular mycorrhizal fungi (AMF) of the Glomeromycots, but extracts have since been conﬁrmed to contain large amounts of soil protein from non-mycorrhizal origin (Gillespie et al., 2011; Rosier et al., 2006). Regardless of origin, the use of Bradford’s assay to measure either GRSP, or protein in gen- eral, generates a false measurement when assayed in the presence of polyphenols which occur both in large and highly variable quantities in soil extracts (Halvorson and Gonzalez, 2006; Roberts and Jones, 2008; Whiffen et al., 2007). Furthermore, the interfer- ence from polyphenols is not quantiﬁed in the assay procedure. This means that no distinction can be made between the protein and polyphenolic content, and thus references to GRSP or even protein quantiﬁed using the Bradford assay can be very misleading (Nannipieri and Eldor, 2009). Table 1 In contrast, the Lowry assay (Lowry et al.,1951) functions in alkaline conditions, and involves two steps: 1) the Biuret reaction: based on the reduction of Cu2þ which then binds to protein forming a Cu1þ peptide complex, and 2) subse- quent reduction of the FolineCiocalteu reagent by this complex (Smith et al., 1985). In the original format proposed by Lowry et al., (1951) the Lowry assay also gave a false indication of protein in the presence of polyphenols, which both reduce the FolineCiocalteu reagent, contributing to absorbance in the same region of the spectrum for protein complexes (w750 nm). We therefore compared the routine Bradford assay with a microplate adaptation of Lowry et al. (1951) including the modiﬁ- cation described by Frolund et al. (1995) which claimed to enable separation of absorbance due to protein and that from the humic fraction. Increases in total protein content were measured by addition of known quantities of BSA to citrate extracts of 3 con- trasting soils. Our aims were to: i) Determine if citrate is problematic in Lowry microplate assays of soil extracts. ii) Compare the accuracy of Bradford and Lowry estimations of protein additions to soil extracts (Bovine serum albumin; BSA), both with, and without increasing polyphenol additions (humic acid; Sigma H1675-2). Colorimetric investigations of soil extracts are also strongly affected by physical interferences (scattering), and physico- chemical effects from suspended clays (sorption). Centrifugation at 3000 g does not completely remove the extra-ﬁne clay frac- tion, which is the most active in these sorption process (Lozzi et al., 2008). In the aforementioned study, the Lowry assay was the only method to give correct protein estimates. Besides being highly sensitive to residual clay content, the Bradford assay is highly time- sensitive, with precipitation of protein-bound-dye occurring about 10 min after contact. This introduces limitations regarding the number of samples measurable per run, reducing throughput and speed. 1. Introduction proteinogenic amino acid residues (e.g. tryptophan and cysteine) are destroyed in the hydrolysis step, and furthermore some peptide bonds are not successfully hydrolysed, particularly bonds of hy- drophobic residues such as valine, isoleucine and leucine (Roberts and Jones, 2008). All methods of total protein estimation are subject to artefacts when analysing extracts of soil and so are best thought of as being ‘semi-quantitative’. The selection of analytical method depends very much on how we choose to deﬁne soil protein, and the analytical resources at our disposal (Gillespie et al., 2011). For example, Roberts and Jones (2008) favoured hydrolysis of protein followed by chromatographic quantiﬁcation of the constituent amino acids. While the analytical stages of hydrolysis approaches are highly tolerant of interference from humic substances, there are several problems. Firstly, partially humiﬁed organic molecules are likely to release some amino acids by hydrolysis, secondly, some In contrast, colorimetric methods suffer interference from hu- mic substances directly, seriously limiting their accuracy (Nannipieri and Eldor, 2009). However, colorimetric methods are still frequently used for relative comparison between treatments as they are rapid and affordable, do not require a hydrolysis step, and frequently show good correlation to more expensive and time- consuming techniques (e.g. Gillespie et al., 2011). Soil extracts usually contain large quantities of humiﬁed organic matter, which is characterised by high polyphenolic content (Martens et al., 2004). This has been demonstrated to erroneously increase esti- mates of extracted protein using the Bradford assay (Whiffen et al., 2007). Furthermore, this interference cannot be assumed to be constant because the size of the polyphenolic pool can be variable under different managements (Martens et al., 2004), increased for M.A. Redmile-Gordon et al. / Soil Biology & Biochemistry 67 (2013) 166e173 167 Table 1 Soil main chemical and physical properties. Soil no. Soil type Management Organic C (mg g1) Total N (mg g1) C/N ratio pH Clay %a 1 Chromic luvisol Arable (wheat) 13.66 1.30 10.5 7.18 18e27 2 Cambric Arenosol Bare fallow 0.30 0.03 10.3 5.53 7.9 3 Cambric Arenosol Grassland 16.86 1.55 10.9 5.95 8.0 a Data from Avery and Catt (1995). Table 1 Soil main chemical and physical properties. 2.8.1. Citrate effect Changes in Absprotein due to BSA and citrate additions were examined by multiple regressions using Residual Maximum Like- lihood (REML). The effect of increasing additions of citrate upon Absprotein over all additions of BSA and citrate is presented for the different analytical matrices. 2. Protein speciﬁc absorbance and humic acid effect Lowry reagents were made from three stock solutions at 3.5 times the concentration of the original Lowry macroassay reagent, i.e.; 3.5 g copper sulphate (CuSO4$5H2O) 100 mL1 H2O, 7 g sodium potassium tartrate 100 mL1 H2O, and 70 g Na2CO3 L1 0.35 N NaOH. The three solutions were combined sequentially in pro- portions of 1:1:100 (v:v:v), respectively (Reagent A). The second reagent (Reagent B) was made in the same way, except the copper sulphate solution was excluded and volume substituted with deionised water. Three replicates of each sample or standard (50 mL) were added to 2 96 well microplates, marked ‘A’ and ‘B’. The volumes of all wells were increased to 100 mL using PBS or standard in PBS, subsequently, 100 mL of reagent A was rapidly injected to wells in plate A using a 12 channel electronic Finnpipette set to speed 9 to ensure good mixing. Reagent B was added to plate B in the same way. Both plates were incubated at room temperature The known additions of BSA were compared with the measured increase in protein concentration of the samples. Regression anal- ysis was used to compare the strength of the impact of HA additions on the intercept and slope of the calibration in buffer and soil extracts. 2.2. Soil extractions Soils were extracted using the ‘easily extractable glomalin’ protocol of Wright and Upadhyaya (1996). Brieﬂy, 8 ml of 20 mM M.A. Redmile-Gordon et al. / Soil Biology & Biochemistry 67 (2013) 166e173 168 in the dark for 10 min. Folin-Phenol reagent was prepared imme- diately before the end of the ﬁrst incubation (2 N diluted 10 fold in H2O), and 100 mL subsequently injected to all wells. The plates were then incubated for a further 30 min (room temperature, dark) before reading at 750 nm for 150 ms per well. sodium citrate at pH 7.0 was dispensed onto 1 g of air-dried soil in 15 ml polypropylene centrifuge tubes and autoclaved (121 C) for 30 min. Immediately after autoclaving, the tubes were cooled on ice and centrifuged in a pre-cooled rotor (4 C) at 3500 g for 20 min. The supernatants were decanted and stored overnight at 4 C for analysis. Two absorbencies per sample were thus obtained: ‘AbsA’ and ‘AbsB’ for the respective reagents. From these absorbencies, theo- retical absorbance due to protein (BSA equivalents) was calculated as ‘Absprotein’. Absorbance due to ‘humic substances’ (speciﬁcally ‘humic acid equivalents’ or HAE) is presented as ‘Abshumic’ as per the following formulae given by Frolund et al. (1995): 2.4. Bradford microplate analysis Samples of 100 mL with standard additions of BSA and citrate were prepared by combining 25 mL of 2.5 diluted soil extract (soil 1) with 25 mL PBS containing 80,160, 240 and 320 ppm BSA, in three replicates, in microplates ‘A’ and ‘B’. Citrate (20 mM) was added to these samples (0, 10, 20 or 40 mL) then made up to 100 mL ﬁnal volumes with PBS buffer. The ﬁnal soil extract dilution was thus 10 fold, with concentrations of protein being þ0, þ20, þ40, þ60, þ80 ppm. The citrate concentrations investigated were therefore 1, 2, 4 and 5 times the typical assay concentration. The modiﬁed Lowry procedure described in paragraph 2.5 was then followed. Citrate additions to PBS buffer (no soil extract), and PBS with 160 ppm humic acid (no soil extract) were also prepared for contrast. Protein measurements in extracts and PBS were made using a Bradford assay kit (Bio Rad Protein Assay; Bio Rad Laboratories). To each 50 mL of dilute extract or PBS (three replicates) in microplate (Nunc 442404) were added 25 mL aliquots of PBS containing sufﬁ- cient BSA and/or HA to give equivalent concentrations (relative to the original 50 mL extract) of 0e100 ppm BSA, and 0e400 ppm HA. Bio-Rad G-250 dye was rapidly mixed with PBS immediately before addition, then immediately and forcefully added with a 12-channel pipette to ensure adequate mixing (Thermo electronic ‘Finnpipette’ set to speed 9). This delivered 50 mL of dye to each well with suf- ﬁcient PBS to reach a ﬁnal well volume of 250 mL. Plates were read 7 min later at 595 nm using a ‘Varioscan’ plate reader (Thermo Scientiﬁc) set to a read duration of 150 ms per well. 2.3. Sample and standard preparation A model polyphenol (humic acid; Sigma H1675-2) and protein (bovine serum albumin; Sigma A7906) were used throughout as standards. Soil extracts 1, 2 and 3 were diluted 7.5, 2 and 12.5 times respectively, with phosphate buffered saline (PBS) to remain within the effective assay range (maximum absorbance < 1.0). BSA stan- dard additions were equal to 0, 20, 40, 60, 80 ppm for the soil ex- tracts, and 0, 25, 50, 75, 100 ppm for standards in PBS alone. These were combined with humic acid (HA) additions of 0, 80, 160, 240, 320 ppm for the soil extracts, and 0, 100, 200, 300, 400 ppm in PBS alone. Absprotein ¼ 1:25ðAbsA AbsbÞ Abshumic ¼ Absb 0:2Absprotein Absprotein ¼ 1:25ðAbsA AbsbÞ Abshumic ¼ Absb 0:2Absprotein 2.5. Lowry microplate analysis The effect of humic acid on protein signal was assessed for each extract of soil (Table 1) and PBS alone, in factorial design, including extract type, BSA and HA addition. A modiﬁcation of the Lowry assay (1951) described by Frolund et al. (1995) was used to separately quantify the proteinaceous and polyphenolic compounds in each soil extract. The principle of this modiﬁcation is that the omission of copper sulphate from the reagent enables determination of the auto-absorbance from humic compounds and chromogenic amino acids. Concentrations and volumes of Lowry and FolineCiocalteu’s phenol reagents (Sigma F9252), were optimised for speed and sensitivity (results not shown) using principles from Miller (1959), Oosta et al. (1978) and Peterson (1979). Ultimately, a more concentrated reagent compared to that of Lowry et al. (1951) was prepared to maximise assay sensitivity, and reduce incubation time as detailed below. 2.8. Statistical methods Table 3 Table 3 Regression coefﬁcients of Absprotein as f(BSA) in the three contrasting matrices, also in the presence of citrate. Analytical matrix Slope of Abs protein as f [BSA] s.e. PBS 0.005573 0.000076 Soil extract in PBS 0.004485 0.000076 PBS spiked with HA (160 ppm) 0.004399 0.000076 well plates. The Lowry-based analytical procedure (including mixing of stock reagents) was achieved in approximately one hour. With the Bradford assay, addition of HA in the complete absence of protein resulted in a false positive estimation of protein (80 ppm protein estimated in the presence of 400 ppm HA) (Fig. 2a, x ¼ 0). Although increasing inclusions of HA caused an additive effect in terms of total absorbance, an increasingly large underestimate of the additional protein also occurred, as seen by the reduction of slope angle with increasing HA content. For example, with 400 ppm of humic acid (Fig. 2a, HA400), the slope of linear regression is 65% less than in the absence of humic acid (HA0). The Lowry microplate method gave more accurate estimates of the quantity of protein added (Fig. 2b). With the Lowry microplate assay, addition of HA alone did not result in a false positive indi- cation of protein (Fig. 2b, x ¼ 0). When combined with protein additions, a systematic underestimate of protein occurred (***P < 0.001). However, this was less than half the suppressive effect seen using the Bradford assay (only a 31% decrease in slope comparing regressions for HA400 and HA0; Fig. 2b). Fig. 1. Negligible effect of citrate upon Lowry estimation of standards in PBS containing 160 ppm HA. the highest and lowest concentrations of citrate tested (Fig. 1). Comparing the linear regressions of Absprotein as a function of BSA in PBS containing neither soil extract nor HA addition, a very small difference is discernible (Fig. S1a). Graphical representation of the effect of all citrate additions upon Absprotein ranging from þ20 mM to þ80 mM over all BSA additions requires three-dimensional representation (inclusion of z axis), and when plotted, the difference is visually indistinguishable. Therefore mean slopes of calibration curves along the z axis are given in Table 2, showing negligible effect of citrate on all three extracts. Using the Bradford method, the mean squared errors of pre- diction (MSEP) of protein added to buffer (affected by additions of HA) were calculated using the method of Wallach and Gofﬁnet (1987). 3.3. Comparison between Bradford and Lowry estimations of standard additions to soil extract The protein concentrations in diluted extracts of soil 1 given by the Bradford and Lowry microplate methods (Fig. 3a and b, respectively), without BSA addition (x ¼ 0) were 21.8 ppm and 12.5 ppm (50 and 100 mL assay concentrations, respectively). These absolute measurements, inclusive of soil protein, with a range of BSA and HA additions show response patterns similar to those observed with PBS alone (Fig. 2a and b). Using the Bradford assay (Fig. 3a), the small addition of humic acid (80 ppm) caused an in- crease in protein estimate of about 75%. In contrast, with the modiﬁed Lowry assay (Fig. 3b) the same quantity of humic acid caused a decrease in soileextract protein estimations of about 25%. Table 3 MSEP increased with HA addition, from 2, to 364, 1152, 1983, and 2778, for HA additions of 0, 100, 200, 300 and 400 ppm, respectively. Using the Lowry-microplate technique, the observed values (protein measured) differ much less from the expected (protein added), with corresponding MSEP’s of 2, 81, 219, 290 and 469, respectively. Thus with increasing additions of HA, the MSEP of the Lowry microplate technique was more than 5-fold smaller than the Bradford assay (Table S1). A mean f [citrate] (Z slope) of 0.000266 for PBS shows that increasing additions of citrate to PBS marginally represses colour development from protein added, as seen by Ji (1973). The extent is negligible however in the range of citrate concentrations likely to be used for soil extraction (Fig. S1b). The negligible effect of citrate is put in perspective by comparison with the matrix effect of HA in PBS (Table 3). It is evident here that the addition of 160 ppm HA to the buffer clearly suppressed Absprotein, changing absorbance from 0.005573 per unit BSA, to 0.004399 per unit BSA (about 20%). Similarly, in soil extract, Absprotein is 0.004485 of BSA addition (again, about 20% less compared to the response of BSA additions to PBS). The suppression of colour development due to protein (Absprotein) was more fully investigated in the following experiment by varying concentration of HA, both in PBS and soil extracts. 3.4. Comparison of estimation accuracy between Bradford and Lowry Table 2 Regression coefﬁcients of multiple regressions for citrate additions given with standard error (s.e.). Analytical matrix Mean Z slope Abs protein as f [citrate] s.e. PBS 0.000266 0.000146 Soil extract in PBS þ0.000294 0.000146 HA spiked PBS (160 ppm) þ0.000052 0.000146 Table 2 Regression coefﬁcients of multiple regressions for citrate additions given with standard error (s.e.). 3.1. Citrate effect on Lowry analysis 3.1. Citrate effect on Lowry analysis Regression analysis of the additions of BSA with increasing ad- ditions of citrate to assay buffer, buffer plus 160 ppm HA, and soil extract revealed negligible impact of citrate upon Absprotein. The resulting calibration curves of Absprotein against BSA additions to soil extract and buffer plus HA were thus almost indistinguishable at M.A. Redmile-Gordon et al. / Soil Biology & Biochemistry 67 (2013) 166e173 169 Fig. 1. Negligible effect of citrate upon Lowry estimation of standards in PBS containing 160 ppm HA. 3.2. Comparison of accuracy between Bradford and Lowry estimations of standards in PBS The modiﬁed two-to-three-reagent Lowry system was less time-sensitive than the Bradford thus permitting full use of the 96 3.4. Comparison of estimation accuracy between Bradford and Lowry Table 2 The Lowry microplate assay thus gave a more linear response than the Bradford assay to both polyphenol and protein additions to soil extracts. to protein occurs, even for soil extract with no HA addition. This would be especially problematic if quantifying extract protein using a calibration curve generated in PBS (common procedure). With the Bradford assay, linear regressions of the responses to added protein, by comparison to the 1:1 theoretical ideal, show underestimations of protein additions equal to 62, 69, 75, 81, and 86% (s.e. 0.8%) for 0, 100, 200, 300 and 400 ppm HA additions respectively. Similarly, linear regressions of absorbance increases with the Lowry microplate assay accounted for 99.71% of the variance in response to protein additions (Fig. 4b). However, although Lowry responses to HA additions to buffer are, strictly speaking, best described by a 2nd order polynomial (Fig. S3b), 99.78% of the variance due to HA additions can be accounted for by a simple straight line (Fig. S3a). The Lowry microplate assay thus gave a more linear response than the Bradford assay to both polyphenol and protein additions to soil extracts. In contrast, using the Lowry microplate assay (Fig. 4b), the un- derestimation of additions was substantially less, with corre- sponding underestimations of 13, 23, 28, 30, and 28% (1.8%) for 0, 100, 200, 300 and 400 ppm HA additions respectively. Protein es- timates assayed in response to BSA additions responded in a similar way between HA spiked PBS buffer and all 3 extracts of soil. Table 2 Using extracts of Soil 1, the increases in protein estimate in response to known additions of BSA are presented in Fig. 4(a and b). Colour development due to pre-existing soil protein or directly from the added polyphenol is excluded. By comparison to the ‘theoretical ideal’ (1:1 line), using estimates given by the Bradford assay (Fig. 4a), substantial suppression of colour development due M.A. Redmile-Gordon et al. / Soil Biology & Biochemistry 67 (2013) 166e173 170 Fig. 2. a) Bradford and b) Lowry estimation of protein as a function of humic acid and protein additions to buffer alone (HA0, 100, 200, 300 and 400 correspond to humic acid additions of 0, 100, 200, 300 and 400 ppm, respectively). Fig. 3. a) Bradford and b) Lowry estimation of protein in soil extract (soil 1) with buffer including analytical responses to additions of BSA and HA. M.A. Redmile Gordon et al. / Soil Biology & Biochemistry 67 (2013) 166 173 170 Fig. 3. a) Bradford and b) Lowry estimation of protein in soil extract (soil 1) with buffer including analytical responses to additions of BSA and HA Fig. 3. a) Bradford and b) Lowry estimation of protein in soil extract (soil 1) with buffer including analytical responses to additions of BSA and HA. Fig. 2. a) Bradford and b) Lowry estimation of protein as a function of humic acid and protein additions to buffer alone (HA0, 100, 200, 300 and 400 correspond to humic acid additions of 0, 100, 200, 300 and 400 ppm, respectively). protein additions (Fig. 4a), with HA additions, only 95.20% of the variance can be accounted for by the straight line model (Fig. S2a), with 99.99% being described by a 3rd order polynomial (Fig. S2b). protein additions (Fig. 4a), with HA additions, only 95.20% of the variance can be accounted for by the straight line model (Fig. S2a), with 99.99% being described by a 3rd order polynomial (Fig. S2b). Similarly, linear regressions of absorbance increases with the Lowry microplate assay accounted for 99.71% of the variance in response to protein additions (Fig. 4b). However, although Lowry responses to HA additions to buffer are, strictly speaking, best described by a 2nd order polynomial (Fig. S3b), 99.78% of the variance due to HA additions can be accounted for by a simple straight line (Fig. S3a). 3.5. Assumptions of linearity The phenolic content of soil is of further contemporary interest as it has been linked to soil organic matter dynamics in the context of land-use change, climate, and CO2 emissions, e.g. (Fernandez et al., 2012). Citrate extracts are best thought to contain a mixture of bio- chemicals from soil microbes, humiﬁed soil organic matter, and reaction products of extraction (Nannipieri and Eldor, 2009). In the current study, depending on soil type, estimates of polyphenol content (shown as HAE) were between 5 and 10 times greater than the protein content. Solid-state 13C DPMAS NMR spectra of various ‘GRSP’ extracts were presented by Schindler et al. (2007) showing high degree of aromaticity but little aliphatic C (41%e51% and 4%e 11%, respectively). The authors summarised this was indicative of a greater proportion of humiﬁed organics as opposed to protein content with BSA showing only 12% aromaticity, and 54% aliphatic content. The protein/HAE ratios we observed thus sit comfortably in the range suggested by NMR. It is not possible to comment precisely upon the accuracy of the determinations of soil extract protein per se (soil extract in the absence of any BSA or polyphenol additions), because there is currently no universally accepted method to measure protein in soils, with each method being subject to idiosyncratic artefacts (Nannipieri and Eldor, 2009). Therefore, in this study, comparison of accuracy is based upon the assumption that BSA, the most commonly used protein reference standard, is a good model for soil protein. Although questions have been raised with regard to the suitability of using a non-microbial protein as a proxy for protein in soils (Criquet et al., 2002), BSA remains the most frequently used model, e.g. Taylor and Williams (2010) and Young et al., (2012) and to our knowledge no replacement is receiving much consideration. The well characterised BSA standard (Wu et al., 2011) is thus still used extensively as a reference protein. Fig. 4. Polyphenol (HA) suppresses colour development from protein in a) Bradford and b) Lowry analyses of soil 1 extract. It is now widely recognised that the extraction procedure of Wright and Upadhyaya (1996) extracts large quantities of non- mycorrhizal proteins from soil with even heat-labile proteins contributing to measures of GRSP (Janos et al., 2008; Rosier et al., 2006). 3.5. Assumptions of linearity However, Bradford determinations of ‘GRSP’ or ‘Bradford reactive soil protein’ (BRSP) are still commonplace, and good cor- relations are repeatedly found with aggregate stability and soil organic C and N, e.g. Emran et al. (2012). It follows that if the Bradford reactive fraction is to serve as a surrogate measure of aggregate stability, or organic matter content, then the de facto GRSP measure may still be useful. However, if the motivation is a better understanding of soil organic matter dynamics, or if we are to continue to estimate protein from extracts (and not only citrate extracts) then simple analytical techniques that are more selective for ‘proteinaceous’ vs. more ‘polyphenolic’ material will be more descriptive. Moreover, confounding these pools through use of the Bradford assay will cloud interpretations of the respective contri- butions of these organic fractions to soil properties. Bradford assay is more suppressed by the presence of polyphenol, it is variably compensated for by the colour development directly from the polyphenol itself. This most likely explains the apparent overestimation of protein in soil 3. In contrast, there appears to be negative bias in soils 1 and 2 by comparison to concentrations given by the Lowry assay. This is most likely due to suppression of colour development from protein as demonstrated in Fig. 4a, i.e. where the protein fraction is proportionally larger, the Bradford assay un- derestimates. Extracts of Soil 1 and Soil 2 (arable, and fallow managements, respectively) exhibit lower polyphenol/protein ra- tios than extract of Soil 3 (grassland). 3.5. Assumptions of linearity Estimates calculated for all extracts (corrected for dilution) are presented in Table 4 for reference. It is important to remember that although colour development from protein per se using the Whereas linear regressions of absorbance increases using the Bradford assay accounted for 99.65% of the variance in response to M.A. Redmile-Gordon et al. / Soil Biology & Biochemistry 67 (2013) 166e173 171 Fig. 4. Polyphenol (HA) suppresses colour development from protein in a) Bradford and b) Lowry analyses of soil 1 extract. the Bradford dye (Fig. S2b). This most likely explains the apparent overestimation of protein in soil 3 by the Bradford assay in com- parison to the Lowry. Overestimation was seen previously with soil extracts containing a large phenolic fraction e.g. Whiffen et al. (2007). The increased HAE/protein ratio indicated by the Lowry assay for soil 3 is in agreement with the ﬁndings of Martens et al. (2004) where the proportion of organic C present as phenolics in grassland was also greater than that in arable soil: grasslands are highly competitive, with phenolics being produced biologically as competitive phytotoxins of allelopathy (Lipinska and Wanda, 2005) and as signalling agents between roots and rhizobia (Cesco et al., 2012), most likely explaining the high HAE/protein ratio found in the present study. The phenolic content of soil is of further contemporary interest as it has been linked to soil organic matter dynamics in the context of land-use change, climate, and CO2 emissions, e.g. (Fernandez et al., 2012). the Bradford dye (Fig. S2b). This most likely explains the apparent overestimation of protein in soil 3 by the Bradford assay in com- parison to the Lowry. Overestimation was seen previously with soil extracts containing a large phenolic fraction e.g. Whiffen et al. (2007). The increased HAE/protein ratio indicated by the Lowry assay for soil 3 is in agreement with the ﬁndings of Martens et al. (2004) where the proportion of organic C present as phenolics in grassland was also greater than that in arable soil: grasslands are highly competitive, with phenolics being produced biologically as competitive phytotoxins of allelopathy (Lipinska and Wanda, 2005) and as signalling agents between roots and rhizobia (Cesco et al., 2012), most likely explaining the high HAE/protein ratio found in the present study. Acknowledgements Lucarini, A.C., Kilikian, B.V., 1999. Comparative study of Lowry and Bradford methods: interfering substances. Biotechnol. Tech. 13, 149e154. Marc Redmile-Gordon acknowledges the BBSRC for funding (Doctoral Training Grant number D527069) and reviewers for helpful comments. Martens, D.A., Reedy, T.E., Lewis, D.T., 2004. Soil organic carbon content and composition of 130-year crop, pasture and forest land-use managements. Global Change Biol. 10, 65e78. Miller, G.L., 1959. Determination of protein: a modiﬁcation of the Lowry method that gives a linear photometric response. Analyt. Biochem. 48, 422e427. that gives a linear photometric response. Analyt. Biochem. 48, 422e427. Nannipieri, P., Eldor, P., 2009. The chemical and functional characterization of soil N and its biotic components. Soil Biol. Biochem. 41, 2357e2369. 4. Discussion Recovery of low molecular weight phenols through solid-phase extraction. Chem. Eng. J. 166, 994e1001. Fl i H C Wi d J 2010 Th bi ﬁl i N R Mi bi l 8 623 Flemming, H.C., Wingender, J., 2010. The bioﬁlm matrix. Nat. Rev. Microbiol. 8, 623e 633. Frolund, B., Griebe, T., Nielsen, P.H., 1995. Enzymatic-activity in the activated-sludge ﬂoc matrix. Appl. Microbiol. Biotechnol. 43, 755e761. Giagnoni, L., Magherini, F., Landi, L., Taghavi, S., Modesti, A., Bini, L., Nannipieri, P., Van der Lelie, D., Renella, G., 2011. Extraction of microbial proteome from soil: potential and limitations assessed through a model study. Eur. J. Soil Sci. 62, 74e 81. 5. Conclusion Gillespie, A.W., Farrell, R.E., Walley, F.L., Ross, A.R.S., Leinweber, P., Eckhardt, K.-U., Regier, T.Z., Blyth, R.I.R., 2011. Glomalin-related soil protein contains non- mycorrhizal-related heat-stable proteins, lipids and humic materials. Soil Biol. Biochem. 43, 766e777. The modiﬁed Lowry assay presented here provided a reasonable estimate of polyphenolic content and a more accurate estimate of protein content in citrate extracts of 3 contrasting soils, and model extracts. It is therefore of potential value in comparative studies of total extractable protein where the polyphenol content is expected to be high. Haider, K., Martin, J.P., Filip, Z., 1975. Humus biochemistry. In: Paul, E.A., McLaren, A.D. (Eds.), Soil Biology and Biochemistry, vol. 4, pp. 195e244. Halvorson, J.J., Gonzalez, J.M., 2006. Bradford reactive soil protein in Appalachian soils: distribution and response to incubation, extraction reagent and tannins. Plant Soil 286, 339e356. Janos, D.P., Garamszegi, S., Beltran, B., 2008. Glomalin extraction and measurement. Soil Biol. Biochem. 40, 728e739. These ﬁndings lead us to add caution against loose usage of the terms ‘glomalin related soil protein’ (GRSP) and ‘Bradford reactive soil protein’ (BRSP) to describe the ‘Bradford reactive fraction’ (BRF) in soil extracts, which itself produces a highly complex analytical response affected by the HAE/protein ratio. Ji, T.H., 1973. Interference by detergents, chelating agents, and buffers with Lowry protein determination. Analyt. Biochem. 52, 517e521. Kuzyakov, Y., Friedel, J.K., Stahr, K., 2000. Review of mechanisms and quantiﬁcation of priming effects. Soil Biol. Biochem. 32, 1485e1498. Lipinska, H., Wanda, H., 2005. Allelopathic effects of Poa pratensis on other grass- land spp. Allelopath. J. 16, 251e259. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin Phenol reagent. J. Biol. Chem. 193, 265e275. 4. Discussion extracellular matrices of entire microbial communities, and not misleadingly attributing extractable protein and humiﬁed organic matter collectively to AMF. Extracellular microbial proteins are produced in vivo with a variety of suspected impacts upon soil physical properties (Or et al., 2007). The structural roles of extra- cellular proteins are currently being explored in related scientiﬁc disciplines and are thought to help impart strength and elasticity to bioﬁlms (Flemming and Wingender, 2010). These extracellular polymers, produced by the living soil biomass (including AMF, saprophytic fungi, bacteria and archaea) are thought to improve aggregate stability, weight for weight, to a greater extent than total SOM (Chenu, 1993; Roberson and Firestone, 1992; Tang et al., 2011; Watts et al., 2005). The same was also originally hypothesised for glomalin (Wright and Upadhyaya, 1998) and links between confounded soil protein/polyphenolic pools and aggregate stability were since reported by many studies. A ﬁrm causal link between AMF and aggregate stability was later established through other methods, e.g. Rillig et al., (2010). A greater understanding of community-wide extracellular proteinaceous material in soils is now required, and besides improved speciﬁcity of extraction methods, the single most important step is likely to be avoiding the largest known artefact currently affecting colorimetric analyses, i.e. the interference from polyphenolic content. Burns, R.G., DeForest, J.L., Marxsen, J., Sinsabaugh, R.L., Stromberger, M.E., Wallenstein, M.D., Weintraub, M.N., Zoppini, A., 2013. Soil enzymes in a changing environment: current knowledge and future directions. Soil Biol. Biochem. 58, 216e234. Cesco, S., Mimmo, T., Tonon, G., Tomasi, N., Pinton, R., Terzano, R., Neumann, G., Weisskopf, L., Renella, G., Landi, L., Nannipieri, P., 2012. Plant-borne ﬂavonoids released into the rhizosphere: impact on soil bio-activities related to plant nutrition. A review. Biol. Fertil. Soils 48, 123e149. Chenu, C., 1993. Clay polysaccharide or sand polysaccharide associations as models for the interface between microorganisms and soil e water related properties and microstructure. Geoderma 56, 143e156. Criquet, S., Farnet, A.M., Ferre, E., 2002. Protein measurement in forest litter. Biol. Fertil. Soils 35, 307e313. Emran, M., Gispert, M., Pardini, G., 2012. Patterns of soil organic carbon, glomalin and structural stability in abandoned Mediterranean terraced lands. Eur. J. Soil Sci. 63, 637e649. Fernandez, I., Carrasco, B., Cabaneiro, A., 2012. Evolution of soil organic matter composition and edaphic carbon efﬂuxes following oak forest clearing for pasture: climate change implications. Eur. J. For. Res. 131, 1681e1693. Ferri, F., Bertin, L., Scoma, A., Marchetti, L., Fava, F., 2011. Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.soilbio.2013.08.017. Oosta, G.M., Mathewson, N.S., Catravas, G.N., 1978. Optimisation of FoilineCiocalteu reagent concentration in an automated Lowry protein assay. Analyt. Biochem. 89, 31e34. Or, D., Smets, B.F., Wraith, J.M., Dechesne, A., Friedman, S.P., 2007. Physical con- straints affecting bacterial habitats and activity in unsaturated porous media e a review. Adv. Water Resour. 30, 1505e1527. 4. Discussion With regard to assayed protein content (Table 4), although colour development from protein per se using the Bradford assay was found to be highly suppressed by the presence of polyphenol (Fig. 4a), this was variably (and nonlinearly) compensated for by the colour development directly from polyphenol complexes with It is likely that in future, a rapid measure of soil protein distinct from highly humiﬁed pools will be called for, e.g. if we are to attempt to quantify the impacts of microbial protein found within M.A. Redmile-Gordon et al. / Soil Biology & Biochemistry 67 (2013) 166e173 172 Table 4 Extract protein concentrations (corrected for dilution). Modiﬁed Lowry also provides estimate of phenolics (as HA equivalent: ‘HAE’). Extract Soil 1 Soil 2 Soil 3 Protein estimate (ppm) Polyphenol estimate (ppm) Protein estimate (ppm) Polyphenol estimate (ppm) Protein estimate (ppm) Polyphenol estimate (ppm) Bradford 163.7 2.4a e 33.1 0.3 e 374.9 2.5 e Lowry 187.5 3.3 1048 11 49.0 0.5 274 2 328.8 10.6 3384 11 HAE/Prot ratio 5.6 5.6 10.3 a Indicates standard error. a Indicates standard error. extracellular matrices of entire microbial communities, and not misleadingly attributing extractable protein and humiﬁed organic matter collectively to AMF. Extracellular microbial proteins are produced in vivo with a variety of suspected impacts upon soil physical properties (Or et al., 2007). The structural roles of extra- cellular proteins are currently being explored in related scientiﬁc disciplines and are thought to help impart strength and elasticity to bioﬁlms (Flemming and Wingender, 2010). These extracellular polymers, produced by the living soil biomass (including AMF, saprophytic fungi, bacteria and archaea) are thought to improve aggregate stability, weight for weight, to a greater extent than total SOM (Chenu, 1993; Roberson and Firestone, 1992; Tang et al., 2011; Watts et al., 2005). The same was also originally hypothesised for glomalin (Wright and Upadhyaya, 1998) and links between confounded soil protein/polyphenolic pools and aggregate stability were since reported by many studies. A ﬁrm causal link between AMF and aggregate stability was later established through other methods, e.g. Rillig et al., (2010). A greater understanding of community-wide extracellular proteinaceous material in soils is now required, and besides improved speciﬁcity of extraction methods, the single most important step is likely to be avoiding the largest known artefact currently affecting colorimetric analyses, i.e. the interference from polyphenolic content. Appendix A. Supplementary data g p p y Nannipieri, P., Eldor, P., 2009. The chemical and functional characterization of soil N and its biotic components. Soil Biol. Biochem. 41, 2357e2369. O t G M M th N S C t G N 1978 O ti i ti f F ili Ci lt Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.soilbio.2013.08.017. Bonmati, M., Ceccanti, B., Nannipieri, P., Valero, J., 2009. Characterization of humuse protease complexes extracted from soil. Soil Biol. Biochem. 41, 1199e1209. Bianchi, D., Golini, P., Bortolo, R., Cesti, P., 1996. Immobilization of penicillin G acylase on aminoalkylated polyacrylic supports. Enzym. Microb. Technol. 18, 592e596. References Peterson, G.L., 1979. Review of the Folin phenol protein quantitation method of Lowry, Rosebrough, Farr and Randall. Analyt. Biochem. 100, 201e220. Bianchi, D., Golini, P., Bortolo, R., Cesti, P., 1996. Immobilization of penicillin G acylase on aminoalkylated polyacrylic supports. Enzym. Microb. Technol. 18, 592e596. Peterson, G.L., 1979. Review of the Folin phenol protein quantitation method of Lowry, Rosebrough, Farr and Randall. Analyt. Biochem. 100, 201e220. Rillig, M.C., Mardatin, N.F., Leifheit, E.F., Antunes, P.M., 2010. Mycelium of arbuscular mycorrhizal fungi increases soil water repellency and is sufﬁcient to maintain water-stable soil aggregates. Soil Biol. Biochem. 42, 1189e1191. Rillig, M.C., Mardatin, N.F., Leifheit, E.F., Antunes, P.M., 2010. Mycelium of arbuscular mycorrhizal fungi increases soil water repellency and is sufﬁcient to maintain water-stable soil aggregates. Soil Biol. Biochem. 42, 1189e1191. Bonmati, M., Ceccanti, B., Nannipieri, P., Valero, J., 2009. Characterization of humuse protease complexes extracted from soil. Soil Biol. Biochem. 41, 1199e1209. M.A. Redmile-Gordon et al. / Soil Biology & Biochemistry 67 (2013) 166e173 173 Roberson, E.B., Firestone, M.K., 1992. Relationship between desiccation and exo- polysaccharide production in a soil pseudomonas sp. Appl. Environ. Microbiol. 58, 1284e1291. Taylor, E.B., Williams, M.A., 2010. Microbial protein in soil: inﬂuence of extraction method and C amendment on extraction and recovery. Microb. Ecol. 59, 390e 399. Wallach, D., Gofﬁnet, B., 1987. Mean squared error of prediction in models for studying ecological and agronomic systems. Biometrics 43, 561e573. Roberts, P., Jones, D.L., 2008. Critical evaluation of methods for determining total protein in soil solution. Soil Biol. Biochem. 40, 1485e1495. Rosier, C.L., Hoye, A.T., Rillig, M.C., 2006. Glomalin-related soil protein: assessment of current detection and quantiﬁcation tools. Soil Biol. Biochem. 38, 2205e2211. Sapan C V Lundblad R L Price N C 1999 Colorimetric protein assay techniques Watts, C.W., Whalley, W.R., Brookes, P.C., Devonshire, B.J., Whitmore, A.P., 2005. Biological and physical processes that mediate micro-aggregation of clays. Soil Sci. 170, 573e583. Rosier, C.L., Hoye, A.T., Rillig, M.C., 2006. Glomalin-related soil protein: assessment of current detection and quantiﬁcation tools. Soil Biol. Biochem. 38, 2205e2211. Sapan, C.V., Lundblad, R.L., Price, N.C., 1999. Colorimetric protein assay techniques. Biotechnol. Appl. Biochem. 29, 99e108. q Sapan, C.V., Lundblad, R.L., Price, N.C., 1999. Colorimetric protein assay techniques. Biotechnol. Appl. Biochem. 29, 99e108. Whiffen, L.K., Midgley, D.J., McGee, P.A., 2007. Polyphenolic compounds interfere with quantiﬁcation of protein in soil extracts using the Bradford method. Soil Biol. Biochem. 39, 691e694. Schindler, F.V., Mercer, E.J., Rice, J.A., 2007. References Chemical characteristics of glomalin- related soil protein (GRSP) extracted from soils of varying organic matter content. Soil Biol. Biochem. 39, 320e329. Wright, S.F., Upadhyaya, A., 1996. Extraction of an abundant and unusual protein from soil and comparison with hyphal protein of arbuscular mycorrhizal fungi. Soil Sci. 161, 575e586. Schmidt, M.W.I., Torn, M.S., Abiven, S., Dittmar, T., Guggenberger, G., Janssens, I.A., Kleber, M., Koegel-Knabner, I., Lehmann, J., Manning, D.A.C., Nannipieri, P., Rasse, D.P., Weiner, S., Trumbore, S.E., 2011. Persistence of soil organic matter as an ecosystem property. Nature 478, 49e56. Wright, S.F., Upadhyaya, A., 1998. A survey of soils for aggregate stability and glo- malin, a glycoprotein produced by hyphae of arbuscular mycorrhizal fungi. Plant Soil 198, 97e107. Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J., Klenk, D.C., 1985. Measurement of pro- tein using bicinchoninic acid. Analyt. Biochem. 150, 76e85. Wu, L.Q., Yang, B., Bi, J.M., Wang, J., 2011. Development of bovine serum albumin certiﬁed reference. Analyt. Bioanal. Chem. 400, 3443e3449. tein using bicinchoninic acid. Analyt. Biochem. 150, 76e85. Tang, J., Mo, Y., Zhang, J., Zhang, R., 2011. Inﬂuence of biological aggregating agents associated with microbial population on soil aggregate stability. Appl. Soil Ecol. 47, 153e159. Young, I.M., Feeney, D.S., O’Donnell, A.G., Goulding, K.W.T., 2012. Fungi in century old managed soils could hold key to the development of soil water repellency. Soil Biol. Biochem. 45, 125e127.
https://openalex.org/W2058213202	https://bmcneurosci.biomedcentral.com/counter/pdf/10.1186/1471-2202-14-121	English	null	Plasticity of primary microglia on micropatterned geometries and spontaneous long-distance migration in microfluidic channels	BMC neuroscience	2,013	cc-by	9,386	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 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. 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]. Background 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. 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. Microglia spontaneously adjust to micropatterned structures 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. 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 Amadio et al. BMC Neuroscience 2013, 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 abou 20 μm, possess numerous very short processes and hav h d d d l l h d h 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. Figure 4 Microfluidic structure adopted for primary microglia culturing. Schematic representation of the microfluid 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. Primary microglia spontaneously migrate in microfluidic channels The maximal length change of individual processes inside the microchannels is 70–80 μm. 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 microfluidi Primary microglia microchannels Primary microglia 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. 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 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 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. Amadio et al. BMC Neuroscience 2013, 14:121 http://www.biomedcentral.com/1471-2202/14/121 Page 7 of 12 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. 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. Moreover, future analysis of parameters such as cell 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. 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, 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) 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 Coating of microfluidic devices with fibronectin 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. Immunofluorescence using microfluidic devices g 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. 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 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). Competing interests The 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. 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. 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. 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. 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 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 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 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). The images are captured with CMOS 1.3 M pixels camera and exported as TIFF files. Image processing Fluorescence microscopy images are imported into Matlab, converted into binary 8-bit images and processed by a code developed according to Kozlowsky and Weimer [45]. The images are then subjected to quantitative analysis of 2. Nimmerjahn A, Kirchhoff F, Helmchen F: Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Science 2005, 308:1314–1318. 3. Rezaie P, Male D: Colonisation of the developing human brain and spinal cord by microglia: a review. Microsc Res Tech 1999, 45:359–382. Page 12 of 12 Amadio et al. BMC Neuroscience 2013, 14:121 http://www.biomedcentral.com/1471-2202/14/121 4. Wake H, Moorhouse AJ, Nabekura J: Functions of microglia in the central nervous system–beyond the immune response. Neuron Glia Biol 2011, 7:47–53. 30. Zhang J, Geula C, Lu C, Koziel H, Hatcher LM, Roisen FJ: Neurotrophins regulate proliferation and survival of two microglial cell lines in vitro. Exp Neurol 2003, 183:469–481. 5. Ransohoff RM, Brown MA: Innate immunity in the central nervous system. J Clin Invest 2012, 122:1164–1171. 31. Ohsawa K, Imai Y, Nakajima K, Kohsaka S: Generation and characterization of a microglial cell line, MG5, derived from a p53-deficient mouse. Glia 1997, 21:285–298. 6. Greter M, Merad M: Regulation of microglia development and homeostasis. Glia 2013, 61:121–127. 6. Greter M, Merad M: Regulation of microglia development an 32. Prinz M, Mildner A: Microglia in the CNS: immigrants from another world. Glia 2011, 59:177–187. homeostasis. Glia 2013, 61:121–127. 7. Hickey WF: Basic principles of immunological surveillance of the normal central nervous system. Glia 2001, 36:118–124. 33. Ousman SS, Kubes P: Immune surveillance in the central nervous system. Nat Neurosci 2012, 15:1096–1101. 8. Davalos D, Grutzendler J, Yang G, Kim JV, Zuo Y, Jung S, Littman DR, Dustin ML, Gan WB: ATP mediates rapid microglial response to local brain injury in vivo. Nat Neurosci 2005, 8:752–758. 34. Carbonell WS, Murase SI, Horwitz AF, Mandell JW: Infiltrative microgliosis: activation and long-distance migration of subependymal microglia following periventricular insults. J Neuroinflammation 2005, 2:5. 9. Hanisch UK, Kettenmann H: Microglia: active sensor and versatile effector 9. Hanisch UK, Kettenmann H: Microglia: active sensor and versatile effector cells in the normal and pathologic brain. Nat Neurosci 2007, 10:1387–1394. 35. Lee S, Chung CY: Role of VASP phosphorylation for the regulation of microglia chemotaxis via the regulation of focal adhesion formation/ maturation. Mol Cell Neurosci 2009, 42:382–390. 10. Kettenmann H, Hanisch UK, Noda M, Verkhratsky A: Physiology of microglia. Physiol Rev 2011, 91:461–553. 36. Image processing Karlstetter M, Lippe E, Walczak Y, Moehle C, Aslanidis A, Mirza M, Langmann T: Curcumin is a potent modulator of microglial gene expression and migration. J Neuroinflammation 2011, 8:125. 11. Tremblay MÈ, Stevens B, Sierra A, Wake H, Bessis A, Nimmerjahn A: The role of microglia in the healthy brain. J Neurosci 2011, 31:16064–16069. 11. Tremblay MÈ, Stevens B, Sierra A, Wake H, Bessis A, Nimmerjahn A: The role of microglia in the healthy brain. J Neurosci 2011, 31:16064–16069. 12. Saijo K, Crotti A, Glass CK: Regulation of microglia activation and y , , , , , j of microglia in the healthy brain. J Neurosci 2011, 31:16064–16069. 12. Saijo K, Crotti A, Glass CK: Regulation of microglia activation and deactivation by nuclear receptors. Glia 2013, 61:104–111. 12. Saijo K, Crotti A, Glass CK: Regulation of microglia activation and deactivation by nuclear receptors. Glia 2013, 61:104–111. 37. Honda S, Sasaki Y, Ohsawa K, Imai Y, Nakamura Y, Inoue K, Kohsaka S: Extracellular ATP or ADP induce chemotaxis of cultured microglia through Gi/o-coupled P2Y receptors. J Neurosci 2001, 21:1975–1982. 13. El-Ali J, Sorger PK, Jensen KF: Cells on chips. Nature 2006, 442:403–411. 14. Zervantonakis IK, Hughes-Alford SK, Charest JL, Condeelis JS, Gertler FB, Kamm RD: Three-dimensional microfluidic model for tumor cell intravasation and endothelial barrier function. Proc Natl Acad Sci U S A 2012, 109:13515–13520. 38. Nasu-Tada K, Koizumi S, Inoue K: Involvement of beta1 integrin in microglial chemotaxis and proliferation on fibronectin: different regulations by ADP through PKA. Glia 2005, 52:98–107. 39. Haynes SE, Hollopeter G, Yang G, Kurpius D, Dailey ME, Gan WB, Julius D: The P2Y12 receptor regulates microglial activation by extracellular nucleotides. Nat Neurosci 2006, 9:1512–1519. 15. Kim BJ, Wu M: Microfluidics for mammalian cell chemotaxis. Ann Biomed Eng 2012, 40:1316–1327. 16. Businaro L, De Ninno A, Schiavoni G, Lucarini V, Ciasca G, Gerardino A, Belardelli F, Gabriele L, Mattei F: Cross talk between cancer and immune cells: exploring complex dynamics in a microfluidic environment. Lab Chip 2013, 13:229–239. 40. Whitesides GM: The origins and the future of microfluidics. Nature 2006, 442:368–373. 40. Whitesides GM: The origins and the future of microfluidics. Nature 2006, 442:368–373. 41. Qin D, Xia Y, Whitesides GM: Soft lithography for micro- and nanoscale patterning. Nat Protoc 2010, 5:491–502. 41. Qin D, Xia Y, Whitesides GM: Soft lithography for micro- and nanoscale patterning. Nat Protoc 2010, 5:491–502. 17. Amadio et al. BMC Neuroscience 2013, 14:121 http://www.biomedcentral.com/1471-2202/14/121 Image processing Taylor AM, Jeon NL: Microfluidic and compartmentalized platforms for neurobiological research. Crit Rev Biomed Eng 2011, 39:185–200. 42. Hosmane S, Yang IH, Ruffin A, Thakor N, Venkatesan A: Circular compartmentalized microfluidic platform: Study of axon-glia interactions. Lab Chip 2010, 10:741–747. 18. Kim HJ, Park JW, Byun JH, Vahidi B, Rhee SW, Jeon NL: Integrated microfluidics platforms for investigating injury and regeneration of CNS axons. Ann Biomed Eng 2012, 40:1268–1276. 43. Park JW, Vahidi B, Taylor AM, Rhee SW, Jeon NL: Microfluidic culture platform for neuroscience research. Nat Protoc 2006, 1:2128–2136. 19. Persheyev S, Fan Y, Irving A, Rose MJ: BV-2 microglial cells sense micro- nanotextured silicon surface topology. J Biomed Mater Res A 2011, 99:135–140. 44. Chen Y, Balasubramaniyan V, Peng J, Hurlock EC, Tallquist M, Li J, Lu QR: Isolation and culture of rat and mouse oligodendrocyte precursor cells. Nat Protoc 2007, 2:1044–1051. 20. Hosmane S, Tegenge MA, Rajbhandari L, Uapinyoying P, Kumar NG, Thakor N, Venkatesan A: Toll/interleukin-1 receptor domain-containing adapter inducing interferon-β mediates microglial phagocytosis of degenerating axons. J Neurosci 2012, 32:7745–7757. 45. Kozlowski C, Weimer RM: An automated method to quantify microglia morphology and application to monitor activation state longitudinally in vivo. PLoS One 2012, 7:e31814. 21. Ransohoff RM, Perry VH: Microglial physiology: unique stimuli, specialized responses. Annu Rev Immunol 2009, 27:119–145. doi:10.1186/1471-2202-14-121 Cite this article as: Amadio et al.: Plasticity of primary microglia on micropatterned geometries and spontaneous long-distance migration in microfluidic channels. BMC Neuroscience 2013 14:121. 22. Tambuyzer BR, Ponsaerts P, Nouwen EJ: Microglia: gatekeepers of central nervous system immunology. J Leukocyte Biol 2009, 85:352–370. 23. Kreutzberg GW: Microglia: a sensor for pathological events in the CNS. Trends Neurosci 1996, 19:312–318. 24. D'Ambrosi N, Finocchi P, Apolloni S, Cozzolino M, Ferri A, Padovano V, Pietrini G, Carrì MT, Volonté C: The proinflammatory action of microglial P2 receptors is enhanced in SOD1 models for amyotrophic lateral sclerosis. J Immunol 2009, 183:4648–4656. 25. Sasaki Y, Hoshi M, Akazawa C, Nakamura Y, Tsuzuki H, Inoue K, Kohsaka S: Selective expression of Gi/o-coupled ATP receptor P2Y12 in microglia in rat brain. Glia 2003, 44:242–250. Image processing 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 26. Marín-Teva JL, Almendros A, Calvente R, Cuadros MA, Navascués J: Tangential migration of ameboid microglia in the developing quail retina: mechanism of migration and migratory behavior. Glia 1998, 22:31–52. 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 27. Khatau SB, Hale CM, Stewart-Hutchinson PJ, Patel MS, Stewart CL, Searson PC, Hodzic D, Wirtz D: A perinuclear actin cap regulates nuclear shape. Proc Natl Acad Sci U S A 2009, 106:19017–19022. 28. Ristanović D, Milosević NT, Stefanović BD, Marić DL, Rajković K: Morphology and classification of large neurons in the adult human dentate nucleus: a qualitative and quantitative analysis of 2D images. Neurosci Res 2010, 67:1–7. 29. Ferrari D, Villalba M, Chiozzi P, Falzoni S, Ricciardi-Castagnoli P, Di Virgilio F: Mouse microglial cells express a plasma membrane pore gated by extracellular ATP. J Immunol 1996, 156:1531–1539.
https://openalex.org/W4210728771	https://ejnmmiphys.springeropen.com/track/pdf/10.1186/s40658-022-00437-3	English	null	Freely available convolutional neural network-based quantification of PET/CT lesions is associated with survival in patients with lung cancer	EJNMMI physics	2,022	cc-by	5,347	© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the mate- rial. 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/. Open Access ORIGINAL RESEARCH Open Access Freely available convolutional neural network‑based quantification of PET/CT lesions is associated with survival in patients with lung cancer Pablo Borrelli1†, José Luis Loaiza Góngora1†, Reza Kaboteh1, Johannes Ulén2, Olof Enqvist2,3, Elin Trägårdh4,5 and Lars Edenbrandt1,6* *Correspondence: lars.edenbrandt@gu.se †Pablo Borrelli and Jose Luis Loaiza Gongora shared first authorship 1 Department of Clinical Physiology, Region Västra Götaland, Sahlgrenska University Hospital, Gothenburg, Sweden Full list of author information is available at the end of the article Borrelli et al. EJNMMI Physics (2022) 9:6 https://doi.org/10.1186/s40658-022-00437-3 Borrelli et al. EJNMMI Physics (2022) 9:6 https://doi.org/10.1186/s40658-022-00437-3 EJNMMI Physics Abstract Background: Metabolic positron emission tomography/computed tomography (PET/CT) parameters describing tumour activity contain valuable prognostic informa- tion, but to perform the measurements manually leads to both intra- and inter-reader variability and is too time-consuming in clinical practice. The use of modern artificial intelligence-based methods offers new possibilities for automated and objective image analysis of PET/CT data. Purpose: We aimed to train a convolutional neural network (CNN) to segment and quantify tumour burden in [18F]-fluorodeoxyglucose (FDG) PET/CT images and to evaluate the association between CNN-based measurements and overall survival (OS) in patients with lung cancer. A secondary aim was to make the method available to other researchers. Methods: A total of 320 consecutive patients referred for FDG PET/CT due to sus- pected lung cancer were retrospectively selected for this study. Two nuclear medicine specialists manually segmented abnormal FDG uptake in all of the PET/CT studies. One-third of the patients were assigned to a test group. Survival data were collected for this group. The CNN was trained to segment lung tumours and thoracic lymph nodes. Total lesion glycolysis (TLG) was calculated from the CNN-based and manual segmentations. Associations between TLG and OS were investigated using a univariate Cox proportional hazards regression model. Results: The test group comprised 106 patients (median age, 76 years (IQR 61–79); n = 59 female). Both CNN-based TLG (hazard ratio 1.64, 95% confidence interval 1.21– 2.21; p = 0.001) and manual TLG (hazard ratio 1.54, 95% confidence interval 1.14–2.07; p = 0.004) estimations were significantly associated with OS. Conclusion: Fully automated CNN-based TLG measurements of PET/CT data showed were significantly associated with OS in patients with lung cancer. This type of meas- urement may be of value for the management of future patients with lung cancer. The CNN is publicly available for research purposes. Page 2 of 10 Page 2 of 10 Borrelli et al. EJNMMI Physics (2022) 9:6 (2022) 9:6 Borrelli et al. EJNMMI Physics MMI Physics (2022) 9:6 Keywords: Computer-assisted analysis, Tumour burden, Total lesion glycolysis, Prognosis Introductionl [18F]fluorodeoxyglucose (FDG) positron emission tomography/computed tomogra- phy (PET/CT) plays an important role in lung cancer, both for small cell and non-small cell cancer diagnosis, staging, response assessment and follow-up [1–4]. Several stud- ies have shown the prognostic value of different metabolic PET parameters [5–10]. The methods to quantify tumour burden are, however, usually based on manually selected lesions by local imaging experts and easily accessible measurements such as maximum or peak standardized uptake value (SUV). An objective way to analyse the PET/CT find- ings would enable effectively comparing the results from different studies and, thereby, facilitate the assessment of the FDG PET/CT findings in patients with suspected lung cancer. The use of modern artificial intelligence (AI)-based methods offers new possibili- ties for automated and objective image analysis [11]. AI-based technology can be trained to assess the entire burden of disease by including both the extent and activity of the tumour and not only the maximum or peak SUV, which represents a very small volume of the tumour [12]. Manual methods of assessing total tumour burden, for example, the segmentation of all tumour lesions and estimation of total lesion glycolysis (TLG), are too time-consuming for clinical use and hampered by low reproducibility. We recently trained a convolutional neural network (CNN) to automatically detect lesions and calculate the TLG from the FDG PET/CT data of patients with lung cancer [13]. That CNN has a sensitivity of 90% and the correlation between the manual and CNN-based automated TLG measurements is strong (r2 = 0.74). These results inspired us to take the next step and train a new CNN using a larger training set and expanding the task of the CNN to segment also thoracic lymph nodes. In addition, a new test group was selected in which survival data were available. Therefore, this study aimed to evalu- ate this new CNN by comparing its automated TLG measurements to corresponding manual measurements and by assessing the association between the TLG measurements and overall survival (OS) in patients with lung cancer. A secondary aim was to make the AI-based method freely available to other researchers. Methods Two groups of consecutive patients referred for FDG PET/CT due to suspected lung cancer were retrospectively selected to develop and evaluate the new CNN. One group of 113 patients underwent PET/CT between April 2008 and December 2010 at the Sahl- grenska University Hospital, Gothenburg, Sweden. This group was used in our first study to train and evaluate a CNN for the detection of lung tumours [13]. The other group of 207 patients underwent PET/CT between November 2017 and November 2018 at the Skåne University Hospital in Lund/Malmö, Sweden. The total study group of 320 patients was divided into a test group of 106 patients (33%) and a training group of 214 patients (67%). Only patients from the Skåne Uni- versity Hospital were selected randomly for the test group since the patients from the Borrelli et al. EJNMMI Physics (2022) 9:6 Page 3 of 10 Sahlgrenska University Hospital were already used to train and test the CNN developed in our previous study. Clinical information and survival data for the test group were col- lected from local medical records and the radiology information system up until Novem- ber 2020. The patient characteristics of the test group are presented in Table 1. h This study was conducted according to the principles expressed in the Declaration of Helsinki and approved by the local research ethics committees at Gothenburg (#295– 08) and Lund Universities (#2016/193 and #2018/753). All patients provided written informed consent. Imaging PET/CT scans were obtained using integrated PET/CT systems (Siemens Biograph 64 Truepoint, Siemens Healthineers, Erlangen, Germany and GE Discovery MI, GE Health- care, Chicago, USA). The patients were injected with 4 MBq/kg (maximum of 400 Mbq) of FDG, fasted for at least 4 h prior to the injection and had adequate glucose levels prior to the injection. The accumulation time was 60 min. Images were acquired at 3 min per bed position (Sahlgrenska) or 1.5 min per bed position (Skåne) from the base of the skull to the mid-thigh. PET images obtained from the Siemens Biograph 64 Truepoint PET/CT scanner were reconstructed with a slice thickness of 3 mm using an iterative ordered subset expec- tation maximization 3D algorithm (four iterations, eight subsets) with a matrix size of 168 × 168. CT-based attenuation and scatter corrections were applied. A low-dose CT scan (64-slice helical, 120 kV, 30 mAs, 512 × 512 matrix) was obtained covering the same area of the patient as the PET scan. The CT was reconstructed using a filtered back-pro- jection algorithm with slice thickness and spacing that matched the PET scan. The PET images obtained from the GE Discovery MI system were reconstructed using the commercially available block-sequential regularized expectation maximiza- tion (BSREM) algorithm Q.Clear (GE Healthcare, Milwaukee, WI, USA) with a beta Table 1 Characteristics of the patients in the test group n Median years (IQR) Age 106 76 (61–79) Sex Female 59 Male 47 Survival status Dead—survival time 51 0.9 (0.54–1.54) Alive—follow-up time 55 2.6 (2.5–2.7) Diagnosis Non-small cell lung carcinoma 85 Lung cancer of unknown type 11 Lung metastases 5 Hamartoma 1 Lymphoma 1 Pneumoconiosis 1 Schwannoma 1 Unknown 1 Table 1 Characteristics of the patients in the test group Borrelli et al. EJNMMI Physics (2022) 9:6 Page 4 of 10 factor of 550. The time-of-flight and point spread functions were used with a 256 × 256 matrix (pixel size 2.7 × 2.7 mm2, slice thickness 2.8 mm). CT images were acquired for attenuation correction and anatomical correlation of the PET images. A diagnostic CT with intravenous and oral contrast medium or a low-dose CT without contrast was per- formed. In our clinical routine, a low-dose CT is performed if a previous diagnostic CT was performed within 4 weeks. Manual segmentations Two nuclear medicine specialists with over 6 and over 12 years of PET/CT experi- ence segmented abnormal FDG uptake in the PET/CT images from the training and test groups. The segmentations were made manually by visual inspection in a consen- sus reading. Abnormal uptakes were classified into one of the following groups: lung tumour, thoracic lymph node, extra-thoracic lymph node, adrenal, bone, liver metas- tasis, inflammatory, high pleura or other high activity. No clinical data, only PET/CT images were available during the segmentation process. A cloud-based annotation tool (RECOMIA, https://www.recomia.org) was used for the manual segmentations [14]. Imaging For diagnostic CTs, tube current modulation was applied by adjusting the tube current for each individual with a noise index of 42.25 and a tube voltage of 100 kV. For the low-dose CT, the tube voltage was 120 kV with a noise index of 45. If a diagnostic CT was performed, it was used for attenuation correction (delayed venous phase of intravenous contrast). The adaptive statistical iterative reconstruction technique (ASiR-V) was applied for all CT reconstructions. Convolutional neural network The CNN was trained to segment lung tumours and thoracic lymph nodes only. The numbers of other abnormal uptake examples were insufficient for CNN training. fi This model uses a CNN with U-net 3D architecture [15]. The final convolutional layer contains three channels with softmax activation, one for background, one for lung tumour and one for the thoracic lymph node. The network has three separate inputs, the CT image, the PET image and a one-hot encoded organ mask constructed using the model from [14]. The purpose of the organ mask is to help the network with a rough anatomical localization for a given uptake; it uses one channel each for bone, liver, lung, heart, aorta and adrenal gland. The model was trained using patches of minimal size, where the patches were chosen with care to provide a good balance between the different classes. All pixels were divided into four groups: background, lung tumour, thoracic lymph node and other abnormal uptake (this group included extra-thoracic lymph nodes, metastases, inflammatory uptake, high pleura uptake and other high uptake). The centre point of each patch was chosen randomly with an equal probability of being inside any of the four groups. The training involved 100 epochs with 10,000 patches per epoch. Categorical cross- entropy was used as the loss function, and the optimization was performed using the Adam method [16] with Nesterov momentum. In order to reduce overfitting, both early stopping (patience 10 epochs with no validation loss decrease) and l2 regularization with weight 0.01 were used. As pre-processing the CT image is clamped to HU range [− 800, 800] and the SUV image to [0, 25], both the CT and SUV images were then rescaled [− 1, 1]. The input patches were augmented using rotations (− 0.15 to 0.15) radians, Borrelli et al. EJNMMI Physics (2022) 9:6 Page 5 of 10 scaling (− 10 to 10%) and intensity shifts of (− 100 to + 100HU) for the CT images and (− 0.5 to + 0.5) for the SUV image. After this phase, the resulting model was applied to the training set. The model was then retrained with 20% of the patches focusing on pixels incorrectly classified by the model. These steps were repeated four times. The resulting model is the last model after these four steps and early stopping (not the model with lowest validation loss). Convolutional neural network Finally, lung tumours and thoracic lymph nodes with TLGs below 0.1 were removed. Statistical analysis Associations between TLG and OS were investigated using a univariate Cox propor- tional hazards regression model. OS was calculated from the date of the PET/CT analy- sis to the date of death or the last follow-up. Hazard ratios (HRs) and 95% confidence intervals (CIs) were estimated. The level of significance was set at 0.05. The TLG meas- urements had a skewed distribution and were log10-transformed after adding 1.0 to handle any zeros. The TLG measurements for the Kaplan–Meier analysis were catego- rized according to the corresponding median value, higher vs. lower than the median. The statistical analysis was performed in R (version 4.0.3) [17]. TLG measurements Uptake from lung tumours and thoracic lymph nodes were used to compute a total TLG for each patient, both from the CNN and manual segmentations. Figure 1 shows a Bland–Altman plot comparing the CNN and manual TLG. Figure 2 plots the rank of each study based on manual TLG against the rank based on CNN-based TLG. The Spearman correlation of the TLG measurements, being the Pearson correlation of these rank values, is 0.95. Both CNN TLG (HR 1.64, 95% CI 1.21–2.21; p = 0.001) and manual TLG (HR 1.54, 95% CI 1.14–2.07; p = 0.004) were significantly associated with OS in univariate propor- tional regression Cox analyses. The Kaplan–Meier curves are shown in Fig. 3. The 53 patients with CNN TLGs above the median value had a significantly shorter survival time than the 53 patients with val- ues below the median. The median survival times for the two groups were 1.57 years 0 1000 2000 3000 4000 5000 −500 0 500 Mean of CNN−based and manual TLG Difference (CNN − manual) MEAN Fig. 1 Bland–Altman plot comparing CNN-based and manual TLG measurements Fig. 1 Bland–Altman plot comparing CNN-based and manual TLG measurements Page 6 of 10 Borrelli et al. EJNMMI Physics (2022) 9 Borrelli et al. EJNMMI Physics 0 20 40 60 80 100 0 20 40 60 80 100 Manual TLG rank CNN TLG rank Fig. 2 The rank of each study based on manual TLG measurements against the rank of each study based on the CNN-based measurements. This serves to show, that in terms of ranking patients with respect to tumour burden the automated method is similar to a manual analysis 0 20 40 60 80 100 0 20 40 60 80 100 Manual TLG rank CNN TLG rank Fig. 2 The rank of each study based on manual TLG measurements against the rank of each study based on the CNN-based measurements. This serves to show, that in terms of ranking patients with respect to tumour burden the automated method is similar to a manual analysis 0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.2 0.4 0.6 0.8 1.0 Years Survival probability a 0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.2 0.4 0.6 0.8 1.0 Years Survival probability b Fig. 3 Kaplan–Meier curves for the groups higher (black line) vs. TLG measurements lower (grey line) than the median TLG values for CNN TLG (a) and manual TLG (b) 0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.2 0.4 0.6 0.8 1.0 Years Survival probability a b b Fig. 3 Kaplan–Meier curves for the groups higher (black line) vs. lower (grey line) than the median TLG values for CNN TLG (a) and manual TLG (b) Borrelli et al. EJNMMI Physics (2022) 9:6 Page 7 of 10 vs. not reached after 3 years of follow-up. The log-rank test showed a significant dif- ference in survival times (p < 0.001). The corresponding median survival values for the two groups stratified using manual TLG were 1.75 years vs. not reached after 3 years of follow-up (p = 0.002). Lesion classification results The CNN and the physicians classified 99/106 (93%) patients similarly regarding the presence of a lung tumour, 94 as positive and 5 as negative. Four patients were classified as positive only by the CNN. The CNN detections in these four cases were classified as thoracic lymph nodes (2), high pleura uptake (1) and inflammatory uptake (1) by the physicians. Three patients were classified as positive only by the physicians. A total of 135 lung tumours were detected by both the CNN and the physicians. These tumours had a median TLG of 13.9 (IQR 2.3–181.7). A total of 56 lesions with a median TLG of 0.4 (IQR 0.1–1.6) were detected as lung tumours by the physicians only. Seven of these lesions were classified as thoracic lymph nodes by the CNN. A total of 153 lesions with a median TLG of 0.6 (IQR 0.3–2.3) were detected as lung tumours by the CNN only. Thirty-five of these lesions were classified as thoracic lymph nodes by the physicians. hii The CNN and the physicians classified 71/106 (67%) patients similarly regarding the presence of thoracic lymph nodes, 59 as positive and 12 as negative. Thirty-five patients were classified as positive only by the CNN. Both the CNN and the physicians detected 170 thoracic lymph nodes. These lesions had a median TLG of 5.8 (IQR 1.5–27.9). A total of 70 lesions with a median TLG of 0.3 (IQR 0.1–0.9) were detected as thoracic lymph nodes by the physicians only. Five of these lesions were classified as lung tumours by the CNN. A total of 274 lesions with a median TLG of 0.8 (IQR 0.3–2.4) were detected as thoracic lymph nodes by the CNN only. Thirty-two of these lesions were classified as lung tumours and 23 as extra-thoracic lymph nodes by the physicians. Discussion In this study, we explored if automated TLG measurements calculated by a CNN were clinically relevant in patients with lung cancer. The results show that a CNN can be trained to automatically segment lung tumours and thoracic lymph nodes and calculate TLG measurements that are significantly associated with OS. The CNN TLG had a simi- lar prognostic performance as the corresponding manual measurements. In a clinical setting, a physician would be able to check CNN-based segmentations and dismiss false-positive lesions. The use of AI support for the calculation of total lesion uptake significantly reduces inter-reader variability in the analysis of prostate lesions and bone metastases with PSMA PET/CT [18]. This process is also more time-effective than a completely manual segmentation process and feasible for a clinical setting. In research, these types of AI tools may facilitate comparisons between studies from different centres, pooling data within multicentre trials and performing meta-analyses when objective evaluation is applied rather than local image readers. Objective AI-based TLG measurements may be useful not only for the prognostic evaluation of patients with lung cancer at the time of diagnosis but also for monitoring treatment response. FDG PET/CT findings have been associated with clinical benefit in patients with non-small cell lung cancer receiving immunotherapy [4]. Borrelli et al. EJNMMI Physics (2022) 9:6 Borrelli et al. EJNMMI Physics (2022) 9:6 Page 8 of 10 AI tools have been used as computer-aided detection (CAD) systems to highlight potential lesions in chest X-rays and lung CTs [19]. The aim is to decrease the likeli- hood of a radiologist missing tumours and the subsequent delay in diagnosis. Such an AI tool needs to have high sensitivity and a low false-positive rate to be of clinical value. The aim of our CNN was not to be a CAD system but a tool for the automated quantification of tumour burden. The sensitivity and false-positive rate of our CNN were not sufficient for use as a clinical CAD system. The disagreements between the physicians and the CNN were, in most cases, related to lesions with low TLG. A com- mon reason for any disagreement was physicians classifying a lesion as a lung tumour and the CNN as a mediastinal/hilar lymph node or vice versa; this is not always an easy decision even for experienced physicians. A limitation of this study is the reference method using the manual segmentations by two nuclear medicine specialists. Discussion A slightly different result would most likely have been found if other, additional image readers had been used given the well-known problems of inter- and intra-observer variability. The comparisons between the CNN- based and manual TLG measurements indicate that the CNN was trained to perform similarly to a nuclear medicine specialist. A strength of this study is that we also used OS as an image-independent reference method. The retrospective design of this study is, on the one hand, a limitation of the study but allowed us to assess the performance of the CNN not only compared with manual segmentation of the same images but also to the independent reference OS. The train- ing material only contained a handful of abnormal uptake other than lung tumours and thoracic lymph nodes, due to this we limited this work to these two uptakes. Extra-thoracic lymph nodes and distant metastases will be added in future versions of our CNN. Further development will also include the localization of lymph nodes into established anatomical definitions and classifications into ipsilateral versus con- tralateral regions [20]. This type of information may improve the prognostic value of AI-based analyses. The development of AI-based analyses depends on the availability of large train- ing and test groups. The training and test groups used in this study were sufficient to show the proof of concept of a fully automated AI-based PET/CT analysis, but we recognize that larger patient groups including patients from several hospitals would improve the CNN-based method and strengthen the results. One approach to achieve this is to invite others researchers to participate in the development and we therefore make our CNN available to others. Another approach is to use methods to maximize the utility of incomplete and missing data as presented by Guo and co-workers [21]. Conclusions Fully automated CNN-based TLG measurements of FDG PET/CT data were signifi- cantly associated with OS in patients with lung cancer. These types of measurements may be of value in the management of future patients with lung cancer. Our CNN is available for research purposes upon request from the RECOMIA platform (https:// recomia.org). Page 9 of 10 Borrelli et al. EJNMMI Physics Borrelli et al. EJNMMI Physics (2022) 9:6 (2022) 9:6 Author details 1 1 Department of Clinical Physiology, Region Västra Götaland, Sahlgrenska University Hospital, Gothenburg, Sweden. 2 Eigenvision AB, Malmö, Sweden. 3 Department of Electrical Engineering, Chalmers University of Technology, Gothen- burg, Sweden. 4 Department of Translational Medicine and Wallenberg Centre for Molecular Medicine, Lund University, Malmö, Sweden. 5 Department of Clinical Physiology and Nuclear Medicine, Skåne University Hospital, Malmö, Sweden. 6 Department of Molecular and Clinical Medicine, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden. Received: 6 June 2021 Accepted: 24 January 2022 Received: 6 June 2021 Accepted: 24 January 2022 Funding g Open access funding provided by University of Gothenburg. The study was financed by grants from the Knut and Alice Wallenberg Foundation, the Medical Faculty at Lund University, Region Skåne and the Swedish state under the agree- ment between the Swedish government and county councils and the ALF agreement (ALFGBG-720751). Availability of data and materials y Our CNN is available for research purposes upon request at the RECOMIA platform (https://recomia.org). The datasets generated and analysed for the current study are not publicly available due to privacy restrictions from the hospitals providing them. References 1. Volpi S, Ali JM, Tasker A, Peryt A, Aresu G, Coonar AS. The role of positron emission tomography in the diagnosis, staging and response assessment of non small cell lung cancer Ann Transl Med 2018 6(5) 95 1. Volpi S, Ali JM, Tasker A, Peryt A, Aresu G, Coonar AS. The role of positron emission tomography in the diagnosis, staging and response assessment of non-small cell lung cancer. Ann Transl Med. 2018;6(5):95. 2. Carretta A, Bandiera A, Muriana P, Viscardi S, Ciriaco P, Samanes Gajate AM, et al. Prognostic role of positron emission tomography and computed tomography parameters in stage I lung adenocarcinoma. Radiol Oncol. 2020;54(3):278–84. 2. Carretta A, Bandiera A, Muriana P, Viscardi S, Ciriaco P, Samanes Gajate AM, et al. Prognostic role of positron emission tomography and computed tomography parameters in stage I lung adenocarcinoma. Radiol Oncol. 2020;54(3):278–84. 3. Martucci F, Pascale M, Valli MC, Pesce GA, Froesch P, Giovanella L, et al. Impact of 18F-FDG PET/CT in staging pat with small cell lung cancer: a systematic review and meta-analysis. Front Med (Lausanne). 2020;6:336. 3. Martucci F, Pascale M, Valli MC, Pesce GA, Froesch P, Giovanella L, et al. Impact of 18F-FDG PET/CT in staging patients with small cell lung cancer: a systematic review and meta-analysis. Front Med (Lausanne). 2020;6:336. 3. Martucci F, Pascale M, Valli MC, Pesce GA, Froesch P, Giovanella L, et al. Impact of 18F-FDG PET/CT in staging patients with small cell lung cancer: a systematic review and meta-analysis. Front Med (Lausanne). 2020;6:336. 4. Humbert O, Cadour N, Paquet M, Schiappa R, Poudenx M, Chardin D, et al. 18FDG PET/CT in the early assessment of non-small cell lung cancer response to immunotherapy: frequency and clinical significance of atypical evolutive 4. Humbert O, Cadour N, Paquet M, Schiappa R, Poudenx M, Chardin D, et al. 18FDG PET/CT in the early assessment of non-small cell lung cancer response to immunotherapy: frequency and clinical significance of atypical evolutive patterns. Eur J Nucl Med Mol Imaging. 2020;47:1158–67. 4. Humbert O, Cadour N, Paquet M, Schiappa R, Poudenx M, Chardin D, et al. 18FDG PET/CT in the early assessment of non-small cell lung cancer response to immunotherapy: frequency and clinical significance of atypical evolutive patterns. Eur J Nucl Med Mol Imaging. 2020;47:1158–67. p g g 5. Chang H, Lee SJ, Lim J, Lee JS, Kim YJ, Lee WW. Authors’ contributions Data collection was performed by RZ. Image segmentation was performed by PB and JLLG. AI tool development was performed by JU and OE. ET and LE contributed to the design and interpretation of the data. All authors read and approved the final manuscript. Consent for publication All patients provided written informed consent. Competing interests The authors declare no conflict of interest. Ethics approval pp The study was conducted according to the principles expressed in the Declaration of Helsinki, approved by the local research ethics committees at Gothenburg (#295-08) and Lund Universities, Sweden (#2016/193 and #2018/753). y p g y g p gy 12. Høilund-Carlsen PF, Piri R, Gerke O, Edenbrandt L, Alavi A. Assessment of total-body atherosclerosis by PET/com- puted tomography. PET Clin. 2021;16:119–28. References Weakly supervised deep learning for determining the prognostic value of 18F-FDG PET/CT in extranodal natural killer/T cell lymphoma, nasal type. Eur J Nucl Med Mol Imaging. 2021;48:3151–61. References Prognostic significance of metabolic parameters measured by 18F- FDG PET/CT in limited-stage small-cell lung carcinoma. J Cancer Res Clin Oncol. 2019;145:1361–7. 6. Chardin D, Paquet M, Schiappa R, Darcourt J, Bailleux C, Poudenx M, et al. Baseline metabolic tumor volume as a strong predictive and prognostic biomarker in patients with non-small cell lung cancer treated with PD1 inhibitors: a prospective study. J Immunother Cancer. 2020;8:e000645. https://doi.org/10.1136/jitc-2020-000645. 7. Jin F, Qu B, Fu Z, Zhang Y, Han A, Kong L, et al. Prognostic value of metabolic parameters of metastatic lymph nodes on 18F-FDG PET/CT in patients with limited-stage small-cell lung cancer with lymph node involvement. Clin Lung Cancer. 2017;19:e101–8. 7. Jin F, Qu B, Fu Z, Zhang Y, Han A, Kong L, et al. Prognostic value of metabolic parameters of metastatic lymph nodes on 18F-FDG PET/CT in patients with limited-stage small-cell lung cancer with lymph node involvement. Clin Lung Cancer. 2017;19:e101–8. 8. Seban RD, Mezquita L, Berenbaum A, Dercle L, Botticella A, Le Pechoux C, et al. Baseline metabolic tumor burden on FDG PET/CT scans predicts outcome in advanced NSCLC patients treated with immune checkpoint inhibitors. Eur J Nucl Med Mol Imaging. 2020;47:1147–57. 8. Seban RD, Mezquita L, Berenbaum A, Dercle L, Botticella A, Le Pechoux C, et al. Baseline metabolic tumor burden on FDG PET/CT scans predicts outcome in advanced NSCLC patients treated with immune checkpoint inhibitors. Eur J Nucl Med Mol Imaging. 2020;47:1147–57. g g 9. Zer A, Domachevsky L, Rapson Y, Nidam M, Flex D, Allen AM, et al. The role of 18F-FDG PET/CT on staging and prog- nosis in patients with small cell lung cancer. Eur Radiol. 2016;26:3155–61.il g g 9. Zer A, Domachevsky L, Rapson Y, Nidam M, Flex D, Allen AM, et al. The role of 1 nosis in patients with small cell lung cancer. Eur Radiol. 2016;26:3155–61. 10. Geredeli C, Artac M, Kocak I, Koral L, Sakin A, Altinok T, et al. The prognostic significance of the 18F-fluorodeoxyglu- cose positron emission tomography/computed tomography in early-stage nonsmall cell lung cancer. J Can Res Ther. 2020;16:816–21.fi 11. Sibille L, Seifert R, Avramovic N, Vehren T, Spottiswoode B, Zuehlsdorff S, et al. 18F-FDG PET/CT uptake classification in lymphoma and lung cancer by using deep convolutional neural networks. Radiology. 2020;294:445–52. 12. Høilund-Carlsen PF, Piri R, Gerke O, Edenbrandt L, Alavi A. Assessment of total-body atherosclerosis by PET/com- puted tomography. PET Clin. 2021;16:119–28. Borrelli et al. References EJNMMI Physics (2022) 9:6 Page 10 of 10 13. Borrelli P, Ly J, Kaboteh R, Ulén J, Enqvist O, Trägårdh E, et al. AI-based detection of lung lesions in18F-FDG PET-CT from lung cancer patients. EJNMMI Phys. 2021;8:32. g p y 14. Trägårdh E, Borrelli P, Kaboteh R, Gillberg T, Ulén J, Enqvist O, et al. RECOMIA-a cloud-based platform for artificial intelligence research in nuclear medicine and radiology. EJNMMI Phys. 2020;7:51. 15. Çiçek Ö, Abdulkadir A, Lienkamp SS, Brox T, Ronneberger O. 3D U-Net: learning dense volumetric segmentation from sparse annotation. In: Ourselin S, Joskowicz L, Sabuncu M, Unal G, Wells W, editors. Medical image computing and computer-assisted intervention—MICCAI 2016. MICCAI 2016. Lecture notes in computer science, vol. 9901. Cham: Springer; 2016. https://doi.org/10.1007/978-3-319-46723-8_49. p g p g 16. Kingma DP, Ba J. Adam: a method for stochastic optimization (2014). arXiv:14126980. 16. Kingma DP, Ba J. Adam: a method for stochastic optimization (2014). arXiv:14126980. 17. R Core Team. R foundation for statistical computing. R: A Language and Environment for Statistical Computing. 2020. https://www.R-project.org/. 17. R Core Team. R foundation for statistical computing. R: A Language and Environment for Statistical Computing. 2020. https://www.R-project.org/. 18. Edenbrandt L, Borrelli P, Ulén J, Enqvist O, Trägårdh E. Automated analysis of PSMA-PET/CT studies using convolu- tional neural networks. https://medrxiv.org/cgi/content/short/2021.03.03.21252818v1. 18. Edenbrandt L, Borrelli P, Ulén J, Enqvist O, Trägårdh E. Automated analysis of PSMA-PET/CT studies using convolu- tional neural networks. https://medrxiv.org/cgi/content/short/2021.03.03.21252818v1. 19. Fazal MI, Patel ME, Tye J, Gupta Y. The past, present and future role of artificial intelligence in imaging. Eur J Radiol. 2018;105:246–50. 19. Fazal MI, Patel ME, Tye J, Gupta Y. The past, present and future role of artificial intelligence in imaging. Eur J Radiol. 2018;105:246–50. 20. UyBico SJ, Wu CC, Suh RD, Le NH, Brown K, Krishnam MS. Lung cancer staging essentials: the new TNM staging system and potential imaging pitfalls. Radiographics. 2010;30:1163–81. 20. UyBico SJ, Wu CC, Suh RD, Le NH, Brown K, Krishnam MS. Lung cancer staging essentials: the new TNM staging system and potential imaging pitfalls. Radiographics. 2010;30:1163–81. y p g g p g p 21. GuoR HuX, Song H, Xu P, Xu H, Rominger A, et al. Weakly supervised deep learning for determining the prognostic value of 18F-FDG PET/CT in extranodal natural killer/T cell lymphoma, nasal type. Eur J Nucl Med Mol Imaging. 2021;48:3151–61. 21. GuoR HuX, Song H, Xu P, Xu H, Rominger A, et al. Publisher’s Note ub s e s ote Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
https://openalex.org/W3114688252	http://www.scielo.br/pdf/aib/v77n3/1808-1657-aib-77-3-0449.pdf	Portuguese	null	PRODUÇÃO DE CONÍDIOS DE METARHIZIUM ANISOPLIAE EM MEIO SÓLIDO À BASE DE RESÍDUOS AGROINDUSTRIAIS	Arquivos do instituto biológico/Arquivos do Instituto Biológico	2,010	cc-by	5,793	2Universidade Estadual do Oeste do Paraná, Centro de Ciências Biológicas e da Saúde, Laboratório de Biotecnologia Agrícola, Cascavel, PR, Brasil. Bolsista de Produtividade em Pesquisa/CNPq. Bolsista de Iniciação Científica/Fundação Araucária. *Bolsista de Iniciação Científica/CNPq. L. Sene1 , L.F.A. Alves1, M.F.P. Lobrigatte1, D. Thomazoni2* L. Sene1 , L.F.A. Alves1, M.F.P. Lobrigatte1, D. Thomazoni2** 1Universidade Estadual do Oeste do Paraná, Centro de Ciências Médicas e Farmacêuticas, Laboratório de Tecnologia das Fermentações, Rua Universitária 2069, CEP 85819-110, Cascavel, PR, Brasil. E-mail: lsene@ unioeste.br DOI: 10.1590/1808-1657v77p4492010 DOI: 10.1590/1808-1657v77p4492010 RESUMO As crescentes despesas com o custo do meio para o cultivo de Metarhizium anisopliae levantam a necessidade de averiguar a eficiência de alguns resíduos agroindustriais que, além de propriedades nutricionais importantes, possuem grande disponibilidade e baixo custo. Os experimentos foram conduzidos no Laboratório de Tecnologia das Fermentações, UNIOESTE, Campus de Cascavel, PR. Neste trabalho, foram avaliados como substratos arroz polido (meio padrão), arroz vermelho, arroz polido suplementado com melaço (2,5%, 5% e 10%), resíduo de cervejaria, resíduo de fecularia, arroz polido + resíduo de cervejaria (1:1) e arroz polido + resíduo de fecularia (1:1). Os experimen tos foram realizados em triplicata, em frascos Erlernmeyer de 250 mL contendo 30 g do substrato (ou 15 g de cada, quando em mistura) e umidade de 70 ± 10%. Os frascos foram inoculados com 0,5 mL da suspensão de conídios (1 × 108 conídios/mL) e incubados em BOD (25 ± 1o C, fotofase 12h, 7 dias). Dentre os meios testados, verificou-se uma elevada produção de conídios no meio composto por arroz + resíduo de cervejaria (11,3 × 108 conídios/g de substrato) e no arroz (9,6 × 108 conídios/g). Os conídios produzidos no meio de arroz + resíduo de cervejaria apresentaram atividade inseticida frente a Spodoptera frugiperda semelhante a dos conídios produzidos no arroz. Este novo meio de cultivo poderá proporcionar uma redução de custo de aproximadamente 50%, representando uma alternativa viável para a produção do fungo. PALAVRAS-CHAVE: Metarhizium anisopliae, resíduos agroindustriais, fermentação em estado sólido. PALAVRAS-CHAVE: Metarhizium anisopliae, resíduos agroindustriais, fermentação em estado sólido INTRODUÇÃO custeio do substrato para o fungo tem acarretado despesas crescentes aos fabricantes. Estudos têm sido realizados desde a década de 80 com o objetivo de avaliar substratos alternativos e mais baratos, incluindo resíduos agroindustriais (Cruz et al., 1983; Alvarenga et al., 1988; Magalhães; Frazão, 1996; Vilas Boas et al., 1996; Alves et al., 1997a; Alves et al., 1997b; Dalla Santa et al., 2005), havendo uma tendência para a regionalização da produção (Alves et al., 1997b). Tal tendência se intensificou nos últimos anos com o apoio do Instituto Biológico, o qual vem prestando consultoria na instalação de biofábricas do fungo M. anisopliae para o controle biológico da cigarrinha da cana-de-açúcar (Leite et al., 2003). Insetos em culturas agrícolas podem provocar perdas consideráveis na produção, acarretando prejuízo ao produtor, sendo que, tradicionalmente, o controle de pragas é realizado com agroquímicos. No entanto, devido aos custos e danos ambientais, medidas alternativas vêm sendo desenvolvidas, destacando-se o controle biológico de insetos a partir de formulações contendo entomopatóge nos (biopesticidas). Recentemente, considerável progresso tem ocorrido com o desenvolvimento de agentes de controle biológico à base de fungos, havendo mais de 30 patentes de produtos biopes ticidas já registrados ou em desenvolvimento em todo o mundo (Kassa, 2003). No entanto, devido aos custos e danos ambientais, medidas alternativas vêm sendo desenvolvidas, destacando-se o controle biológico de insetos a partir de formulações contendo entomopatóge nos (biopesticidas). Recentemente, considerável progresso tem ocorrido com o desenvolvimento de agentes de controle biológico à base de fungos, havendo mais de 30 patentes de produtos biopes ticidas já registrados ou em desenvolvimento em todo o mundo (Kassa, 2003). O resíduo úmido da produção de cerveja, também denominado farelo ou bagaço de cevada, pode ser descrito como uma massa resultante da aglutinação da casca com resíduos do processo de mosturação. Segundo Cabral Filho (1999), os altos valores de pro teína e açúcares resultantes das reações de hidrólise do conteúdo amiláceo do cereal são os maiores atra tivos neste resíduo, embora ocorra variação na sua composição bromatológica em função do processo industrial no qual foi gerado. ( ) Embora muitos micro-organismos sejam utiliza dos no controle de pragas da agricultura, estima-se que os fungos sejam mais frequentemente envolvi dos nas doenças de insetos. Dentre os mais usados, salientam-se os gêneros Metarhizium, Beauveria, Lecanicillium, Nomuraea, Hirsutella, Entomophthor e Asckersonia (Melo; Azevedo, 1998). INTRODUÇÃO A espécie Metarhizium anisopliae encontra-se distribuída de forma ampla na natureza, sendo encontrada no solo, sobrevivendo por longos períodos. Acredita-se que este entomopatógeno ocorra naturalmente em mais de 300 espécies de insetos das diferentes ordens, incluindo pragas importantes como gafanhotos, Mahanarva posticata (cigarrinha-de-folhas da cana-de- açúcar) e Mahanarva fimbriolata (cigarrinha-da-raiz da cana-de-açúcar) (Alves, 1998). De acordo com Bueno (2010), no Brasil, o fungo M. anisopliae vem sendo usado com sucesso para o controle das cigarrinhas das pastagens e de cana-de-açúcar. O resíduo úmido de fecularia ou farelo ou bagaço de mandioca é gerado durante o processo de extração da fécula de mandioca e, após seco, possui um teor de amido de aproximadamente 64% e 61%, respec tivamente, para os processos de extração manual e mecânico (Rocha, 2005). O arroz vermelho, de menor valor comercial que o arroz branco, é um biótipo pertencente à mesma espécie do arroz comum (Oryza sativa L.), entretanto, é considerado uma planta invasora, pois infesta as lavouras e compete com o arroz branco, limitando o seu potencial de produtividade (Agostinetto et al., 2001). As estruturas mais produzidas e comercializa das de M. anisopliae são os conídios, produzidos na superfície de meio de cultura sólido, dentro de diferentes recipientes conforme o objetivo e escala de produção (Almeida; Batista Filho, 2006), sendo o arroz o substrato mais utilizado para a produção conidial. Isto se deve, provavelmente, à combinação de fatores como balanço nutricional, custo, ampla disponibilidade mundial, características físicas como tamanho e forma do grão, propriedades de hidratação e integridade estrutural mesmo após a colonização pelo fungo (Jenkins et al., 1998). Neste contexto, o presente trabalho visou deter minar o potencial de utilização de resíduos agroin dustriais e do arroz vermelho, disponíveis na região oeste do Estado do Paraná, na composição de meios de cultura sólidos para a produção de conídios de M. anisopliae. ABSTRACT PRODUCTION OF CONIDIA OF METARHIZIUM ANISOPLIAE IN SOLID MEDIA BASED ON AGROINDUSTRIAL RESIDUES. Increasing costs of substrate for production of Metarizhium anisopliae raise the necessity of investigating the efficiency of some agro-industrial residues that, in addition to important nutritional properties, are widely available at low cost. The assays were conducted at the Fermentation Technology Laboratory, UNIOESTE, Cascavel, PR, Brazil. In this work, substrates such as polished rice (standard media), red rice, polished rice + sugar molas ses (2.5%, 5% and 10%), brewery residue, cassava residue, polished rice + brewery residue (1:1) and polished rice + cassava residue (1:1) were evaluated. The experiments were carried out in triplicate, in Erlernmeyer flasks of 250 mL, containing 30 g of the substrate (or 15 g of each one, when as mixture) and moisture content of 70 ± 10%. The flasks were inoculated with 0.5 mL of conidia suspension (1 × 108 conidia/mL) and then incubated in controlled temperature and light (25 ± 1o C, 12 hours photophase, 7 days). High conidial production was verified in rice + brewery residue media (11.30 × 108 conidia/g of substrate and in the polished rice (9.59 × 108 conidia/g of substrate). The conidia produced in rice + brewery residue media showed an insecticidal activ ity against Spodoptera frugiperda similar to that attained with the conidia produced in rice. This new culture media may provide a reduction of approximately 50% in the production cost, thus presenting a viable alternative for the fungus production. KEY WORDS: Metarhizium anisopliae, agro-industrial residues, solid-state fermentation. Arq. Inst. Biol., São Paulo, v.77, n.3, p.449-456, jul./set., 2010 450 L. Sene et al. Micro-organismo e preparo do inóculo A produção de fungos entomopatogênicos representa uma etapa crítica e limitante no desen volvimento de um programa de controle microbiano para uma determinada praga. A pesquisa de novas metodologias de sistemas de produção é muito importante para tornar o controle microbiano de pragas economicamente viável para ser aplicado em grandes áreas (Tanzini, 2002). Os experimentos foram conduzidos no Laboratório de Tecnologia das Fermentações da UNIOESTE, Campus de Cascavel, PR. Empregou-se o fungo M. anisopliae (isolado E9), armazenado na forma de conídios puros, em frascos Eppendorf a -4o C. Para a produção inicial de conídios utilizaram-se placas de Petri contendo meio para conidiogênese (MC) com posto de (g/1.000 mL de água destilada): 1,58 NaNO3; Segundo Ottati-de-Lima (2007), devido ao elevado preço que o arroz vem alcançando no comércio, o Arq. Inst. Biol., São Paulo, v.77, n.3, p.449-456, jul./set., 2010 451 Produção de conídios de Metarhizium anisopliae em meio sólido à base de resíduos agroindustriais. O experimento foi realizado em frascos Erlern meyer de 250 mL, contendo 30 g do substrato com teor de umidade de 70 ± 10%, sendo preparados 3 frascos para cada meio de cultura. Para avaliação das misturas de substratos, foram utilizados 15 g de cada. Os frascos foram autoclavados a 1 atm, 121o C por 20 minutos e resfriados. Em seguida, em câmara de fluxo laminar vertical e com o auxílio de um bastão de vidro estéril, fez-se a desagregação dos meios sólidos para permitir que o fungo se de senvolvesse uniformemente sobre todo o substrato. Após inoculação de 0,5 mL da suspensão de conídios (1 ×108 conídios/mL), os frascos foram fechados com tampão de algodão e gaze e incubados 25 ± 1o C e fotofase de 12 horas durante 7 dias. Para a avaliação da perda de umidade, foram incubados frascos não inoculados, contendo o substrato e 0,5 mL de água + espalhante adesivo (Tween 80) a 0,01%, os quais foram pesados antes e após o período de incubação e após secagem em estufa a 80° C até peso constante. 0,36 KH2PO4; 0,60 MgSO4.7H2O; 1,05 NaHPO4.7H2O; 1,00 KCl; 5,0 extrato de levedura; 10,0 glicose; 20,0 ágar (Alves, 1998). A inoculação dos conídios nas placas foi realizada em câmara de fluxo laminar vertical. Em seguida, as placas foram incubadas a 25 ± 1o C e fotofase de 12 horas, por um período de 7 a 10 dias para crescimento e conidiogênese do fungo. Teste preliminar para ajuste da umidade dos meios para 70 ± 10% Após o período de incubação, cada fraco recebeu 120 mL de água destilada mais espalhante adesivo (Tween 80) a 0,01% e foi submetido à agitação com bastão de vidro estéril até a visualização do despren dimento total dos conídios da matriz sólida por, pelo menos, cinco minutos. Desta suspensão, coletou-se uma alíquota de 0,1 mL a qual foi adicionada a 0,9 mL de água + espalhante adesivo (Tween 80) a 0,01%, obtendo-se uma diluição 1:10 para realização da contagem em câmara de Neubauer sob microscópio de luz (aumento de 400×). O teste consistiu na adição de diferentes volumes de água destilada em 30 g de cada substrato, contidos em frascos Erlenmeyer de vidro (250 mL). Os frascos foram autoclavados e, após resfriamento, realizou-se a pesagem. Em seguida, os frascos foram levados à estufa (80º C) para secagem dos materiais até peso constante. Realizou-se nova pesagem e, após a sub- tração da massa seca, foram calculados os volumes de água a serem adicionados a cada substrato de forma a resultar numa umidade em torno de 70 ± 10% após a autoclavagem, conforme descrito na Tabela 1. Micro-organismo e preparo do inóculo Após este período, os conídios foram coletados, ras pando a superfície dos meios de cultura nas placas e transferidos para tubos de vidro fechados com filme PVC e armazenados sob refrigeração a 4o C por um período não superior a 10 dias. Para o preparo das suspensões, os conídios foram adicionados em água estéril + espalhante adesivo (Tween 80®) a 0,01%. Em seguida, foi estimada a concentração dos conídios em câmara de Neubauer e as suspensões foram padroni zadas em 1 × 108 conídios/mL. Teste preliminar para ajuste da umidade dos meios para 70 ± 10% Avaliação da produção de Metarhizium anisopliae em meio sólido à base de resíduos agroindustriais Amostras dos conídios produzidos nos diferen tes meios foram tomadas e submetidas a diluições seriadas, até se obter uma concentração de 1 × 106 conídios/mL. Desta suspensão, inoculou-se, em triplicata, 100 mL em placas de Petri contendo meio BDA (Batata-Ágar-Dextrose), que em seguida foram incubadas por 18 horas a 25 ± 1° C e fotofase de 12 horas. Após esse período, foram realizadas as con tagens em cada placa, de 200 a 300 conídios viáveis ou não, sob microscópio de luz (aumento de 400×). Foram avaliados substratos ricos em carboidratos como arroz polido (meio padrão), arroz vermelho, arroz polido suplementado com melaço (resíduo da produção de açúcar) a 2,5%, 5% e 10%, resíduo da cervejaria, resíduo da fecularia, arroz polido e resíduo de cervejaria (1:1) e arroz polido e resíduo de fecularia (1:1). Tabela 1 - Volumes de água destilada e de solução de melaço (em diferentes concentrações) necessários para se obter 70 ± 10% de umidade em 30 g de substrato. Arq. Inst. Biol., São Paulo, v.77, n.3, p.449-456, jul./set., 2010 Avaliação da atividade inseticida Substrato (30 g) Volume (mL) Arroz 60 Arroz vermelho 60 Arroz + melaço (2,5%) 60 Arroz + melaço (5%) 60 Arroz + melaço (10%) 60 Res. fecularia 10 Arroz + resíduo de fecularia (1:1) 40 Res. cervejaria 0 Arroz + resíduo de cervejaria (1:1) 30 Os meios selecionados nas etapas anteriores foram novamente empregados para a produção de conídios, seguindo os mesmos procedimentos. Após a produção, os frascos contendo meio de cultura e conídios foram armazenados em freezer (4o C) até a disponibilização de insetos para os bioensaios. Para tal, foram utilizadas lagartas de Spodoptera frugiperda, obtidas a partir de criação de insetos no próprio laboratório, mantidas em copos plásticos de 50 mL com tampa e contendo dieta artificial (Parra, 1996) Arq. Inst. Biol., São Paulo, v.77, n.3, p.449-456, jul./set., 2010 452 L. Sene et al. desde a eclosão dos ovos, à temperatura ambiente. As lagartas encontravam-se no terceiro ínstar, com aproximadamente 1 cm de comprimento e foram divididas em três repetições de 15 indivíduos para cada tratamento. Os insetos foram imersos por 10 segundos na suspensão de conídios contendo 1 × 109 conídios/mL em água destilada + espalhante adesivo (Tween 80) a 0,01%. Na testemunha, as lagartas foram imersas apenas em água destilada estéril + espalhante adesivo (Tween 80) a 0,01%. Após a inoculação, as lagartas foram acondicionadas individualmente em placas de Petri contendo a dieta artificial e foram mantidas a 25 ± 1o C e fotofase de 12 horas. e o arroz polido (9,6 × 108 conídios/g) (Tabela 2). A elevada produção de conídios verificada na mistura arroz + resíduo da cervejaria pode ser atribuída ao suporte de celulose (casca da cevada), semelhante ao verificado por Dalla Santa et al. (2005), em estudos utilizando batata e bagaço de cana de açúcar para a produção de conídios de Beauveria bassiana e também por Magalhães; Frazão (1996), que verificaram que a palha de arroz + farelo de arroz e o arroz parboilizado foram os melhores substratos para a produção de M. flavoviride. Segundo Lonsane et al. (1985), a utilização de um substrato fibroso favorece a aeração, causa meno res problemas de compactação e maior superfície de crescimento durante um cultivo em substrato sólido. Análise estatística Os experimentos foram realizados segundo o de lineamento experimental inteiramente aleatorizado, sendo os dados obtidos analisados estatisticamente quanto à variância (teste F) e as médias comparadas entre si (teste Tukey), ambos no nível de 5% de proba bilidade, utilizando-se o programa computacional Sisvar (Ferreira, 2003). Avaliação da atividade inseticida Os insetos foram observados diariamente durante 10 dias, sendo que os mortos foram retirados, imersos em solução de álcool 70% por 10 segundos e depois em água destilada e mantidos em câmara úmida para confirmação da mortalidade pelo fungo. Para tal, as lagartas foram colocadas individualmente em placas de Petri contendo papel-filtro umedecido e mantidas em potes plásticos fechados com tampa e com fundo recoberto por espuma de poliuretano umedecida até a saturação. Assim, foram incubados nas mesmas condições descritas anteriormente, por mais 10 dias. O resíduo da cervejaria empregado isoladamente, no entanto, proporcionou uma conidiogênese média de apenas 1,9 × 108 conídios/g (Tabela 2), provavel mente por razões nutricionais, não tendo exercido o duplo papel de suporte e substrato, como se esperava. Neste meio, o teor de umidade foi o maior verificado (aproximadamente 77%), porém, a perda de umidade ao longo do cultivo foi também elevada (7,63%) (Tabela 3), não sendo possível afirmar se este foi outro fator que contribuiu para a baixa produção de conídios. O teor ótimo de umidade para o crescimento de fungos e produção de conídios precisa ser identificado para cada combinação de substrato/isolado, pois o conteúdo de umidade tem um papel essencial na produção de coní dios, embora a otimização deste parâmetro possa ser complexa (Jenkins et al., 1998). Segundo Pandey (2003), alta umidade resulta numa diminuição da porosidade do substrato, a qual impede a penetração do oxigênio, enquanto que um baixo conteúdo de umidade pode levar a uma pobre disponibilidade dos nutrientes, comprometendo o crescimento microbiano. Estimativa dos custos de produção A análise de custos foi realizada apenas com base no preço da matéria-prima para composição do meio, obtido em supermercados e também junto aos fornecedores do resíduo de cervejaria e de fecularia. Não foram considerados gastos com transporte e de mais custos do processo de produção (investimento e depreciação de equipamentos consumo de energia, mão-de-obra etc). Tabela 2 - Quantidades médias de conídios de M. anisopliae obtidas em diferentes substratos após incubação por 7 dias (25º C e fotofase de 12h) Substrato Concentração (x108 conídios/g)* Arroz 9,59 a Arroz vermelho 6,92 b Arroz + melaço(2,5%) 4,63 c Arroz + melaço(5%) 5,09 b c Arroz + melaço(10%) 4,19 c Res. fecularia 0,20 d Arroz + resíduo de fecularia (1:1) 4,14 c Resíduo cervejaria 1,91 d Arroz + resíduo de cervejaria (1:1) 11,30 a CV (%) 13,27 Médias (+ EP) seguidas pela mesma letra minúscula nas colunas não diferem entre si no nível de 5% de probabili dade pelo Teste de Tukey. Tabela 2 - Quantidades médias de conídios de M. anisopliae obtidas em diferentes substratos após incubação por 7 dias (25º C e fotofase de 12h) Avaliação da produção de Metarhizium anisopliae em meio sólido à base de resíduos agroindustriais Avaliação da produção de Metarhizium anisopliae em meio sólido à base de resíduos agroindustriais Entre os diferentes meios testados, os que pro porcionaram maior produção de conídios foram o meio composto por arroz + resíduo da cervejaria (1:1), obtendo-se 11,3 × 108 conídios/g de substrato, Arq. Inst. Biol., São Paulo, v.77, n.3, p.449-456, jul./set., 2010 453 Produção de conídios de Metarhizium anisopliae em meio sólido à base de resíduos agroindustriais. manecendo ainda, no final do período, com umidade de 68%, indicando que a baixa produção de conídios neste meio pode não estar associada diretamente ao seu conteúdo de umidade, mas provavelmente, ao problema de compactação observado. A produção de conídios de M. anisopliae no arroz vermelho foi inferior à produção no arroz branco, usualmente empregado na produção de conídios deste fungo. Constatou-se que, após cozimento em autoclave, o arroz vermelho apresentou maior agregação entre os grãos que o arroz branco, com consequente redução de sua área superficial, além de uma maior dificuldade para desprendimento dos conídios da matriz sólida. Com base nos resultados obtidos e considerando que a contagem de conídios não deve ser usada como parâmetro único da adequabilidade de um meio, o meio de arroz + resíduo de cervejaria (1:1) foi selecionado para novos testes, tomando-se como comparação o arroz, meio padrão para cultivo de M. anisopliae. Segundo Jackson (1997), após a etapa de seleção de um meio que proporcione uma boa produção da forma desejada do fungo, é importante avaliar sua capacidade de suportar processos de secagem, sua estabilidade e sua virulência. A proporção carbono e nitrogênio (C:N) do meio é um importante fator a ser considerado no desen volvimento de meios de cultura, pois sua composição pode ter uma estreita relação com o custo e a quali dade do fungo produzido, podendo influenciar no tipo, formato e quantidade de propágulo, sendo que meios ricos em C, mas deficientes em N, tendem a produzir maior quantidade de conídios (Leite et al., 2003). O melaço possui cerca de 50% de açúcares e 3% de matérias nitrogenadas (Lima et al., 2001), o que poderia contribuir para o favorecimento da conidiogênese. No entanto, a suplementação do ar roz branco com solução de melaço a 2,5%, 5% e 10% não favoreceu a produção de conídios em relação ao meio padrão, ou seja, o arroz branco. Teste de viabilidade dos conídios Não foi verificada diferença estatística entre a porcentagem de germinação (18 horas de incubação) de conídios de M. anisopliae produzidos no arroz e na mistura de arroz + resíduo de cervejaria, as quais apresentaram-se em torno de 85% (Tabela 4). No entanto, os valores encontrados foram ligeiramente inferiores aos verificados por Vilas Boas et al. (1996) ao avaliarem o cultivo de M. anisopliae em arroz e meios alternativos tais como fava, feijão, sorgo e caupi. Vale ressaltar que diferindo do autor citado, neste trabalho, os conídios foram armazenados por 60 dias, em condições ideais para a preservação de fungos (-4o C), porém na ausência de agentes crioprotetores. j Embora os farelos de fecularia apresentem em sua composição média 75% amido, 15% de fibras, 1,6% de cinzas, 2% de proteínas, 1% de açúcares e 0,8% de matéria graxa, expressos na base seca (Cereda, 1996), dentre todos meios estudados neste trabalho, o resíduo da fecularia foi o substrato que proporcio nou ao fungo a menor taxa de conidiogênese. Este meio apresentou uma perda de umidade de 4,86% durante os 7 dias de incubação (Tabela 2), mas per Tabela 3 - Variação da umidade dos substratos empregados para o cultivo de M. anisopliae após incubação por 7 dias (25º C e fotofase de 12h) Substrato Umidade inicial (%) Umidade final (%) Arroz 69,50 66,41 Arroz vermelho 66,78 64,03 Arroz + melaço(2,5%) 67,77 66,17 Arroz + melaço (5%) 67,24 62,58 Arroz + melaço(10%) 64,49 62,61 Res. fecularia 72,89 68,03 Arroz + resíduo de fecularia(1:1) 72,83 69,99 Resíduo de cervejaria 76,91 69,28 Arroz + resíduo de cervejaria (1:1) 71,26 67,72 CV(%) 5,52 4,13 Tabela 3 - Variação da umidade dos substratos empregados para o cultivo de M. anisopliae após incubação por 7 dias (25º C e fotofase de 12h) Substrato Umidade inicial (%) Umidade final (%) Arroz 69,50 66,41 Arroz vermelho 66,78 64,03 Arroz + melaço(2,5%) 67,77 66,17 Arroz + melaço (5%) 67,24 62,58 Arroz + melaço(10%) 64,49 62,61 Res. fecularia 72,89 68,03 Arroz + resíduo de fecularia(1:1) 72,83 69,99 Resíduo de cervejaria 76,91 69,28 Arroz + resíduo de cervejaria (1:1) 71,26 67,72 CV(%) 5,52 4,13 ade dos substratos empregados para o cultivo de M. anisopliae após incubação por 7 dias Tabela 4 - Comportamento de M. anisopliae produzido em diferentes substratos quanto à viabilidade de conídios e à mortalidade de larvas de S. Arq. Inst. Biol., São Paulo, v.77, n.3, p.449-456, jul./set., 2010 Arq. Inst. Biol., São Paulo, v.77, n.3, p.449-456, jul./set., 2010 Valores seguidos por letras iguais nas colunas não diferem entre si ao nível de 5% de probabilidade pelo Teste de Tukey. AGRADECIMENTOS Os autores agradecem à FINEP pelo financia mento deste trabalho, parte integrante do Projeto BIOAGROPAR, e à Fundação Araucária e ao CNPq pela concessão das bolsas de iniciação científica e produtividade aos autores. CONCLUSÕES Observou-se que a atividade inseticida dos conídios produzidos no meio formulado com arroz + resíduo de cervejaria frente a S. frugiperda não diferiu estatisticamente dos valores verificados para os conídios produzidos no arroz, na concentração empregada neste estudo (109 conídios/mL), sendo, para ambos os meios testados, inferior a 25% (Ta bela 4). Dados da literatura têm demonstrado uma baixa susceptibilidade de S. frugiperda ao fungo M. anisopliae. Segundo Rodrigues; Pratissoli (1989), M. anisopliae não mostrou nenhum efeito letal nas três concentrações testadas (103, 105 e 107), enquanto que Lecuona; Díaz (2001) verificaram que 6 das 15 cepas estudadas não foram patogênicas e que somente 3 cepas superaram 80% de mortalidade. O arroz + resíduo úmido de cervejaria poderá representar um meio de cultura alternativo para a produção de conídios de M. anisopliae, visto que neste meio, o fungo manteve elevada conidiogênese, sem perda da atividade inseticida. No entanto, fazem-se necessários estudos com outros isolados do fungo e bioensaios com outros insetos. Ressalta-se que este meio poderá ser utilizado em grande escala, levando-se em consideração as importantes reduções no custo de produção e sua disponibilidade na região Oeste do Paraná em todas as épocas do ano. p p A atividade inseticida dos fungos entomopa togênicos está relacionada à produção de proteases degradadoras de cutícula. No caso de M. anisopliae, proteases do tipo Pr1 e Pr2 foram identificadas e caracterizadas, sendo a presença de suas isoformas influenciada pelo meio, ou seja, a expressão dos genes pr1 e pr2 depende do nível das fontes de carbono e nitrogênio e da presença de proteínas indutoras no meio (St. Leger et al., 1988; St. Leger et al., 1994; Shah et al., 2005). A depleção de reservas endógenas e níveis de nutrientes insuficientes para promover a repressão catabólica de proteases favorecem o processo de infecção do hospedeiro (St. Leger et al., 1988). Segundo Alves et al. (1996), fatores bióticos e abióticos estão envolvidos na perda da atividade inseticida de vários agentes de controle microbiano, podendo haver uma correlação entre taxa ou tempo de germinação e virulência, dependendo do inseto alvo. Como já mencionado anteriormente, não houve diferença na porcentagem de germinação de conídios de M. anisopliae produzidos em ambos os meios, o que viria a explicar, juntamente com fatores nutri cionais, os resultados obtidos para a mortalidade de S. frugiperda. Teste de viabilidade dos conídios frugiperda Substrato Viabilidade média (%) Mortalidade confirmada (%) Arroz puro 84,46 a 13,33 a Arroz + resíduo de cervejaria (1:1) 85,55 a 24,44 a Testemunha - 2,22 a CV(%) 0,83 71,1 V l id l t i i l ã dif t i í l d 5% d b bilid d l T t d T k tamento de M. anisopliae produzido em diferentes substratos quanto à viabilidade de conídios e à vas de S. frugiperda Valores seguidos por letras iguais nas colunas não diferem entre si ao nível de 5% de probabilidade pelo Teste de Tukey. *Valores seguidos por letras iguais nas colunas não diferem entre si ao nível de 5% de probabilidade pelo Teste de Tukey. 454 L. Sene et al. Estimativa do custo de produção Segundo Hong et al. (1997), Alves et al. (1996 e Alves et al. (2002), a temperatura de estocagem, o conteúdo de umidade e o tipo de formulação são os principais fatores que influenciam na lon gevidade dos conídios. Em estudo realizado com B. bassiana, conídios armazenados em congelador e freezer mantiveram 100% da viabilidade após um período de armazenamento de 7 anos, no entanto, estes autores observaram que 100% da germinação somente ocorreu após 36-72 horas, enquanto que os conídios recém-produzidos germinaram em apenas 12 horas (Alves et al., 1996). No presente estudo, o reduzido tempo de incubação utilizado para o fungo armazenado pode ter sido o motivo para o resultado obtido. Considerando-se apenas o custo do meio de cul tura e a produção final obtida tanto no arroz puro quanto no arroz + resíduo de cervejaria, as quais foram as melhores e não diferiram significativamente entre si, verificou-se redução de aproximadamente 50% no custo de produção de conídios no meio con stituído de arroz + resíduo de cervejaria em relação à produção no arroz, já que o resíduo é vendido por, aproximadamente, R$ 30,00/t, ou seja, R$ 0,03/kg, enquanto que o preço médio do arroz é R$ 2,00/kg. Além disto, o resíduo de cervejaria é empregado in natura, sem necessidade de cozimento, o que pode vir a reduzir ainda mais os custos e a mão-de-obra durante o preparo. REFERÊNCIAS Revista de Investigaciones Agropecuarias, v.30, n.1, p.25-42, 2001. LECUONA, R.E.; DÍAZ, B. M. Susceptibilidad de Spo doptera frugiperda (J. E. Smith) a los hongosentomopató genos Nomuraea rileyi, Metarhizium anisopliae y Beauveria bassiana. Revista de Investigaciones Agropecuarias, v.30, n.1, p.25-42, 2001. ALVES, R.T.; BATEMAN, R.P.; GUNN, J.; PRIOR, C.; LEATHER, S.R. Effects of different formulations on viability and medium-term storage of Metarhizium anisopliae conidia. Neotropical Entomology, v.31, n.1, p.91- 99, 2002. LEITE, L.G.; BATISTA FILHO, A.; ALMEIDA, J.E.M; ALVES, S.B. (Ed.). Produção de fungos entomopatogênicos. Ribeirão Preto: A.S. Pinto, 2003. 92p. BUENO, V.P. Controle biológico na região sudeste. Revista G.Bio, v.1, p.4-6, 2010. LIMA, U.A.; AQUARONE, E.; BORZANI, W.; SCHMI DELL, W. Biotecnologia industrial – processos fermentati vos e enzimáticos. São Paulo: Edgard Blücher, 2001. v.3, 593p. CABRAL FILHO, S.L.S. Avaliação do resíduo de cerveja ria em dietas de ruminantes através de técnicas nucleares e correlatas. 1999. 68f. Dissertação (Mestrado em Ciências - Área de Tecnologia Nuclear na Agricultura) - Centro de Energia Nuclear na Agricultura, Universidade de São Paulo, Piracicaba, 1999. LONSANE, B.K.; GHILDYAL, N.P.; BUDIATMAN, S.; RAMAKRISHNA, S.V. Engineering aspects of solid- state fermentation. Enzyme and Microbial Technology, v.7, n.6, p.258-265, 1985. CEREDA, M.P. (Ed.). Caracterização, usos e tratamentos de resíduos da industrialização da mandioca. Botucatu: Centro de Raízes Tropicais, Universidade Estadual Paulista, 1996. 56p. MAGALHÃES, B.P.; FRAZÃO, H.S. Effects of temperature, water content and substrate on conidial production of Metharhizium flavoviride. Revista de Microbiologia, v.27, p.242- 246, 1996. CRUZ, B.P.B; ABREU, O.C.; OLIVEIRA, D.A.; CHIBA, S. Crescimento de Metarhizium anisopliae (Metsch.) So rokin em meios de cultura naturais, líquidos. Biológico, São Paulo, v.49, p.111-116, 1983. MELO, I. S.; AZEVEDO, J.L. Controle biológico. Jaguariú na: EMBRAPA, 1998. v.1, 264p. OTTATI-DE-LIMA, E.L. Produção de Metarhizium ani sopliae (Metsch.) Sorok e Beauveria bassiana (Bals.) Vuill. em diferentes substratos e efeito da radiação ultravioleta e da temperatura sobre estruturas infectivas desses entopamopatô genos. 2007. 92f. Tese (Doutorado em Agronomia – Área de Proteção de Plantas) - Faculdade de Ciências Agro nômicas UNESP, Botucatu, 2007. DALLA SANTA, H.S.; DALLA SANTA, O.R.; BRAND, D.; VANDENBERGHE, L.P.S.; SOCCOL, C.R. Spore production of Beauveria bassiana from agro-industrial residues. Brazilian Archieves of Biology and Technology, v.48, 51-60, 2005. FERREIRA, D.F. (Ed.). Sisvar: versão 4.3. Lavras: DEX/ UFLA, 2003. PANDEY, A. Solid-state fermentation. Biochemical Engi neering Journal, v.13, n.2/3, p.81-84, 2003. HONG, T.D., R.H. ELLIS; D. MOORE. REFERÊNCIAS AGOSTINETTO, D.; FLECK, N.G.; RIZZARDI, M.A.; MEROTTO JUNIOR, A.; VIDAL, R.A. Arroz vermelho: ecofisiologia e estratégias de controle. Ciência Rural, v.31, n.2, p.341-349, 2001. ALMEIDA, J.E.M; BATISTA FILHO, A. Controle bi ológico da cigarrinha-da-raiz da cana-de-açúcar com o fungo Metarhizium anisopliae. Boletim Técnico do Instituto Biológico. São Paulo, n.16, p.1-19, 2006. ALVARENGA, A.R.M.; CRUZ, B.P.B.; OLIVEIRA, D.A.; SILVEIRA, A.P.; BULISANI, E.A. Novos testes de cul Arq. Inst. Biol., São Paulo, v.77, n.3, p.449-456, jul./set., 2010 455 Produção de conídios de Metarhizium anisopliae em meio sólido à base de resíduos agroindustriais. tivo de fungos utilizados em controle biológico usando meios de cultura naturais líquidos. Arquivos do Instituto Biológico, São Paulo, v.55, n.1/4, p.31-35, 1988. JACKSON, M.A. Optimizing nutritional conditions for the liquid culture production of effective fungal biologi cal control agents. Journal of Industrial Microbiology & Biotechnology, v.19, n.3, p.180-187, 1997. ALVES, S.B.; PEREIRA, R.M.; STIMAC, J.L.; VIEIRA, S.A. Delayed germination of Beauveria bassiana conidia after prolonged storage at low, above-freezing temperatures. Biocontrol Science and Technology, v.6, 575-581, 1996. JENKINS, N.E.; HEVIOFO, G.; LANGEWALD, J.; CHERRY, A.J.; LOMER, C.J. Development of mass production technology for aerial conidia for use as mycopesticides. Biocontrol News and Information, v.31, n.2, p.21-31, 1998. ALVES, L.F.A.; ALVES, S.B.; PEREIRA, R.M.; CAPAL BO, D.M.F. Production of Bacillus thuringiensis Berliner var. kurstaki grown in alternative media. Biocontrol Science and Technology, v.7, 377-83, 1997a. KASSA, A. Development and testing of mycoinsecticides based on submerged spores and aerial conidia of the entomo pathogenic fungi Beauveia bassiana and Metarhizium anisopliae (Deuteromycotina: Hyphomycetes) for control of locusts, grasshoppers and storage pests. 2003. 27f. Thesis (Doctoral of Agricultural Sciences) - Faculty of Agricul tural Sciences, George-August, University Göttingen. Göttingen, 2003. KASSA, A. Development and testing of mycoinsecticides based on submerged spores and aerial conidia of the entomo pathogenic fungi Beauveia bassiana and Metarhizium anisopliae (Deuteromycotina: Hyphomycetes) for control of locusts, grasshoppers and storage pests. 2003. 27f. Thesis (Doctoral of Agricultural Sciences) - Faculty of Agricul tural Sciences, George-August, University Göttingen. Göttingen, 2003. ALVES, L.F.A.; ALVES, S.B.; CAPALBO, D.M.F. Seleção de matéria-prima para a elaboração de meio de cultura para a produção de Bacillus thuringiensis var. kurstaki Berliner. Anais da Sociedade Entomológica do Brasil, v.26, n.2, 379-382, 1997b. ALVES. S.B. (Ed.). Controle Microbiano de Insetos. 2.ed. Piracicaba: FEALC, 1998. 1163p. LECUONA, R.E.; DÍAZ, B. M. Susceptibilidad de Spo doptera frugiperda (J. E. Smith) a los hongosentomopató genos Nomuraea rileyi, Metarhizium anisopliae y Beauveria bassiana. VILAS BOAS, A.M.; ANDRADE, R.M.; OLIVEIRA, J.V. Diversificação de meios de cultura para a produção de fungos entomopatogênicos. Arquivos de Biologia e Tecno logia, v.39, 123-128, 1996. Arq. Inst. Biol., São Paulo, v.77, n.3, p.449-456, jul./set., 2010 REFERÊNCIAS Development of a model to predict the effect of temperature and mois ture on fungal spore longevity. Annals of Botany, v.79, p.121-128, 1997. PARRA, J.R.P. Técnicas de criação de insetos para progra mas de controle biológico. 3.ed. Piracicaba: FEALQ, 1996. 137p. Arq. Inst. Biol., São Paulo, v.77, n.3, p.449-456, jul./set., 2010 456 L. Sene et al. St. LEGER, R.J.; BIDOCHKA, M.J.; ROBERTS, D.W. Isoforms of the cuticle-degrading Pr1 proteinase and production of a metalloproteinase by Metarhizium anisopliae. Archives of Biochemistry and Biophysics, v.313, p.1-7, 1994. St. LEGER, R.J.; BIDOCHKA, M.J.; ROBERTS, D.W. Isoforms of the cuticle-degrading Pr1 proteinase and production of a metalloproteinase by Metarhizium anisopliae. Archives of Biochemistry and Biophysics, v.313, p.1-7, 1994. RODRIGUES, C.; PRATISSOLI, D. Avaliação da pato genidade dos fungos entomógenos Beauveria bassiana e Metarhizium anisopliae sobre lagartas de Spodoptera frugiperda. Revista de Cultura UFES, v.14, n.39/40, p.85- 89, 1989. TANZINI, M.R. Controle do percevejo-de-renda-da- seringueira (Leptopharsa heveae) com fungos entomopa togênicos. 2002. 140f. Tese (Doutorado em Ciências – Área de Entomologia) - Escola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo, Piraci caba, 2002. ROCHA, A.S. Caracterização e aproveitamento do farelo re sidual no processamento de fécula de mandioca na elaboração de biscoitos. 2005. 48f. Dissertação (Mestrado em Ciências Agrárias – Área de Fitotecnia) - Universidade Federal da Bahia, Cruz das Almas, 2005. SHAH, F.A.; CHENG, S.W.; BUTT, T.M. Nutrition influ ences growth and virulence of the insect-pathogenic fungus Metarhizium anisopliae. FEMS Microbiology Let ters, v.251, p.259-266, 2005. VILAS BOAS, A.M.; ANDRADE, R.M.; OLIVEIRA, J.V. Diversificação de meios de cultura para a produção de fungos entomopatogênicos. Arquivos de Biologia e Tecno logia, v.39, 123-128, 1996. St. LEGER, R.J.; DURRANDS, P.K.; COOPER, R.M.; CHARNLEY, A.K. Regulation of production of proteolitic enzymes by entomopathogenic fungus Metarhizium anisopliae. Archives of Microbiology, v.150, p.413-416, 1988. Recebido em 11/5/09 Aceito em 10/8/10
https://openalex.org/W3091912534	https://www.nature.com/articles/s41598-020-73452-y.pdf	English	null	The crystal structure of mycobacterial epoxide hydrolase A	Scientific reports	2,020	cc-by	9,833	The crystal structure of mycobacterial epoxide hydrolase A Eike C. Schulz1,2, Sara R. Henderson2,7, Boris Illarionov3, Thomas Crosskey2, Stacey M. Southall2,8, Boris Krichel4, Charlotte Uetrecht4,5, Markus Fischer3 & Matthias Wilmanns2,6 Eike C. Schulz1,2, Sara R. Henderson2,7, Boris Illarionov3, Thomas Crosskey2, Stacey M. Southall2,8, Boris Krichel4, Charlotte Uetrecht4,5, Markus Fischer3 & Matthias Wilmanns2,6 The human pathogen Mycobacterium tuberculosis is the causative agent of tuberculosis resulting in over 1 million fatalities every year, despite decades of research into the development of new anti-TB compounds. Unlike most other organisms M. tuberculosis has six putative genes for epoxide hydrolases (EH) of the α/β-hydrolase family with little known about their individual substrates, suggesting functional significance for these genes to the organism. Due to their role in detoxification, M. tuberculosis EH’s have been identified as potential drug targets. Here, we demonstrate epoxide hydrolase activity of M. thermoresistibile epoxide hydrolase A (Mth-EphA) and report its crystal structure in complex with the inhibitor 1,3-diphenylurea at 2.0 Å resolution. Mth-EphA displays high sequence similarity to its orthologue from M. tuberculosis and generally high structural similarity to α/β-hydrolase EHs. The structure of the inhibitor bound complex reveals the geometry of the catalytic residues and the conformation of the inhibitor. Comparison to other EHs from mycobacteria allows insight into the active site plasticity with respect to substrate specificity. We speculate that mycobacterial EHs may have a narrow substrate specificity providing a potential explanation for the genetic repertoire of epoxide hydrolase genes in M. tuberculosis. Infectious diseases pose a major threat to societies in the twenty-first century as many bacteria have managed to evolve mechanisms that protect them from antibiotics1. Of particular concern is infection with Mycobacterium tuberculosis (M. tuberculosis) the causative agent of tuberculosis, which caused approximately 10.4 million cases in 2017, resulting in 1.3 million deaths in HIV-negative patients. Alarmingly, in excess of 4% of the cases in 2017 were found to be multi-drug resistant2. In addition to this, there have been several reports of totally drug resistant tuberculosis in recent years3–5. In spite of decades of intense research simple, effective solutions to treat tuberculosis are still unavailable6. These facts demonstrate that it is of paramount importance to gain a more comprehensive understanding of the biological foundations of M. tuberculosis to be able to treat tuberculosis in the future. Substantial insight into M. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports/ hydrolases ephA–ephF (EHs; E.C. 3.3.2.3; open reading frames Rv3617, Rv1938, Rv1124, Rv2214c, Rv3670 and Rv0134) could be identified7. Later another open reading frame (Rv2740) was shown to produce an atypical epoxide hydrolase (E.C. 3.3.2.8)10. At present, four of these genes have been confirmed to produce proteins with resulting enzymatic activity of an epoxide hydrolase (ephAthis work, ephB11,12, ephD13, ephG10). The idea to use mycobacterial EHs as potential drug targets was recently emphasized by screening of a large compound library in a whole cell phenotypic minimum inhibitory concentration (MIC) assay, which pointed towards EHs as the main target of the lead compound14. g p EHs are essential enzymes that convert toxic epoxides to less reactive and more water soluble trans-dihydrodi- ols. They are generally implicated in detoxification of genotoxic epoxides but are also involved in more specialized processes like carbon catabolism and signal transduction. To date, three main classes of EHs have been identified; bacterial limonene EH (LEH), LTA4 hydrolases and α/β-hydrolase fold EH15. LEHs are essential for bacteria that use limonene as their only carbon source. The structural analyses of LEH from Rhodococcus erythropolis and an atypical EH from M. tuberculosis showed that these EHs do not belong to the family of α/β-hydrolases but rather consists of an α/β-barrel fold and follow a different reaction mechanism10,16. The second group EHs, the LTA4 hydrolases are Zn-dependent and show structural similarity to the bacterial protease thermolysin17. Similar to the bacterial LEHs, the LTA4 hydrolases display a specifically evolved active site and reaction mechanism15.h i The majority of mycobacterial EHs have been predicted to be members of the α/β-hydrolase fold family, presenting the third and largest class of EHs. Significant insight into the reaction mechanism of this EH-family was gained by several crystal structures of bacterial and mammalian EHs18–22. The structures of the α/β-hydrolase family EHs can be divided into two subdomains; the catalytic ‘core-domain’ and the so-called ‘cap-domain’. The active site is located in a cleft between these two domains. The core domain contributes a conserved catalytic triad to the active site, while two strictly conserved tyrosine residues are located in the cap-domain. These tyros- ine residues are suggested to affect epoxide polarization and facilitate ring opening23,24. The two-step reaction mechanism starts with an attack by the nucleophilic active site aspartate at one of the substrates epoxide carbons forming a covalent substrate-enzyme intermediate. www.nature.com/scientificreports/ Subsequently, a charge-relay pair between a general acid (aspartate/glutamate) and a general base histidine activates a water molecule. This leads to the hydrolysis of the covalent enzyme–substrate intermediate. A conserved histidine-glycine-X-proline (HGXP) motif that is gener- ally found in α/β-hydrolases, together with the nitrogen atom of the residue following the nucleophile forms an oxyanion hole that is suggested to stabilize the intervening tetrahedral intermediate23–27. Computational evidence supports that this catalytic strategy is conserved for enzymes within this family28. EHs of the α/β-hydrolase family are known to accept a broad spectrum of structurally diverse substrates. In mammals, the soluble EHs hydrolyze gem-di-, trans-di-, cis-di-, tri- and tetra-substituted epoxides, while the microsomal EHs hydrolyze mono- and cis- di-substituted epoxides. On a molecular level, this broad sub- strate spectrum could be explained by the nucleophile elbow, a sharp turn in the active site, which harbours the nucleophilic aspartate that is responsible for the initial substrate attack11,15,18,23,24. It has been suggested that an increased flexibility of this aspartate enables adaptation to the position of the substrate in the active site15. Due to the high structural similarity between mammalian and bacterial EHs, it would seem conceivable that myco- bacterial α/β-hydrolase EHs show a similar versatility. However, the position of the nucleophilic aspartate and the other active site residues of M. tuberculosis epoxide hydrolase B (Mtb-EphB, Rv1938) remain unaltered in the inhibitor bound and ligand-free protein11. g In combination with the comparably large number of Ehs in mycobacteria this lets us speculate about an individual substrate selectivity, presumably targeting classes of different substrates9. In support of this hypothesis, the structure of Mtb-EphB has revealed a relatively small and hydrophobic active site and an altered substrate selectivity in comparison to mammalian or plant EHs11. However, our understanding of a particular active site geometry in relation to substrate specificity is limited by the available structural information, limited to Mtb- EphB, to date. Several EH inhibitors have not only shown antitubercular activity and low cytotoxicity but also to target several different EHs. However, these inhibitors have also targeted the human enzymes14. Therefore, in the design of epoxide hydrolase inhibitors for the treatment of TB, it is desirable to increase their specificity selecting only mycobacterial targets, which would be facilitated by structural knowledge of all mycobacterial EHs. From the six putative α/β-hydrolase EHs in M. tuberculosis only the structure of Mtb-EphB has been solved to date11. www.nature.com/scientificreports/ Although the crystallization of M. tuberculosis Epoxide hydrolase A (Mtb-EphA, Rv3617) has been reported, its limited X-ray diffraction prevented structure determination12. To enhance our structural understanding myco- bacterial epoxide hydrolases, we have solved the crystal structure of the M. thermoresistibile Epoxide hydrolase A (Mth-EphA) in complex with a urea-based inhibitor at 2.0 Å resolution. The structure clearly displays an α/β- hydrolase EH fold but in comparison to other EHs shows substantial differences in the substrate binding channel. The crystal structure of mycobacterial epoxide hydrolase A tuberculosis biology was provided by sequencing of its complete genome, which revealed that a comparably large fraction of all genes codes for enzymes involved in lipogenesis and lipolysis7,8. While epoxide hydrolases (EHs) are found in about 20% of all organisms with sequenced genomes, most of them occur in organisms with large genomes (> 8 Mb). The mycobacterial genome harbors less than 4 Mb but contains on average five epoxide hydrolases compared to approximately 2 found in other organisms. Since evolutionary pressure drives bacteria to deleting unneeded genes, this comparably large number of EHs supports the idea of functional significance of these enzymes to M. tuberculosis biology9. Initially, six putative copies of epoxide 1Max Planck Institute for the Structure and Dynamics of Matter, Luruper Chausee 149, 22761 Hamburg, Germany. 2European Molecular Biology Laboratory, Hamburg Unit, Notkestrasse 85, 22603 Hamburg, Germany. 3Hamburg School of Food Science, Institute of Food Chemistry, Universität Hamburg, Grindelallee 117, 20146 Hamburg, Germany. 4Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Martinistraße 52, 20251 Hamburg, Germany. 5European XFEL GmbH, Holzkoppel 4, 22869 Schenefeld, Germany. 6University of Hamburg Medical Center Hamburg-Eppendorf, Martinistraße 52, 20246 Hamburg, Germany. 7Present address: Norwich Medical School, Rosalind Franklin Road, Norwich Research Park, Norwich, Norfolk NR4 7UQ, UK. 8Present address: Sosei Heptares, Steinmetz Building, Granta Park, Great Abington, Cambridge CB21 6DG, UK. *email: eike.schulz@mpsd.mpg.de \| https://doi.org/10.1038/s41598-020-73452-y Scientific Reports \| (2020) 10:16539 www.nature.com/scientificreports/ Results i Protein characterization and enzyme activity assay. A sequence comparison of epoxide hydrolase A from M. tuberculosis (Mtb-EphA) and its temperature tolerant relative Mycobacterium thermoresistibile (Mth- EphA) shows that the two orthologs share approximately 75% sequence identity. All residues suspected to be involved in catalysis are identical (Figure S1). This demonstrates that Mth-EphA may serve well as a model to understand the molecular details of Mtb-EphA. Consequently, the gene for Mth-EphA was cloned and expressed in E. coli, the recombinant protein was purified to homogeneity. i To qualitatively determine whether Mth-EphA has indeed epoxide hydrolase activity we incubated the protein with following compounds: cis-stilbene oxide (1), trans-stilbene oxide (2), or trans-1,3-diphenyl-2,3-epoxypro- pan-1-one (4) (Fig. 1) and analyzed its reaction products by liquid chromatography mass-spectrometry (LC–MS) (Table 1). Treatment of 1 or 2 with Mth-EphA did not reduce the concentration of any of those two compounds in the assay samples and did not give rise to hydrobenzoin (3) (Fig. S5–S7). However, treatment of 4 with Mth-EphA Scientific Reports \| (2020) 10:16539 \| https://doi.org/10.1038/s41598-020-73452-y www.nature.com/scientificreports/ Figure 1. Mth-EphA has epoxide hydrolase activity. 2D-projections of compounds (1)–(5): cis-stilbene oxide (1), trans-stilbene oxide (2), hydrobenzoin (3), trans-1,3,diphenyl-2,3-epoxypropan-1-one (4) and 2,3dihydroxy-1,3diphenyl-1-propanone (5). LC–MS based activity assays demonstrate that Mth-EphA converts (4) into (5) but not (1) or (2) into (3). Figure 1. Mth-EphA has epoxide hydrolase activity. 2D-projections of compounds (1)–(5): cis-stilbene oxide (1), trans-stilbene oxide (2), hydrobenzoin (3), trans-1,3,diphenyl-2,3-epoxypropan-1-one (4) and 2,3dihydroxy-1,3diphenyl-1-propanone (5). LC–MS based activity assays demonstrate that Mth-EphA converts (4) into (5) but not (1) or (2) into (3). Figure 1. Mth-EphA has epoxide hydrolase activity. 2D-projections of compounds (1)–(5): cis-stilbene oxide (1), trans-stilbene oxide (2), hydrobenzoin (3), trans-1,3,diphenyl-2,3-epoxypropan-1-one (4) and 2,3dihydroxy-1,3diphenyl-1-propanone (5). LC–MS based activity assays demonstrate that Mth-EphA conv (4) into (5) but not (1) or (2) into (3). reduced the concentration of its adducts (H+, NH4 + and Na+, retention time 37.5 min) in the measured samples approximately to half of the initial values and gave rise to three signals with m/z values of 243Th, 260Th and 265Th, all with identical retention time of 15 min. We concluded that these molecular species must be three adducts (H+, NH4 + and Na+, respectively) of the chemical compound with molecular weight of 242 Da, which is characteristic to the hydroxylation product of 4, 2,3-dihydroxy-1,3-diphenyl-1-propanone (5) (Table 1 and Fig. S8–S11). The latter is commercially not available. Results i Therefore, for fragmentation of those three molecular spe- cies (5-H+, 5-Na+, 5-NH4 +) and the three adducts of 4 (4-H+, 4-Na+, 4-NH4 +) the MRM parameters established for 4 have been used. The species 4-Na+ and 5-Na+ as expected did not give any measurable fragments because sodium adducts normally tend to produce very low fragmentation. The molecular species 4-H+, 4-NH4 +, 5-H+ and 5-NH4 +, all produced three fragments with m/z of 51Th, 77Th and 105Th (cf. Table 1, fragments 4-1, 4-2, 4-3, 5-1, 5-2, 5-3). While the first is characteristic for fragmentation of phenols, the second and third correspond to fragmentation results of substituted phenols. For the molecular species 5-H+ and 5-NH4 + fragments with m/z of 119Th and 225Th could be observed. The first species can be easily identified as 2-oxo-2-phenylethylium (5-4), while the second species (5-5) has an m/z value identical with 4-H+. In other words 5 showed neutral loss of water which is a typical fragmentation pattern of alcohols. A loss of a second molecule of water by fragmentation of the 5-5 would be conceivable if the 5-5 was an alcohol, too. But the putative product of such fragmentation with m/z 197Th (C15H11O1 +) was not observed. Thus, we concluded that under neutral loss of one water molecule the diol (5) is converted to a dicarbonyl compound (5-5) in a way similar to the pinacol rearrangement. Therefore the 5-5 must be a protonated 1,3-diphenylpropanedione and not 1,3-diphenylhydroxypropanone. p p y p p p y y yp p In conclusion, the LC–MS data are consistent with 5 being 2,3-dihydroxy-1,3-diphenyl-1-propanone indicat- ing that Mth-EphA can convert 4 to 5 under described reaction conditions. By contrast, the hydroxylation of the two other potential EH substrates 1 and 2 was not observed. Structure determination. The purified protein was used in crystallization experiments which following optimization, yielded crystals diffracting to 2.0 Å resolution (Table 2, Figure S1). The structure was solved by molecular replacement using a homology model as template. Despite the challenging diffraction properties of the crystals, the data yielded an electron-density map with clearly interpretable features allowing accurate model building for residues 4–321 of the protein construct. All residues lie within allowed Ramachandran areas, with the exception of the active site residue Asp103. The strained conformation of Asp103 can likely be explained by its position at the tip of the nucleophilic elbow. Results i Notably, Asp103 displays very well-defined electron density and its unfavorable conformation is held in position by interactions to the surrounding residues. Asp103 forms backbone hydrogen bonds to Ala106, Leu107 as well as Leu126 and its side chain carboxyl group contacts the backbone nitrogens of Phe35 and Trp104. Although four monomers were observed in the asymmetric unit, no Scientific Reports \| (2020) 10:16539 \| https://doi.org/10.1038/s41598-020-73452-y www.nature.com/scientificreports/ Compounda Retention time, min Adduct or fragment m/z (Th) Chemical formula Chemical name of the compound/ fragment ion CE, eV CXP, V 1 32 1-H+ 197 C14H13O+ Cis-stilbene epoxide, H+ adduct 5 10 1-Na+ 219 C14H12ONa+ Cis-stilbene epoxide, Na+ adduct 5 10 1-NH4 + 215 C14H16ON+ Cis-stilbene epoxide, NH4 + adduct 5 10 1-1 51 C4H3 + Cyclobutadienylium 91 0 1-2 77 C6H5 + Phenylium 49 4 1-3 91 C7H6 + Tropylium 19 6 1-4 105 C7H5O+ or C8H9 + Oxo(phenyl)methy- lium or phenylethy- lium 25 6 2 32 2-H+ 197 C14H13O+ Trans-stilbene epox- ide, H+ adduct 5 10 2-Na+ 219 C14H12ONa+ Trans-stilbene epox- ide, Na+ adduct 5 10 2-NH4 + 215 C14H16ON+ Trans-stilbene epox- ide, NH4 + adduct 5 10 2-1 51 C4H3 + Cyclobutadienylium 85 14 2-2 77 C6H5 + Phenylium 51 12 2-3 91 C7H6 + Tropylium 19 6 2-4 105 C7H5O+ or C8H9 + Oxo(phenyl)methy- lium or phenylethy- lium 29 6 3 13 3-H+ 215 C14H15O2 + Hydrobenzoin, H+ adduct 5 10 3-Na+ 237 C14H14O2Na+ Hydrobenzoin, Na+ adduct 5 10 3-NH4 + 232 C14H18O2N+ Hydrobenzoin, NH4 + adduct 5 10 3-1 51 C4H3 + Cyclobutadienylium 93 8 3-2 77 C6H5 + Phenylium 57 6 3-3 91 C7H6 + Tropylium 25 6 3-4 105 C7H5O+ or C8H9 + Oxo(phenyl)methy- lium or phenylethy- lium 27 5 4 37.5 4-H+ 225 C15H13O2 + 1,3-diphenyl- 2,3-epoxy-1-pro- panone, H+ adduct 5 10 4-Na+ 247 C14H12O3Na+ 1,3-diphenyl- 2,3-epoxy-1-pro- panone, Na+ adduct 5 10 4-NH4 + 242 C14H16O3N+ 1,3-diphenyl- 2,3-epoxy-1-pro- panone, NH4 + adduct 5 10 4-1 51 C4H3 + Cyclobutadienylium 89 6 4-2 77 C6H5 + Phenylium 49 4 4-3 105 C7H5O+ Oxo(phenyl)methy- lium or phenylethy- lium 13 8 Continued https://doi.org/10.1038/s41598-020-73452-y Scientific Reports \| (2020) 10:16539 \| www.nature.com/scientificreports/ Table 1. Adducts of compounds cis-stilbene oxide (1), trans-stilbene oxide (2), hydrobenzoin (3), trans-1,3- diphenyl-2,3-epoxypropan-1-one (4), 2,3-dihydroxy-1,3-diphenyl-1-propanone (5), the MRM parameters optimized for their fragmentation and the resulting fragmentation products. CE collision energy, CXP collision cell exit potential. Results i a Declustering potential values (V): 56 (1), 66 (2), 44 (3), 31 (4), 31 (5). Compounda Retention time, min Adduct or fragment m/z (Th) Chemical formula Chemical name of the compound/ fragment ion CE, eV CXP, V 5 15.0 5-H+ 243 C15H15O3 + 2,3-dihydroxy- 1,3-diphenyl-1-pro- panone, H+ adduct 5 10 5-Na+ 265 C15H14O3Na+ 2,3-dihydroxy- 1,3-diphenyl-1-pro- panone, Na+ adduct 5 10 5-NH4 + 260 C15H17O3N+ 2,3-dihydroxy- 1,3-diphenyl- 1-propanone, NH4 + adduct 5 10 5-1 51 C4H3 + Cyclobutadienylium 89 6 5-2 77 C6H5 + Phenylium 49 4 5-3 105 C7H5O+ or C8H9 + Oxo(phenyl)methy- lium or phenylethy- lium 13 8 5-4 119 C8H7O+ 2-Oxo-2-phenyleth- ylium 11 10 5-5 225 C15H13O2 + 1,3-diphenylpropan- edione, H+ adduct 5 10 Table 1. Adducts of compounds cis-stilbene oxide (1), trans-stilbene oxide (2), hydrobenzoin (3), trans-1,3- diphenyl-2,3-epoxypropan-1-one (4), 2,3-dihydroxy-1,3-diphenyl-1-propanone (5), the MRM parameters optimized for their fragmentation and the resulting fragmentation products. CE collision energy, CXP collision cell exit potential. a Declustering potential values (V): 56 (1), 66 (2), 44 (3), 31 (4), 31 (5). Table 1. Adducts of compounds cis-stilbene oxide (1), trans-stilbene oxide (2), hydrobenzoin (3), trans-1,3- diphenyl-2,3-epoxypropan-1-one (4), 2,3-dihydroxy-1,3-diphenyl-1-propanone (5), the MRM parameters optimized for their fragmentation and the resulting fragmentation products. CE collision energy, CXP collision cell exit potential. a Declustering potential values (V): 56 (1), 66 (2), 44 (3), 31 (4), 31 (5). Table 2. Data collection and refinement statistics. Data collection Refinement Wavelength (Å) 0.9752 Resolution limits (Å) 47.82–2.0 Cell dimensions (Å) No. reflections 93,380 a 61.80 No. atoms 10,371 b 105.01 Macromolecules 9898 c 108.95 Ligands 64 α 90 Water 408 β 90 RWork (%)/RFree (%) 20.86/25.08 γ 90 Space group P 1 21 1 B-factors Resolution range (Å) 47.82 -2.0 Macromolecules 22.00 No. reflections 316,649 (32,211) Solvent 21.30 I/σ 7.68 (2.42) Ligands 18.80 Completeness (%) 96.7(97.5) R.m.s. deviations Redundancy 3.4 (3.5) Bond lengths (Å) 0.014 RMeas (%) 13.63 Bond angles (°) 1.5 CC(1/2) 0.994 (0.83) Table 2. Data collection and refinement statistics. Table 2. Data collection and refinement statistics. significant dimer interface that would lead to the formation of a stable quaternary structure was determined in PISA analysis29. Results i (A) Mth-EphA belongs to the α/β-hydrolase fold family of enzymes. The α/β-hydrolase domain features its characteristic central β-sheet with α-helices aligned to its sides. Clearly the structure is organized into two separated domains: the all-α cap domain closing over the α/β-hydrolase fold domain. (B) The course of the substrate tunnel is indicated as a blue sphere inside of Mth-EphA; black arrowheads indicate solvent accessible openings; a yellow arrowhead marks the position of the constriction site. (C) Side chains lining the tunnel are indicated as grey stick, catalytic residues are shown in yellow. (D) APBS surface charge representation of Mth-EphA. The surface charges clearly show the acidic (red) and alkaline (blue) charges on the surface of Mth-EphA. While the cap-domain is predominantly acidic the core-domain has less pronounced charges. Particularly interesting are the entry sites to the substrate channel, which display opposite charges on the surface of the protein. It is conceivable that these features are involved in substrate orientation or selection. The inhibitor in the active site is visible as yellow spheres. Figure 2. Structural overview of Mth-EphA. Cartoon ribbon representation of Mth-EphA, α-helices are colored in blue, β-strands are colored in red, loops are in grey. (A) Mth-EphA belongs to the α/β-hydrolase fold family of enzymes. The α/β-hydrolase domain Figure 2. Structural overview of Mth-EphA. Cartoon ribbon representation of Mth-EphA, α-helices are colored in blue, β-strands h Figure 2. Structural overview of Mth-EphA. Cartoon ribbon representation of Mth-EphA, α-helices are colored in blue, β-strands are colored in red, loops are in grey. (A) Mth-EphA belongs to the α/β-hydrolase fold family of enzymes. The α/β-hydrolase domain features its characteristic central β-sheet with α-helices aligned to its sides. Clearly the structure is organized into two separated domains: the all-α cap domain closing over the α/β-hydrolase fold domain. (B) The course of the substrate tunnel is indicated as a blue sphere inside of Mth-EphA; black arrowheads indicate solvent accessible openings; a yellow arrowhead marks the position of the constriction site. (C) Side chains lining the tunnel are indicated as grey stick, catalytic residues are shown in yellow. (D) APBS surface charge representation of Mth-EphA. The surface charges clearly show the acidic (red) and alkaline (blue) charges on the surface of Mth-EphA. While the cap-domain is predominantly acidic the core-domain has less pronounced charges. Results i Thi i i i h i (MS) l i d i h M h E hA i significant dimer interface that would lead to the formation of a stable quaternary structure was determined in PISA analysis29.h This is consistent with native mass spectrometry (MS) analysis demonstrating that Mth-EphA is a monomer in solution (Figure S1). When sprayed from a nano-ESI (electrospray ionization) source the peaks appeared in a narrow charge state distribution with mainly + 10 and + 11 charges indicating a well-defined fold. A dissocia- tion constant (KD) can be estimated from the relative peak intensities of apo Mth-EphA (100%) compared to Mth-EphA bound to 1,3-diphenylurea (DPU) (5%). However, the intensity ratios will not reflect the KD of such a hydrophobic interaction. Generally, non-polar interactions are poorly preserved in the gas phase and unspecific clustering rarely occurs. Detecting this interaction suggests that it is in fact specific, and that it would likely be much stronger in solution. Moreover, such a small hydrophobic ligand would be unlikely to detect at all if not buried inside the molecule30. Overall structure. The overall structure of Mth-EphA clearly displays the globular fold of the α/β-hydrolase EHs. It is separated into two domains; the α/β-hydrolase core-domain (residues 1–136 and 247–321) with its central, eight-stranded β–sheet and the all-α-helical cap-domain (residues 137–246) (Fig. 2). The interface Scientific Reports \| (2020) 10:16539 \| https://doi.org/10.1038/s41598-020-73452-y https://doi.org/10.1038/s41598-020-73452-y www.nature.com/scientificreports/ nature.com/scientificreports/ Figure 2. Structural overview of Mth-EphA. Cartoon ribbon representation of Mth-EphA, α-helices are colored in blue, β-strands are colored in red, loops are in grey. (A) Mth-EphA belongs to the α/β-hydrolase fold family of enzymes. The α/β-hydrolase domain features its characteristic central β-sheet with α-helices aligned to its sides. Clearly the structure is organized into two separated domains: the all-α cap domain closing over the α/β-hydrolase fold domain. (B) The course of the substrate tunnel is indicated as a blue sphere inside of Mth-EphA; black arrowheads indicate solvent accessible openings; a yellow arrowhead marks the position of the constriction site. (C) Side chains lining the tunnel are indicated as grey stick, catalytic residues are shown in yellow. (D) APBS surface charge representation of Mth-EphA. The surface charges clearly show the acidic (red) and alkaline (blue) charges on the surface of Figure 2. Structural overview of Mth-EphA. Cartoon ribbon representation of Mth-EphA, α-helices are colored in blue, β-strands are colored in red, loops are in grey. Results i Particularly interesting are the entry sites to the substrate channel, which display opposite charges on the surface of the protein. It is conceivable that these features are involved in substrate orientation or selection. The inhibitor in the active site is visible as yellow spheres. Scientific Reports \| (2020) 10:16539 \| https://doi.org/10.1038/s41598-020-73452-y www.nature.com/scientificreports/ Figure 3. Inhibitor binding. (A) The 1,3-diphenylurea ligand is indicated by yellow spheres in the active site between the cap-domain and the α/β-hydrolase domain. (B) The inhibitor 1,3-diphenylurea binds in the active site residues of Mth-EphA. Catalytic residues are shown in yellow. (C) A simulated annealing OMIT map of the inhibitor (\|Fo-Fc\|, shown at 2 σ), is displayed as a green mesh. Figure 3. Inhibitor binding. (A) The 1,3-diphenylurea ligand is indicated by yellow spheres in the active site between the cap-domain and the α/β-hydrolase domain. (B) The inhibitor 1,3-diphenylurea binds in the active site residues of Mth-EphA. Catalytic residues are shown in yellow. (C) A simulated annealing OMIT map of the inhibitor (\|Fo-Fc\|, shown at 2 σ), is displayed as a green mesh. between cap-domain and core-domain harbors the active site of Mth-EphA, with the conserved residues of the catalytic triad (Asp103, His297 and Asp268) and the epoxide polarizing tyrosines (Tyr153, Tyr238) (Fig. 2). The cap-domain closes over the core-domain leaving space for a characteristic ‘L’-shaped tunnel with a hydrophobic interior (Fig. 2). The tunnel has an overall length of approximately 30 Å and bulky cavities close to its two distinct solvent openings, with radii of 2.7 Å and 3.4 Å, respectively (Figure S2). However, at its center, in proximity to the active site residues, it shows a constriction with a radius of only 1.2 Å. This constriction is imposed by three hydrophobic residues (Leu107, Met241, Met274) and presumably influences the substrate selection by Mth- EphA. A surface charge calculation shows a generally acidic surface for the cap-domain of Mth-EphA, while the surface charges at the core-domain are less pronounced. Interestingly, the two openings to the substrate-channel display opposite charges. While one opening has a strongly acidic surrounding the other shows clear alkaline properties (Fig. 2). It is conceivable that these properties influence substrate selection or guide substrate orienta- tion and binding. Inhibitor binding. The previously established EH inhibitor 1,3-diphenylurea was co-crystallized with Mth- EphA. The inhibitor is deeply buried within the substrate tunnel, where it binds to the conserved active site resi- dues (Fig. 3). Figure 4. Comparison to other α/β-hydrolase EHs. EHs display a high degree of structural conservation, that is not only limited to their main-chain atoms but can also be seen on the side chain level in the active site. However, substantial differences can be observed in the size, shape and propagation of the substrate channels. (A) A superposition of Mth-EphA with Mtb-EphB, human sEH and the EH from A. radiobacter displays their global similarity. (B) A close-up to the catalytic residues of the superposed EHs illustrates the structural conservation in the active site. (C) In spite of the global similarity of the EHs their substrate channels clearly adopt different sizes, shapes and propagate into different directions. For clarity the N-terminal residues of human sHE are not shown. The surface area of the substrate channels is shown below the structures. Figure 4. Comparison to other α/β-hydrolase EHs. EHs display a high degree of structural conservation, that is not only limited to their main-chain atoms but can also be seen on the side chain level in the active site. However, substantial differences can be observed in the size, shape and propagation of the substrate channels. (A) A superposition of Mth-EphA with Mtb-EphB, human sEH and the EH from A. radiobacter displays their global similarity. (B) A close-up to the catalytic residues of the superposed EHs illustrates the structural conservation in the active site. (C) In spite of the global similarity of the EHs their substrate channels clearly adopt different sizes, shapes and propagate into different directions. For clarity the N-terminal residues of human sHE are not shown. The surface area of the substrate channels is shown below the structures. Results i The conformation of the inhibitor binding could be unambiguously determined from the electron density maps. While both of the amine nitrogens form hydrogen bonds with Asp103, the phenyl groups are stabilized by van der Waals interactions (Phe35, Met178, Leu182, Phe200, Trp298 and Trp104, Leu107, Ile154, Met241). The carbonyl oxygen of the inhibitor is hydrogen bonded by Tyr153 and Tyr238 mimicking the posi- tion of the epoxide oxygen. Interestingly, the inhibitor adopts a slightly bent conformation with the carbonyl group and both phenyl-rings oriented towards Tyr238. In proximity of Asp103 and His297 a water molecule shows electron density and is stabilized by hydrogen bonds to the side chains of those residues, to Glu39 as well as to the main chain of Phe35 (Fig. 3). https://doi.org/10.1038/s41598-020-73452-y Scientific Reports \| (2020) 10:16539 \| www.nature.com/scientificreports/ Figure 4. Comparison to other α/β-hydrolase EHs. EHs display a high degree of structural conservation, that is not only limited to their main-chain atoms but can also be seen on the side chain level in the active site. However, substantial differences can be observed in the size, shape and propagation of the substrate channels. (A) A superposition of Mth-EphA with Mtb-EphB, human sEH and the EH from A. radiobacter displays their global similarity. (B) A close-up to the catalytic residues of the superposed EHs illustrates the structural conservation in the active site. (C) In spite of the global similarity of the EHs their substrate channels clearly adopt different sizes, shapes and propagate into different directions. For clarity the N-terminal residues of human sHE are not shown. The surface area of the substrate channels is shown below the structures. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Similar to the mycobacterial EHs, the human sEH channel also has two openings to the solvent. However, clearly distinct from the mycobacterial EHs, the channel of the human orthologue appears to be wide enough to accept a variety of different and bulky substrates. This is consistent with its general versatility accepting dif- ferentially substituted epoxy fatty acids as substrates16,23,31. In contrast to the former structures, the EH from A. radiobacter has only one large opening on its surface and a shallow connection to the active site that is not fully shielded from the solvent (Fig. 4, Figure S4). It appears to be better suited to accommodate smaller substrates with epoxides on terminal positions consistent with its primary, natural substrate epichlorohydrin32,33.i p p p y p y Unfortunately, to the best of our knowledge the actual substrate specificity of mycobacterial EHs are unknown. However, a comparison of the affinities of inhibitors for human sHE and Mtb-EphB indicates substantial differ- ences between the two enzymes. Both of them can bind smaller inhibitors like 1,3-diphenylurea with high affin- ity. In consistence with its narrow substrate channels, Mtb-EphB shows much lower affinity to those inhibitors that exhibit long or bulky side-chains11. This suggests that its natural substrates could also be smaller molecules. The substrate channel of Mth-EphA is clearly distinct from the one in Mtb-EphB, showing two bulky cavities, a constriction at its center and highly polar entry sites (Fig. 2). It is conceivable that these features could play a role in substrate orientation and selection. In line with these findings Mtb-EphB shows activity against both cis- and trans-stilbene oxide while Mth-EphA did not show any activity against either of the compounds in our assay11. In summary, the properties of the active site channels as well as the results of our activity assay support the view that the comparably large genetic repertoire in mycobacterial epoxide hydrolases may be related to the substrate specificity of the individual enzymes. In line with this a sequence alignment reveals that the residues that line the substrate channel predominantly map to the most variable parts of the EHs, that is the α-helical cap-domain (Fig. 5). Similar to Mth-EphA and Mtb-EphB, the remaining mycobacterial EHs also show substantial sequence variability in their putative substrate channel locations. This further supports our view that mycobacterial epoxide hydrolases may have varying substrate specificities. www.nature.com/scientificreports/ Thus, we speculate that the large genetic repertoire of EHs in M. tuberculosis may have evolved to address different substrate classes.h yf The nature of the substrate of EHs is likely to have important kinetic consequences: human soluble EHs are able to process a large number of structurally diverse substrates following a 2-step reaction mechanism whereas the LEH from Rhodococcus erythropolis has a very narrow substrate specificity and enantio-selectivity. How- ever, this narrow specificity is linked to a high turnover number of its main substrate maintaining its efficient hydrolysis15,16,34,35. By analogy, it is conceivable that a narrow substrate specificity of mycobacterial EHs could be linked to higher turnover rates. Therefore, the evolution of a large number of mycobacterial EHs could be an adaptation for the efficient hydrolysis of different substrate classes.h pfi y yf This is in good agreement with other physiological properties of M. tuberculosis. Since a large fraction of its genome is dedicated to the lipid metabolism it is reasonable to assume that it also requires an increased number of lipid-modifying enzymes. Moreover, the degradation of host cell lipids is vital to M. tuberculosis, as they serve as precursors for metabolic processes and are utilized as components of the mycobacterial cell wall7. This hypothesis gains support from recent findings that show up-regulated expression of epoxygenases in human macrophages, the natural habitat of M. tuberculosis36,37. Thus, it is conceivable that due to its genetic repertoire in EHs, M. tuberculosis is able to exploit the modified fatty acids present in human macrophages. Moreover, epoxygenases were also recently implicated in infection with intracellular bacteria underlining the potential importance for mycobacterial EHs during infection38. p y g Clearly, further research is required to fully understand the importance of EHs and the role they play during mycobacterial infections. Their overall structure is highly similar to the human soluble EH rendering an inhibi- tor design that targets the catalytic residues a challenging task. However, the structure of Mth-EphA described here furthers our understanding of the mechanism by which EHs might achieve substrate specificity and points to new opportunities for modulation of their activity. Further crystal structures of the remaining EHs (EphC-F) would add to our understanding of the nuances of mycobacterial EHs and facilitate design of EH-inhibitors that target other features of the proteins. www.nature.com/scientificreports/ The subtype specific substrate channels appear to be an interesting goal for future studies—not only limited to inhibitor design but also to better understand mycobacterial EH substrate specificity and thus their interaction with the host cell. Discussion M. thermoresistibile epoxide hydrolase A has high sequence similarity to its orthologue from M. tuberculosis and its crystal structure described here displays overall high structural similarity to α/β-hydrolase EHs. To gain insight into its substrate binding properties and shed light on its potential substrate specificity, we compare its substrate channel with that of another bacterial EH and the human orthologue. The structure of Mth-EphA presented here can be superimposed onto the structures of Mtb-EphB (2ZJF), to the EH from Agrobacterium radiobacter (1EHY) and to the catalytic domain of human soluble epoxide hydrolase (sEH; 1S8O) with an average r.m.s.d. of less than 0.5 Å. Moreover, the global similarity of the EHs is not limited to their main chains but also extends to the catalytic residues in the active site, which directly overlap upon superposition (Fig. 4). f h d f b l d ff h b h l However, in spite of the conserved active site conformation, substantial differences in the substrate channels can be observed. Mth-EphA features a wide double cavity that is connected by a narrow constriction at its center (Figs. 2, 4, Figure S2 and S3). The channel has two major openings to the solvent that exhibit opposite surface charges, while the other EHs compared here do not feature opposite charges at their active site entry sites. Mtb- EphB, which shares 40% sequence identity with Mth-EphA, on the other hand has a much narrower substrate channel with only two minor connections to the solvent. Moreover, upon superposition of the structures it is evident that the individual substrate channels not only have different shapes and sizes but also propagate along different directions. Scientific Reports \| (2020) 10:16539 \| https://doi.org/10.1038/s41598-020-73452-y Methods P i Protein purification. The gene encoding Mycobacterium thermoresistibile epoxide hydrolase A (Mth-EphA) (gi 490022613) was cloned into the pETM-11 vector (EMBL); protein expression was conducted in Escherichia coli Rosetta 2 (DE3) in LB medium at 20 °C for 18 h. The Mth-EphA construct used in this study contained all amino acids of the 321-residue protein. At the N-terminus it contained 4 additional residues (Gly-Ala-Met-Ala) linked to a TEV protease cleavage site and His6-tag. The His6-fusion protein was purified via affinity chroma- tography on a Ni–NTA resin (Quiagen) following the manufacturer’s instructions (50 mM Tris/HCl pH 7.5, 300 mM NaCl, 5% (v/v) glycerol). The His tag was removed by addition of TEV protease and dialysis at 4 °C for 18 h (50 mM Tris/HCl pH 7.5, 50 mM NaCl, 5% (v/v) glycerol). To remove the cleaved His6-tag and the TEV protease the solution was further purified via a MonoQ 10/100 ion exchange column (50 mM Tris/HCl pH 7.5, 5% (v/v) glycerol, 50–300 mM NaCl). EphA was further purified by Superdex S-75 size exclusion chromatogra- phy (50 mM Tris/HCl pH 7.5, 150 mM NaCl). Protein purity is not only reflected in a sharp, mono-dispersed elution profile in size exclusion chromatography but also in clear bands on SDS–polyacrylamide gels (Figure S1). Purified EphA was concentrated to 18 mg/ml and stored at − 80°°C until further use. Native mass spectrometry (MS). Purified Mth-EphA was exchanged into 150 mM ammonium acetate (AmAc) (PN431311, 99.99% purity, Sigma-Aldrich, MO, USA), pH 7.5, via centrifugal filter units (Vivaspin 500, MWCO 10,000, Sartorius, Germany) at 4 °C. The near physiological conditions allow on one hand preservation Scientific Reports \| (2020) 10:16539 \| https://doi.org/10.1038/s41598-020-73452-y www.nature.com/scientificreports/ Figure 5. Sequence alignments of mycobacterial EHs. Sequence and secondary structure alignment Mth-EphA, the six putative M. tuberculosis α/β-hydrolase family EHs, A. radiobacter EH and human EH. Secondary structure annotations are derived from Mth-EphA and human sHE. Substrate channe residues are colored in green, those that deviate between Mth-EphA and Mtb-EphA are highlighted i catalytic residues are indicated with a blue asterisk. Clearly the substrate channel lining residues (gree to the variable regions in the α-helical cap-domain, between strands β6 and β7. Most of the substrate lining residues are identical in Mth-EphA and Mtb-EphA, however, species–specific mutation can be for ~ 27% of the residues. Figure 5. Sequence alignments of mycobacterial EHs. Sequence and secondary structure alignment of Mth-EphA, the six putative M. www.nature.com/scientificreports/ of the native fold of the protein and on the other hand prevent adducts by nano-ESI. For binding experiments a 500 mM DPU stock in DMSO was diluted 50:1 in 150 mM AmAc in order to produce a saturated solution, considering the maximum DPU solubility in aqueous solutions of 710 µM/L (The Merck Index. 9th ed. Rahway, New Jersey: Merck & Co., Inc., 1976., p. 227) with and without 10% (v/v) butanediol, since it was also used for crystallization. Prior to measurements the inhibitor mix was diluted and incubated with EphA, resulting in 6 μM EphA, 71 μM DPU and 0.2% DMSO. Native MS was carried out in positive ion mode on a QToF 2 (Micromass/ Waters, UK) modified for high masses (MS Vision, the Netherlands)39. The gas pressures were 10 mbar in the source region and 1.2 × 10–2 mbar Argon in the collision cell. Raw data were calibrated with CsI (25 mg/mL) and analyzed using MassLynx (Waters). Peak deconvolution and determination of relative intensity was performed using UniDec40. LC–MS based enzyme activity assay. Stock solutions of cis-stilbene oxide (1), trans-stilbene oxide (2), hydrobenzoin (1,2-diphenyl-1,2-ethandiol) (3), trans-1,3-diphenyl-2,3-epoxypropane-1-one (4) were prepared in methanol LC–MS grade (each 20 mM) and kept at − 80 °C. Assay and control samples were prepared as fol- lows: To 200 µl of the assay buffer (25 mM Tris-formic acid pH 7.2) 4 µl of one of the potential EH substrates (1, 2 or 4, final concentration in the assay 400 µM) was added. The reaction in the assay samples was started by addition of 0.5 µl of 20 µM enzyme solution (final concentration 0.025 µM). To control samples no enzyme was added. The assay and control a samples were incubated 60 min at 30 °C. Thereafter the samples were chilled on ice for 3 min and the reaction was quenched by addition of 800 µl of ice-cold methanol. After incubation on ice for 10 min the samples were centrifuged 30 min, 13,000 rpm, 0 °C. The supernatant was collected and stored at − 80 °C. The supernatant samples were analyzed on HPLC1200 (Agilent) equipped with PRP C18 column (150 mm × 2.1 mm, Hamilton) and coupled to API4000 Q-Trap Triple Quad mass spectrometer (SCIEX). Mobile phase A was 3 mM ammonium formate in water:methanol (1:1, v/v), mobile phase B was 3 mM ammonium for- mate in water:methanol (1:995, v/v). www.nature.com/scientificreports/ Aliquots (20 µl) were injected onto the column equilibrated with the mobile phase A. The column was developed with a gradient of the mobile phase A: 100%, at 0 min; 0% at 10 min; 0% at 45 min; 100%, at 46 min; 100%, at 70 min. The flow rate was 200 µL/min. MRM transition parameters (electro- spray ionization, positive mode) for four available standard compounds were optimized using the Compound Optimization tool of the software Analyst (SCIEX). The m/z values, transition parameters and chemical formula of five compounds as well as their most populated fragmentation products are shown in the Table 1. The MRM chromatograms are shown in the supplemental material (Fig. S5–S11). Protein crystallization. Mth-EphA displayed high sensitivity towards DMSO. Thus, an inhibitor complex was generated by diluting a 1,3-diphenylurea solution (100 mM in 100% DMSO) 1:50 (v/v) with the protein solution, the resulting precipitate was removed by centrifugation, the supernatant was used for crystallization trials. Mth-EphA crystallized in multiple conditions, however, best diffracting crystals were only obtained in presence of low concentrations of 2,3-butanediol (1–3% v/v). Crystals were grown at 18 °C in sitting drop vapour diffusion plates combining equal volumes of precipitant (0.1 M BisTris pH 7.5; 2.15 M (NH4)2SO4; 4.5% (v/v) PEG 400; 1–3% (v/v) 2,3-butanediol) and the protein solution. The crystals appeared infrequently, as thin and often coadunate plates within 2–3 weeks and grew to a size of approximately 100 × 80 × 10 µm (Fig. S1). Crystals were cryo-protected by soaking in precipitant solution containing additionally 12% (v/v) 2,3-butanediol. Crys- tals were flash frozen in liquid nitrogen prior to data collection. Structure determination. X-ray data collection was performed at 100 K; diffraction images were collected at beamline ID30A-2 at ESRF, Grenoble, using the automated MASSIF data collection program41,42. Diffraction images were indexed, integrated and scaled using XDS and XSCALE, respectively43,44. Initial indexing of the dif- fraction patterns indicated an orthorhombic space group. However, analysis with PHENIX.XTRIAGE revealed a strong off-origin Patterson peak of ~ 56% origin intensity indicating pseudo-translational symmetry45. Moreo- ver, it also revealed that the data are twinned by pseudo-merohedry (α ~ 33%; twin law h, -k, -l), which was later accounted for during structure refinement. These features suggested a lower symmetry space group, which was confirmed by an analysis with ZANUDA46. Methods P i tuberculosis α/β-hydrolase family EHs, A. radiobacter EH and human soluble EH. Secondary structure annotations are derived from Mth-EphA and human sHE. Substrate channel lining residues are colored in green, those that deviate between Mth-EphA and Mtb-EphA are highlighted in yellow, catalytic residues are indicated with a blue asterisk. Clearly the substrate channel lining residues (green) map to the variable regions in the α-helical cap-domain, between strands β6 and β7. Most of the substrate channel lining residues are identical in Mth-EphA and Mtb-EphA, however, species–specific mutation can be observed for ~ 27% of the residues. https://doi.org/10.1038/s41598-020-73452-y Scientific Reports \| (2020) 10:16539 \| www.nature.com/scientificreports/ References 1. Nikaido, H. Multidrug resistance in bacteria. Annu. Rev. Biochem. 78, 119–146 (2009). 1. Nikaido, H. Multidrug resistance in bacteria. Annu. Rev. Biochem. 78, 119–146 (2009). g . World Health Organization. Global tuberculosis report 2018 (20 g p 3. Parida, S. K. et al. Totally drug-resistant tuberculosis and adjunct therapies. J. Intern. M g p 3. Parida, S. K. et al. Totally drug-resistant tuberculosis and adjunct therapies. J. Intern. Med. 277, 388–405 (2015). h d l Th d l h d d f l d 4. Dheda, K. et al. The epidemiology, pathogenesis, transmission, diagnosis, and management of multidrug-resistant, extensively drug resistant and incurable tuberculosis Lancet Respir Med 5 291 360 (2017) 4. Dheda, K. et al. The epidemiology, pathogenesis, transmission, diagnosis, and management of multidrug-res drug-resistant, and incurable tuberculosis. Lancet Respir. Med. 5, 291–360 (2017). g p 5. Nguyen, L. Antibiotic resistance mechanisms in M. tuberculosis: an update. Arch. Toxicol. 90, 1585–1604 (2016). 6. Sacchettini, J. C., Rubin, E. J. & Freundlich, J. S. Drugs versus bugs: in pursuit of the persistent predator Mycobacterium tuberculosis Nat. Rev. Microbiol. 6, 41–52 (2008). 7. Cole, S. T. et al. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393, 537–544 (1998).ii 8. Gordon, S. V. et al. Identification of variable regions in the genomes of tubercle bacilli using bacterial artificial chromosome arrays Mol. Microbiol. 32, 643–655 (1999). 9. van Loo, B., Kingma, J., Arand, M., Wubbolts, M. G. & Janssen, D. B. Diversity and biocatalytic potential of epoxide hydrolases identified by genome analysis. Appl. Environ. Microbiol. 72, 2905–2917 (2006). i y g y pp 10. Johansson, P. et al. Structure of an atypical epoxide hydrolase from Mycobacterium tuberculosis gives insights into its function. J. Mol. Biol. 351, 1048–1056 (2005).h 1. Biswal, B. K. et al. The molecular structure of epoxide hydrolase B from Mycobacterium tuberculosis and its complex with a urea- based inhibitor. J. Mol. Biol. 381, 912 (2008).i 12. Biswal, B. K., Garen, G., Cherney, M. M., Garen, C. & James, M. N. G. Cloning, expression, purification, crystallization and pre- liminary X-ray studies of epoxide hydrolases A and B from Mycobacterium tuberculosis. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 62, 138 (2006). d k l f h d h d l hd h b l f l d b l h 3. Madacki, J. et al. a availability dinates and structure factors have been deposited in the Protein Data Bank under accession codes 5CW2. Received: 30 September 2019; Accepted: 16 September 2020 References Impact of the epoxide hydrolase ephd on the metabolism of mycolic acids in mycobacteria. J. Biol. Chem. 293 5172–5184 (2018).h 14. Brown, J. R. et al. The structure–activity relationship of urea derivatives as anti-tuberculosis agents. Bioorg. Med. Chem. 19, 5585–5595 (2011).h 5. Arand, M., Cronin, A., Oesch, F., Mowbray, S. L. & Jones, T. A. The telltale structures of epoxide hydrolases. Drug Metab. Rev. 35 365–383 (2003). 6. Arand, M. et al. Structure of Rhodococcus erythropolis limonene-1,2-epoxide hydrolase reveals a novel active site. EMBO J. 22 2583–2592 (2003).h 17. Thunnissen, M. M., Nordlund, P. & Haeggström, J. Z. Crystal structure of human leukotriene A(4) hydrolase, a bifunctional enzyme in inflammation. Nat. Struct. Biol. 8, 131–135 (2001). l 8. Nardini, M. & Dijkstra, B. W. α/β Hydrolase fold enzymes: the family keeps growing. Curr. Opin. Struct. Biol. 9, 732–737 (1999). 9 N di i M t l Th t t f id h d l f A b t i di b t AD1 A t d t if h f 18. Nardini, M. & Dijkstra, B. W. α/β Hydrolase fold enzymes: the family keeps growing. Curr. Opin. Struct. Biol. 9, 732–737 (1999). 19. Nardini, M. et al. The x-ray structure of epoxide hydrolase from Agrobacterium radiobacter AD1. An enzyme to detoxify harmful epoxides. J. Biol. Chem. 274, 14579–14586 (1999). 9. Nardini, M. et al. The x-ray structure of epoxide hydrolase from Agrobacterium radiobacter AD1. An enzyme to detoxify harmfu epoxides. J. Biol. Chem. 274, 14579–14586 (1999). 0. Argiriadi, M. A. et al. Binding of alkylurea inhibitors to epoxide hydrolase implicates active site tyrosines in substrate activation J. Biol. Chem. 275, 15265–15270 (2000).i 1. Argiriadi, M. A., Morisseau, C., Hammock, B. D. & Christianson, D. W. Detoxification of environmental mutagens and carcinogens structure, mechanism, and evolution of liver epoxide hydrolase. Proc. Natl. Acad. Sci. 96, 10637–10642 (1999). 2. Ferrandi, E. E. et al. New thermophilic α/β class epoxide hydrolases found in metagenomes from hot environments. Front. Bioeng Biotechnol. 6, 1–16 (2018). p β p y g g Biotechnol. 6, 1–16 (2018). 23. Morisseau, C. & Hammock, B. D. Epoxide hydrolases: mechanisms, inhibitor designs, and biological roles. Annu. Rev. Pharmacol. Biotechnol. 6, 1–16 (2018). 23. Morisseau, C. & Hammock, B. D. Epoxide hydrolases: mechanisms, inhibitor designs, and biological roles. Annu. Rev. Pharmacol. Toxicol 45 311 333 (2005) 3. Morisseau, C. & Hammock, B. D. Epoxide hydrolases: mechanisms, inhibitor designs, and biological roles. References Annu. Rev. Pharmacol Toxicol. 45, 311–333 (2005). 4. Armstrong, R. N. & Cassidy, C. S. New structural and chemical insight into the catalytic mechanism of epoxide hydrolases. Drug Metab. Rev. 32, 327–338 (2000). 5. Lacourciere, G. M. & Armstrong, R. N. Microsomal and soluble epoxide hydrolases are members of the same family of C-X bond hydrolase enzymes. Chem. Res. Toxicol. 7, 121–124 (1994).h 6. Lacourciere, G. M. & Armstrong, R. N. The catalytic mechanism of microsomal epoxide hydrolase involves an ester intermediate J. Am. Chem. Soc. 115, 10466–10467 (1993). 7. Arand, M. et al. Sequence similarity of mammalian epoxide hydrolases to the bacterial haloalkane dehalogenase and other related proteins. Implication for the potential catalytic mechanism of enzymatic epoxide hydrolysis. FEBS Lett. 338, 251–256 (1994). 28. Rabi, S., Patel, A. H. G., Burger, S. K., Verstraelen, T. & Ayers, P. W. Exploring the substrate selectivity of human sEH an tuberculosis EHB using QM/MM. Struct. Chem. 28, 1501–1511 (2017). g 29. Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007) l S h & l S d h b l d d h 30. Liu, L., Bagal, D., Kitova, E. N., Schnier, P. D. & Klassen, J. S. Hydrophobic protein-ligand interactions preserved in the gas p J. Am. Chem. Soc. https://doi.org/10.1021/ja9060454 (2009). g j 1. Wixtrom, R. N. & Hammock, B. D. Membrane-Bound and Soluble-Fraction Epoxide Hydrolases: Methodological Aspects (Biochemi cal pharmacology and toxicology, 1985). 32. Rink, R., Fennema, M., Smids, M., Dehmel, U. & Janssen, D. B. Primary structure and catalytic mechanism of the epoxide hydrolase from Agrobacterium radiobacter AD1. J. Biol. Chem. 272, 14650–14657 (1997). g 3. Jacobs, M. H., Van den Wijngaard, A. J., Pentenga, M. & Janssen, D. B. Characterization of the epoxide hydrolase from an epichlo- rohydrin-degrading Pseudomonas sp. Eur. J. Biochem. 202, 1217–1222 (1991). y g g 34. van der Werf, M. J., Overkamp, K. M. & de Bont, J. A. Limonene-1,2-epoxide hydrolase from Rhodococcus erythropolis DCL14 belongs to a novel class of epoxide hydrolases. J. Bacteriol. 180, 5052–5057 (1998).ii 35. van der Werf, M. J. et al. Substrate specificity and stereospecificity of limonene-1,2-epoxide hydrolase from Rhodococcus erythro- polis DCL14; an enzyme showing sequential and enantioconvergent substrate conversion. Appl. Microbiol. Biotechnol. 52, 380–385 (1999). 36. Frömel, T. et al. Cytochrome P4502S1: a novel monocyte/macrophage fatty acid epoxygenase in human atherosclerotic plaques. Basic Res. Cardiol. www.nature.com/scientificreports/ The crystals obtained in this study belonged to space group P21 (a = 61.8, b = 105, c = 108.9; α, β, γ = 90°) with four monomers in the asymmetric unit and diffracted up to a reso- lution of 2 Å. The structure was solved by molecular replacement (PHASER) using a homology model generated with the PHYRE2 server47,48. Model building was performed in COOT49. Coordinates were refined to reasonable stereochemistry using PHENIX.REFINE (Table 2)45. Alternating steps of refinement and structure adjustments were performed until the R-values converged. The structure was validated using MOLPROBITY50. It was refined to Rwork and Rfree values of 20.86% and 25.08%, respectively (Table 2). All Mth-EphA monomers can be super- imposed to each other with an r.m.s.d. of less than 0.3 Å, demonstrating their structural equivalence. Refined coordinates were deposited in the RCSB as entry 5CW2. Structure and sequence comparison. Protein sequences were derived from Tuberculist and aligned using TCOFFEE51,52. Sequence alignments were subsequently visualized using ESPRIPT253. Structure analysis was conducted using tools from the CCP4 suite as well as using software from the PHENIX package45,54. Sub- strate tunnels were calculated using the CAVER3.0 plugin to PYMOL. After superimposition of the individual structures the carbonyl oxygen from the 1,3-diphenylurea inhibitor was used as a common starting point, using default values for optimization. The probe radius was set to 1, the shell depth to 4 and the number of approximat- ing balls to 20 for all protein structures. The shell radius and the clustering threshold were chosen individually to retrieve the most prominent tunnels (shell radius/clustering threshold: 1S8O-4/7; 1EHY-4/3.5; 2ZJF-3/3.5; Mth-EphA-3/3.5)55. Overlapping tunnels have been merged for visualization. 11 https://doi.org/10.1038/s41598-020-73452-y Scientific Reports \| (2020) 10:16539 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ The Phyre2 web portal for protein modeling, prediction and analysis. Nat. Protoc. 10, 845–858 (2015). y 9. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. Sect. D Biol. Crystallogr. 60, 2126–2132 (2004). 50. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. Sect. D Biol. Crystallogr. 66, 12–21 (2010).t y g 51. Lew, J. M., Kapopoulou, A., Jones, L. M. & Cole, S. T. TubercuList—10 years after. Tuberculosis 91, 1–7 (2011).f t 52. Notredame, C., Higgins, D. G. & Heringa, J. T-Coffee: a novel method for fast and accurate multiple sequence alignment. J. Mol. Biol. 302, 205–217 (2000). 53. Gouet, P., Robert, X. & Courcelle, E. ESPript/ENDscript: extracting and rendering sequence and 3D information from atomic structures of proteins. Nucleic Acids Res. 31, 3320–3323 (2003).h p 54. Collaborative, C. P. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. Sect. D Biol. Crystallogr. 50, 760 (1994). p 4. Collaborative, C. P. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. Sect. D Biol. Crystallogr. 50, 760–763 (1994). 55. Chovancova, E. et al. CAVER 3.0: a tool for the analysis of transport pathways in dynamic protein structures. PLoS Comput. Biol. 8, e1002708 (2012). 55. Chovancova, E. et al. CAVER 3.0: a tool for the analysis of transport pathways in dynamic protein structures. PLoS Comput. Biol. 8, e1002708 (2012). Acknowledgements g We are indebted to our colleagues Vivian Pogenberg and Annabel Parret for their continuous support, helpful discussions and critical reading of the manuscript. Moreover, we would like to thank the beamline staff of ID-30 from ESRF for their support during data collection. Author contributions M.W., S.M.S., E.C.S. designed the experiment; S.M.S., S.R.H. cloned, the protein; S.M.S., S.R.H., T.C. expressed and purified the proteins; S.R.H., E.C.S. crystallized the protein; B.K., C.U. performed native MS analysis; E.C.S. determined the crystal structure, analysed the data, prepared the images, wrote the manuscript; B.I., M.F. were involved in protein characterization and the enzyme activity assays; all authors discussed and contributed to the manuscript. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 38. Bystrom, J. et al. Inducible CYP2J2 and its product 11,12-EET promotes bacterial phagocytosis: a role for CYP2J2 deficiency in the pathogenesis of Crohn’s disease?. PLoS ONE 8, e75107 (2013).l p g 9. Van Den Heuvel, R. H. H. et al. Improving the performance of a quadrupole time-of-flight instrument for macromolecular mass spectrometry. Anal. Chem. https://doi.org/10.1021/ac061039a (2006). p y p g ( ) 40. Marty, M. T. et al. Bayesian deconvolution of mass and ion mobility spectra: from binary interactions to polydisperse ensem Anal Chem https://doi org/10 1021/acs analchem 5b00140 (2015) Marty, M. T. et al. Bayesian deconvolution of mass and ion mobili 0. Marty, M. T. et al. Bayesian deconvolution of mass and ion mobility spectra: from binary interactions to polydisperse ensembles Anal. Chem. https://doi.org/10.1021/acs.analchem.5b00140 (2015).f y y Anal. Chem. https://doi.org/10.1021/acs.analchem.5b00140 (2015 p g 1. Bowler, M. W. et al. Diffraction cartography: applying microbeams to macromolecular crystallography sample evaluation and data collection. Acta Crystallogr. Sect. D Biol. Crystallogr. 66, 855–864 (2010).hl 41. Bowler, M. W. et al. Diffraction cartography: applying microbeams to macromolecular cr collection. Acta Crystallogr. Sect. D Biol. Crystallogr. 66, 855–864 (2010).hl f g y y g collection. Acta Crystallogr. Sect. D Biol. Crystallogr. 66, 855–86 y g y g 2. Brockhauser, S. et al. The use of workflows in the design and implementation of complex experiments in macromolecular crystal- lography. Acta Crystallogr. Sect. D Biol. Crystallogr. 68, 975–984 (2012).f hl g p graphy. Acta Crystallogr. Sect. D Biol. Crystallogr. 68, 975–984 (201 g p y y g y g 3. Kabsch, W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J Appl. Crystallogr. 26, 795–800 (1993).i pp y g 4. Kabsch, W. Integration, scaling, space-group assignment and post-refinement. Acta Crystallogr. Sect. D Biol. Crystallogr. 66, 133–144 (2010). 45. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010). y g 6. Lebedev, A. A. & Isupov, M. N. Space-group and origin ambiguity in macromolecular structures with pseudo-symmetry and its treatment with the program Zanuda. Acta Crystallogr. Sect. D Biol. Crystallogr. 70, 2430–2443 (2014).t cCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallo y J y g pt J pp y g ( ) 48. Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N. & Sternberg, M. J. E. References 108, 319 (2013).i 7. Behmoaras, J. et al. Macrophage epoxygenase determines a profibrotic transcriptome signature. J. Immunol. (Baltimore, Md 1950) 194, 4705–4716 (2015). Scientific Reports \| (2020) 10:16539 \| https://doi.org/10.1038/s41598-020-73452-y Competing interests h p g The authors declare no competing interests. Funding g Open Access funding enabled and organized by Projekt DEAL. This work was supported by an BMBF ERA-NET Grant Pathogenomic GeMoA to M.W. with Reference Number 0315908. The Heinrich Pette Institute, Leibniz Institute for Experimental Virology is supported by the Free and Hanseatic City of Hamburg and the Federal Ministry of Health. BK and CU are funded by the Leibniz Association through SAW-2014-HPI-4 Grant. This work was supported by the Max Planck Society, the Joachim Herz Foundation (Biomedical Physics of Infection) and the Excellence Cluster “CUI: Advanced Imaging of Matter” of the Deutsche Forschungsgemeinschaft (DFG), EXC 2056, project ID 390715994. 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https://openalex.org/W4254041217	https://discovery.ucl.ac.uk/10081967/1/The%20effects%20of%20high%20versus%20low%20talker%20variability%20and%20individual%20aptitude%20on%20phonetic%20training%20of%20Mandarin%20lexical%20tones.pdf	English	null	The effects of high versus low talker variability and individual aptitude on phonetic training of Mandarin lexical tones	null	2,019	cc-by	25,980	The effects of high versus low talker variability and individual aptitude on phonetic training of Mandarin lexical tones Submitted 26 July 2018 Accepted 26 May 2019 Published 9 August 2019 Corresponding authors Hanyu Dong, hanyu.dong.10@ucl.ac.uk Elizabeth Wonnacott, e.wonnacott@ucl.ac.uk Academic editor Vesna Stojanovik Additional Information and Declarations can be found on page 41 DOI 10.7717/peerj.7191 Copyright 2019 Dong et al. Distributed under Creative Commons CC-BY 4.0 Submitted 26 July 2018 Accepted 26 May 2019 Published 9 August 2019 Corresponding authors Hanyu Dong, hanyu.dong.10@ucl.ac.uk Elizabeth Wonnacott, e.wonnacott@ucl.ac.uk Academic editor Vesna Stojanovik Additional Information and Declarations can be found on page 41 DOI 10.7717/peerj.7191 Copyright 2019 Dong et al. Distributed under Creative Commons CC-BY 4.0 Subjects Psychiatry and Psychology, Human-Computer Interaction, Computational Science Keywords L2 phonetic contrasts, Phonetic training, Lexical tone learning, Second language Subjects Psychiatry and Psychology, Human-Computer Interaction, Computational Science Keywords L2 phonetic contrasts, Phonetic training, Lexical tone learning, Second language How to cite this article Dong H, Clayards M, Brown H, Wonnacott E. 2019. The effects of high versus low talker variability and individual aptitude on phonetic training of Mandarin lexical tones. PeerJ 7:e7191 DOI 10.7717/peerj.7191 The effects of high versus low talker variability and individual aptitude on phonetic training of Mandarin lexical tones Hanyu Dong1, Meghan Clayards2,3, Helen Brown4 and Elizabeth Wonnacott1 1 Division of Psychology and Language Sciences, University College London, London, UK 2 Department of Linguistics, McGill University, Montreal, QC, Canada 3 School of Communication Sciences and Disorders, McGill University, Montreal, QC, Canada 4 Department of Psychology, Nottingham Trent University, Nottingham, UK High variability (HV) training has been found to be more effective than low variability (LV) training when learning various non-native phonetic contrasts. However, little research has considered whether this applies to the learning of tone contrasts. The only two relevant studies suggested that the effect of HV training depends on the perceptual aptitude of participants (Perrachione et al., 2011; Sadakata & McQueen, 2014). High variability (HV) training has been found to be more effective than low variability (LV) training when learning various non-native phonetic contrasts. However, little research has considered whether this applies to the learning of tone contrasts. The only two relevant studies suggested that the effect of HV training depends on the perceptual aptitude of participants (Perrachione et al., 2011; Sadakata & McQueen, 2014). The present study extends these ﬁndings by examining the interaction between individual aptitude and input variability using natural, meaningful second language input (both previous studies used pseudowords). A total of 60 English speakers took part in an eight session phonetic training paradigm. They were assigned to high/low/high-blocked variability training groups and learned real Mandarin tones and words. Individual aptitude was measured following previous work. Learning was measured using one discrimination task, one identiﬁcation task and two production tasks. All tasks assessed generalization. All groups improved in both the production and perception of tones which transferred to untrained voices and items, demonstrating the effectiveness of training despite the increased complexity compared with previous research. Although the LV group exhibited an advantage with the training stimuli, there was no evidence for a beneﬁt of high-variability in any of the tests of generalisation. Moreover, although aptitude signiﬁcantly predicted performance in discrimination, identiﬁcation and training tasks, no interaction between individual aptitude and variability was revealed. Additional Bayes Factor analyses indicated substantial evidence for the null for the hypotheses of a beneﬁt of high-variability in generalisation, however the evidence regarding the interaction was ambiguous. We discuss these results in light of previous ﬁndings. INTRODUCTION Distributed under Creative Commons CC-BY 4.0 One challenging aspect of learning a second language (L2) is learning to accurately perceive non-native phonetic categories. This task is particularly difﬁcult when the L2 relies on the same acoustic dimensions as the ﬁrst language (L1), but for different purposes (Bygate, Swain & Skehan, 2013), suggesting that it is challenging to adjust existing acoustic properties in the L1 to learn new L2 categories. This challenge is compounded by the fact that speech is highly variable in the natural linguistic environment. Variability comes not only from the phonetic context but also from differences between speakers. Thus, learners must learn to distinguish the new L2 categories despite all the variability present in the learning input. There is evidence that native listeners can process this variability in speech faster and more accurately than non-native listeners (Bradlow & Pisoni, 1999), indicating that variability is indeed a challenge for L2 learners. Despite this, it has been suggested that input variability may be beneﬁcial for L2 learning and generalisation (Barcroft & Sommers, 2005; Lively, Logan & Pisoni, 1993). However, recent evidence suggests that the ability to beneﬁt from variability may depend on individual learner aptitude (Perrachione et al., 2011; Sadakata & McQueen, 2014), at least in the learning of lexical tones (i.e. the distinctive pitch patterns carried by the syllable of a word which, in certain languages, distinguish meaningful lexical contrasts). The current paper further explores how and when variability supports or impedes learning of new L2 phonetic categories, focusing on English learners of Mandarin tone contrasts. Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 High variability L2 phonetic training for non-tonal contrasts A substantial body of literature has explored whether phonetic training can be used to improve identiﬁcation and discrimination of non-native phonetic contrasts in L2 learners. An early study by Strange & Dittmann (1984) attempted to train Japanese speakers on the English /r/-/l/ distinction, a phoneme contrast that does not exist in Japanese. Participants were trained on stimuli from a synthetic rock-lock continuum. The key result was that although performance increased both for trained and novel synthetic items, participants failed to show any improvement for naturally produced minimal pair items. Later research suggested that a key factor which prevented generalisation to natural speech tokens was a lack of variability in the training materials: Variability was present in the form of the ambiguous intermediate stimuli along the continuum, however, there was a single phonetic context and a single (synthesized) speaker. Logan, Lively & Pisoni (1991) also trained Japanese learners on the English /r/-/l/ contrast, but included multiple natural exemplars spoken by six speakers, with the target speech sounds appearing in a range of phonetic contexts. In contrast to Strange and Dittman, they found that participants successfully generalised both to new speakers and new words at test. This was the ﬁrst study to indicate the importance of variability within the training materials. A follow up study by Lively, Logan & Pisoni (1993) provided further evidence for this by contrasting a condition with high variability (HV) input to one with low variability (LV) input in which the stimuli were spoken by a single speaker (although still exempliﬁed in multiple phonetic contexts). Participants in the LV group improved during the training sessions but failed to generalise this learning to a new speaker. Following Lively, Logan & Pisoni (1993) high variability phonetic training (HVPT) has become standard in L2 phonetic training. This methodology has been successfully extended to training a variety of contrasts in various languages such as learning of Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 2/45 the English /u/-/ʊ/ distinction by Catalan/Spanish bilinguals (Aliaga-García & Mora, 2009), learning of the English /i/-/ɪ/ contrast by native Greek speakers (Giannakopoulou, Uther & Ylinen, 2013; Lengeris & Hazan, 2010), and learning of the English /w/-/v/ distinction by native German speakers (Iverson et al., 2008). There is also some evidence that this type of perceptual training beneﬁts production in addition to perception. Bradlow et al. High variability L2 phonetic training for non-tonal contrasts (1999) found that production of the /r/-/l/ contrast improved in Japanese speakers following HVPT, with this improvement being retained even after 3 months. Similar improvement on the production of American English mid to low vowels by Japanese speakers following HVPT was also reported by Lambacher et al. (2005). However, the evidence here is mixed: A recent study by Alshangiti & Evans (2014) employed HVPT to train Arabic learners on non-native English vowel contrasts and found no improvements in production, although participants receiving additional explicit production training did show some limited improvement. Although the studies reviewed above all used HVPT, only the original work by Lively, Logan & Pisoni (1993) directly contrasted the use of high and low variability materials. It is notable these seminal experiments used small samples (the tests of generalisation were administered to only three of the participants). Since then, few studies have explicitly contrasted high and low variability training. One such study was Sadakata & McQueen (2013), who trained native Dutch speakers with geminate and singleton variants of the Japanese fricative /s/. Participants were trained with either a limited set of words recorded by a single speaker (LV) or with a more variable set of words recorded by multiple speakers (HV). Both types of training led to increases in generalisation to untrained fricatives and speakers. However, in an identiﬁcation task, the improvement was greater for participants receiving HV training than those receiving LV training. Similar results were reported by Wong (2014) who trained native Cantonese speakers with the English /e/-/æ/ contrast. Both LV (one speaker) and HV (six speakers) training lead to increased performance from pre- to post-test, but the improvement was greater for the HV group. This was found in tests of generalisation to new speakers and new items, and from perception to production. In contrast, a recent phonetic training study did not ﬁnd the same beneﬁt. Giannakopoulou et al. (2017) compared matched HV (four speakers) and LV (one speaker) training for adult and child (8-year-old) native Greek speakers who were trained on the English /i/-/ɪ/ contrast. This study did not show a beneﬁt for HV compared to LV training in either age group, even for generalisation items. However, for adult participants, it is unclear the extent to which this was due to ceiling effects. Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 Phonetic training of L2 lexical tones Th di d h i h l h HV i i Each of the phonetic training studies discussed above involved training a segmental contrast (consonantal or vocalic). Lexical tone is another type of phonological contrast in some natural languages, whereby the pitch contour is used to distinguish lexical or grammatical meanings (Yip, 2002). For example, Mandarin Chinese has four lexical tones: level-tone (Tone 1), rising-tone (Tone 2), dipping-tone (Tone 3) and falling-tone (Tone 4). These pitch contours combine with syllables to distinguish meanings. For instance, the syllable ba combines with the four tones to mean: eight (bā, Tone 1), pluck (bá, Tone 2), grasp (bǎ, Tone 3) and father (bà, Tone 4). Each of these words thus forms a minimal pair with each of the others. Note that while non-tonal languages such as English use pitch information extensively for intonation (e.g. forming a question, or for emphasis), and that pitch plays a role in marking stress at the lexical level in (e.g. IMport/imPORT), this is quite different from a lexical tone system, causing difﬁculties for L2 learners of Mandarin. q y g The ﬁrst study examining lexical tone training was conducted by Wang et al. (1999). A similar paradigm to that used by Logan, Lively & Pisoni (1991) was adopted using four speakers for training. Training materials were all real monosyllabic Mandarin words that varied in the consonants, vowels and syllable structure. During training participants heard a syllable whilst viewing two of the four standard diacritic representations (i.e. /, ↗, ∨, ↘, which are iconic in nature). They were asked to pick out the picture of the arrow that corresponded to the tone. At test, participants chose which tone they had heard out of a choice of all four diacritics. There were also two generalisation tasks, one testing generalisation to untrained items and one testing generalisation to a new speaker. Native American English speakers showed signiﬁcant improvement in the accuracy of tone identiﬁcation after eight sessions of HV training over 2 weeks, and this generalised to both new words and a new speaker. In a follow up study, Wang, Jongman & Sereno (2003) used the same training paradigm to test whether learning transferred to production. They recruited participants taking Mandarin courses and asked them to read through a list of 80 Mandarin words written in Pinyin (an alphabetic transcription) before and after training. High variability L2 phonetic training for non-tonal contrasts To our knowledge, the only other previous studies that speciﬁcally manipulated variability during learning of non-native phonetic categories are those by Perrachione et al. (2011) and Sadakata & McQueen (2014), which both looked at the learning of lexical tone. We discuss these studies in more detail in the following section. Although there is a relatively small evidence base regarding a beneﬁt of high over low phonetic training for non-native phoneme categories, there is further evidence for this beneﬁt in related areas of speech and language learning, speciﬁcally accent categorization and adaptation (Bradlow & Bent, 2008; Clopper & Pisoni, 2004), and L2 vocabulary learning (Barcroft & Sommers, 2005, 2014; Sommers & Barcroft, 2007, 2011). Beneﬁts of Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 HVPT are generally seen in tasks of generalisation, suggesting that exposure to variation across speakers and/or items boosts the ability to generalise across these dimensions. This intuitively sensible result is in line with the predictions of computational models in which irrelevant contextual/speaker identity cues compete with phonetically relevant cues, so that dissociation of these irrelevant cues is the key mechanism which underpins generalisation (Apfelbaum & McMurray, 2011; Ramscar & Baayen, 2013; Ramscar et al., 2010). Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 Phonetic training of L2 lexical tones g Each of the phonetic training studies discussed above involved training a segmental contrast (consonantal or vocalic). Lexical tone is another type of phonological contrast in some natural languages, whereby the pitch contour is used to distinguish lexical or grammatical meanings (Yip, 2002). For example, Mandarin Chinese has four lexical tones: level-tone (Tone 1), rising-tone (Tone 2), dipping-tone (Tone 3) and falling-tone (Tone 4). These pitch contours combine with syllables to distinguish meanings. For instance, the syllable ba combines with the four tones to mean: eight (bā, Tone 1), pluck (bá, Tone 2), grasp (bǎ, Tone 3) and father (bà, Tone 4). Each of these words thus forms a minimal pair with each of the others. Note that while non-tonal languages such as English use pitch information extensively for intonation (e.g. forming a question, or for emphasis), and that pitch plays a role in marking stress at the lexical level in (e.g. IMport/imPORT), this is quite different from a lexical tone system, causing difﬁculties for L2 learners of Mandarin. The ﬁrst study examining lexical tone training was conducted by Wang et al. (1999). A similar paradigm to that used by Logan, Lively & Pisoni (1991) was adopted using four speakers for training. Training materials were all real monosyllabic Mandarin words that varied in the consonants, vowels and syllable structure. During training participants heard a syllable whilst viewing two of the four standard diacritic representations (i.e. /, ↗, ∨, ↘, which are iconic in nature). They were asked to pick out the picture of the arrow that corresponded to the tone. At test, participants chose which tone they had heard out of a choice of all four diacritics. There were also two generalisation tasks, one testing generalisation to untrained items and one testing generalisation to a new speaker. Native American English speakers showed signiﬁcant improvement in the accuracy of tone identiﬁcation after eight sessions of HV training over 2 weeks, and this generalised to both new words and a new speaker. In a follow up study, Wang, Jongman & Sereno (2003) used the same training paradigm to test whether learning transferred to production. They recruited participants taking Mandarin courses and asked them to read through a list of 80 Mandarin words written in Pinyin (an alphabetic transcription) before and after training. They found improvements in production, although these were mainly seen in pitch contour rather than pitch height. Phonetic training of L2 lexical tones They found improvements in production, although these were mainly seen in pitch contour rather than pitch height. These studies suggested that as with segmental phoneme contrasts, HV training may also facilitate the learning of tone contrasts. However, Wang et al. (1999) and Wang, Jongman & Sereno (2003) did not directly contrast high and low variability training materials. Perrachione et al. (2011) investigated this contrast directly. They trained native American English speakers with no previous knowledge of Mandarin (or any other tonal Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 4/45 language), using English monosyllabic pseudowords combined with Mandarin tones 1, 2, and 4 (/, ↗& ↘). The training task used either LV (one speaker) or HV (four speaker) input. During the training, participants matched the sound they heard with one of three pictures of concrete objects presented, where the three words associated with these pictures were minimal trios that differed only in tone. Participants were tested on their ability to generalise their learning to new speakers. Importantly, Perrachione et al. (2011) were also interested in the role of individual differences in learning. Therefore, they also determined participants’ baseline ability to perceive the tone contrasts prior to training using a Pitch Contour Perception Test. In this task, participants heard a vowel produced with either Mandarin tone 1, 2 or 4 whilst viewing pictures of standard diacritics associated with these tones (/, ↗& ↘), and were asked to select the arrow that corresponded to the tone. Based on performance in this task, the researchers grouped participants into high and low aptitude groups. The results showed that whilst the LV group outperformed the HV group during training (presumably due to accommodation to a repeated speaker throughout the task), there were no differences between the high and LV groups during test. Critically however, there was an interaction between an individuals’ aptitude categorization and the type of variability training: Only participants with high aptitude beneﬁtted from HV training, while those with low aptitude actually beneﬁtted more from LV training. It is important to note that this interaction was seen in a task which relied on participants’ ability to generalise their learning1 of tones to an untrained speaker. That is, in a task where we would expect that exposure to multiple speakers would be beneﬁcial since it should allow learners to better dissociate the tones from the particular speakers used in training. Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 Phonetic training of L2 lexical tones a novel position), and (4) items where the tone was embedded in a sentence context. As in the study by Perrachione et al. (2011), Sadakata & McQueen (2014) also tested individual aptitude but with a different method. They employed a categorization task using stimuli from a six step Tone 2 to Tone 3 continuum (created using natural productions of the two tones with the Mandarin vowel /a/ as endpoints and linearly interpolating between these endpoints). Participants were asked to identify if the sound they heard was more like Tone 2 or Tone 3, and a categorization slope was obtained for each participant providing a measure of their ability to discriminate this contrast, which is generally found to be the most challenging tone contrast for L2 learners of Mandarin. Participants were grouped according to their slopes, and this grouping was entered as a factor in the analyses of tests of learning, along with the effect of training condition (high-medium- low) and the interaction between factors. For the test with trained speakers and items, there was no group level effect of variability condition, however there was an interaction between variability and aptitude similar to that reported by Perrachione et al.: Participants with high aptitude beneﬁtted from HV training, while those with lower aptitude beneﬁtted more from LV training. For the generalisation tests, participants showed above chance performance in all but the new position condition, demonstrating an ability to generalise their learning of tone across different dimensions. However, they did not demonstrate an overall beneﬁt of higher variability in any of the transfer tests, nor, did variability interaction with aptitude. Note that the overall lack of a HV beneﬁt is again surprising, particularly for test items with untrained talkers and novel items, since the manipulations in training should speciﬁcally work to increase generalisation along these dimensions. In sum, the two studies which have directly compared high and low variability input in training Mandarin tone contrasts have not found the predicted beneﬁt of HV on generalisation, either when varying just speakers or when varying speakers and items. However, both of these studies found an interaction between participant aptitude and variability condition. Phonetic training of L2 lexical tones These results, therefore, suggest that only the high aptitude learners can take advantage of this beneﬁt. Another training study by Sadakata & McQueen (2014) also explored the relationship between input variability and individual aptitude in lexical tone training, though using different training and testing materials. They trained native Dutch speakers (with no prior knowledge of Mandarin or any other tonal language) using naturally produced bisyllabic Mandarin pseudowords. The two syllables in each word either had Tone 2 followed by Tone 1, or Tone 3 followed by Tone 1, and each tone pair was randomly assigned one of two numeric labels (e.g. for one participant Tone 2-Tone 1 was labelled ‘1’, Tone 3-Tone 1 was labelled ‘2’). During the training task, participants identiﬁed the tone pair type of each stimulus by choosing the correct numeric label (e.g. hear /pasa/ with Tone 2-Tone 1, correct response is 1). Thus, in contrast to the study by Perrachione et al. (2011), participants did not need to learn the meaning of each word. Input variability was manipulated, with three levels (low/medium/high). In contrast to the work by Perrachione et al. where the HV and LV conditions differed only in terms of the number of speakers, in this study variability was increased both by including more speakers and more items. Speciﬁcally, the number of different vowels used in the bi-syllabic sequences was manipulated: the LV group 1 In their paper, Perrachione et al. (2011) do not refer to this task as a general- isation task. Instead they report a gen- eralisation measure which is a ratio of performance on this test with novel speakers to performance in training (test- performance/training-performance). Note that this ratio will increase not only if participants are better at test, but also if they are worse in training. Using this measure, Perrachione et al. found a beneﬁt of high variability training. However on inspection of the means, it seems that this relationship is driven by the poorer performance in training in the high variability condition, rather than by better performance in the test with novel speakers. We therefore do not see the ratio measure as providing evidence for an overall beneﬁt of HV training on generalisation. Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 5/45 at (1) trained items spoken by an untrained talker; (2) pseudowords containing untrained vowels (3) pseudowords in which the order of tones in the bi-syllables were reversed (i.e. Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 The current study The fact that neither of the tone training studies found an overall beneﬁt of high over LV in tone generalisation is surprising in light of the phonetic literature and the predictions of the computational model (Apfelbaum & McMurray, 2011; Ramscar & Baayen, 2013) mentioned above. Moreover, as the previous authors point out, if it is actually the case that learning from multiple voices is more or less effective for different groups of learners, this has important implications for the design of L2 training tools. For this to be the case, it is important to establish the generalisability of the ﬁndings to different contexts and materials, particularly those which are relevant in an L2 learning context. We suggest that what L2 learners are most interested in developing is their ability to use tone when mapping a word’s phonological form to its meaning (and vice versa). In this light, the paradigm used by Sadakata & McQueen (2014) lacks ecological validity in looking only at mapping to abstract tone categories. On the other hand, Perrachione et al. (2011) do train form-meaning mappings, yet, unlike Sadakata & McQueen (2014) they use English pseudo-word stimuli, which has the consequence that learners do not simultaneously have to deal with non-native segments and tones, as in a real world L2 learning situation. Furthermore, although there is limited data on the differences between words and non-words in production, it has been noted that non-words may have different properties from real words even within the same language (Scarborough, 2012) and may be more clearly articulated (Hay, Drager & Thomas, 2013; Maxwell et al., 2015). Thus, using non-words might make stimuli slightly easier to learn than if real words were used. The current training study addresses these issues in a partial replication of the previous work: We use stimuli produced by native Mandarin speakers which are real words in that language. This design choice follows earlier studies such as Wang et al. (1999) using a paradigm in which participants are trained to identify word meaning on the basis of tone. In contrast to the previous studies, we also trained the contrasts between all four tones (six tone contrasts) rather than just three (on the assumption that learners are interested in learning the complete set of contrasts within a particular language). We note that these design choices potentially increase the difﬁculty of our training materials compared to previous work. Phonetic training of L2 lexical tones The results of these studies thus provide mutually corroborating evidence—using somewhat different training and testing methods—that the ability to learn from HV input is dependent on learner aptitude, although it should be noted that this interaction was found in a task with untrained speakers in one study (Perrachione et al., 2011), but in a task with trained stimuli in the other (Sadakata & McQueen, 2014). Why might the ability to beneﬁt from varied training materials depend on participant aptitude? Perrachione et al. (2011) suggest that one reason why low aptitude participants may struggle with multi-speaker input is that the speakers were intermixed during training: This requires trial-by-trial adaptation to each speaker, which was not required in the corresponding single speaker LV conditions. This may place a burden on learners (see Mattys & Wiget, 2011; Nusbaum & Morin, 1992, for evidence that intermixed multi-speaker stimuli are difﬁcult even for L1 processing and that this interacts with constraints on working memory and attention). To test this, Perrachione et al. (2011) conducted a second experiment in which items from each speaker were presented in separate Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 blocks (as is more common in HVPT). This improved performance during the training task compared with unblocked training for low aptitude learners only, conﬁrming the hypothesis that switching between speakers on a trial-by-trial basis during training interferes with learning for low aptitude learners. On the other hand, Sadakata & McQueen (2014) employed a training paradigm in which speakers were blocked in the HV condition, yet they still found the interaction with aptitude. However, recall that in their experiment they also manipulated item variability, yet only speakers were blocked by session, not items. Thus, it remains possible that trial-by-trial inconsistency at the level of items could explain some of the greater difﬁculty of low aptitude learners in their study. Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 The current study A key question was whether these choices would impact the interaction between learner aptitude and the beneﬁts of more variable training materials. We followed Perrachione et al. (2011) in varying variability along one dimension only—speaker variability, keeping training items identical across conditions. We also Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 followed Perrachione et al. (2011) in comparing HV input which was blocked by speaker, with input that was not, making three training conditions: LV (one speaker), HV (four speakers intermixed within each training session) and blocked training (four speakers each presented in separate blocks). Note that our choice to manipulate only talker-variability means that the HV blocked condition is matched to the LV condition in terms of trial-by-trial inconsistency, unlike in Sadakata & McQueen (2014) where, even though they blocked by speaker, the HV condition contained more trial-by-trial variability in terms of items. We predicted that the difﬁculty of HV input for lower aptitude participants would be greater in the unblocked condition, thus potentially increasing the likelihood of seeing the predicted interaction between variability and learner aptitude. On the other hand, blocked input is more usual of HVPT (Iverson, Hazan & Bannister, 2005; Logan, Lively & Pisoni, 1991) and may increase the possibility of seeing an overall beneﬁt of speaker variability on generalisation. We used two perceptual tasks designed to tap individual aptitude. These were adapted from those used in Perrachione et al. (2011) and Sadakata & McQueen (2014). However, while the previous studies grouped participants into one of two categories (high aptitude vs low aptitude) based on the aptitude score, in the current study they were used as continuous measures. This allowed us to avoid assigning an arbitrary ‘cut off’ for high vs low aptitude groups, and the loss of information which occurs when an underlying continuous variable is turned into a binary measure. Note that the statistical approach used in the current paper (logistic mixed effect models) allowed us to include continuous predictors and look at their interactions with other factors. A further extension in the current study is that we use several new outcome measures to test learning and generalisation. First, most similar to the task used in Perrachione et al. (2011) was a picture identiﬁcation task which was a version of the training task (2AFC picture identiﬁcation) without feedback. Following Perrachione et al. 2 If we wished to use trained speakers, in order to be able to the use the same test with the low variability condition, we would have to use a single speaker across all three test trials. Our pilot work sug- gested that participants performed at ceiling on a single-speaker version of this task, even at pre-test. The current study (2011) we included untrained-speaker items, where beneﬁts of speaker variability in training should be most apparent. However, bearing in mind that Sadakata & McQueen (2014) actually found the key interaction with aptitude only in the test with trained stimuli, we also included trained-speaker test items. We also included a second perceptual task which did not involve knowing speciﬁc form-meaning mappings and thus had the beneﬁt that it could be conducted both pre- and post-test. This was a three interval oddity task which required participants to pick the odd-one-out after hearing three words spoken aloud, each by a different speaker. Two of the tokens were productions of the same word and the third differed only in the tone (e.g. bā, Tone 1; bā, Tone 1; bà, Tone 4). Because all three tokens are physically different, it requires the listener to focus on the phonological level ignoring irrelevant acoustic differences. Furthermore, the use of three speakers forces the listener to ignore irrelevant speaker-speciﬁc differences, making it especially challenging (Strange & Shafer, 2008). This task used untrained speakers in every trial, so that every test-item required generalisation to new speakers2. In addition, here it was possible to use both trained and untrained items. Note that even though the variability over items is matched across conditions, it is possible that varying speaker speciﬁc cues might also thus promote Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 generalisation across this dimension. If this is the case, a HV beneﬁt may be stronger for untrained items than trained items. Table 1 Mean age range, average number of languages learned and mean starting age of learning the ﬁrst L2 for participants in each condition. Condition Mean age Age range Languages learned Average staring age Low variability 26.15 (2.2) 19–53 2.7 (0.5) 13.8 (1.1) High variability 25.65 (0.7) 19–47 2.5 (0.6) 12.2 (0.5) High variability blocked 22.05 (1.4) 19–30 2.0 (1.3) 11.8 (0.4) Table 1 Mean age range, average number of languages learned and mean starting age of learning the ﬁrst L2 for participants in each condition. generalisation across this dimension. If this is the case, a HV beneﬁt may be stronger for untrained items than trained items. generalisation across this dimension. If this is the case, a HV beneﬁt may be stronger for untrained items than trained items. The current study Finally, we also tested production using a picture naming task at post-test, in which participants were required to name the pictures used in training in Mandarin. We also conducted a word repetition task, which had the beneﬁt that it could also be conducted at pre-test, and that we could use both trained and untrained words (as for the three-interval oddity task discussed above). Although there is evidence HVPT can beneﬁt the production of tones (Wang, Jongman & Sereno, 2003), there has been no direct examination of whether HV training materials are more effective than LV training materials for production. However, more generally in the L2 vocabulary learning literature, training with multiple speakers has been found to lead to better recall in a picture naming task (Barcroft & Sommers, 2005), suggesting that the HVPT advantage should extend to production measures. In sum, the current experiment assessed whether individuals beneﬁt from high over LV perceptual training when learning novel L2 tone contrasts, and whether this interacts with learner aptitude. We used measures of aptitude taken from previous studies, but a training paradigm with real Mandarin stimuli embedded in a vocabulary learning task, which trained discrimination of all six Mandarin tone contrasts. Learning and generalisation were measured in multiple tests of both perception and production. In general, the current design increased ecological validity and likely also increased the difﬁculty of the learning task relative to previous work. It is possible that increasing difﬁculty could exacerbate differences between learners of different aptitudes, potentially increasing the effect. On the other hand, it is also possible that the increased difﬁculty might make HV input much harder for all participants, decreasing or removing the speciﬁc beneﬁt of HVPT for high aptitude learners. Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 Participants A total of 60 adults recruited from UCL Psychology Subject Pool participated in the experiment, 20 in each of the three conditions (LV, HV, high variability blocked (HVB)). Participant information is summarized in Table 1. There was no difference between these groups in age, F (2,57) = 1.95, p = 0.15. Participants had no known hearing, speech, or language impairments. Written consent was obtained from participants prior to the ﬁrst session. Each participant was paid £45 at the end of the study. Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 Table 2 Use of trained and untrained items and voices in different tasks. Task Items Voice Picture identiﬁcation Trained One trained voice (counterbalanced, see Table 3) One untrained voice (counterbalanced, see Table 3) Three interval oddity (Pre and Post) Trained and untrained Four new voices Picture naming Trained NA Word repetition (Pre and Post) Trained and untrained One trained voice (counterbalanced, see Table 3) Individual aptitude test 1 Pitch contour perception test (Pre and post) Vowels Four untrained voices Individual aptitude test 2 Categorisation of synthesised tonal continua (Pre and Post) Synthesised voice Synthesised voice All participants except three were native English speakers. Of the remaining three, one participant (LV condition) was a native bilingual of English and Hindi, one participant (HV condition) was a native French speaker, and one participant (HV condition) was a native Finnish speaker. Critically, participants had no prior experience of Mandarin Chinese or any other tonal language. On average, participants had learned 2.4 (SD = 0.8) languages and the average age for starting to learn the ﬁrst L2 was 12.6 years (SD = 1.3). Ethical approval was given by the UCL Research Ethics Committee with the approval number 6176/002. Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 Stimuli used in training and in the picture identification, three interval oddity, word repetition and picture naming tests This ensured that any difference found between the low and HV conditions, and between trained and untrained voices, were not due to idiosyncratic difference between speakers. There was no counterbalancing of speaker in other tasks. All words were edited into separate sound ﬁles, and peak amplitude was normalised using Audacity (Audacity Team, 2015, http://audacity.sourceforge.net/). Any background noise was also removed. All recordings were perceptually natural and highly distinguishable as judged by native Chinese speakers. Clipart pictures of the 72 words were selected from free online clipart databases. Stimuli used in training and in the picture identification, three interval oddity, word repetition and picture naming tests Table 3 Counterbalancing of voices across training conditions in the picture identiﬁcation task (the only test in which trained and untrained voices are directly contrasted) and the Word Repetition tests. Task Voice Version 1 Version 2 Version 3 Version 4 Version 5 Training, LV F1 F2 F3 M1 M2 Training, HV/HVB F1 F2 F3 M1 M2 F3 F1 M2 F1 F2 M1 M1 F1 F2 F3 M2 M2 F2 F3 M1 Picture Identiﬁcation Trained voice F1 F2 F3 M1 M2 Untrained voice F2 F3 M1 M2 F1 Word repetition F1 F2 F3 M1 M2 Table 3 Counterbalancing of voices across training conditions in the picture identiﬁcation task (the only test in which trained and untrained voices are directly contrasted) and the Word Repetition tests. Table 3 Counterbalancing of voices across training conditions in the picture identiﬁcation task (the only test in which trained and untrained voices are directly contrasted) and the Word Repetition tests. Table 3 Counterbalancing of voices across training conditions in the picture identiﬁcation task (the only test in which trained and untrained voices are directly contrasted) and the Word Repetition tests. items in the picture identiﬁcation test. In the HV conditions, four speakers (Trained voice 1 plus three others) were used in training. Only one of these speakers (Trained voice 1) was used in the word repetition test and for trained items in the picture identiﬁcation test. In all conditions, a further speaker (Untrained voice 1) was assigned to the untrained test items in the picture identiﬁcation test. The assignment of speakers was rotated across participants, resulting in ﬁve counterbalanced versions of each condition (see Table 3). This ensured that any difference found between the low and HV conditions, and between trained and untrained voices, were not due to idiosyncratic difference between speakers. There was no counterbalancing of speaker in other tasks. items in the picture identiﬁcation test. In the HV conditions, four speakers (Trained voice 1 plus three others) were used in training. Only one of these speakers (Trained voice 1) was used in the word repetition test and for trained items in the picture identiﬁcation test. In all conditions, a further speaker (Untrained voice 1) was assigned to the untrained test items in the picture identiﬁcation test. The assignment of speakers was rotated across participants, resulting in ﬁve counterbalanced versions of each condition (see Table 3). Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 Stimuli used in training and in the picture identification, three interval oddity, word repetition and picture naming tests These stimuli consisted of 36 minimal pairs of Mandarin words (six minimal pairs for each of the six tone contrasts generated by the four Mandarin tones). The words in each pair contained the same phonemes, differing only in tone (e.g. māo, Tone 1 (cat) vs mào, Tone 4 (hat)). All words were picturable and started with a wide range of phonemes (see Appendix A). In order to examine generalisation across items, half of the word pairs (three per tone contrast) were designated ‘trained’ words and other half were designated ‘untrained’ words. Trained words were encountered in both training and test tasks; untrained words were only encountered in the three interval oddity and word recognition tests. The full set of 72 Mandarin words was recorded by two groups of native Mandarin speakers using a Sony PCM-M10 handheld digital audio recorder. The ﬁrst group consisted of three female and two male speakers. These stimuli were used in the Training, Word Repetition and Picture Identiﬁcation tasks. The second group consisted of three new female speakers and two new male speakers. These stimuli were used in the three interval oddity task (making all new speakers in that task). See Table 2 for a summary of the manipulation of item and speaker novelty across the different test tasks, and Table 3 for the tasks in which speakers are counterbalanced. In the LV condition only one speaker (Trained voice 1) was used in training, and this same speaker was also used as the test voice in the Word Repetition test and for trained Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 10/45 items in the picture identiﬁcation test. In the HV conditions, four speakers (Trained voice 1 plus three others) were used in training. Only one of these speakers (Trained voice 1) was used in the word repetition test and for trained items in the picture identiﬁcation test. In all conditions, a further speaker (Untrained voice 1) was assigned to the untrained test items in the picture identiﬁcation test. The assignment of speakers was rotated across participants, resulting in ﬁve counterbalanced versions of each condition (see Table 3). This ensured that any difference found between the low and HV conditions, and between trained and untrained voices, were not due to idiosyncratic difference between speakers. There was no counterbalancing of speaker in other tasks. Stimuli used in the aptitude tests Pitch Contour Perception Test: Six Mandarin vowels (/a/, /o/, /e/, /i/, /u/, /y/) were repeated in the four Mandarin tones by two male and two female native Mandarin speakers from talker set 2, making 96 stimuli in total. Stimuli were identical across conditions and participants. Categorization of Synthesised Tonal Continua: Natural endpoints were chosen from a native Mandarin male speaker producing the word ‘wan’ with both Tone 2 and Tone 3. A neutral vowel was also recorded by a native male English speaker producing the ‘father vowel’ /a/. This vowel was edited slightly to remove portions containing creaky voice at the end. The three syllables (wan (Tone 2), wan (Tone 3), /a/) were then manipulated in Praat (Boersma & Weenink, 2015). All three syllables were normalised to be approximately 260 ms long using the Pitch Synchronous Overlap and Add method. The neutral vowel was manipulated to have a ﬂat fundamental frequency (148 Hz) and a ﬂat intensity contour (75 dB). The pitch contours of the two natural endpoints were extracted and a Pitch Contour Perception Test: Six Mandarin vowels (/a/, /o/, /e/, /i/, /u/, /y/) were repeated in the four Mandarin tones by two male and two female native Mandarin speakers from talker set 2, making 96 stimuli in total. Stimuli were identical across conditions and participants. Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 Figure 1 Tasks completed in each of the eight sessions. This ﬁgure describes all tasks arranged through session 1–8. Full-size  DOI: 10.7717/peerj.7191/ﬁg-1 Figure 1 Tasks completed in each of the eight sessions. This ﬁgure describes all tasks arranged through session 1–8. Full-size  DOI: 10.7717/peerj.7191/ﬁg-1 six-step pitch continuum (Step 1: Tone 2, Step 6: Tone 3) was generated by linearly interpolating between the endpoints. These six pitch contours were then each superimposed on a copy of the neutral vowel using the PSOLA method. Stimuli were identical across participants and conditions. six-step pitch continuum (Step 1: Tone 2, Step 6: Tone 3) was generated by linearly interpolating between the endpoints. These six pitch contours were then each superimposed on a copy of the neutral vowel using the PSOLA method. Stimuli were identical across participants and conditions. Procedure The experiment involved three stages (see Fig. 1): Pre-test (session 1), training (sessions 2–7), and post-test (session 8). Participants were required to complete all eight sessions within 2 weeks, with the constraint of one session per day at most. The majority of sessions took place in a quiet, soundproof testing room in Chandler House, UCL. The remaining sessions took place in a quiet room in a student house. Participants were given a brief introduction about the aim of the study and told that they were going to learn some Mandarin tones and words. They were explicitly told that Mandarin has four tones (ﬂat, rising, dipping and falling) and that the tonal differences were used to distinguish meanings. The experiment ran on a Dell Alienware 14R laptop with a 14-inch screen. The experiment software was built using a custom-built software package developed at the University of Rochester. The speciﬁc instructions for each task were displayed on-screen before the task started. After each task, participants had the opportunity to take a 1-min break. The tasks completed in each session are listed in Fig. 1 and described in more detail below. Note that the Pitch Contour Perception Test and Categorisation of Synthesised Tonal Continua were carried out at the beginning of the ﬁrst session as they provided the measure of individual aptitude prior to exposure to any Mandarin stimuli. There was no time limit for making responses in any of the tasks. Participants wore a pair of HD 201 Sennheiser headphones throughout the experiment with audio stimuli presented at a comfortable listening level. Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 Categorisation of synthesised tonal continua This test was based on Sadakata & McQueen (2014). Participants ﬁrst practiced listening to Tone 2 and Tone 3 while viewing the corresponding picture of an arrow depicting the pitch change. Each tone was repeated 10 times. In each test trial, participants then decided whether the sound they heard was closer to Tone 2 or Tone 3 by clicking on the corresponding arrow. No feedback was provided. The speech continua consisted of six steps (Step 1: Tone 2, Step 6: Tone 3) with each step repeated 10 times per block. Participants completed two blocks, with an optional 1 min break in the middle, resulting in 120 trials in total. This task provided a measure of individual differences in tone perception prior to training. In line with Sadakata and McQueen’s procedure, participants completed the task both before and after training and we conducted analyses to explore whether there was improvement from pre to post-test (section ‘The Pitch Contour Perception Test’). Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 Individual aptitude measures Individual aptitude measures The pitch contour perception test This test was based on the work of Wong & Perrachione (2007). Participants heard a tone (e.g. /a/ (Tone 1)), while viewing pictures of four arrows indicating the different pitch contours. Participants clicked on the arrow that they thought matched the tone heard. The pitch contour perception test This test was based on the work of Wong & Perrachione (2007). Participants heard a tone (e.g. /a/ (Tone 1)), while viewing pictures of four arrows indicating the different pitch contours. Participants clicked on the arrow that they thought matched the tone heard. Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 No feedback was provided. There were 96 stimuli in total (four speakers four tones four vowels). This task provided another measure of individual differences in tone perception prior to training. Although Perrachione et al. only conducted this task at pre-test, for consistency with the Categorization of Synthesised Tonal Continua (described below) we also repeated the test at post-test and conducted analyses to identify whether performance on this task was itself improved as a result of training (see section ‘Categorisation of Synthesised Tonal Continua’). Training task Participants completed the training task in Session 2–7. On each trial, participants heard a Mandarin word and selected one of two candidate pictures displayed on the computer screen. The two pictures always belonged to the same minimal pair. Feedback was provided about whether the answer was correct (a green happy face appeared) or incorrect (a red sad face appeared). If the correct choice was made, a picture of a coin also appeared in a box on the left-hand side of the screen, with the aim of motivating participants to try to earn more coins in each subsequent session of training. After that, everything but the correct picture was removed from the screen and the participant heard the correct word again. In the lower right corner of the screen a trial indicator of X/288 was displayed where X indicated the number of trials completed. This tool helped participants to keep track of their performance (see Fig. 2). There were 18 picture/word pairs used. Each word was used as the target four times. Thus, each picture pair appeared eight times, resulting in 288 trials per session. Participants were assigned to one of the following conditions: LV, HV and HVB (with the assignment of speakers counterbalanced—see Table 3). Each training session lasted for approximately 30 min. In the LV condition, only one speaker was used. In the HV conditions, four speakers were used. For each participant, each of their six training sessions was identical. In the HV condition without blocking, all of the speakers were heard in each of the training sessions, with the order randomised so that speaker varied from trial to trial. In contrast, Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 13/45 Figure 2 Screen shot from the Training task. The stimuli heard is ‘dì’, tone 4, (earth). The foil picture on the right is ‘dí’ tone 2, (siren). Full-size  DOI: 10.7717/peerj.7191/ﬁg-2 Figure 2 Screen shot from the Training task. The stimuli heard is ‘dì’, tone 4, (earth). The foil picture on the right is ‘dí’ tone 2, (siren). Full-size  DOI: 10.7717/peerj.7191/ﬁg-2 in the HV blocked condition, from Day 1 to Day 4 of training (i.e. Session 2–5), only one speaker was involved on each day’s training session, (with the trained speaker that was used in the test tasks (e.g. F1 for Version 1) always occurring on Day 3 (i.e. Training task Session 4)); on Days 5 and 6 of training (i.e. Sessions 6 and 7), participants heard all four speakers, each in a separate block, with each word being repeated twice in each voice on these days. In all three conditions, the order of items was randomised within each session. Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 Perceptual tests Three interval oddity test (pre-post test) Production test All 72 Mandarin words from the main stimulus set (18 trained pairs, 18 untrained pairs) set were presented one at a time in a randomised order. They were always spoken by the same speaker and this speaker was also used in their training stimuli (training voice 1; see Table 3). After each word, 2 s of white noise was played. This was included to make sure that participants had to encode the stimulus they were repeating and could not access the information in echoic storage (Flege, Takagi & Mann, 1995). Participants were instructed to listen carefully to the word and then to repeat the word aloud after the white noise. Verbal responses were digitally recorded and were later transcribed and rated by native speakers of Mandarin (see section ‘Coding and Inter-rater Reliability Analyses’). This task was completed once in the pre-test and once in the post-test. Picture naming test (post-only test) All 36 pictures from the training words were presented in a randomised order. Participants were instructed to try to name the picture using the appropriate Mandarin word. Verbal responses were recorded and were later transcribed and rated by native Mandarin speakers (see section ‘Coding and Inter-rater Reliability Analyses’). This task was completed only in the post-test. Three interval oddity test (pre-post test) This task required participants to identify the odd one out (i.e. the stimulus with a different tone) from a choice of three Mandarin words, each spoken by a different speaker. Four untrained speakers were used (three female, one male). Each trial used one of the 36 minimal pairs from the main stimuli set (18 trained pairs, 18 untrained pairs). Preliminary work suggested that trials differed in difﬁculty depending on whether the ‘different’ stimulus was spoken by the single male speaker, or one of the three female speakers. We therefore ensured that there were equal numbers of the following trial types: (i) ‘Neutral’—all three words were spoken by female speakers (ii) ‘Easy’—the ‘different’ word was spoken by a male speaker and the other two were spoken by female speakers; (iii) ‘Hard’—the ‘different’ word was spoken by a female speaker and the other two were spoken by one male speaker and one female speaker. Each of the words in the minimal pair was used once as the target (‘different’) word, making 72 trials in total. During the task, three frogs were displayed on the screen. Participants heard three words (played with ISIs of 200 ms) and indicated which word was the odd one out by clicking on the appropriate frog, which could be in any of the three positions. They could not make their response until all three words had been heard, at which point a red box containing the instruction ‘Click on the frog that said the different word’ appeared at the bottom of the screen. No feedback was provided. Participants completed this task twice—once in the pre-test, and once in the post-test. Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 Picture identiﬁcation test (post-only test) ﬁ (p y ) This task was the same as the training task with the following changes. Firstly, each word was only repeated twice, once by a trained speaker (trained voice 1) and once by an untrained speaker (Untrained voice 1), making 72 trials in total. Secondly, no feedback was given. This task was completed only in the post-test. This task was the same as the training task with the following changes. Firstly, each word was only repeated twice, once by a trained speaker (trained voice 1) and once by an untrained speaker (Untrained voice 1), making 72 trials in total. Secondly, no feedback was given. This task was completed only in the post-test. RESULTS Statistical approach English introduction task This task was included in the batch of tasks administered at pre-test in case the meaning of some pictures were ambiguous (not all items were concrete nouns—for example, ‘to paint’). Participants saw each of the 36 pictures from the training set presented once each in a random order and heard the corresponding English word. No response was recorded. Participants completed this task only once, at the end of the pre-test session. Questionnaires Participants completed a language background questionnaire after the experiment. Participants were asked to list all the places they had lived for more than 3 months and any languages that they had learned. For each language the participant was asked: (a) to state how long they learned the language for and their starting age; (b) to rate their own current proﬁciency of the language. Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 3 This differs from the default coding of contrasts in the lme4 package. It was achieved by replacing the three-way fac- tor ‘condition’ with two centred dummy variables and using the main ﬁxed effects from the output of this model. Statistical approach Three different sets of frequentist analyses are reported. First, we conducted the analysis on two individual aptitude measures Categorisation of Synthesised Tonal Continua and Pitch Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 Contour Perception Test. The primary aim of these analyses was to ensure that the three groups did not differ at pre-test, however we also looked for possible differences at post-test. Second, separate analyses are reported on data from the tests administered pre- and post-training (i.e. Word Repetition task and Three Interval Oddity task), the data collected during Training and the data from the two tasks administered only at post-test (i.e. the Picture Identiﬁcation task and Picture Naming task). These analyses explored the effects of our experimentally manipulated conditions on the various measures of Mandarin tone learning. Third, analyses were conducted exploring the role of aptitude in each of these tasks (section ‘Analyses with Individual Aptitude’). Speciﬁcally, we wanted to see whether aptitude interacted with variability-condition in predicting the beneﬁts of training, in line with the predictions of previous research (Perrachione et al., 2011; Sadakata & McQueen, 2014). Except where stated, analyses used logistic mixed effect models (Baayen, Davidson & Bates, 2008; Jaeger, 2008; Quené & Van den Bergh, 2008) using the package lme4 (Bates et al., 2013) for the R computing environment (R Development Core Team, 2010). Logistic mixed effect models allow binary data to be analysed with logistic models rather than as proportions, as recommended by Jaeger (2008). In each of the analyses, the factor variability-condition has three levels (LV, HV and HVB) which we coded into two contrasts with LV as the baseline (LV vs HV, LV vs HVB). An exception to this is the training data, where a model containing all three conditions would not converge and we took a different approach, as described in the section ‘Training’. We also included the interactions between these contrasts and the other factors. We used centred coding which ensured that other effects were evaluated as averaged over all three levels of variability-condition (rather than the reference level of LV3). Similarly, for the Three Interval Oddity task, we included a trial-type factor. The purpose of this was to control for the fact that participants were likely to ﬁnd some trial types easier than others due to the gender of the speakers producing the stimuli (see section ‘Three Interval Oddity Test (Pre-Post Test)’). Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 Statistical approach We therefore coded a factor trial-type with three levels (neutral, easy, hard–see method) and included contrasts with neutral (‘neutral vs easy’ and ‘neutral vs hard’) using centred coding. In order to perform the analysis comparing pre- and post-test performance, test-session was coded as a factor with two levels (pre-test/post-test) with ‘pre-test’ set as the reference level. This allowed us to look at the (accidental) possible differences between the experimental conditions at the pre-test stage, as well as whether post-test performance differed from this baseline. All other predictors, including both discrete factor codings with two levels (item-novelty in the Word Repetition and Three Interval Oddity tasks, and voice-novelty in the Picture Identiﬁcation task) and numeric predictors (training-session) in the Training data analyses and the individual difference measures in the models reported in the section ‘Analyses with Individual Aptitude’), were centred (i) to reduce the effects of collinearity between main effects and interactions, and (ii) so that the main effects were evaluated as the average effects over all levels of the other predictors (rather than at a speciﬁed reference level for each factor). We automatically put experimentally manipulated variables and all of their interactions into the model, without using model selection (except for trial-type in the Three Interval Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 Oddity task which works as a control factor and for this factor we only used its main effect and the interaction with test-session). However, we did not inspect the models for all main effects and interactions. Instead, we report the statistics which were necessary to look for accidental differences at pre-test, and those related to our hypotheses. We aimed to examine whether the training improved participants’ performance on both untrained items and untrained voices and whether such improvement was modulated by their individual aptitudes. Participant is included as a random effect and a full random slope structure was used (i.e. by-subject slopes for all experimentally manipulated within-subject effects (test-session, voice-novelty, item-novelty) and interactions, as recommended by Barr et al. (2013). In some cases the models did not converge and in those cases correlations between random slopes were removed. Models converged with bound optimization by quadratic approximation (BOBYQA optimization; Powell, 2009). R scripts showing full model details can be found here: https://osf.io/wdh8a/. In addition to the frequentist analyses, in order to aid interpretation of key null results we also included Bayes factor analyses. The pitch contour perception test The predicted variable was whether a correct response was given (1/0) on each trial. The predictors were the contrasts between variability-conditions (LV vs HV; LV vs HVB) and test-session (pre-test, post-test). There was no signiﬁcant difference between the LV and HV groups (β = -0.35, SE = 0.26, z = -1.38, p = 0.17) or between the LV and HVB groups (β = 0.17, SE = 0.26, z = 0.66, p = 0.51) at pre-test on this measure. Participants showed signiﬁcant improvement after training (β = 0.21, SE = 0.05, z = 4.13, p < 0.001), which can be seen in Fig. 3. Thus, the three participant groups did not differ in their pre-test performance and the groups showed equivalent improvement from pre- to post-test. Given that this measure is affected by training, we used participants scores at pre-test as our measure of individual differences in the analyses reported in the section ‘Analyses with Individual Aptitude’. Statistical approach Our approach for these is described within the relevant section (Section ‘Bayes Factor Analyses’). Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 Training A model containing data from all three conditions did not converge; however two separate models, one including the LV and HV conditions, and the other the LV and HVB conditions (with condition as a factor with two levels), did converge. In each case the predicted variable was whether a correct response was given (1/0) on each trial. The predictors were the numeric factor training-session (1:6) and the factor variability- condition which had two levels (Model 1: LV vs HV; Model 2, LV vs HVB). The mean accuracy is displayed in Fig. 4. A model containing data from all three conditions did not converge; however two separate models, one including the LV and HV conditions, and the other the LV and HVB conditions (with condition as a factor with two levels), did converge. In each case the predicted variable was whether a correct response was given (1/0) on each trial. The predictors were the numeric factor training session (1:6) and the factor variability conditions (with condition as a factor with two levels), did converge. In each case the predicted variable was whether a correct response was given (1/0) on each trial. The predictors were the numeric factor training-session (1:6) and the factor variability- condition which had two levels (Model 1: LV vs HV; Model 2, LV vs HVB). The mean accuracy is displayed in Fig. 4. The predictors were the numeric factor training-session (1:6) and the factor variability- condition which had two levels (Model 1: LV vs HV; Model 2, LV vs HVB). The mean accuracy is displayed in Fig. 4. In both models, there was an effect of training-session (Model 1: β = 0.49, SE = 0.04, z = 11.52, p < 0.001; Model 2: β = 0.53, SE = 0.04, z = 12.17, p < 0.001): Participants’ performance increased signiﬁcantly over time, with additional training sessions. Overall, the LV group performed better than both the HV group (β = -0.79, SE = 0.16, z = -5.03, p < 0.001) and the HVB group (β = -0.83, SE = 0.32, z = -2.61, p < 0.01). However, the LV vs HV contrast was also modulated by an interaction with test-session (β = -0.19, SE = 0.04, z = -4.59, p < 0.001), as was the LV vs HVB contrast (β = -0.35, SE = 0.08 z = -4.33, p < 0.001). From Fig. Categorisation of synthesised tonal continua We estimated individual’s performance on the Categorisation of Synthesised Tonal Continua task following Sadakata & McQueen (2014). We used the Logistic Curve Fit function in SPSS to calculate a slope coefﬁcient for each participant (Joanisse et al., 2000). The slope (standardised β) indicates individual differences in tone perception. The smaller the slope, the better the performance. Sadakata and McQueen, removed data from participants with a slope measuring greater than 1.2. Using this threshold 43/60 participants failed the threshold in the current study. This is consistent with the observation that most of the participants were not able to consistently categorise the endpoints of the continua, indicating that this was not a good test of aptitude. We do not report further analyses involving this aptitude variable however they can be found in the supplemental materials (https://osf.io/wdh8a/). Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 Figure 3 Mean accuracy for the LV (low variability), HV (high variability) & HVB (high variability blocked) groups in Pitch Contour Perception Task. Error bars represents the 95% conﬁdence intervals. Full-size  DOI: 10.7717/peerj.7191/ﬁg-3 Figure 3 Mean accuracy for the LV (low variability), HV (high variability) & HVB (high variability blocked) groups in Pitch Contour Perception Task. Error bars represents the 95% conﬁdence intervals. Full-size  DOI: 10.7717/peerj.7191/ﬁg-3 Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 Training 4 it can be seen that the LV and the HVB group did not differ in the ﬁrst session (i.e. where they get identical input) but the difference gradually Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 Figure 4 Mean accuracy in the Training task for the LV (Low Variability), HV (High Variability) and HVB (High Variability Blocked) training groups in each session. Y-axis starts from chance level. Error bars show 95% conﬁdence intervals. Full-size  DOI: 10.7717/peerj.7191/ﬁg-4 Figure 4 Mean accuracy in the Training task for the LV (Low Variability), HV (High Variability) and HVB (High Variability Blocked) training groups in each session. Y-axis starts from chance level. Error bars show 95% conﬁdence intervals. Full-size  DOI: 10.7717/peerj.7191/ﬁg-4 increased over the next few sessions. For the LV and the HV group, they differed starting from the ﬁrst session and this difference continued to increase throughout trainin increased over the next few sessions. For the LV and the HV group, they differed starting from the ﬁrst session and this difference continued to increase throughout training. Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 Three interval oddity task The predicted variable was whether a correct response was given (1/0) on each trial. The predictors were test-session (pre-test, post-test), variability-condition (LV vs HV, LV vs HVB), trial-type (neutral vs easy, neutral vs hard) and item-novelty (trained item, untrained item). The mean accuracy is displayed in Fig. 5. At pre-test, there was no signiﬁcant difference between the LV and HV groups (β = -0.002, SE = 0.14, z = -0.01, p = 0.99) nor between the LV and HVB groups (β = 0.12, SE = 0.14, z = 0.86, p = 0.39), suggesting that the groups started at a similar level. However, performance with the ‘untrained’ was signiﬁcantly greater than performance on the ‘trained’ items at pre-test (β = -0.31, SE = 0.06, z = -4.95, p < 0.01), suggesting incidental differences between item sets. As expected, at pre-test participants performed signiﬁcantly better on ‘easy’ trials (where the target speaker had a different gender) than ‘neutral’ trials (where all three speakers had the same gender, β = 0.40, SE = 0.08, z = 5.09, p < 0.01) and ‘neutral’ trials were marginally easier than ‘hard’ trials (where one of the foil speakers had the odd gender out, β = -0.14, SE = 0.08, z = -1.81, p = 0.07). g β p Overall, participants’ performance increased signiﬁcantly after training (Mpre = 0.59, SDpre = 0.21, Mpost = 0.66, SDpost = 0.19, β = 0.31, SE = 0.05, z = 6.54, p < 0.001). Th i t ti b t t t i d it lt t i iﬁ t (β 0 14 Overall, participants’ performance increased signiﬁcantly after training (Mpre = 0.59, SDpre = 0.21, Mpost = 0.66, SDpost = 0.19, β = 0.31, SE = 0.05, z = 6.54, p < 0.001). The interaction between test-session and item-novelty was not signiﬁcant (β = 0.14, SE = 0.09, z = 1.49, p = 0.14), suggesting no evidence that training had a greater effect SDpre = 0.21, Mpost = 0.66, SDpost = 0.19, β = 0.31, SE = 0.05, z = 6.54, p < 0.001). The interaction between test-session and item-novelty was not signiﬁcant (β = 0.14, SE = 0.09, z = 1.49, p = 0.14), suggesting no evidence that training had a greater effect Dong et al. Three interval oddity task (2019), PeerJ, DOI 10.7717/peerj.7191 Figure 5 Mean accuracy in Three Interval Oddity task for LV (low variability), HV (high variability) and HVB (high variability blocked) training groups in Pre- and Post-tests for trained and untrained items. Error bars show 95% conﬁdence intervals. Full-size  DOI: 10.7717/peerj.7191/ﬁg-5 Figure 5 Mean accuracy in Three Interval Oddity task for LV (low variability), HV (high variability) and HVB (high variability blocked) training groups in Pre- and Post-tests for trained and untrained items. Error bars show 95% conﬁdence intervals. Full-size  DOI: 10.7717/peerj.7191/ﬁg-5 for trained words than for untrained words. Critically, there was no interaction with test-session for either the contrast between the LV vs the HV conditions (β = -0.01, SE = 0.12, z = -0.12, p = 0.90) or the contrast between the LV vs the HVB conditions (β = 0.01, SE = 0.12, z = 0.11, p = 0.91) and they were not qualiﬁed by any higher level interactions with item-novelty (LV vs HV: β = -0.1, SE = 0.22, z = -0.64, p = 0.52; LV vs HVB: β = 0.13, SE = 0.22, z = 0.57, p = 0.57). This suggests no evidence that the extent to which participants improved on this task between pre and post-test differed according to variability-conditions, or that this differed for trained vs untrained items. for trained words than for untrained words. Critically, there was no interaction with test-session for either the contrast between the LV vs the HV conditions (β = -0.01, SE = 0.12, z = -0.12, p = 0.90) or the contrast between the LV vs the HVB conditions (β = 0.01, SE = 0.12, z = 0.11, p = 0.91) and they were not qualiﬁed by any higher level interactions with item-novelty (LV vs HV: β = -0.1, SE = 0.22, z = -0.64, p = 0.52; LV vs HVB: β = 0.13, SE = 0.22, z = 0.57, p = 0.57). This suggests no evidence that the extent to which participants improved on this task between pre and post-test differed according to variability-conditions, or that this differed for trained vs untrained items. Although not part of our key predictions, we also looked to see if there was evidence that participants improved more with the easier or harder trials. Three interval oddity task In fact, the interaction between test-session and the contrast between ‘easy’ and ‘neutral’ was signiﬁcant (β = -0.27, SE = 0.11, z = -2.39, p = 0.02) while the contrast between ‘neutral’ and ‘hard’ was not (β = 0.12, SE = 0.11, z = 1.06, p = 0.29). This was due to the fact that there was improvement for ‘neutral’ (Mpre = 0.57, SDpre = 0.14, Mpost = 0.65, SDpost = 0.15) and ‘hard’ trials (Mpre = 0.54, SDpre = 0.16, Mpost = 0.65, SDpost = 0.15) but not for ‘easy’ trials (Mpre = 0.66, SDpre = 0.16, Mpost = 0.68, SDpost = 0.15). Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 Picture identification The predicted variable was whether a correct response was given (1/0) on each trial. The predictors were the factor voice-novelty (Trained voice, Untrained voice) and the factor variability-condition which had two contrasts (LV vs HV, LV vs HVB). The mean accuracy is displayed in Fig. 6. Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 20/45 Figure 6 Mean accuracy of Picture Identiﬁcation for LV (low variability), HV (high variability) and HVB (high variability blocked) training groups for untrained voices and trained voices. Error bars show 95% conﬁdence intervals. Full-size  DOI: 10.7717/peerj.7191/ﬁg-6 Figure 6 Mean accuracy of Picture Identiﬁcation for LV (low variability), HV (high variability) and HVB (high variability blocked) training groups for untrained voices and trained voices. Error bars show 95% conﬁdence intervals. Full-size  DOI: 10.7717/peerj.7191/ﬁg-6 There was a main effect of voice-novelty (β = 1.07, SE = 0.16, z = 6.53, p < 0.001) reﬂecting higher performance in trials with trained voices. Although participants in the LV group performed better than those in the HV group (β = -0.71, SE = 0.32, z = -2.23, p = 0.03), there was no signiﬁcant difference between the LV and the HVB group (β = -0.14, SE = 0.32, z = -0.44, p = 0.66) and there was a signiﬁcant interaction between voice-novelty and both the LV-HV contrast (β = -1.19, SE = 0.35, z = -3.43, p < 0.01) and the LV-HVB contrast (β = -1.11, SE = 0.36, z = -3.08, p < 0.01). Breaking this down by variability-condition: for each condition there was signiﬁcantly better performance with trained than untrained voices (LV: β = 1.83, SE = 0.29, z = 6.42, p < 0.001; HV: β = 0.64, SE = 0.23, z = 2.86, p < 0.01; HVB: β = 0.73, SE = 0.26, z = 2.82, p < 0.01), indicating greater ease with the familiar voice. Breaking down by voice-novelty: For the trained voice, performance was higher in the LV condition than in either the HV or HVB conditions, although this was only signiﬁcant for the LV vs HV contrast (LV vs HV: β = -1.30, SE = 0.44, z = -2.97, p < 0.01; LV vs HVB: β = -0.70, SE = 0.45, z = -1.55, p = 0.12). Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 Coding and inter-rater reliability analyses The same methods were used for both production tests. The ﬁles were combined into a single set, along with the 360 stimuli which were used in the experiment (and which were produced by native Mandarin speakers). The latter items were included in order to examine whether the raters were reliable. All stimuli were rated by two raters: Rater 1 was the ﬁrst author and Rater 2 was recruited from the UCL MA Linguistics program and was naïve to the purposes of the experiment. Raters were presented with recordings in blocks in a random sequence (blind to test-type, condition, whether the stimulus was from pre-test or post-test and whether it was produced by a participant or was one of the experimental stimuli). For each item, raters were asked to (i) identify the tone, (ii) give a rating quantifying how native-like they thought the pronunciation was compared (one to seven with one as not recognisable and seven as native speaker level), and (iii) transcribe the pinyin (segmental pronunciation) produced by the participants. The same methods were used for both production tests. The ﬁles were combined into a single set, along with the 360 stimuli which were used in the experiment (and which were produced by native Mandarin speakers). The latter items were included in order to examine whether the raters were reliable. All stimuli were rated by two raters: Rater 1 was the ﬁrst author and Rater 2 was recruited from the UCL MA Linguistics program and was naïve to the purposes of the experiment. Raters were presented with recordings in blocks in a random sequence (blind to test-type, condition, whether the stimulus was from pre-test or post-test and whether it was produced by a participant or was one of the experimental stimuli). For each item, raters were asked to (i) identify the tone, (ii) give a rating quantifying how native-like they thought the pronunciation was compared (one to seven with one as not recognisable and seven as native speaker level), and (iii) transcribe the pinyin (segmental pronunciation) produced by the participants. If there was no sound or the tone was unrecognizable, the rater coded 0 when identifying the tone. Data from these trials were removed from the dataset before analyses were conducted. In addition, all of the data from one participant was removed from the analyses due to bad recording quality resulting from a technical error. Picture identification Importantly, for untrained voices, neither of the contrasts between conditions was signiﬁcant (LV vs HV: β = -0.12, SE = 0.26, z = -0.45, p = 0.65; LV vs HVB β = 0.41, SE = 0.27, z = 1.51, p = 0.13), indicating no evidence for greater generalisation following HV training. Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 21/45 Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 Coding and inter-rater reliability analyses In total, this resulted in 3.38% (359/10,620) of production trials being removed from analysis (Word Repetition: Pre-test 1.98% (84/4,248); Post-test 3.72% (158/4,248); Picture Naming 5.51% (117/2,124)). Three measurements were taken from the production tasks: mean accuracy of tone identiﬁcation (Tone accuracy), mean tone rating (Tone rating) and mean accuracy of production in pinyin (derived by coding each production as correct (1 = the entire string is correct) or incorrect (0 = at least one error in the pinyin)). As a ﬁrst test of rater reliability, performance with the native speaker stimuli was examined–these were near ceiling: Rater 1: Tone accuracy = 98%, Tone rating = 6.7, Pinyin accuracy = 80%; Rater 2: Tone accuracy = 87%, Tone rating = 6.5, Pinyin accuracy = 80%). Furthermore, for the remaining data (i.e. the experimental data) inter-rater reliability was examined for all three measures for the two production tasks. For the binary measures (Tone accuracy and Pinyin accuracy), kappa statistics were calculated using the ‘fmsb’ package in R (Cohen, 2014). For the Word Repetition data, for Tone accuracy kappa = 0.39 (‘fair agreement’), and for Pinyin accuracy kappa = 0.33 (‘fair agreement’; Landis & Koch, 1977). For the Picture Naming test, for Tone accuracy kappa = 0.67 (‘substantial agreement’) and for Pinyin accuracy kappa = 0.53 (‘moderate agreement’); For the Tone rating, the package ‘irr’ in R was used to assess the intra-class correlation (McGraw & Wong, 1996) based on an average-measures, two-way mixed-effects model. For Word Repetition, ICC = 0.22 and for Picture Identiﬁcation ICC = 0.37; according to Cicchetti (1994), values less than 0.40 are regarded as ‘poor’. Given this, we do not include analyses with Tone Rating as the dependent variable (though these data are included in the data set https://osf.io/wdh8a/). All of the analyses presented in the sections ‘Word Repetition’ and ‘Picture Naming’ were based on Rater 2 (the naive rater). Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 22/45 Figure 7 Accuracy of Word Repetition for LV (low variability), HV (high variability) and HVB (high variability blocked) training groups in Pre- and Post-tests for trained and untrained items. Error bars show 95% conﬁdence intervals. Full-size  DOI: 10.7717/peerj.7191/ﬁg-7 Figure 7 Accuracy of Word Repetition for LV (low variability), HV (high variability) and HVB (high variability blocked) training groups in Pre- and Post-tests for trained and untrained items. Error bars show 95% conﬁdence intervals. Coding and inter-rater reliability analyses Full-size  DOI: 10.7717/peerj.7191/ﬁg-7 Figure 7 Accuracy of Word Repetition for LV (low variability), HV (high variability) and HVB (high variability blocked) training groups in Pre- and Post-tests for trained and untrained items. Error bars show 95% conﬁdence intervals. Full-size  DOI: 10.7717/peerj.7191/ﬁg-7 Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 Word repetition Tone accuracy Tone accuracy The predicted variable was whether a correct response was given (1/0) on each trial (as identiﬁed by the coder). The predictors were test-session (pre-test, post-test), variability- condition (LV vs HV, LV vs HVB) and item-novelty (trained, untrained). The mean accuracy, split by test-session and training condition, is shown in Fig. 7. At pre-test, there was no signiﬁcant difference between the LV and the HV group (β = 0.01, SE = 0.18, z = 0.06, p = 0.95) nor between the LV and the HVB group (β = 0.11, SE = 0.18, z = 0.64, p = 0.53), suggesting the groups started at a similar level. There was also no difference between trained and untrained words at pre-test (β = -0.02, SE = 0.07, z = -0.26, p = 0.80). Across the three groups, participants’ performance increased signiﬁcantly after training (Mpre = 0.71, SDpre = 0.09, Mpost = 0.79, SDpost = 0.09, β = 0.40, SE = 0.08, z = 5.29, p < 0.001). There was no signiﬁcant difference in the improvement for trained and untrained items (word-type by test-session interaction: β = 0.13, SE = 0.10, z = 1.22 p = 0.22). Critically, the interactions between the variability contrasts and test-session were not signiﬁcant (LV vs HV: β = -0.10, SE = 0.18, z = -0.55, p = 0.58; LV vs HVB: β = -0.11, SE = 0.18, z = -0.62, p = 0.54), and they were not qualiﬁed by any higher level interactions with item-novelty (LV vs HV: β = 0.15, SE = 0.25, z = 0.61, p = 0.54; LV vs HVB: β = -0.31, SE = 0.26, z = -1.21, p = 0.23). This suggests there is no Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 23/45 Figure 8 Mean pinyin accuracy of Word Repetition for LV (low variability), HV (high variability) and HVB (high variability blocked) training groups in Pre- and Post-tests for trained and untrained items. Error bars show 95% conﬁdence intervals. Full-size  DOI: 10.7717/peerj.7191/ﬁg-8 Figure 8 Mean pinyin accuracy of Word Repetition for LV (low variability), HV (high variability) and HVB (high variability blocked) training groups in Pre- and Post-tests for trained and untrained items. Error bars show 95% conﬁdence intervals. Full-size  DOI: 10.7717/peerj.7191/ﬁg-8 evidence that participants’ improvement in their production of tones was affected by their variability-condition, or that this differed for trained vs untrained items. Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 Pinyin accuracy The predicted variable was whether the participants produced the correct string of phonemes (1/0) in each trial and there was a single predictor variability-condition (LV vs HV, LV vs HVB). For both models there was no signiﬁcant difference between variability conditions (LV vs HV: β = 0.09, SE = 0.23, z = 0.41, p = 0.68; LV vs HVB: β = 0.12, SE = 0.23, z = 0.51, p = 0.61). This suggests there is no evidence that participants’ pinyin accuracy differed according to their variability-condition. Tone accuracy The predicted variable was whether a correct response was given (1/0) on each trial (as identiﬁed by the coder). There was only one predictor, variability-condition (LV vs HV, LV vs HVB) for both models. The descriptive statistics are displayed in Fig. 9. Participants in the LV group showed no signiﬁcant difference compared with the HV group (β = -0.34 SE = 0.19, z = -1.81, p = 0.07) and the HVB group (β = -0.10, SE = 0.19, z = -0.52, p = 0.61. This suggests there is no evidence that participants’ ability to produce the tones accurately differed according to their variability-condition. Pinyin accuracy Full-size  DOI: 10.7717/peerj.7191/ﬁg-9 Figure 9 Tone accuracy and Pinyin accuracy of Picture Naming for LV (low variability), HV (high variability) and HVB (high variability blocked) training groups. Error bars show 95% conﬁdence intervals. (A) Mean accuracy of Picture Naming, tone accuracy measure. (B) Mean accuracy of Pic- ture Naming, pinyin accuracy measure. Full-size  DOI: 10.7717/peerj.7191/ﬁg-9 there is no evidence that participants’ improvement in pinyin accuracy was affected by their variability-condition, or that this differed for trained vs untrained items. there is no evidence that participants’ improvement in pinyin accuracy was affected by their variability-condition, or that this differed for trained vs untrained items. Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 Picture naming Tone accuracy Pinyin accuracy The predicted variable was whether the participants produced the correct string of phonemes (1/0) in each trial (as determined by Rater 2). The predictors were test-session (pre-test, post-test), variability-condition (LV vs HV, LV vs HVB) and item-novelty (trained, untrained). Mean pinyin accuracy is displayed in Fig. 8. At pre-test, there was no signiﬁcant difference between the LV and the HV group (β = -0.01, SE = 0.11, z = -0.11, p = 0.91) nor between the LV and the HVB group (β = -0.03, SE = 0.11, z = -0.24, p = 0.81), suggesting that the groups started at a similar level. However, participants did better on untrained words than trained words at pre-test (β = 0.21, SE = 0.07, z = 3.11, p < 0.01), suggesting potential accidental differences in these items. Participants showed signiﬁcant improvement after training (Mpre = 0.54, SDpre = 0.09, Mpost = 0.58, SDpost = 0.19, β = 0.15, SE = 0.05, z = 3.38, p < 0.01). However, there was no evidence that different variability conditions resulted in different amounts of improvement (test-session by LV vs HV: β = 0.05, SE = 0.11, z = 0.46, p = 0.65; test-session by LV vs HVB: β = -0.12, SE = 0.11, z = -1.08, p = 0.28) or any interaction between variability condition, test-session and item-novelty (LV vs HV: β = 0.11, SE = 0.22, z = 0.51, p = 0.61; LV vs HVB: β = -0.14, SE = 0.22, z = -0.64, p = 0.52). This suggests Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 24/45 there is no evidence that participants’ improvement in pinyin accuracy was affected by their variability-condition, or that this differed for trained vs untrained items. Figure 9 Tone accuracy and Pinyin accuracy of Picture Naming for LV (low variability), HV (high variability) and HVB (high variability blocked) training groups. Error bars show 95% conﬁdence intervals. (A) Mean accuracy of Picture Naming, tone accuracy measure. (B) Mean accuracy of Pic- ture Naming, pinyin accuracy measure. Full-size  DOI: 10.7717/peerj.7191/ﬁg-9 Figure 9 Tone accuracy and Pinyin accuracy of Picture Naming for LV (low variability), HV (high variability) and HVB (high variability blocked) training groups. Error bars show 95% conﬁdence intervals. (A) Mean accuracy of Picture Naming, tone accuracy measure. (B) Mean accuracy of Pic- ture Naming, pinyin accuracy measure. Analyses with individual aptitude Data set Coefﬁcient name Statistics Word repetition: Tone accuracy (Pre/post) Aptitude β = 0.07, SE = 0.03, z = 2.35, p = 0.019 Aptitude by Test-Session β = 0.03, SE = 0.04, z = 0.72, p = 0.473 Aptitude by LV-HV Contrast by Test-Session β = 0.05, SE = 0.11, z = 0.47, p = 0.639 Aptitude by LV-HVB Contrast by Test-Session β = 0.13, SE = 0.10, z = 1.35, p = 0.176 Aptitude by LV-HV Contrast by Test-Session by Item-Novelty β = -0.14, SE = 0.15, z = -0.97, p = 0.334 Aptitude by LV-HVB Contrast by Test-Session by Item-Novelty β = 0.07, SE = 0.13, z = 0.50, p = 0.61 Three interval oddity (Pre/post) Aptitude β = 0.07, SE = 0.03, z = 2.19, p = 0.029 Aptitude by Test-Session β = 0.01, SE = 0.23, z = 0.31, p = 0.757 Aptitude by LV-HV Contrast by Test-Session β = 0.05, SE = 0.07, z = 0.77, p = 0.443 Aptitude by LV-HVB Contrast by Test-Session β = 0.05, SE = 0.06, z = 0.83, p = 0.410 Aptitude by LV-HV Contrast by Test-Session by Item-Novelty β = -0.12, SE = 0.13, z = -0.94, p = 0.346 Aptitude by LV-HVB Contrast by Test-Session by Item-Novelty β = 0.06, SE = 0.11, z = 0.52, p = 0.604 Training Aptitude β = 0.13, SE = 0.048, z = 2.70, p = 0.007 Aptitude by LV-HV Contrast β = -0.04, SE = 0.11, z = -0.332, p = 0.740 Aptitude by LV-HVB Contrast β = 0.03, SE = 0.10, z = 0.26, p = 0.795 Picture identiﬁcation (Post only) Aptitude β = 1.48, SE = 0.08, z = 1.96, p = 0.050 Aptitude by Voice Novelty β = -0.03, SE = 0.07, z = -0.33, p = 0.745 Aptitude by LV-HV Contrast β = -0.02, SE = 0.19, z = -0.12, p = 0.901 Aptitude by LV-HVB Contrast β = 0.01, SE = 0.17, z = 0.09, p = 0.932 Aptitude by LV-HV Contrast by Voice-Novelty β = 0.35, SE = 0.21, z = 1.63, p = 0.103 Aptitude by LV-HVB Contrast by Voice-Novelty β = -0.11, SE = 0.19, z = -0.58, p = 0.566 Picture naming: tone accuracy Aptitude β = 0.08, SE = 0.04, z = 1.89, p = 0.059 Aptitude by LV-HV Contrast β = -0.09, SE = 0.11, z = -0.84, p = 0.402 Aptitude by LV-HVB Contrast β = 0.12, SE = 0.10, z = 1.22, p = 0.224 Aptitude by LV-HVB Contrast Perception test) was centred and used as a continuous predictor (aptitude) and added to each of the models reported above. Analyses with individual aptitude In order to look at the effect of learner aptitude and the interaction between this factor and variability condition, we ﬁrst calculated the mean accuracy at pre-test on the Pitch Contour Perception Test for each participant. This score (scaled by a factor of 10, so that each one unit increase in aptitude corresponded to a 10% higher performance in the Pitch Contour Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 Table 4 Statistics obtained when adding in participant aptitude (as measured by performance on the Pitch Contour Perception Test task at pre-test) into the models predicting performance on the test and training tasks. Statistics marked in bold are signiﬁcant (0.05) results. Table 4 Statistics obtained when adding in participant aptitude (as measured by performance on the Pitch Contour Perception Test task at pre-test) into the models predicting performance on the test and training tasks. Statistics marked in bold are signiﬁcant (0.05) results. Table 4 Statistics obtained when adding in participant aptitude (as measured by performance on the Pitch Contour Perception Test task at pre-test) into the models predicting performance on the test and training tasks. Statistics marked in bold are signiﬁcant (0.05) results. Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 Analyses with individual aptitude In addition, we added the interaction between this factor and key experimental factors (see Table 4). Based on Perrachione et al. (2011) and Sadakata & McQueen (2014), for our measures of tone-learning, HV should beneﬁt high aptitude participants only, while LV would beneﬁt low aptitude participants only. In our design, we used a continuous measure of individual ability rather than a binary division of high and LV. We therefore predicted a stronger positive correlation between aptitude and amount of learning in the HV condition than in the LV condition. In the tests administered only post training (i.e. Picture Identiﬁcation and Picture Naming) this would show up as an interaction between aptitude and condition. In the models for the pre- and post-test data (i.e. Three Interval Oddity and Word Repetition) this would show up as a three-way interaction between condition, test-session and aptitude. We also looked at the interactions between these factors and voice-novelty (Picture Identiﬁcation) and item-novelty (Three Interval Oddity and Word Repetition). Note that there are no clear directional hypotheses here: Perrachione et al. (2011) found the interaction in a test with untrained voices and trained items, and Sadakata & McQueen (2014) found Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 26/45 the interaction in a test with trained voices and trained items. For training, in principal both the two-way interaction of aptitude by condition and the three-way interaction of aptitude by condition by training-session are of interest. However, it was not possible to ﬁt a converging model containing the three-way factor4. Each model reported in Table 4 contained all the ﬁxed effects included in the original models in addition to the ﬁxed effects listed in the table (note that to avoid convergence issues due to over complex models, we did not attempt to include the complete set of interactions for every combination of experimental variables with aptitude—only those for which we had predictions). We attempted to have full random effects structure for these ﬁxed effects however in some cases we had to remove correlations between slopes due to problems with convergence and for one of the models with the training data we had to remove the random slope for training session). Note that we don’t include models for the pinyin measures, since our measure of aptitude is relevant to tone learning only. 4 This was the case even if we split the data into two models, as we did in the Section ‘Training’. Analyses with individual aptitude For each of the new models we ﬁrst conﬁrmed that adding in the new effects and interactions with the individual measures did not change any of the previously reported patterns of signiﬁcance for the experimental effects (see script https://osf.io/wdh8a/) for full models. The results are shown in Table 4. Aptitude is a positive predictor of performance in each of the tests and in training, with p-values signiﬁcant or marginal in each case. However there was no interaction between aptitude and any other factor. Thus, there was no evidence that this measure of aptitude correlated with participants ability to beneﬁt from training (no interaction with test-session), nor—critically for our hypothesis—did this differ by training condition (no interaction with condition or with test-session by condition). Although the analyses use a continuous measure of Pitch Contour Perception Test, for the purposes of visualisation, Fig. 10 (Three Interval Oddity task and Training task), Fig. 11 (Picture Naming and Picture Identiﬁcation) and Fig. 12 (Word Repetition) use the mean accuracy for participants split into aptitude groups using a median split based on their Pitch Contour Perception Test score. In sum, participants with higher aptitude measures were better at the tasks, but there is no evidence either that this affected their improvement due to training, or, critically, their ability to beneﬁt from the different variability exposure sets. Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 Bayes factor analyses In the analyses reported above, we did not ﬁnd evidence—in any of our tests—for either of two key hypotheses: (1) the hypothesis that training with multiple speakers leads to greater generalisation to new speakers than training with a single speaker or (2) the hypothesis that there is an interaction between the variability of the training materials and participant aptitude, such that higher aptitude participants beneﬁt more from training with multiple speakers while lower aptitude participants beneﬁt more from training with a single speaker. However, there is a difﬁculty in interpreting these null results since a non-signiﬁcant result (p > 0.05) does not tell us whether we have evidence for the null, as opposed to no evidence for any conclusion at all, or even evidence against the null. Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 Figure 10 Accuracy in Three Interval Oddity and Training for LV (low variability), HV (high variability) and HVB (high variability blocked) training groups. Error bars show 95% conﬁdence interval. (A)Mean accuracy of Three Interval Oddity, split by high (HA) vs low (LA) aptitude in the Pitch Contour Perception Test (B) Mean accuracy of Training, split by high (HA) vs low (LA) aptitude in the Pitch Contour Perception Test. Full-size  DOI: 10.7717/peerj.7191/ﬁg-10 Figure 10 Accuracy in Three Interval Oddity and Training for LV (low variability), HV (high variability) and HVB (high variability blocked) training groups. Error bars show 95% conﬁdence interval. (A)Mean accuracy of Three Interval Oddity, split by high (HA) vs low (LA) aptitude in the Pitch Contour Perception Test (B) Mean accuracy of Training, split by high (HA) vs low (LA) aptitude in the Pitch Contour Perception Test. Full-size  DOI: 10.7717/peerj.7191/ﬁg-10 Figure 10 Accuracy in Three Interval Oddity and Training for LV (low variability), HV (high variability) and HVB (high variability blocked) training groups. Error bars show 95% conﬁdence interval. (A)Mean accuracy of Three Interval Oddity, split by high (HA) vs low (LA) aptitude in the Pitch Contour Perception Test (B) Mean accuracy of Training, split by high (HA) vs low (LA) aptitude in the Pitch Contour Perception Test. Full-size  DOI: 10.7717/peerj.7191/ﬁg-10 Thus, we should not reduce our conﬁdence in either of our hypotheses on the basis of the null results reported above (despite the fact that reducing conﬁdence in a theory following non-signiﬁcant results is common practice)—see Dienes (2014) for discussion. Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 Bayes factor analyses An alternative statistic is a Bayes Factor, which are used to assess the strength of evidence for one theory (H1) over another (the null hypothesis). We therefore supplemented the analyses above by computing Bayes factors for contrasts relating to these two key hypotheses. These are reported in the sections ‘H1: Greater generalization—to Novel Voices and in Production—in the Multiple Speaker Conditions (HV and HVB) than in the LV Condition’ and H1: There is an Interaction Between an Individual’s Tone-Aptitude and Variability-Condition, Such That Participants with Greater Tone-Aptitude Show Greater Performance Following the Multiple Speaker Conditions (HV and HVB) and Thus, we should not reduce our conﬁdence in either of our hypotheses on the basis of the null results reported above (despite the fact that reducing conﬁdence in a theory following non-signiﬁcant results is common practice)—see Dienes (2014) for discussion. An alternative statistic is a Bayes Factor, which are used to assess the strength of evidence for one theory (H1) over another (the null hypothesis). We therefore supplemented the analyses above by computing Bayes factors for contrasts relating to these two key hypotheses. These are reported in the sections ‘H1: Greater generalization—to Novel Voices and in Production—in the Multiple Speaker Conditions (HV and HVB) than in the LV Condition’ and H1: There is an Interaction Between an Individual’s Tone-Aptitude and Variability-Condition, Such That Participants with Greater Tone-Aptitude Show Greater Performance Following the Multiple Speaker Conditions (HV and HVB) and Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 28/45 Figure 11 Accuracy in Picture Naming and Picture Identiﬁcation for LV, HV and HVB training groups, split by high (HA) vs low (LA) aptitude in the Pitch Contour Perception Test. Error bars show 95% conﬁdence interval. (A) Mean accuracy of Picture Naming tone accuracy measure (B) Scatter plot contrasting Mean accuracy of Picture Naming tone accuracy measure and corresponding aptitude measure from Picture Contour Perception Test (C) Mean accuracy of Picture Naming Pinyin accuracy measure (D) Scatter plot contrasting Mean accuracy of Picture Naming Pinyin accuracy measure and corresponding aptitude measure from Picture Contour Perception Test (E) Mean accuracy of Picture Identiﬁcation (F) Scatter plot contrasting Mean accuracy of Picture Identiﬁcation and corresponding aptitude measure from Picture Contour Perception Test. Full-size  DOI: 10.7717/peerj.7191/ﬁg-11 Figure 11 Accuracy in Picture Naming and Picture Identiﬁcation for LV, HV and HVB training groups, split by high (HA) vs low (LA) aptitude in the Pitch Contour Perception Test. Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 Bayes factor analyses Error bars show 95% conﬁdence interval. (A) Mean accuracy of Picture Naming tone accuracy measure (B) Scatter plot contrasting Mean accuracy of Picture Naming tone accuracy measure and corresponding aptitude measure from Picture Contour Perception Test (C) Mean accuracy of Picture Naming Pinyin accuracy measure (D) Scatter plot contrasting Mean accuracy of Picture Naming Pinyin accuracy measure and corresponding aptitude measure from Picture Contour Perception Test (E) Mean accuracy of Picture Identiﬁcation (F) Scatter plot contrasting Mean accuracy of Picture Identiﬁcation and corresponding aptitude measure from Picture Contour Perception Test. Full-size  DOI: 10.7717/peerj.7191/ﬁg-11 Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 Those with Lesser Tone Aptitude Show Greater Performance in the Single Speaker Condition (LV)’ below. Figure 12 Accuracy in Word Repetition for LV, HV and HVB training groups, split by high (HA) vs low (LA) aptitude in the Pitch Contour Perception Test. Error bars show 95% conﬁdence intervals. (A) Mean accuracy of Word Repetition tone accuracy measure (B) Mean accuracy of Word Repetition Pinyin accuracy measure. Full-size  DOI: 10.7717/peerj.7191/ﬁg-12 Figure 12 Accuracy in Word Repetition for LV, HV and HVB training groups, split by high (HA) vs low (LA) aptitude in the Pitch Contour Perception Test. Error bars show 95% conﬁdence intervals. (A) Mean accuracy of Word Repetition tone accuracy measure (B) Mean accuracy of Word Repetition Pinyin accuracy measure. Full-size  DOI: 10.7717/peerj.7191/ﬁg-12 Figure 12 Accuracy in Word Repetition for LV, HV and HVB training groups, split by high (HA) vs low (LA) aptitude in the Pitch Contour Perception Test. Error bars show 95% conﬁdence intervals. (A) Mean accuracy of Word Repetition tone accuracy measure (B) Mean accuracy of Word Repetition Pinyin accuracy measure. Full-size  DOI: 10.7717/peerj.7191/ﬁg-12 Those with Lesser Tone Aptitude Show Greater Performance in the Single Speaker Figure 12 Accuracy in Word Repetition for LV, HV and HVB training groups, split by high (HA) vs low (LA) aptitude in the Pitch Contour Perception Test. Error bars show 95% conﬁdence intervals. (A) Mean accuracy of Word Repetition tone accuracy measure (B) Mean accuracy of Word Repetition Pinyin accuracy measure. Full-size  DOI: 10.7717/peerj.7191/ﬁg-12 Those with Lesser Tone Aptitude Show Greater Performance in the Single Speaker Condition (LV)’ below. Those with Lesser Tone Aptitude Show Greater Performance in the Single Speaker Condition (LV)’ below. H1: Greater generalisation—to novel voices and in production—in the multiple speaker conditions (HV and HVB) than in the low variability condition (LV) H1: Greater generalisation—to novel voices and in production—in the multiple speaker conditions (HV and HVB) than in the low variability condition (LV) We aimed to compute Bayes Factors comparing this hypothesis to the null for each of our data sets. To have maximum evidence, we pool the HV and HVB conditions and contrast this with the LV condition. For the post-tests we are interested in the evidence for a main effect of this contrast. For the pre-post tests, we are interested in the interaction between this contrast and session. To further maximise evidence, for the Three Interval Oddity test and Word Repetition tests we look at trained and untrained items combined (since both types of item involve generalisation to an untrained voice and thus should beneﬁt from HV training), however in the Picture Identiﬁcation test we excluded trained voice test items, since the beneﬁt of HV training was not predicted for these items. For the production measures, we are interested in whether there is a HV beneﬁt for our tone learning measure and our pinyin measure (the latter given that Barcroft & Sommers (2014), found a beneﬁt of multi-speaker training in their vocabulary recall task). We computed Bayes factors following Dienes (2014) and Dienes, Coulton & Heather (2018). To compute a Bayes factor (B) it is necessary to have both a model of the data and a model of H1. The model of the data is an estimate of the mean difference for the contrast in question, and of the standard error. Here, we get these estimates by running logistic mixed models and taking the betas and standard errors for the relevant coefﬁcients (note that this allows us to meet normality assumptions by continuing to work within log-odds space). The models we ran here were similar to the previous Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 analyses but with variability-condition coded as a centred contrast between LV and the HV+HVB conditions, and other factors combined/excluded as described in the previous paragraphs. The full set of models is in https://osf.io/wdh8a/. We model H1 using a half-normal distribution with a mode of 0 and a standard deviation x which is set to be a rough estimate of the predicted difference for this contrast. This allows for possible effects between 0 and twice the predicted effect, with values closer to 0 being more likely (Dienes, 2014). H1: Greater generalisation—to novel voices and in production—in the multiple speaker conditions (HV and HVB) than in the low variability condition (LV) g y In the absence of any prior data using sufﬁciently similar materials, and since we did not wish to use unprincipled default values, we estimated x for each contrast using the scale and/or values from elsewhere in the data (see Dienes, 2014, 2015 for a related approach). Speciﬁcally, for each of the cases where we predicted a main effect (Picture Identiﬁcation and Picture Naming), we set x as the difference between the grand mean (the Intercept—since we use a centred coding) and an estimate of minimal possible performance on the task. The logic is as follows5: The maximum difference between conditions is seen if LV participants show baseline performance and HV participants show performance greater than baseline. In this case, if performance on this test is p (so the grand mean is p) and the baseline is b, the difference in p between the two conditions will be equal to: 2(p b). This gives us an estimate of the maximum value of x; since we are using a half normal distribution with a mean of zero, we assume the maximum value is equal to approximately 2SD, so we can set our estimate x of the standard deviation to be equal to half of this value (i.e. x ¼ p b). Baseline performance depends on the task: for the 2AFC Picture Identiﬁcation task it is chance (50% = 0 in log odds space); for the Picture Naming, tone measure, we assume a ¼ chance of identifying the correct one (25% = -1.099 in log odds space); for Picture Naming, Pinyin measure, there is no chance and we therefore took minimal performance as making one correct response in the test6 (i.e. 1/72 = -4.263 in log odds space). For the cases where we are estimating an interaction between test-session and variability-condition we set x as equal to the mean increase in performance from pre- and post-test across conditions (main effect of test- session). The logic is as follows: the maximum difference is seen if LV participants show no effect of test-session (no improvement) and HV participants show a positive effect of test-session. In this case, if the mean effect of test-session ist, the difference in t between the two conditions will be equal to 2t. Again, we can set our estimate of x to be half this value (i.e., x ¼ t). 5 Further details of the logic of these computations is spelt out in the script available at https://osf.io/wdh8a/. 6 Note that we cannot compute log-odds of 0. Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 H1: Greater generalisation—to novel voices and in production—in the multiple speaker conditions (HV and HVB) than in the low variability condition (LV) We interpret BFs using the following conventions: B < 1/3 indicates substantial evidence for the null, B > 3 indicates substantial evidence for H1, values between 1/3 and 3 indicate that the data collected do not sensitively distinguish H0 from H1 (Jeffreys, 1998; Dienes, 2008). Since there is subjectivity in how the values for H1 are determined, we indicate the robustness of Bayesian conclusions by reporting a robustness region for each B, which gives the range of values of the scale factor x that qualitatively support the same conclusion (i.e. evidence as supporting H0, or as supporting H1, or there not being much evidence at all). Note that for evidence for H0, the maximum x is always inﬁnity. The results are reported in Table 5. It can be seen we have substantial or strong evidence for the null for every test except for the Word Repetition test for the Pinyin accuracy measure, where 31/45 31/45 Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 Table 5 Bayes Factor results testing the hypothesis that there is greater generalisation following either of the high variability training conditions than the low variability condition. he hypothesis that there is greater generalisation following either of the high variability training dition. y g yp g g g g y g conditions than the low variability condition. Contrast Mean difference Stand. Error H1 estimate x Bayes factor (B) Robustness region Picture ID (Novel voice only) HV+ HVB > LV 0.13 0.228 1.71 0.219 1.11 : ∞ Picture naming, (Tone accuracy) HV+ HVB > LV -0.225 0.168 1.076 0.067 0.202 : ∞ Picture naming (Pinyin Accuracy) HV+ HVB > LV 0.104 0.196 4.05 0.08 0.101 : ∞ Word repetition (Tone accuracy) test-session by HV+ HVB > LV -0.108 0.157 0.395 0.239 0.303 : ∞ Word repetition (Pinyin accuracy) test-session byHV+ HVB > LV 0.095 -0.034 0.152 0.421 0 : 0.202 Three interval odditytest-session by HV+ HVB > LV -0.001 0.1 0.31 0.303 0.303 : ∞ the evidence is ambiguous, and that the robustness regions indicate that we would continue to have evidence for the null even with smaller estimates of the scale factor x. Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 7 An alternative which would be more equivalent to the other BF analyses would be to inform the effect using the value of the two-way interaction of aptitude: test-session. We do not do this since we did not ﬁnd an effect of this two-way interaction in either data set. ID, (Tone accuracy) aptitude by HV+ HVB > LV The logic is as follows: The maximal effect of the interaction would be seen if participants in the LV condition showed the same baseline effect of aptitude at pre-test and at post-test (ba), whereas participants in the HV condition showed maximal improvement at post-test (maxa). In this case, the interaction between aptitude and session for the HV group would be equal to: maxa—ba. Again, we can set our estimate of x—the SD of the half normal—to be half this maximum value, that is, x ¼ maba 2 . The maximum effect of aptitude was computed from the scale and the length of the aptitude predictor. Speciﬁcally, we assumed that the maximal effect of aptitude would be obtained if participants with maximal aptitude were at ceiling (71/72 correct—log odds 4.263) and those with minimal aptitude were at chance (25% in Word Repetition, Tone Accuracy, log odds= 1.099; 33.33% in Three Interval Oddity, log odds = 0.693). We divided this range by the length of the aptitude predictor to obtain a measure of a one-step change in aptitude. The results are summarised in Table 6. It can be seen that although there is more evidence for the null than H1 in each case (i.e. BF < 1) we do not have substantial evidence for the null over H1 in any case. Thus, we cannot draw any inferences about the interaction from this data. Note that, in most cases, the robustness regions indicate that even if the scale factor x was twice as large, that is, corresponding to the maximum value we might expect, the B would be ambiguous. ID, (Tone accuracy) aptitude by HV+ HVB > LV effect of aptitude across conditions (main effect of aptitude)7. The logic is as follows: The maximum difference is seen if LV participants show no effect of aptitude and the HV participants show a positive effect of aptitude (note that a negative effect of aptitude is not expected in any condition). In this case, if the mean effect of aptitude is a, the difference in a between the two conditions will be equal to 2a. Again, we can set our estimate of x—the SD of the half normal—to be half this maximum value, that is, x ¼ a. For the cases where we are interested in the three-way interaction between aptitude, test-condition and test-session, we based our estimate on half the difference between the maximal effect of aptitude (maxA—taken from the scale) and their actual aptitude score at pre-test (baselineA—taken from the data). The logic is as follows: The maximal effect of the interaction would be seen if participants in the LV condition showed the same baseline effect of aptitude at pre-test and at post-test (ba), whereas participants in the HV condition showed maximal improvement at post-test (maxa). In this case, the interaction between aptitude and session for the HV group would be equal to: maxa—ba. Again, we can set our estimate of x—the SD of the half normal—to be half this maximum value, that is, x ¼ maba 2 . The maximum effect of aptitude was computed from the scale and the length of the aptitude predictor. Speciﬁcally, we assumed that the maximal effect of aptitude would be obtained if participants with maximal aptitude were at ceiling (71/72 correct—log odds 4.263) and those with minimal aptitude were at chance (25% in Word Repetition, effect of aptitude across conditions (main effect of aptitude)7. The logic is as follows: The maximum difference is seen if LV participants show no effect of aptitude and the HV participants show a positive effect of aptitude (note that a negative effect of aptitude is not expected in any condition). In this case, if the mean effect of aptitude is a, the difference in a between the two conditions will be equal to 2a. Again, we can set our estimate of x—the SD of the half normal—to be half this maximum value, that is, x ¼ a. H1: Greater generalisation—to novel voices and in production—in the multiple speaker conditions (HV and HVB) than in the low variability condition (LV) (2019), PeerJ, DOI 10.7717/peerj.7191 32/45 Table 6 Bayes Factor results testing the hypothesis that there is an interaction between aptitude and variability-condition greater generalisation following either of the high variability training conditions than the low variability condition. Table 6 Bayes Factor results testing the hypothesis that there is an interaction between aptitude and variability-condition greater generalisation following either of the high variability training conditions than the low variability condition. Table 6 Bayes Factor results testing the hypothesis that there is an interaction between aptitude and variability-condition greater generalisation following either of the high variability training conditions than the low variability condition. Table 6 Bayes Factor results testing the hypothesis that there is an interaction between aptitude and variability-condition greater generalisation following either of the high variability training conditions than the low variability condition. Table 6 Bayes Factor results testing the hypothesis that there is an interaction between aptitude and variability-condition greater generalisation following either of the high variability training conditions than the low variability condition. Contrast Mean difference Stand. Error H1 estimate x Bayes factor (B) Robustness region ID, (Tone accuracy) aptitude by HV+ HVB > LV 0.006 0.127 0.171 0.617 0 : 0.354 Picture naming, (Tone accuracy) aptitude by HV+ HVB > LV 0.042 0.083 0.099 0.904 0 : 0.354 Three interval oddity (Tone accuracy) aptitude by test-session by HV+ HVB > LV 0.048 0.05 0.345 0.371 0 : 0.354 Word Repetition (Tone accuracy) aptitude by test-session by HV+ HVB > LV 0.091 0.082 0.379 0.654 0 : 0.758 Training aptitude by HV > LV -0.037 0.119 0.129 0.572 0 : 0.253 Training aptitude by HVB > LV 0.026 0.101 0.129 0.732 0 : 0.354 H1: Greater generalisation—to novel voices and in production—in the multiple speaker conditions (HV and HVB) than in the low variability condition (LV) H1: There is an interaction between an individual's tone-aptitude and variability- condition, such that participants with greater tone-aptitude show greater performance following the multiple speaker conditions (HV and HVB) and those with lesser tone aptitude show greater performance in the single speaker condition (LV) We aimed to compute Bayes Factors comparing this hypothesis to the null for each of our data sets. We take the same approach as above except that we also compute Bayes factors for Training data, and for the Picture Identiﬁcation test we look at both trained voice and untrained voice data—pooling the two in order to maximise available evidence. This is because this interaction has been reported with trained items (Sadakata & McQueen, 2013) as well as untrained items (Perrachione et al., 2011). We again combine the HV and HVB conditions except for training where we look at the LV vs HV and LV vs HVB contrasts separately, since we have seen in our previous analyses that HV and HVB are quite different (HVB participants show higher performance).We again combine the evidence from trained and untrained items in the pre-post tests. For the post-session only tests, we are interested in the evidence for an interaction between the variability-condition contrast and aptitude. For the tests which appeared both pre- and post-training, we are interested in the interaction between the variability-condition contrast, aptitude and test-session. For training we look at the evidence for an interaction between each variability-condition contrast and aptitude (a more complex model containing the interaction with training-session did not converge). As in our frequentist analyses of aptitude, for the production measures—Word Repetition and Picture Naming—we do not look at the pinyin measures since our aptitude measure is relevant only to tone learning. We computed Bayes factors following the same procedure as in the section ‘H1: Greater Generalization—to Novel Voices and in Production—in the Multiple Speaker Conditions (HV and HVB) than in the LV Condition’ and again derived our estimates of the scale factor x—the difference predicted under H1—using the scale and/or values from elsewhere in the data. Speciﬁcally, for each of the cases where we predicted a two-way interaction between variability-condition and aptitude we set x as equal to the mean Dong et al. Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 ID, (Tone accuracy) aptitude by HV+ HVB > LV For the cases where we are interested in the three-way interaction between aptitude, test-condition and test-session, we based our estimate on half the difference between the maximal effect of aptitude (maxA—taken from the scale) and their actual aptitude score at pre-test (baselineA—taken from the data). The logic is as follows: The maximal effect of the interaction would be seen if participants in the LV condition showed the same baseline effect of aptitude at pre-test and at post-test (ba), whereas participants in the HV condition showed maximal improvement at post-test (maxa). In this case, the interaction between aptitude and session for the HV group would be equal to: maxa—ba. Again, we can set our estimate of x—the SD of the half normal—to be half this maximum value, that is, x ¼ maba 2 . effect of aptitude across conditions (main effect of aptitude)7. The logic is as follows: The maximum difference is seen if LV participants show no effect of aptitude and the HV participants show a positive effect of aptitude (note that a negative effect of aptitude is not expected in any condition). In this case, if the mean effect of aptitude is a, the difference in a between the two conditions will be equal to 2a. Again, we can set our estimate of x—the SD of the half normal—to be half this maximum value, that is, x ¼ a. For the cases where we are interested in the three-way interaction between aptitude, effect of aptitude across conditions (main effect of aptitude)7. The logic is as follows: The maximum difference is seen if LV participants show no effect of aptitude and the HV participants show a positive effect of aptitude (note that a negative effect of aptitude is not expected in any condition). In this case, if the mean effect of aptitude is a, the difference in a between the two conditions will be equal to 2a. Again, we can set our estimate of x—the SD of the half normal—to be half this maximum value, that is, x ¼ a. For the cases where we are interested in the three-way interaction between aptitude, y p test-condition and test-session, we based our estimate on half the difference between the maximal effect of aptitude (maxA—taken from the scale) and their actual aptitude score at pre-test (baselineA—taken from the data). DISCUSSION The current study investigated the effect of different types of phonetic training on English speakers’ learning of novel Mandarin words and tones. To our knowledge, this is the Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 ﬁrst study to train naive participants on all four Mandarin tones, using real language stimuli embedded in a word learning task. Learning was examined using a range of perception and production tasks. Following previous literature, we compared three training conditions: LV (single speaker), HV (four speakers, presented intermixed) and HV blocked (four speakers, presented in blocks). We also administered tests designed to tap individual aptitude in the perception of pitch contrasts, adapted from the previous literature. The results indicated that participants’ performance increased during training and that training also led to improved performance on pre- to post-tests of discrimination and production, with evidence of generalisation to untrained voices and items. Participants also showed some ability to recall trained words—including their tones—in a picture naming task administered at post-test. However, the only place where we saw any effect of the variability manipulation was in the training task (and with trained items in the picture identiﬁcation task, which was highly similar to training), where the low variability group outperformed both of the HV groups. Critically, we found no evidence in any of our tests that HV input beneﬁtted learning or generalisation, nor did we ﬁnd any evidence of an interaction between individual aptitude and the ability to beneﬁt from HV training. In the following discussion, we ﬁrst consider the ﬁndings from each task in turn before turning to a more general discussion of our ﬁndings in relation to the predicted beneﬁt of HV input. Tests of individual aptitude In the current work, we conducted two tests with the purpose of capturing individual aptitude: The Pitch Contrast Perception Test (following Perrachione et al., 2011) and the Categorisation of Synthesised Tonal Continua (following Sadakata & McQueen, 2014). Although our goal was to measure participants’ baseline aptitude, the tests were conducted both at pre- and post-test, following Sadakata and McQueen, who did not ﬁnd differences from pre- to post-tests with their categorisation measure, and who used combined data from pre- and post-test to compute participants slopes. Unfortunately, the performance of our own participants suggested that the Categorisation of Synthesised Tonal Continua test was not a good test of aptitude, with the majority of participants failing to meet the slope threshold used in Sadakata and McQueen, and most being unable to consistently categorise the end points of the continua. It is unclear why our results differ from the previous study (we aimed to follow their procedures), but this meant that we were unable to use this as an aptitude measure in our later analyses. The scores on the Pitch Contrast Perception Test alone therefore served as our measure of individual aptitude. Interestingly, preliminary analyses (section ‘The Pitch Contour Perception Test’) demonstrated that performance in this test improved from pre- to post-training. This suggests that this measure is not a ‘pure’ measure of individual differences since it also appears to be affected by experience. Given this, we only used participants’ scores on this test from pre-test as the measure of aptitude in subsequent analyses. Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 Performance in training The training task employed in this study was a 2AFC task, where participants had to identify the correct meaning of a Mandarin word based on its tone. The results from Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 34/45 34/45 training indicate that participants performed better in the single speaker LV training than in either the multiple speaker HV or HVB groups. This difference was present from the ﬁrst session for the LV-HV contrast, and from the second session for the LV-HVB contrast (i.e. the ﬁrst session where the two conditions differ), and increased over time for both contrasts. Greater difﬁculty with multiple speaker input is in line with the ﬁndings of Perrachione et al. (2011), although the differences did not emerge so rapidly in that study, possibly due to there being fewer trials per session. Intuitively, repeated exposure to the single speaker in the LV condition allows for greater adaptation to speaker speciﬁc cues, whereas in the HV conditions participants have to adapt to multiple speakers. This is particularly difﬁcult in the unblocked HV condition, where trial-by-trial adaptation is needed, which is effortful for participants (Magnuson & Nusbaum, 2007). Importantly, however, for all three groups, their performance gradually increased over each session. In combination with the fact that their performance on the other tasks increased after training, this indicates that the training task and materials were effective. We also explored the role of learner aptitude in this task (as measured by performance on the Pitch Contour Perception Test at pre-test) and whether this inﬂuenced participant’s performance differently in the different variability conditions. Overall, aptitude was found to be a signiﬁcant predictor of performance during training. However, there was no evidence for an interaction with training condition, although our Bayes Factor Analyses suggests that the data here are inconclusive. We return to this ﬁnding in the section ‘The Role of High Variability Materials in Training and Generalisation’ below. Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 Perception tests We included two perceptual tasks which tapped learning and generalisation due to training: A Picture Identiﬁcation task administered at post-test and a Three Interval Oddity task administered at both pre- and post-test. The Picture Identiﬁcation task was a version of the training task without feedback, and is the most similar to the tests used by Perrachione et al. (2011), and Sadakata & McQueen (2014). We used this test to look at learning of the trained stimuli, comparing trained and untrained voices. The three interval oddity task had not been used in the previous studies, but allowed us to use a pre-/post-test design, and also to look at participants’ performance with untrained items. These tests provided evidence that participants improved in their perception of tones following training: They were above chance in using the tone to identify the correct picture in the picture identiﬁcation task at post-test, and they improved in their ability to discriminate tones in the three interval oddity task (59% performance prior to training, 66% post training). There was also evidence of generalisation across both voices and items: Participants were above chance in identifying the correct pictures even with an untrained voice (although they did show signiﬁcantly weaker performance than with the trained voice) and they improved in their ability to discriminate the between minimal pair items in the three interval oddity task, even for untrained items. Our key questions concerned the role of variability in training. First, we were interested in whether there was evidence that exposure to multiple voices during training led to greater ability to generalise across voices at test—that is, greater performance with novel Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 35/45 voices in the HV conditions than in the LV condition. We did not see this. In fact, the only effect of variability in this data was a low variability beneﬁt, which we saw in the Picture Identiﬁcation task for the trained-voice items (seen in the contrast between LV and HV conditions). This mirrors what we saw in training and reﬂects the greater exposure to this particular speaker in the LV training. However, in the tests tapping generalisation to a novel speaker—that is, in untrained voice trials in the Picture Identiﬁcation task, and with all of the test-items in the Three Interval Oddity task, there was no difference between variability-training conditions. Perception tests Bayes factor analyses indicate that in both cases, there was substantial evidence for the null. The second hypothesis was that there would be an interaction between learner aptitude (as measured by the Pitch Contour Perception Test at pre-test) and variability training condition, such that high aptitude participants would beneﬁt more from HV training. Note that previous work had found this interaction both in tests involving generalisation (Perrachione et al., 2011) and with trained items (Sadakata & McQueen, 2014) so we considered both in our analyses here. There was no evidence of such an interaction in either the Picture Identiﬁcation or Three Interval Oddity tasks. However, Bayes Factor analyses suggest that the data are inconclusive. We return to these points in the section ‘The Role of High Variability Materials in Training and Generalisation’ below. Another ﬁnding from the Three Interval oddity test that is worth noting, although it did not concern our hypotheses, is that some trial types were harder than others. Recall that this test involved participants hearing three different stimuli each produced by a different speaker, which makes noting the similarity across two of the stimuli much harder—something we discovered in pilot work, where even before training participants were near ceiling with an equivalent task in which the same speaker produced all three stimuli within a single trial. However, analyses of trial-type demonstrated that participants were additionally affected by the gender of the three speakers producing each of the stimuli. Speciﬁcally, at pre-test, participants showed best performance for trials where one of the speakers was male and the other two were female, and the target ‘odd man’ was the male speaker (‘easy’ trials). In contrast, they showed worst performance if there was one male and two female speakers, but the ‘odd man’ was one of the female speakers (‘hard’ trials). Middle level performance was shown for trials where all three speakers were female (‘neutral’ trials). This is presumably due to participants relying on perceptual cues associated with speaker gender to do the task. Interestingly, our analyses showed that performance only increased for the trials where the odd one was not the lone male (the ‘neutral’ and ‘hard’ ones), but not for those where the male was the odd man. Given that participants are not near ceiling at pre-test (67%), it is perhaps surprising that their trained knowledge of the tone contrasts does not boost their performance. Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 Perception tests One possibility is although they are now better able to use tone cues, they are also less likely to use gender based cues, which they may now realise are less reliable, masking improvement based on tone for these particular test items. Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 Production tasks The role of high variability materials in training and generalisation In the current study, across all of the different tests, we did not ﬁnd either an overall beneﬁt of exposure to HV training materials for generalisation, or any interaction between such a beneﬁt and individual aptitude. Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 Production tasks In this study, we used two production tasks, a word repetition task administered pre and post training, in which participants repeated back Mandarin words, and a Picture Naming task testing vocabulary recall, which was administered at post-test only. HV perceptual training for tones has been previously found to transfer to production (Bradlow & Pisoni, 1999; Zeromskaite, 2014), however the beneﬁts of HV and LV training have not been contrasted. In the Word Repetition task, there was a signiﬁcant, though relatively modest improvement in participants’ ability to reproduce the tone of the stimuli, such that it could be identiﬁed by a native speaker (from pre- to post-test: 70–76%) and in the Picture Naming task, participants showed an ability to recall and reproduce the correct tone, although unsurprisingly with less accuracy than in the repetition task (50%). For Word Repetition, we were also able to look at transfer to untrained words: As in the perception tasks, there was once again equivalent improvement for both trained and untrained items. Together, these results provide evidence that purely perceptual training on tone contrast can transfer to production, as well as to novel items. In addition to looking at the production of tones, we also looked at participants’ ability to produce the correct segmental phonology (pinyin-score). Participants showed a small but signiﬁcant improvement on this measure in Word Repetition (54% correct at pre-test, 58% at post-test), and some ability to recall the segments in the Picture Naming test (50% correct). This indicates some learning of segmental phonology due to training, despite the fact that the focus of the training task was on training tonal information through the presentation of tonal minimal-pairs. Turning to the role of variability, the predicted beneﬁt of HV training was not evident in any of the measures in either of the production tasks, with Bayes factor analyses indicating substantial evidence for the null except for the Word Repetition pinyin-measure, where the evidence was ambiguous. With regard to aptitude, although performance on the Pitch Contour Perception Test at pre-test was predictive of participants’ ability to produce tones in both tasks (indicating a relationship between participants perceptual and production ability), we did not ﬁnd the predicted interaction between aptitude and variability condition in either task. Here however, Bayes Factor analyses suggests that the results are inconclusive. We return to these points about variability below. The role of high variability materials in training and generalisation In the current study, across all of the different tests, we did not ﬁnd either an overall beneﬁt of exposure to HV training materials for generalisation, or any interaction between such a beneﬁt and individual aptitude. We consider ﬁrst the lack of overall variability beneﬁt for generalisation. Importantly, in addition to ﬁnding a pattern of null results (i.e. p < 0.05) in the frequentist analyses, additional Bayes Factor analyses also found substantial evidence for the null (BF < 0.33) in all but one of the test measures (Word Repetition, Pinyin, where BF = 0.421). Thus, there is good evidence that, at least for these training and test materials, exposure to stimuli from multiple speakers does not lead to greater generalisation in either Thus, there is good evidence that, at least for these training and test materials, exposure to stimuli from multiple speakers does not lead to greater generalisation in either Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 37/45 perception or production. This ﬁnding is consistent with the lack of a main effect of variability condition in the transfer tasks in either Sadakata & McQueen (2014) or Perrachione et al. (2011) (see also footnote 1). However it is at odds with other phonetic training studies focused on segmental contrasts (Clopper & Pisoni, 2004; Logan, Lively & Pisoni, 1991; Lively, Logan & Pisoni, 1993; Sadakata & McQueen, 2013) and with the literature demonstrating a HV beneﬁt in vocabulary learning (Barcroft & Sommers, 2005, 2014; Sommers & Barcroft, 2007, 2011). This suggests that this overall variability beneﬁt may be restricted to segmental rather than tonal phonetic learning, at least for speakers of a non-tonal L1. It is difﬁcult to reconcile the lack of beneﬁt for vocabulary learning in the picture naming task, given the ﬁndings of Barcroft & Sommers (2005, 2014) and Sommers & Barcroft (2007, 2011), since this test is quite similar to that used in their experiments. However, one possibility is that this is due to differences in our training set up (i.e. focused on training tonal contrasts) compared with the earlier vocabulary studies. Nonetheless it remains unclear why tone learning should be different from other types of phonetic learning in terms of beneﬁting from talker-variability. Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 The role of high variability materials in training and generalisation Theoretically speaking, in a framework where all cues compete, variation in idiosyncratic speaker-speciﬁc cues would be expected to provide key evidence as to which cues are irrelevant to the phonetic contrast in question (Apfelbaum & McMurray, 2011; Ramscar & Baayen, 2013; Ramscar et al., 2010). This raises the question of how participants in our LV condition are able to generalise at all—that is, how can they identify the phonetically relevant cues compared with the idiosyncratic cues associated with the single speaker to which they were exposed? One possibility is that other variation in our materials aided generalisation, in particular in our real word stimuli, each tone-contrast is encountered with multiple consonants and vowels. If item variability also aids generalisation to new speakers, this might explain why we found equivalent generalisation across conditions instead of seeing greater generalisation in the HV conditions (i.e. even the LV condition is really a HV condition, because of the item variability). On the other hand, Sadakata & McQueen (2014) also saw generalisation even for their LV condition, and in their study this condition lacked variation in terms of both speakers and phonetic contexts. This suggests that the relevant cues for the tone contrasts may be sufﬁciently acoustically salient for learners to identify them, even when exposure occurs in limited contexts. Another possibility—and the one suggested by the ﬁndings of Sadakata & McQueen (2014) and Perrachione et al. (2011)—is that beneﬁts of HV for generalisation are masked by individual differences. In their studies, only high aptitude participants showed a HV beneﬁt, while low aptitude participants did not. It is possible that for lower aptitude participants, the beneﬁts of exposure to varying, idiosyncratic cues are offset by the greater difﬁculty that these participants have in attuning to the different speakers during training, as discussed above (section ‘Tests of Individual Aptitude’). This explanation is supported by evidence from a study by Goldinger, Pisoni & Logan (1991) who explored the effect of increasing the processing cost of multi-speaker input in the context of word recall (in the L1). Speciﬁcally, they exposed participants to single vs multi-speaker word Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 38/45 lists, manipulating presentations rates. They found that single-speaker lists produced better word recall than multiple-speaker lists at short inter-word intervals (less than 2,000 ms) whereas this effect was reversed for longer inter-word intervals. The role of high variability materials in training and generalisation This suggests that increasing encoding difﬁculty can remove the beneﬁts of multi-speaker exposure. Relatedly, Sinkeviciute et al. (2019) found that young learners have greater difﬁculty processing multi-speaker training materials in L2 vocabulary learning, and subsequently fail to show a speaker-variability beneﬁt at test. One interpretation of these ﬁndings is that age-related capacity limitations may constrain the ability to beneﬁt from speaker variability, supporting the notion that differences in capacity limitations can affect an individual’s ability to beneﬁt from multi-talker training. Returning to the current study, we did not ﬁnd an interaction between variability- training and learner aptitude. However, it is important to acknowledge the results of our Bayes factor analyses, which did not ﬁnd substantial evidence in support of the null over H1 (or H1 over H0) for any of the test tasks. This means that we cannot draw conclusions about this hypothesis from the current data. In theory, we could continue collecting data until we had substantial evidence for either H0 or H1. To explore the feasibility of this, we conducted supplementary analyses to estimate the sample size that might be needed to see substantial evidence for the null (based on the assumption that the error term would reduce in proportion to √SE). Taking the Picture Identiﬁcation test (the test most similar to previous studies) our results suggests that it would require N > 300—that is, over ﬁve times our current sample size. This suggests that this experimental paradigm is not sufﬁciently sensitive to address this hypothesis. Given the ambiguity of our ﬁndings with regard to the interaction, it is not appropriate to extensively interpret why we do not ﬁnd the interaction while the previous studies did. However, we note that there are a variety of differences across the studies which could underpin the different ﬁndings, if it holds true. For example, the test of individual differences which we use is harder than that used by Sadakata & McQueen (2014) since it uses all six Mandarin vowels (whereas the original study used ﬁve, without /y/) and all of the Mandarin tones (where Perrachione et al. used three, without Tone 3). This change also means that that we cannot easily contrast the range of participant scores in the two studies and it may be that the spread of ability of our participant is different from theirs. Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 The role of high variability materials in training and generalisation In addition, our training task is potentially harder than both of the previous studies, that is, involving all four tones in the context of natural Mandarin stimuli in the context of a word learning tasks. Finally, we also note that our statistical analyses are different from both of the previous studies in that they took their continuous aptitude measures and turned these into binary factors using a ‘cut off’, whereas our statistical approach allows us to use them as continuous variables. However, this should in principle make our approach more powerful than in previous studies. Future directions Future directions If the interaction between aptitude and training condition reported in Sadakata & McQueen (2014) and Perrachione et al. (2011) is to have implications for educational materials, Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 39/45 it is important to establish whether it extends to other more naturalistic materials. Given the relatively small samples in these original studies, and the increasing recognition that psychology experiments have been routinely underpowered (Maxwell, Lau & Howard, 2015; and see Vasishth et al. (2018) for a recent demonstration in the area of reading) and that can lead to increases in both type 1 and type 2 error, we suggest that it would be useful to implement a direct, high powered replication of these previous studies. We note that having a sufﬁcient sample to provide substantial evidence for H1/H0 using Bayesian methods, or to obtain 90% power for frequentist methods, would likely require a much larger sample than is standard in these types of studies. Given the time-consuming nature of these multiple session training studies, moving to online testing may be necessary to make this feasible (see Xie et al., 2018 for an example of an acoustic training study done over the web), or alternately multi-lab collaboration may be necessary. Note that this would also allow us to see whether the fact that Perrachione et al., (2011) found their interaction with untrained voices, whereas Sadakata & McQueen (2014) found it only for trained voices, is a true difference (due to the different paradigms) or due to power. Critically, successful replication would allow us to then extend the paradigms in such a way as to explore the factors above. For example, would increasing the number of tones to use all four Mandarin tones and/or using natural Mandarin stimuli affect the interaction between variability in the input and learner aptitude? Although direct replication will play a useful role in establishing these effects, we believe that ultimately it will also be important to develop a more nuanced approach to measuring the factors leading to different levels of aptitude both in tone learning, and in other types of phonetic learning. We note that here in addition to not seeing the predicted interaction with variability, we also didn’t see interactions between aptitude and training session in any of our tasks, suggesting that our aptitude measure predicted baseline performance on the task and not the ability to improve due to training. Future directions In addition, the tasks used to measure ‘aptitude’ are quite similar in nature to the training and test tasks, decreasing their explanatory value. Our ongoing work explores the combined predictive value of a range of measures including measures of attention, working memory and musical ability. Identifying factors which are predictive of aptitude for tone learning has clear implications for teaching and the personalisation of teaching methods. Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 Competing Interests The authors declare that they have no competing interests. Human Ethics The following information was supplied relating to ethical approvals (i.e. approving body and any reference numbers): UCL Research Ethics Committee approved the study (Project ID Number: 6176/002). Data Availability The following information was supplied regarding data availability: The raw data and analytic code are available in the Supplemental Files. CONCLUSION We trained naive participants on all four Mandarin tones, using real language stimuli embedded in a word learning task. We found improvements in both production and perception of tones which transferred to novel voices and items. We found that learning was greatest for training with a single voice but that training with a single voice vs four voices (whether intermixed or blocked) lead to equal amounts of generalisation. Although learner aptitude predicted performance in most tasks, there was no evidence that different levels of aptitude lead to better or worse learning from different types of training input. 40/45 Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 Author Contributions Hanyu Dong conceived and designed the experiments, performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, prepared ﬁgures and/or tables, authored or reviewed drafts of the paper, approved the ﬁnal draft. y g y prepared ﬁgures and/or tables, authored or reviewed drafts of the paper, approved the ﬁnal draft. Meghan Clayards analyzed the data, contributed reagents/materials/analysis tools, prepared ﬁgures and/or tables, authored or reviewed drafts of the paper, approved the ﬁnal draft. Helen Brown authored or reviewed drafts of the paper, approved the ﬁnal draft. Elizabeth Wonnacott conceived and designed the experiments, analyzed the data, contributed reagents/materials/analysis tools, authored or reviewed drafts of the paper, approved the ﬁnal draft. Funding This work was supported by the British Academy Small Grant, a grant awarded to Elizabeth Wonnacott and Meghan Clayards (SG111965) and a Social Sciences and Humanities Research Council of Canada grant (SSHRC #435-2016-0747) to Meghan Clayards and Elizabeth Wonnacott. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Grant Disclosures The following grant information was disclosed by the authors: The following grant information was disclosed by the authors: British Academy Small Grant: SG111965. Social Sciences and Humanities Research Council of Canada grant: SSHRC #435-2016- 0747. Social Sciences and Humanities Research Council of Canada grant: SSHRC #435-2016- 0747. Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 REFERENCES Aliaga-García C, Mora JC. 2009. Assessing the effects of phonetic training on L2 sound perception and production. In: Watkins MA, Rauber AS, Baptista BO, eds. Recent Research in Second Language Phonetics/Phonology: Perception and Production. Newcastle upon Tyne: Cambridge Scholars Publishing, 2–31. Alshangiti W, Evans BG. 2014. Investigating the domain-speciﬁcity of phonetic training for second language learning: comparing the effects of production and perception training on the acquisition of English vowels by Arabic learners of English. In: The Proceedings of the International Seminar for Speech Production. Cologne. Apfelbaum KS, McMurray B. 2011. Using variability to guide dimensional weighting: associative mechanisms in early word learning. Cognitive Science 35(6):1105–1138 DOI 10.1111/j.1551-6709.2011.01181.x. Audacity Team. 2015. Audacity. Version 2.1.1. Computer program. Available at http://audacityteam.org/ (accessed May 2015). Baayen RH, Davidson DJ, Bates DM. 2008. Mixed-effects modeling with crossed random effects for subjects and items. Journal of Memory and Language 59(4):390–412 DOI 10.1016/j.jml.2007.12.005. Barcroft J, Sommers MS. 2005. Effects of acoustic variability on second language vocabulary learning. Studies in Second Language Acquisition 27(3):387–414 DOI 10.1017/S0272263105050175. Barcroft J, Sommers MS. 2014. Effects of variability in fundamental frequency on L2 vocabulary learning: a comparison between learners who do and do not speak a tone language. Studies in Second Language Acquisition 36(3):423–449. Barr DJ, Levy R, Scheepers C, Tily HJ. 2013. Random effects structure for conﬁrmatory hypothesis testing: keep it maximal. Journal of Memory and Language 68(3):255–278 DOI 10.1016/j.jml.2012.11.001. Bates D, Maechler M, Bolker B, Walker S. 2013. lme4: Linear mixed-effects models using Eigen and S4. R package Version 1.0–5. Available at https://cran.r-project.org/web/packages/lme4/ index.html. Boersma P, Weenink D. 2015. Praat: doing phonetics by computer [Computer program]. Version 5.4.14. Available at http://www.praat.org/ (accessed 24 July 2015). Bradlow AR, Akahane-Yamada R, Pisoni DB, Tohkura YI. 1999. Training Japanese listeners to identify English/r/and/l: Long-term retention of learning in perception and production. Perception & Psychophysics 61(5):977–985 DOI 10.3758/BF03206911. Bradlow AR, Bent T. 2008. Perceptual adaptation to non-native speech. Cognition 106(2):707–729 DOI 10.1016/j.cognition.2007.04.005. Bradlow AR, Pisoni DB. 1999. Recognition of spoken words by native and non-native listeners: Talker-, listener-, and item-related factors. Journal of the Acoustical Society of America 106(4):2074–2085 DOI 10.1121/1.427952. Bygate M, Swain M, Skehan P. 2013. Researching pedagogic tasks: Second language learning, teaching, and testing. London: Routledge. Cicchetti DV. 1994. Guidelines, criteria, and rules of thumb for evaluating normed and standardized assessment instruments in psychology. Psychological Assessment 6(4):284–290 DOI 10.1037/1040-3590.6.4.284. Clopper CG, Pisoni DB. 2004. Supplemental Information Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.7191#supplemental-information. Supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.7191#supplemental-information. Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 REFERENCES Some acoustic cues for the perceptual categorization of American English regional dialects. Journal of Phonetics 32(1):111–140 DOI 10.1016/S0095-4470(03)00009-3. Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 42/45 Cohen AD. 2014. Strategies in learning and using a second language. London: Routledge. Dienes Z. 2008. Understanding psychology as a science: an introduction to scientiﬁc and statistical inference. London: Macmillan International Higher Education. Dienes Z. 2014. Using Bayes to get the most out of non-signiﬁcant results. Frontiers in psychology 5:781 DOI 10.3389/fpsyg.2014.00781. Dienes Z. 2015. How Bayesian statistics are needed to determine whether mental states are unconscious. In: Overgaard M, ed. Behavioural Methods in Consciousness Research. Oxford: Oxford University Press, 199–220. Dienes Z, Coulton S, Heather N. 2018. Using Bayes factors to evaluate evidence for no effect: examples from the SIPS project. Addiction 113(2):240–246 DOI 10.1111/add.14002. Flege JE, Takagi N, Mann V. 1995. Japanese adults can learn to produce English /I/ and /l/ accurately. Language and Speech 38(1):25–55 DOI 10.1177/002383099503800102. Giannakopoulou A, Brown H, Clayards M, Wonnacott E. 2017. High or low? Comparing high and low-variability phonetic training in adult and child second language learners. PeerJ 5:e3209 DOI 10.7717/peerj.3209. Giannakopoulou A, Uther M, Ylinen S. 2013. Enhanced plasticity in spoken language acquisition for child learners: evidence from phonetic training studies in child and adult learners of English. Child Language Teaching and Therapy 29(2):201–218 DOI 10.1177/0265659012467473. Goldinger SD, Pisoni DB, Logan JS. 1991. On the nature of talker variability effects on recall of spoken word lists. Journal of Experimental Psychology: Learning, Memory, and Cognition 17(1):152–162. Hay J, Drager K, Thomas B. 2013. Using nonsense words to investigate vowel merger. English Language & Linguistics 17(2):241–269 DOI 10.1017/s1360674313000026. Iverson P, Ekanayake D, Hamann S, Sennema A, Evans BG. 2008. Category and perceptual interference in second-language phoneme learning: an examination of English /w/-/v/ learning by Sinhala, German, and Dutch speakers. Journal of Experimental Psychology: Human Perception and Performance 34(5):1305–1316 DOI 10.1037/0096-1523.34.5.1305. Iverson P, Hazan V, Bannister K. 2005. Phonetic training with acoustic cue manipulations: A comparison of methods for teaching English /r/-/l/ to Japanese adults. Journal of the Acoustical Society of America 118(5):3267–3278 DOI 10.1121/1.2062307. Jaeger TF. 2008. Categorical data analysis: Away from ANOVAs (transformation or not) and towards logit mixed models. Journal of Memory and Language 59(4):434–446 DOI 10.1016/j.jml.2007.11.007. Jeffreys H. 1998. The theory of probability. Oxford: Oxford University Press. Joanisse MF, Manis FR, Keating P, Seidenberg MS. 2000. Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 REFERENCES Language deﬁcits in dyslexic children: Speech perception, phonology, and morphology. Journal of Experimental Child Psychology 77(1):30–60 DOI 10.1006/jecp.1999.2553. Lambacher SG, Martens WL, Kakehi K, Marasinghe CA, Molholt G. 2005. The effects of identiﬁcation training on the identiﬁcation and production of American English vowels by native speakers of Japanese. Applied Psycholinguistics 26(2):227–247 Landis JR, Koch GG. 1977. The measurement of observer agreement for categorical data. Biometrics 33(1):159–174 DOI 10.2307/2529310. Lengeris A, Hazan V. 2010. The effect of native vowel processing ability and frequency discrimination acuity on the phonetic training of English vowels for native speakers of Greek. Journal of the Acoustical Society of America 128(6):3757–3768 DOI 10.1121/1.3506351. Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 43/45 Lively SE, Logan JS, Pisoni DB. 1993. Training Japanese listeners to identify English /r/ and /l/. II: the role of phonetic environment and talker variability in learning new perceptual categories. Journal of the Acoustical Society of America 94(3):1242–1255 DOI 10.1121/1.408177. Logan JS, Lively SE, Pisoni DB. 1991. Training Japanese listeners to identify English /r/ and /l/: a ﬁrst report. Journal of the Acoustical Society of America 89(2):874–886 DOI 10.1121/1.1894649. Magnuson JS, Nusbaum HC. 2007. Acoustic differences, listener expectations, and the perceptual accommodation of talker variability. Journal of Experimental Psychology: Human Perception and Performance 33(2):391–409 DOI 10.1037/0096-1523.33.2.391. Mattys SL, Wiget L. 2011. Effects of cognitive load on speech recognition. Journal of Memory and Language 65(2):145–160 DOI 10.1016/j.jml.2011.04.004. Maxwell O, Baker B, Bundgaard-Nielsen R, Fletcher J. 2015. A comparison of the acoustics of nonsense and real word stimuli: coronal stops in Bengali. In: Proceedings of the Meeting of the International Congress of Phonetic Sciences. Glasgow. Maxwell SE, Lau MY, Howard GS. 2015. Is psychology suffering from a replication crisis? What does “failure to replicate” really mean? American Psychologist 70(6):487–498 Maxwell SE, Lau MY, Howard GS. 2015. Is psychology suffering from a replication crisis? What does “failure to replicate” really mean? American Psychologist 70(6):487–498 DOI 10.1037/a0039400. McGraw KO, Wong SP. 1996. Forming inferences about some intraclass correlation coefﬁcients. Psychological Methods 1(1):30–46 DOI 10.1037/1082-989X.1.1.30. Nusbaum HC, Morin TM. 1992. Paying attention to differences among talkers. In: Tohkura K, Vatikiotis-Bateson E, Sagisaka Y, eds. Speech Perception, Production and Linguistic Structure. Amsterdam: IOS Press, 113–134. Perrachione TK, Lee J, Ha LY, Wong PC. 2011. Learning a novel phonological contrast depends on interactions between individual differences and training paradigm design. Journal of the Acoustical Society of America 130:461–472. Powell MJ. 2009. Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 REFERENCES The BOBYQA algorithm for bound constrained optimization without derivatives. Cambridge NA Report NA2009/06. Cambridge: University of Cambridge, 26–46. Quené H, Van den Bergh H. 2008. Examples of mixed-effects modeling with crossed random effects and with binomial data. Journal of Memory and Language 59(4):413–425 DOI 10.1016/j.jml.2008.02.002. R Development Core Team. 2010. R: a language and environment for statistical computing. Version R 3.3.2. Vienna: The R Foundation for Statistical Computing. Available at http://www.R-project.org/ (accessed September 2017). Ramscar M, Baayen H. 2013. Production, comprehension, and synthesis: a communicative perspective on language. Frontiers in Psychology 4:233 DOI 10.3389/fpsyg.2013.00233. Ramscar M, Yarlett D, Dye M, Denny K, Thorpe K. 2010. The effects of feature-label-order and their implications for symbolic learning. Cognitive science 34(6):909–957 DOI 10.1111/j.1551-6709.2009.01092.x. Sadakata M, McQueen JM. 2013. High stimulus variability in nonnative speech learning supports formation of abstract categories: evidence from Japanese geminates. Journal of the Acoustical Society of America 134(2):1324–1335 DOI 10.1121/1.4812767. Sadakata M, McQueen JM. 2014. Individual aptitude in Mandarin lexical tone perception predicts effectiveness of high-variability training. Frontiers in Psychology 5:1318 O /f Scarborough R. 2012. Lexical similarity and speech production: neighborhoods for nonwords. Lingua 122(2):164–176 DOI 10.1016/j.lingua.2011.06.006. Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 44/45 Sinkeviciute R, Brown H, Brekelmans G, Wonnacott E. 2019. The role of input variability and learner age in second language vocabulary learning. Epub ahead of print 20 June 2019. Studies in Second Language Acquisition DOI 10.1017/S0272263119000263. Sommers MS, Barcroft J. 2007. An integrated account of the effects of acoustic variability in ﬁrst language and second language: evidence from amplitude, fundamental frequency, and speaking rate variability. Applied Psycholinguistics 28(2):231–249 DOI 10.1017/S0142716407070129. Sommers MS, Barcroft J. 2011. Indexical information, encoding difﬁculty, and second language vocabulary learning. Applied Psycholinguistics 32(2):417–434 DOI 10.1017/S0142716410000469. Strange W, Dittmann S. 1984. Effects of discrimination training on the perception of /r-l/ by Japanese adults learning English. Perception & Psychophysics 36(2):131–145 DOI 10.3758/BF03202673. Strange W, Shafer VL. 2008. Speech perception in second language learners: the re-education of selective perception. In: Edwards JGH, Zampini ML, eds. Phonology and Second Language Acquisition. Amsterdam: John Benjamins Publishing Company, 153–192. Vasishth S, Mertzen D, Jäger LA, Gelman A. 2018. The statistical signiﬁcance ﬁlter leads to overoptimistic expectations of replicability. Journal of Memory and Language 103:151–175 DOI 10.1016/j.jml.2018.07.004. Wang Y, Jongman A, Sereno JA. 2003. Acoustic and perceptual evaluation of Mandarin tone productions before and after perceptual training. Dong et al. (2019), PeerJ, DOI 10.7717/peerj.7191 Zeromskaite I. 2014. The potential role of music in second language learning: a review article. Journal of European Psychology Students 5(3):78–88 DOI 10.5334/jeps.ci. REFERENCES Journal of the Acoustical Society of America 113(2):1033–1043 DOI 10.1121/1.1531176. Wang Y, Spence MM, Jongman A, Sereno JA. 1999. Training American listeners to perceive Mandarin tones. Journal of the Acoustical Society of America 106(6):3649–3658 DOI 10.1121/1.428217. Wong J. 2014. The Effects of High and Low Variability Phonetic Training on the Perception and Production of English Vowels /e/-/æ/ by Cantonese ESL Learners with High and Low L2 Proﬁciency Levels. In: proceedings of INTERSPEECH 2014, the 15th Annual Conference of the International Speech Communication Association, Singapore. ISCA Archive, 524–528. Available at http://www.isca-speech.org/archive/interspeech_2014. Wong P, Perrachione TK. 2007. Learning pitch patterns in lexical identiﬁcation by native English-speaking adults. Applied Psycholinguistics 28(4):565–585 DOI 10.1017/S0142716407070312. Xie X, Weatherholtz K, Bainton L, Rowe E, Burchill Z, Liu L, Jaeger TF. 2018. Rapid adaptation to foreign-accented speech and its transfer to an unfamiliar talker. Journal of the Acoustical Society of America 143(4):2013–2031 DOI 10.1121/1.5027410. g p J f Society of America 143(4):2013–2031 DOI 10.1121/1.5027410. Yip M. 2002. Tone. Cambridge textbooks in linguistics. Cambridge: Cambridge University Press. Zeromskaite I. 2014. The potential role of music in second language learning: a review article. J l f E P h l S d (3) 8 88 DOI 10 334/j i Yip M. 2002. Tone. Cambridge textbooks in linguistics. Cambridge: Cambridge University Press. Zeromskaite I. 2014. The potential role of music in second language learning: a review article. Journal of European Psychology Students 5(3):78–88 DOI 10.5334/jeps.ci. 45/45
https://openalex.org/W3046079573	https://www.aclweb.org/anthology/2020.figlang-1.32.pdf	English	null	Go Figure! Multi-task transformer-based architecture for metaphor detection using idioms: ETS team in 2020 metaphor shared task	null	2,020	cc-by	6,372	Abstract In the NLP universe, there’s been substantial re- cent interest in automated detection of metaphor (Dankers et al., 2019; Mikhalkova et al., 2019; Mao et al., 2019; Igamberdiev and Shin, 2018; Marhula et al., 2019; Markert, 2019; Saund et al., 2019). This paper describes the ETS entry to the 2020 Metaphor Detection shared task. Our contri- bution consists of a sequence of experiments using BERT, starting with a baseline, strength- ening it by spell-correcting the TOEFL cor- pus, followed by a multi-task learning set- ting, where one of the tasks is the token-level metaphor classiﬁcation as per the shared task, while the other is meant to provide additional training that we hypothesized to be relevant to the main task. In one case, out-of-domain data manually annotated for metaphor is used for the auxiliary task; in the other case, in- domain data automatically annotated for id- ioms is used for the auxiliary task. Both multi- task experiments yield promising results. This paper describes the ETS entry to the 2020 Metaphor Detection shared task held as a part of the 2nd Workshop on Processing Figurative Lan- guage, at ACL 20201. The shared tasks consists of four tracks: all content parts of speech – nouns, verbs, adjectives, and adverbs (AllPOS) and a verbs-only track (Verbs) for two corpora – (a) a corpus of well-edited BNC articles from a variety of genres annotated using the MIP-VU protocol, and (b) a corpus of medium to high quality timed, non-native essays written for the Test of English as a Foreign Language annotated under a different protocol. We participated in all the four tracks. 1https://sites.google.com/view/ﬁglang2020/home Go Figure! Multi-task transformer-based architecture for metaphor detection using idioms: ETS team in 2020 metaphor shared task Xianyang Chen, Chee Wee (Ben) Leong, Michael Flor, and Beata Beigman Kleb Educational Testing Service xchen002,cleong,mflor,bbeigmanklebanov@ets.org 3 Baseline system We use the VU Amsterdam Metaphor Corpus (VUA) (Steen et al., 2010) as provided by the shared task organizers. The dataset consists of 117 fragments sampled across four genres from the British National Corpus: Academic, News, Con- versation, and Fiction. Each genre is represented by approximately the same number of tokens, al- though the number of texts differs greatly, where the news archive has the largest number of texts. The data is annotated using the MIP-VU proce- dure with a strong inter-annotator reliability of κ > 0.8. It is based on the MIP procedure (Prag- glejaz, 2007), extending it to handle metaphoric- ity through reference (such as marking did as a metaphor in As the weather broke up, so did their friendship) and allow for explicit coding of difﬁ- cult cases where a group of annotators could not arrive at a consensus. Note that we only consid- ered words marked as metaphors decided as such by the shared task organizers. The VUA dataset and annotations is the same as the one used in the ﬁrst shared task on metaphor detection (Leong et al., 2018). We build our baseline system based on BERT (De- vlin et al., 2018). BERT (Bidirectional Encoder Representations from Transformers) is a trans- former (Vaswani et al., 2017) model that is pre- trained on a large quantity of texts, and obtained state-of-the-art performance on many NLP bench- marks (Wang et al., 2018; Zellers et al., 2018; Ra- jpurkar et al., 2016). Since its introduction, there have been many improvements over the original BERT model, such as RoBERTa (Liu et al., 2019), ERNIE (Sun et al., 2019), XLNet (Yang et al., 2019); we use the most basic model (bert-base- uncased). We ﬁne-tune the BERT model as a standard to- ken classiﬁcation task, that is, after obtaining the contextualized embeddings of a sentence, we ap- ply a linear layer followed by softmax on each token to predict whether it is metaphorical or not. Fig 1 shows the architecture of the baseline model. We tune the hyperparameters based on cross-validation on training data. The fold parti- tions for the VUA corpus are the same as the ones used for experiments in Beigman Klebanov et al. (2016). For the TOEFL corpus, we obtained the folds information from the shared task organiz- ers directly. 1 Introduction We use metaphors in our everyday life as a means of relating our experiences to other subjects and contexts (Lakoff and Johnson, 2008); it is com- monly used to help us understand the world in a structured way, and oftentimes in an unconscious manner while we speak and write. It sheds light on the unknown using the known, explains the com- plex using the simple, and helps us to emphasize the relevant aspects of meaning resulting in effec- tive communication. Our contribution consists of a sequence of experiments using BERT, starting with a base- line, then strengthening it by spell-correcting the TOEFL corpus (section 4). We then devised a multi-task learning setting, where one of the tasks is the token level metaphor classiﬁcation as per the shared task, while the other is meant to provide additional training that we hypothesized to be rel- evant to the main task (section 5). The ﬁrst multitask learning is the utilization out-of-domain data annotated for metaphor, al- beit under a different annotation protocol, by us- ing data from the other competition corpus. Thus, we use metaphor prediction on the VUA corpus as an auxiliary task for the main TOEFL task, and vice versa. We show that this setup resulted in an improved performance on the TOEFL test data but not on VUA data. A sanity-check experi- ment where the two training datasets were simply merged together yielded performance that was in- ferior to the baseline for all tracks (section 5.1). There is a large body of work in the litera- ture that discusses how metaphor has been used in the context of political communication, marketing, mental health, teaching, assessment of English proﬁciency, among others (Beigman Klebanov et al., 2018; Gutierrez et al., 2017; Littlemore et al., 2013; Thibodeau and Boroditsky, 2011; Ka- viani and Hamedi, 2011; Kathpalia and Carmel, 2011; Landau et al., 2009; Beigman Klebanov et al., 2008; Zaltman and Zaltman, 2008; Little- more and Low, 2006; Cameron, 2003; Lakoff, 2010; Billow et al., 1997; Bosman, 1987); see chapter 7 in Veale et al. (2016) for a recent review. 235 Proceedings of the Second Workshop on Figurative Language Processing, pages 235–243 July 9, 2020. c⃝2020 Association for Computational Linguistics https://doi.org/10.18653/v1/P17 Proceedings of the Second Workshop on Figurative Language Processing, pages 235–243 July 9, 2020. c⃝2020 Association for Computational Linguistics https://doi.org/10.18653/v1/P17 metaphors. 1 Introduction Average inter-annotator agreement was κ = 0.56 – 0.62, for multiple passes of the annotation (see (Beigman Klebanov et al., 2018) for more details). For the experiments, we used the metaphor annotations marked as such by the organizers. We used 180 essays for training and 60 essays for testing, as provided by the shared task organizers. Tables 1 and 2 show some de- scriptive characteristics of the data: the number of texts, sentences, tokens, and class distribution in- formation for Verbs and AllPOS tracks for the two corpora – VUA and TOEFL. The second auxiliary task is utilization of a large in-domain corpus that we automatically tagged for occurrence of a different type of ﬁgurative lan- guage phenomenon – idioms. We hypothesize that the afﬁnity between the two ways of ﬁguration might help the system become more sensitive to metaphor by learning to attend to idioms. Our results provide support to the hypothesis, as this setting yielded our best result on the VUA dataset (section 5.2). We provide a discussion of our ﬁnd- ings in section 7. 3 Baseline system We select batch size in {16, 32, 64}, number of training epochs in {2, 3, 4, 5}, and use a ﬁxed learning rate of 3 × 10−5. We also apply the learning rate scheduler known as slanted triangu- lar (Howard and Ruder, 2018). Due to the imbal- anced class distribution in our data (see Table 2), the positive class is up-weighted by a factor of 3. The same setting applies to experiments described in all the following sections. 2https://catalog.ldc.upenn.edu/LDC2014T06 4 Experiment 1: Spell correction system Proper automatic detection of lexically-anchored phenomena in text often depends on availabil- ity of correct spelling in the text. The contri- bution of spelling correction to other tasks has been documented previously, especially for En- glish texts produced by non-native learners of En- glish (Rozovskaya and Roth, 2016; Granger and Wynne, 1999). Essays written by TOEFL test- takers are known to contain a considerable amount of spelling errors (Flor et al., 2015). To alleviate this, we used a state-of-the-art automatic spelling corrections system (Flor et al., 2019) to correct spelling in the TOEFL dataset. Speciﬁcally, for the training partition of the TOEFL dataset, the 5 Multi-task system As we ﬁne-tune BERT on relatively small datasets, we attempted to enrich the learning with partially relevant additional materials through a multi-task setting - adding auxiliary tasks and train the metaphor detection task with them. The auxiliary tasks are described in sections 5.1 and 5.2. The model we use for multi-task learning is as follows: Instead of directly making predictions based on the output embeddings of BERT, the em- beddings are ﬁrst projected to a lower-dimensional representation by a linear layer; each task then has its own classiﬁer on top of that linear layer. The architecture of the model is shown in Fig. 2. Figure 1: Baseline system architecture. The output is a pair of probabilities – the ﬁrst for class 0 (non- metaphor) and the second for class 1 (metaphor). During training, the data in batches of the metaphor task (our main task) and the auxiliary tasks are mixed and trained on in an interleaved manner; the speciﬁcs will be described for each of the auxiliary tasks separately. In order for the main task to dominate the learning, we also scale the gradient of the auxiliary tasks by a factor of 0.1. The hyperparameters are selected in the same way as described in section 3. 2.2 TOEFL corpus This data labeled for metaphor was sampled from the publicly available ETS Corpus of Non- Native Written English2 (Blanchard et al., 2013) and was ﬁrst introduced by (Beigman Klebanov et al., 2018). The annotated data comprises es- say responses to eight persuaisve/argumentative prompts, for three native languages of the writer (Japanese, Italian, Arabic), and for two proﬁciency levels – medium and high. The data was annotated using the protocol in Beigman Klebanov and Flor (2013), that emphasized argumentation-relevant 2https://catalog.ldc.upenn.edu/LDC2014T06 236 Datasets VUA TOEFL Train Test Train Test #texts 90 27 180 60 #sents 12,123 4,081 2,741 968 Table 1: Number of texts and sentences for both VUA and TOEFL datasets. Table 1: Number of texts and sentences for both VUA and TOEFL datasets. Table 1: Number of texts and sentences for both VUA and TOEFL datasets. Datasets VUA TOEFL Verbs All POS Verbs All POS Train Test Train Test Train Test Train Test #tokens 17,240 5,873 72,611 22,196 7,016 2,301 26,737 9,014 %M 29% − 18% − 13% − 7% − Table 2: Number of tokens and percentage of metaphors breakdown for both VUA and TOEFL datasets, grouped by Verbs and AllPOS. Table 2: Number of tokens and percentage of metaphors breakdown for both VUA and TOEFL datasets, grouped by Verbs and AllPOS. Table 2: Number of tokens and percentage of metaphors breakdown for both VUA and TOEFL datasets, grouped by Verbs and AllPOS. Figure 1: Baseline system architecture. The output is a pair of probabilities – the ﬁrst for class 0 (non- metaphor) and the second for class 1 (metaphor). system corrected 1553 errors in 180 essays, and 510 errors in 60 essays of the test partition. 5.1 Experiment 2: Learning from out-of-domain data ferent genres (well-edited BNC text in academic, news, conversation and ﬁction genres vs relatively short English language learner essays), and since the guidelines under which the two datasets were annotated are different, it is possible that each corpus is only partially or indirectly relevant to the other. We experimented with both a straight- forward merging of the training sets of the two datasets (as a preview – this did not produce good results) and with a multi-task setting where the other corpus is used for the auxiliary task. Although there exists considerable prior re- search on automatic detection of idioms (for a brief review see Flor and Beigman Klebanov (2018)), idiom detection systems are typically constrained to very small sets of idioms or to par- ticular types of expressions (e.g. verb-noun con- structions). We opted to use a system that marks candidate expressions but does not verify their id- iomaticity in the given context. The advantage of this particular system is that it has very wide cov- erage. We assume that many of the idioms found in a particular corpus might be well-known idioms that are listed in various dictionaries. Our system (Flor and Beigman Klebanov, 2018) is equipped with a dictionary of about 5000 English idiomatic expressions (culled from Wiktionary), and per- forms a ﬂexible search for idioms and their syn- tactic and lexical variants in running text. In fact, it performs a simultaneous ﬂexible pattern match- ing. The idiom detection system looks only for expressions that have more than one word, and excludes common greeting phrases (e.g. ’have a nice day’), phrasal verbs and verb+preposition constructions (unless they are part of a larger id- iom). The system marks expressions that poten- tially might be instances of idioms, but it does not perform idiom/non-idiom classiﬁcation. For the present experiment we used this system with rather conservative settings that yielded precision We use the same batch size and learning rate for the main task and the auxiliary task. The batches from the two tasks are interleaved uniformly; as there are roughly four times more sentences in the VUA corpus than in the TOEFL corpus, there are ﬁve batches of the VUA task following every batch of the TOEFL task. We do not sub-sample the VUA corpus or over-sample the TOEFL corpus. 5.1 Experiment 2: Learning from out-of-domain data Since both the VUA and the TOEFL corpora are annotated for metaphors, using one to help the other during learning could potentially provide ad- ditional relevant training data. However, since the data is from different types of texts and dif- 237 Figure 2: Multi-task system architecture. The output is a pair of probabilities – the ﬁrst for class 0 (non- metaphor) and the second for class 1 (metaphor). in psycholinguistic literature (Gibbs and O’Brien, 1990; Nunberg et al., 1994; Glucksberg, 2001). in psycholinguistic literature (Gibbs and O’Brien, 1990; Nunberg et al., 1994; Glucksberg, 2001). in psycholinguistic literature (Gibbs and O’Brien, 1990; Nunberg et al., 1994; Glucksberg, 2001). The main idea here is that one or more of the words participating in idiomatic expressions are often used metaphorically. Thus, in CUT- TING EDGE both the words are used metaphori- cally; in PAY attention, false STEP, helping HAND, GOLDEN opportunity, and social LADDER, the capitalized word is a metaphor while the other is not. There are also idioms where none of the words are used metaphorically such as matter of fact, other than, and once in a while. Still, it ap- pears likely that the preponderance of metaphors within idiomatic expressions would be higher than in non-idiomatic language. It is also possible that learning to detect idioms – a different but re- lated type of ﬁgurative language – could help with metaphor detection, as these might tend to be used in similar contexts. Experiment 3 is an attempt to explore these observations by setting idiom de- tection as an auxiliary task for the main metaphor detection task. Figure 2: Multi-task system architecture. The output is a pair of probabilities – the ﬁrst for class 0 (non- metaphor) and the second for class 1 (metaphor). ferent genres (well-edited BNC text in academic, news, conversation and ﬁction genres vs relatively short English language learner essays), and since the guidelines under which the two datasets were annotated are different, it is possible that each corpus is only partially or indirectly relevant to the other. We experimented with both a straight- forward merging of the training sets of the two datasets (as a preview – this did not produce good results) and with a multi-task setting where the other corpus is used for the auxiliary task. 7 Discussion First, we observe that comparative results across the different systems are highly consistent for All- POS and Verbs-only settings; we therefore focus on AllPOS in the discussion. We ran the idiom-candidate marking system on TOEFL-113 essays and on the BNC corpus (ex- cluding texts of the shared task). In total, the sys- tem detected 3,581 different idiom types in the BNC, with 179,967 instances of (candidate) id- ioms; in the TOEFL-11 data, we found 504 dif- ferent idiom types, with 3,908 instances. There is somewhat more idiom usage per sentence in the BNC than in the TOEFL data: The system identi- ﬁed an idiom in 3% of all BNC sentences and in 2.2% for of TOEFL-11 sentences. Table 3 shows the 20 most frequently found (candidate, or un- veriﬁed) idioms in the BNC and TOEFL data; the lists contain a mix of idioms that contain and do not contain metaphors. Combining the training data from TOEFL and VUA sets does not result in better performance on either test set (see Dall in Tables 4, 5). This could be due to both out-of-domain nature of the two corpora with respect to each other, to the dif- ference in the guidelines under which the two cor- pora were annotated, and/or to the difference in the distribution of metaphors vs non-metaphors in the two corpora (see Table 2 for class distribution information). However, when set up as a multi-task system with a shared representation, using data from VUA as part of the training process results in better per- formance on TOEFL test data, with a 2.6 points F1 score gain for AllPOS (0.666 vs 0.692 in Table 4). Thus, it appears that using the VUA data as part of the training process through the shared representa- tion but without the TOEFL training process sus- taining a loss for mis-classifying instances from VUA (as was the case when the training sets were merged), the system has apparently successfully acquired useful information that helped boost per- formance on TOEFL test data. The idiom detection auxiliary task is also for- malized as a token classiﬁcation task: Given a sentence, predict for each token whether it is part of an idiom. Given the size of the BNC cor- pus, we only sample a small subset of it for train- ing: 10,000 sentences with idioms and 10,000 sen- tences without idioms. 7 Discussion For the TOEFL-11 data, we keep all sentences with idioms and sample the same number (3,908) of sentences without idioms. It is interesting to note that the multi-task ver- sion of the setting for using out-of-domain data did not result in improvements on VUA test data (F1 score of 0.717 vs 0.715). The drop in re- call and increase in precision observed for the Dmt model on VUA data is consistent with the direction of the results where the two training datasets were simply merged into a bigger train- ing dataset (Dall). It appears that under the guide- lines in which TOEFL data was annotated where argumentation-relevant metaphors are detected in- tuitively, without recourse to a standard dictionary, the annotation outcomes are more conservative: Some instances that the system trained on VUA data considered metaphorical were not considered so in a system that was exposed to TOEFL data 5.2 Experiment 3: Learning from another type of ﬁgurative language Differently from Experiment 2, where we utilized an out-of-domain dataset annotated for the same phenomenon (albeit under somewhat different an- notation protocol), in Experiment 3 we are at- tempting to make use of a different but related phe- nomenon – a different type of ﬁgurative language, namely, idioms. The metaphorical underpinnings of many idiomatic expressions have been noted 238 Verbs-only subset of the test data, and (b) train- ing and testing on Verbs only subsets. Version (a) yielded better results, which are reported here. of 0.571 in our previous evaluations on a subset of the TOEFL data; see the leftmost column in Fig- ure 1-A in Flor and Beigman Klebanov (2018) for the details of the conﬁguration. Based on the prior evaluation, for this system conﬁguration, most of the errors were cases where the expression is iden- tiﬁed correctly but it is used literally rather than idiomatically. 3https://catalog.ldc.upenn.edu/LDC2014T06 6 Results Overall, the drop in recall was not sufﬁciently offset by the increase in pre- cision (although there is a small improvement in F1 score for the Verbs only data – from 0.756 to 0.762, see Table 5). Still, our results suggest that if one is interested in a precision-focused system, using TOEFL data in a multi-task setting when training and testing on VUA could be beneﬁcial, as Dmt achieved the best precision on the VUA dataset among all the compared systems. during training. For example, the three underlined words in the following sentence were classiﬁed as metaphors by the version that was trained on VUA data only, but were classiﬁed as non-metaphors af- ter augmentation with the TOEFL data: “A less di- rect measure which is applicable only to the most senior management is to observe the fall or rise of the share price when a particular executive leaves or joins a company.” Of these, senior and leaves are metaphors according to VUA ground truth, while observe is not. Overall, the drop in recall was not sufﬁciently offset by the increase in pre- cision (although there is a small improvement in F1 score for the Verbs only data – from 0.756 to 0.762, see Table 5). Still, our results suggest that if one is interested in a precision-focused system, using TOEFL data in a multi-task setting when training and testing on VUA could be beneﬁcial, as Dmt achieved the best precision on the VUA dataset among all the compared systems. F1 score – with no penalty in precision, the sys- tem gained about 3.5 points in recall (0.713 vs 0.749 on AllPOS; 0.790 vs 0.823 on Verbs). This conﬁrms the usefulness of attending to a related type of ﬁgurative language through an auxiliary task – even though the identiﬁcation of idioms was done using an automated procedure and therefore is quite noisy. To examine the impact of the idiom auxiliary task, we used one of the cross-validation folds as development set. Looking at instances tagged as non-metaphor by the baseline model and as metaphor by the current model, there are two ob- servations. First, of the 236 VUA sentences with newly tagged metaphors, only 9 sentences con- tained an idiom, according to our idiom detection system. 6 Results Tables 4 and 5 show performance of the various systems on AllPOS and Verbs-only tasks, respec- tively, for both VUA and TOEFL data. Since it is clear that spelling correction is useful for improv- ing performance on TOEFL data, we used the spell corrected version of the data for all the systems from experiments 2 and 3 on TOEFL data. Our best-performing systems reported here are also benchmarked against other participating systems in the shared task summary report (Leong et al., 2020). We obtained a ranking of 2nd and 4th in the VUA and TOEFL tasks, respectively. Since VUA data contains well-edited BNC text, we did not run spelling correction on VUA data. For the Verbs tracks, we experimented with both (a) training on AllPOS data and evaluating on the 239 All POS Sys- VUA TOEFL tem P R F P R F BL .721 .713 .718 .701 .563 .624 Sp − − − .656 .676 .666 Dall .728 .676 .701 .576 .637 .605 Dmt .741 .692 .715 .669 .717 .692 Imt .721 .749 .734 .718 .616 .663 Table 4: AllPOS performance. BL = baseline BERT system; Sp = baseline BERT system trained and tested on spell-corrected TOEFL data; Dall = baseline BERT system trained on combined TOEFL and VUA data; Dmt = a multi-task system using out-of-domain metaphor annotated data; Imt = multi-task system us- ing idiom detection as an auxiliary task. BNC TOEFL 11 ﬁnd oneself long time other than need-to-know long time pay attention great deal matter of fact once again other than ups and downs day-to-day once more ﬁnd oneself much less long run come through stay at home old woman play games bear in mind great deal cup of tea jack of all trades day-to-day side effect ask the question much less let alone change one’s mind need-to-know ask the question common law again and again close one’s eyes well and good blue-eyed tell the truth change one’s mind once again All POS Sys- VUA TOEFL tem P R F P R F BL .721 .713 .718 .701 .563 .624 Sp − − − .656 .676 .666 Dall .728 .676 .701 .576 .637 .605 Dmt .741 .692 .715 .669 .717 .692 Imt .721 .749 .734 .718 .616 .663 Table 4: AllPOS performance. 6 Results BL = baseline BERT system; Sp = baseline BERT system trained and tested on spell-corrected TOEFL data; Dall = baseline BERT system trained on combined TOEFL and VUA data; Dmt = a multi-task system using out-of-domain metaphor annotated data; Imt = multi-task system us- ing idiom detection as an auxiliary task. Verbs Sys- VUA TOEFL tem P R F P R F BL .725 .790 .756 .624 .694 .657 Sp − − − .674 .694 .684 Dall .747 .733 .740 .614 .664 .638 Dmt .754 .772 .762 .747 .661 .702 Imt .732 .823 .775 .705 .631 .667 Table 5: Verbs performance. BL = baseline BERT system; Sp = baseline BERT system trained and tested on spell-corrected TOEFL data; Dall = baseline BERT system trained on combined TOEFL and VUA data; Dmt = a multi-task system using out-of-domain metaphor annotated data; Imt = multi-task system us- ing idiom detection as an auxiliary task. Verbs Sys- VUA TOEFL tem P R F P R F BL .725 .790 .756 .624 .694 .657 Sp − − − .674 .694 .684 Dall .747 .733 .740 .614 .664 .638 Dmt .754 .772 .762 .747 .661 .702 Imt .732 .823 .775 .705 .631 .667 Table 3: Top 20 most frequently observed (unveriﬁed) idioms in the BNC and TOEFL 11 corpora. Note: Hy- phens are treated as between-word delimiters and are optionally matched. Thus, both “stay-at-home” and “day to day” will be matched, even though these are not the canonical forms of the idioms on the list. Table 5: Verbs performance. BL = baseline BERT system; Sp = baseline BERT system trained and tested on spell-corrected TOEFL data; Dall = baseline BERT system trained on combined TOEFL and VUA data; Dmt = a multi-task system using out-of-domain metaphor annotated data; Imt = multi-task system us- ing idiom detection as an auxiliary task. during training. For example, the three underlined words in the following sentence were classiﬁed as metaphors by the version that was trained on VUA data only, but were classiﬁed as non-metaphors af- ter augmentation with the TOEFL data: “A less di- rect measure which is applicable only to the most senior management is to observe the fall or rise of the share price when a particular executive leaves or joins a company.” Of these, senior and leaves are metaphors according to VUA ground truth, while observe is not. 6 Results Thus, it does not appear to be the case that it is speciﬁcally metaphors within known id- ioms that the system has now learned to ﬁnd; this We next turn to the experiments with an aux- iliary idiom detection task. We observe that on VUA data this resulted in a 2-point increase in 240 is a tentative conclusion, however, as it is also pos- sible that these sentences did contain idioms that were either not on the list of the 5,000 the system is searching for, or are on the list and are present in the text but are not detected by the system. Secondly, it appears that the system has learned some sentence-level characteristics of sentences that contain ﬁgurative language, in that quite often multiple words in the same sentence got tagged as metaphors: the 236 sentences contained 323 newly tagged metaphors. The most extreme case is that of 4 new words in the same sentence being tagged as metaphors (italicized): “This desire that can not ﬁnd its name (though it would dare speak, if it could) is pleasurable.” for occurrence of a different type of ﬁgurative language phenomenon – idioms. This manipula- tion resulted in an improved performance on VUA data, but not on TOEFL data. Given the promis- ing results with idiom auxiliary task, we intend to continue work in this direction by improving au- tomatic detection of idioms and by a ﬁner-grained analysis of the contribution of various types of id- ioms to improve metaphor detection. References Beata Beigman Klebanov, Daniel Diermeier, and Eyal Beigman. 2008. Lexical cohesion analysis of politi- cal speech. Political Analysis, 16(4):447–463. Using idioms for an auxiliary task did not help with TOEFL data. We also tried using the BNC id- iom data instead of the TOEFL 11 idiom data; this resulted in comparable performance, still without improvement over the spell-checked single-task version. Since results on VUA suggest that idioms could provide useful information for metaphor de- tection, we intend to further pursue this line of work by attending more closely to the different types of idiomatic expressions that might be more or less useful for metaphor detection, and by im- proving the idiom detection mechanism. Beata Beigman Klebanov and Michael Flor. 2013. Argumentation-relevant metaphors in test-taker es- says. In Proceedings of the First Workshop on Metaphor in NLP, pages 11–20. Beata Beigman Klebanov, Chee Wee Leong, and Michael Flor. 2018. A corpus of non-native writ- ten English annotated for metaphor. In Proceed- ings of the 2018 Conference of the North Ameri- can Chapter of the Association for Computational Linguistics: Human Language Technologies, Vol- ume 2 (Short Papers), pages 86–91, New Orleans, Louisiana. Association for Computational Linguis- tics. Beata Beigman Klebanov, Chee Wee Leong, E Dario Gutierrez, Ekaterina Shutova, and Michael Flor. 2016. Semantic classiﬁcations for detection of verb metaphors. In Proceedings of the 54th Annual Meet- ing of the Association for Computational Linguistics (Volume 2: Short Papers), volume 2, pages 101–106. 8 Conclusion This paper describes the ETS entry to the 2020 Metaphor Detection shared task held as a part of the 2nd Workshop on Processing Figurative Lan- guage, at ACL 2020. We participated in all four tracks – Verbs and AllPOS for each of VUA and TOEFL datasets. Our contribution consists of a se- quence of experiments using BERT, starting with a baseline, then strengthening it by spell-correcting the TOEFL corpus, followed by a multi-task learn- ing setting, where one of the tasks is the token- level metaphor classiﬁcation as per the shared task, while the other is meant to provide additional training that we hypothesized to be relevant to the main task. Richard M Billow, Jeffrey Rossman, Nona Lewis, De- berah Goldman, and Charles Raps. 1997. Observ- ing expressive and deviant language in schizophre- nia. Metaphor and Symbol, 12(3):205–216. Daniel Blanchard, Joel Tetreault, Derrick Higgins, Aoife Cahill, and Martin Chodorow. 2013. Toeﬂ11: A corpus of non-native english. ETS Research Re- port Series, 2013(2):i–15. Jan Bosman. 1987. Persuasive effects of political metaphors. Metaphor and Symbol, 2(2):97–113. Lynne Cameron. 2003. Metaphor in educational dis- course. A&C Black. The ﬁrst multitask learning is the utilization of out-of-domain data annotated for metaphor, albeit under a different annotation protocol, by using data from the other competition corpus; this ma- nipulation helped improve F1 scores for metaphor class on TOEFL test data, but not on VUA data. The second auxiliary task is utilization of a large in-domain corpus that we automatically tagged Verna Dankers, Marek Rei, Martha Lewis, and Eka- terina Shutova. 2019. Modelling the interplay of metaphor and emotion through multitask learning. In Proceedings of the 2019 Conference on Empirical Methods in Natural Language Processing and the 9th International Joint Conference on Natural Lan- guage Processing (EMNLP-IJCNLP), pages 2218– 2229. 241 Mark J Landau, Daniel Sullivan, and Jeff Green- berg. 2009. Evidence that self-relevant motives and metaphoric framing interact to inﬂuence polit- ical and social attitudes. Psychological Science, 20(11):1421–1427. Jacob Devlin, Ming-Wei Chang, Kenton Lee, and Kristina Toutanova. 2018. Bert: Pre-training of deep bidirectional transformers for language understand- ing. arXiv preprint arXiv:1810.04805. Michael Flor and Beata Beigman Klebanov. 2018. Catching idiomatic expressions in EFL essays. In Proceedings of the Workshop on Figurative Lan- guage Processing, pages 34–44, New Orleans, LA. Chee Wee Leong, Beata Beigman Klebanov, Chris Hamill, Egon Stemle, Rutuja Ubale, and Xianyang Chen. 2020. 8 Conclusion A report on the 2020 vua and toeﬂ metaphor detection shared task. In Proceedings of the Second Workshop on Figurative Language Pro- cessing, Seattle, WA. Michael Flor, Michael Fried, and Alla Rozovskaya. 2019. A benchmark corpus of English misspellings and a minimally-supervised model for spelling cor- rection. In Proceedings of the Fourteenth Workshop on Innovative Use of NLP for Building Educational Applications, page 76–86, Florence, Italy. Chee Wee Leong, Beata Beigman Klebanov, and Eka- terina Shutova. 2018. A report on the 2018 vua metaphor detection shared task. In Proceedings of the Workshop on Figurative Language Processing, pages 56–66. Michael Flor, Yoko Futagi, Melissa Lopez, and Matthew Mulholland. 2015. Patterns of mis- spellings in L2 and L1 English: a view from the ETS spelling corpus. Bergen Language and Linguistics Studies, 6. Jeannette Littlemore, Tina Krennmayr, James Turner, and Sarah Turner. 2013. An investigation into metaphor use at different levels of second language writing. Applied linguistics, 35(2):117–144. R. W. Gibbs and J. E. O’Brien. 1990. Idioms and men- tal imagery: The metaphorical motivation for id- iomatic meaning. Cognition, 36:35–68. Jeannette Littlemore and Graham Low. 2006. Metaphoric competence, second language learning, and communicative language ability. Applied linguistics, 27(2):268–294. Sam Glucksberg. 2001. Understanding Figurative Language: from metaphors to idioms. Oxford Uni- versity Press, New York, NY. Yinhan Liu, Myle Ott, Naman Goyal, Jingfei Du, Man- dar Joshi, Danqi Chen, Omer Levy, Mike Lewis, Luke Zettlemoyer, and Veselin Stoyanov. 2019. Roberta: A robustly optimized bert pretraining ap- proach. arXiv preprint arXiv:1907.11692. Sylviane Granger and Martin Wynne. 1999. Optimis- ing measures of lexical variation in EFL learner cor- pora. In Corpora Galore, pages 249–257, Amster- dam. Rodopi. E Dario Gutierrez, Guillermo Cecchi, Cheryl Corco- ran, and Philip Corlett. 2017. Using automated metaphor identiﬁcation to aid in detection and pre- diction of ﬁrst-episode schizophrenia. In Proceed- ings of the 2017 Conference on Empirical Methods in Natural Language Processing, pages 2923–2930. Rui Mao, Chenghua Lin, and Frank Guerin. 2019. End-to-end sequential metaphor identiﬁcation in- spired by linguistic theories. In Proceedings of the 57th Annual Meeting of the Association for Compu- tational Linguistics, pages 3888–3898. Jeremy Howard and Sebastian Ruder. 2018. Universal language model ﬁne-tuning for text classiﬁcation. arXiv preprint arXiv:1801.06146. Joanna Marhula, Justyna Polak, Maria Janicka, and Aleksander Wawer. 2019. Recognizing metaphor: how do non-experts and machines deal with a metaphor identiﬁcation task? In BOOK OF AB- STRACTS, page 70. Timour Igamberdiev and Hyopil Shin. 2018. 8 Conclusion Metaphor identiﬁcation with paragraph and word vectoriza- tion: An attention-based neural approach. In Pro- ceedings of the 32nd Paciﬁc Asia Conference on Language, Information and Computation. Katja Markert. 2019. Literature list ps/hs ss2019 ﬁgu- rative language resolution. Sujata S Kathpalia and Heah Lee Hah Carmel. 2011. Metaphorical competence in ESL student writing. RELC Journal, 42(3):273–290. Elena Mikhalkova, Nadezhda Ganzherli, Vladislav Maraev, Anna Glazkova, and Dmitriy Grigoriev. 2019. A comparison of algorithms for detection of “ﬁgurativeness” in metaphor, irony and puns. In In- ternational Conference on Analysis of Images, So- cial Networks and Texts, pages 186–192. Springer. Hossein Kaviani and Robabeh Hamedi. 2011. A quan- titative/qualitative study on metaphors used by per- sian depressed patients. Archives of Psychiatry and Psychotherapy, 4(5-13):110. Geoffrey Nunberg, Ivan A. Sag, and Thomas Wasow. 1994. Idioms. Language, 70(3):491–538. George Lakoff. 2010. Moral politics: How liberals and conservatives think. University of Chicago Press. Pragglejaz. 2007. MIP: A method for identifying metaphorically used words in discourse. Metaphor and symbol, 22(1):1–39. George Lakoff and Mark Johnson. 2008. Metaphors we live by. University of Chicago press. 242 Pranav Rajpurkar, Jian Zhang, Konstantin Lopyrev, and Percy Liang. 2016. Squad: 100,000+ questions for machine comprehension of text. arXiv preprint arXiv:1606.05250. Alla Rozovskaya and Dan Roth. 2016. Grammatical error correction: Machine translation and classiﬁers. In Proceedings of the 54th Annual Meeting of the As- sociation for Computational Linguistics (Volume 1: Long Papers), pages 2205–2215, Berlin, Germany. Association for Computational Linguistics. Carolyn Saund, Marion Roth, Mathieu Chollet, and Stacy Marsella. 2019. Multiple metaphors in metaphoric gesturing. In 2019 8th International Conference on Affective Computing and Intelligent Interaction (ACII), pages 524–530. IEEE. Gerard J Steen, Aletta G Dorst, J Berenike Herrmann, Anna Kaal, Tina Krennmayr, and Trijntje Pasma. 2010. A method for linguistic metaphor identiﬁca- tion: From MIP to MIPVU, volume 14. John Ben- jamins Publishing. Yu Sun, Shuohuan Wang, Yukun Li, Shikun Feng, Xuyi Chen, Han Zhang, Xin Tian, Danxiang Zhu, Hao Tian, and Hua Wu. 2019. Ernie: Enhanced rep- resentation through knowledge integration. arXiv preprint arXiv:1904.09223. Paul H Thibodeau and Lera Boroditsky. 2011. Metaphors we think with: The role of metaphor in reasoning. PloS one, 6(2):e16782. Ashish Vaswani, Noam Shazeer, Niki Parmar, Jakob Uszkoreit, Llion Jones, Aidan N Gomez, Łukasz Kaiser, and Illia Polosukhin. 2017. Attention is all you need. In Advances in neural information pro- cessing systems, pages 5998–6008. Tony Veale, Ekaterina Shutova, and Beata Beigman Klebanov. 2016. 8 Conclusion Metaphor: A com- putational perspective. Synthesis Lectures on Human Language Technologies, 9(1):1–160. Alex Wang, Amanpreet Singh, Julian Michael, Felix Hill, Omer Levy, and Samuel R Bowman. 2018. Glue: A multi-task benchmark and analysis platform for natural language understanding. arXiv preprint arXiv:1804.07461. Zhilin Yang, Zihang Dai, Yiming Yang, Jaime Car- bonell, Russ R Salakhutdinov, and Quoc V Le. 2019. Xlnet: Generalized autoregressive pretraining for language understanding. In Advances in neural in- formation processing systems, pages 5754–5764. Gerald Zaltman and Lindsay H Zaltman. 2008. Mar- keting metaphoria: What deep metaphors reveal about the minds of consumers. Harvard Business Press. Rowan Zellers, Yonatan Bisk, Roy Schwartz, and Yejin Choi. 2018. Swag: A large-scale adversarial dataset for grounded commonsense inference. arXiv preprint arXiv:1808.05326. 243
https://openalex.org/W3210256952	https://www.e3s-conferences.org/10.1051/e3sconf/202131202014/pdf	English	null	The choice of appropriate generator systems to enhance the renewable energy share in buildings. A comparison between PV-assisted heat pumps and biomass boilers	E3S web of conferences	2,021	cc-by	8,576	1. Introduction Because in recent years the issue related to the global warming caused by the use of fossil fuels have increased, it has become mandatory to expand the use of renewable energy source in the building sector. Indeed, currently the world housing account for 2,109,205 thousand tons of oil equivalent of consumed energy (21.2% of total energy consumption), with a correspondent amount of CO2 emission of 2033 Mton per year [1]. For this purpose, in Europe the promulgation of the Directive 28/2009/CE has promoted the use of renewable energy in the building sector to reduce the percentage of 40% on the final energy consumption that contributes for the 36% of GHG emissions [2]. Recently, in October 2020 the regulation amending the Energy Performance of Buildings Directive (2018/844/EU) has introduced new targets as part of the so-called “European Green Deal”, by presenting an action plan with concrete regulatory, financing and promoting measures to boost the penetration of renewable technologies in the building sector [3]. Among the several generation systems employed in residential buildings for heating and DHW production purposes, electric heat pumps assisted by PV generators and biomass boilers are able to allow a rational exploitation of the renewable sources [4]. Indeed, the firsts use the renewable energy contained in the air, water or ground to supply appropriate emitters for the air-conditioning and to produce Domestic Hot Water (DHW) by absorbing electricity from the grid, that could be properly limited by the self- consumed PV electricity [5,6]. Biomass boilers, instead, benefit of the neutral CO2 emission that allows for considering the majority of the embodied primary energy as fully renewable [7]. Consequently, it is interesting to compare these generations systems in terms of exploited renewable energy, in order to determine how they act on the limitation of the fossil primary energy and on the external environment. p y gy The comparison is justified by the fact that modern biomass boilers have performance indexes considerably lower than those detectable in heat pumps, therefore they could seem naturally penalized. On the other hand, in heat pumps the advantage could be largely counterbalanced by the electricity absorbed from the grid, whose conversion factor required to quantify the fossil primary energy share is almost 10 times greater than that employed in biomass boilers [8]. The choice of appropriate generator systems to enhance the renewable energy share in buildings. A comparison between PV-assisted heat pumps and biomass boilers Abstract. Low-energy buildings are generally equipped with generation systems driven by renewable sources. Regarding heating and DHW production, two choices appear appropriate: PV assisted heat pumps and biomass boilers. In this paper, by means of TRNSYS dynamic simulations, the non-renewable primary energy was determined for two buildings located in different climatic contexts by varying the PV size to consider the actual self-consumed electricity of commercial devices. Results showed that in cold climates biomass boilers are more suggested, especially in unfavourable climatic zones, whereas the COP of air- water heat pumps is strongly penalized by the outdoor temperatures and in many cases the self-consumed PV electricity does not limit the grid intervention adequately. However, in building with limited thermal energy demands and in favorable climates, suitable PV sizes make heat pumps more performant than biomass boilers. The same calculations were conducted with the quasi-steady approach, in accordance with the Italian building energy certification procedure, observing a favorable scenario in a heating plant equipped with a PV assisted heat pump because it assumes the renewable electricity entirely absorbed, while the accounting of the actual self-consumed share produces a greater demand of non-renewable energy. © 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/). https://doi.org/10.1051/e3sconf/202131202014 https://doi.org/10.1051/e3sconf/202131202014 E3S Web of Conferences 312, 02014 (2021) 76° Italian National Congress ATI 1. Introduction Moreover, when heat pumps use the outdoor air as thermal source, the absorbed electricity strongly depends on the external temperature levels [9], therefore the actual performances have to be evaluated in different climatic contexts. https://doi.org/10.1051/e3sconf/202131202014 E3S Web of Conferences 312, 02014 (2021) 76° Italian National Congress ATI A preliminary comparison between biomass boilers and heat pumps was conducted in [10] in which authors compared the generation systems exclusively in economic and environmental terms for small-detached houses located in Poland. They determined that the operating cost savings of the heat pump did not compensate the large installation costs in an acceptable period, moreover the CO2 emission level were larger than biomass boilers. In [11], heat pumps and biomass boilers were analyzed in terms of LCA, in order to compare these generation systems with the other available for tertiary buildings. Biomass boilers and other devices supplied by fossil fuels were compared in [12], whereas the limitations of air-water heat pumps in cold climate was deeply investigated in [13]. Some studies suggest to use rationally the rejected heat from biomass boilers as thermal source for heat pumps in hybrid systems [14], whereas no studies were found to compare the renewable primary energy share attainable in different plant configurations equipped with these devices. Therefore, regarding heating and DHW production, in this paper the generation systems were compared in terms of non-renewable primary energy by referring to two different buildings located in different climatic zones of the Italian territory. In particular, the buildings have different thermal energy requirements, whereas the PV size was varied in presence of heat pumps to quantify the benefits that could be achieved in terms of reduction of the electricity absorbed from the grid. Dynamic simulations of the building-plant systems were conducted in TRNSYS to determine in detail the electricity absorbed by a commercial air-water heat pump as function of the sources temperature and of the operation in part-load mode [15]. Furthermore, for the renewable electricity, it was assumed to give the priority to drive the generation systems, whereas the remaining part to supply internal appliances and the surpluses used to charge a thermal storage system or transferred to the grid. Regarding the biomass boilers, the primary energy was determined by varying the device efficiency as a linear function of the capacity ratio, in accordance with values listed in commercial datasheets. 1. Introduction In the considered climatic zones, the simulated buildings are the same from the geometrical point of view whereas the thermal characteristics of the dispersing elements were changed in function of heating degree-day (HDD). Specifically, the insulation thickness in opaque walls was increased whereas the thermal transmittance of the windowed surfaces was reduced with the HDD growth, in order to meet the national regulations in terms of minimum energy performance requirements for new buildings [8]. The same calculations were repeated by adopting the quasi-steady approach [16–18], because currently in Italy it is used for the building energy certification and often used as a reference to establish building energy performances, allowing also to quantify the deviances between the results provided by dynamic and simplified procedures. 2.1 Buildings description Energy evaluations were conducted by dynamic simulations carried out in TRNSYS on the detached houses of Fig. 1, with gross surface on the ground of 100 m² and net heated surfaces equal to 57.7 m² and 115.4 m² respectively for the single-storey and the double-storey building. The first structure represents a modular unit and the second building was designed by superimposing two modular units, so that the aspect ratio (ratio between the gross dispersing surface S and gross heated volume V) is equal to respectively 0.75 m-1 and 0.625 m-1. The first was simulated in TRNSYS with a single air node (ground floor), whereas for the second one two air nodes (ground and first floor) have been used and connected to the same generation system [19]. Both the buildings have the same Window to Wall Ratio (WWR) on each exposure, equal to 13%, 10% and 4% for North/South, East and West [20]. Internal gains (332 W per floor, whose 50% is radiative) [21,22] and natural ventilation (0.5 air-change per hour) were set in accordance to Italian standards. These structures, in fact, have been thermally designed in order to meet the minimal energy requirements regulated by the national legislation [8] In particular the following aspects were imposed: [8]. In particular, the following aspects were imposed: [8]. In particular, the following aspects were imposed: 8]. 2.1 Buildings description In particular, the following aspects were imposed: a) mean global thermal exchange coefficient of the envelope lower than a specific threshold, the latter defined as function of the building aspect ratio and the HDD; a) mean global thermal exchange coefficient of the envelope lower than a specific threshold, the latter defined as function of the building aspect ratio and the HDD; g p ; heating and cooling thermal requirements lower than the correspondent values determined for a referenc building with the same geometry but with different envelope thermal parameters; b) heating and cooling thermal requirements lower than the correspondent values d building with the same geometry but with different envelope thermal parameters; g g y p p c) mean seasonal global efficiency for the heating and the DHW systems greater than a limit value, d i d f i f h i d l d f h h f c) mean seasonal global efficiency for the heating and the DHW systems greater than a determined as function of heating and DHW plants and of the heat generators features; c) mean seasonal global efficiency for the heating and the DHW systems greater than a limit value, the latter determined as function of heating and DHW plants and of the heat generators features; d) global primary energy requirements, determined as sum of the correspondent terms for heating and DHW, lower than the correspondent value for the reference building including the share provided by renewable systems; p g g e) 50% of the domestic hot water produced by a renewable system; f) 2 kWp of installed PV per 100 m² of gross surface area on the ground. f) 2 kWp of installed PV per 100 m² of gross surface area on the ground. Because in the parametric study the number of HDD changes, the respect of the points a), b) and d) has imposed the variation of the insulation thickness (EPS, = 0.041 W/mK [23]) inside the dispersing opaque surfaces (vertical walls, ground floor and flat roof) and the change of the windows thermal transmittances (Uw) and of the normal solar factor (g┴), in accordance with the values listed in Tab. 1. It can be noted that the buildings were located in five different climatic zone, uniformly distanced by about 500-550 HDD. 2 https://doi.org/10.1051/e3sconf/202131202014 E3S Web of Conferences 312, 02014 (2021) 76° Italian National Congress ATI 76° Italian National Congress ATI Fig. 2.1 Buildings description 1- Perspective views of the single-storey and double-storey building involved in TRNSYS simulations Fig. 1- Perspective views of the single-storey and double-storey building involved in TRNSYS simulations Tab. 1 – Insulation thickness in dispersing opaque walls and windows thermal transmittance set in the simulated buildings, as function of the climatic zone Tab. 1 – Insulation thickness in dispersing opaque walls and windows thermal transmittance set in the simulated buildings, as function of the climatic zone EPS Insulation thickness [mm] Uw [W/m²K] and g┴ [-] HDD Ground floor Flat roof Vertical walls Windows 772 60 80 50 1.69 – 0.66 (low- double pane) 1317 60 90 100 1.69 – 0.66 (low- double pane) 1861 100 110 100 1.69 – 0.66 (low- double pane) 2394 110 140 130 1.40 – 0.70 (clear triple pane) 2897 110 140 130 1.40 – 0.70 (clear triple pane) 3445 120 160 160 1.1 – 0.62 (low- triple pane) The localities with the HDD listed in Tab. 1 have the position depicted in Fig. 2; it can be appreciated that these have more or less similar latitudes in order to attain similar PV producibility, between 1300-1400 kWh/kWp assuming a polycrystalline technology [24,25]. The PV generator is facing South and tilted of 30°C, made of modules with main electric data listed in Tab. 2. The size of the PV was varied between 2 kWp (required minimum installed peak power) to 6 kWp (to avoid three-phase installations). The remaining components of the PV plant have been considered in simplified way by setting a BOS (Balance of System) efficiency of 0.85 throughout the year. Regarding the DHW production, the required water flow rate was determined as function of the net plan surface, in accordance to Italian standard [17], obtaining 100 and 172 liters per day respectively for the single and the double-storey building, and scheduled at daily level as indicated in Tab. 3. For the weather data, TMYs were used and simulations were conducted for the whole year with a timestep of 0.125 h, setting a continuous functioning situation of the heating plant in order to compare the TRNSYS results with those provided by the quasi-steady procedure. Fig. 2 – Position of the considered localities considered for the buildings energy performance analysis HDD 772 HDD 1317 HDD 1861 HDD 2394 HDD 2897 HDD 3445 Fig. 2 – Position of the considered localities considered for the buildings energy performance analysis Tab. 2.1 Buildings description 2 – Main electric features of the polycrystalline PV panels considered in simulations Short-circuit current at STC 9.17 A Current at max power point in STC 8.80 A Open-circuit voltage in STC 35.17 V Voltage at max power point in STC 28.49 V Temperature coefficient of short-circuit current 0.0052 A/K Temperature coefficient of open-circuit voltage -0.0081 V/K Tab. 2 – Main electric features of the polycrystalline PV panels considered in simulations Tab. 2 – Main electric features of the polycrystalline PV panels considered in simulations Short-circuit current at STC 9.17 A Current at max power point in STC 8.80 A Open-circuit voltage in STC 35.17 V Voltage at max power point in STC 28.49 V Temperature coefficient of short-circuit current 0.0052 A/K Temperature coefficient of open-circuit voltage -0.0081 V/K 3 https://doi.org/10.1051/e3sconf/202131202014 E3S Web of Conferences 312, 02014 (2021) E3S Web of Conferences 312, 02014 (2021) 76° Italian National Congress ATI 76° Italian National Congress ATI Tab. 3 – Scheduled water flow rate (in liters per hour) requirements for the calculation of the annual DHW energy needs in the considered buildings DHW flow rate (l/h) 00:00- 07:00 07:00- 10:00 10:00- 12:00 12:00- 14:00 14:00- 18:00 18:00- 21:00 21:00- 00:00 Single-storey 0 12.5 0 12.5 0 12.5 0 Double-storey 0 21.5 0 21.5 0 21.5 0 Tab. 3 – Scheduled water flow rate (in liters per hour) requirements for the calculation of the annual DHW energy needs in the considered buildings Tab. 3 – Scheduled water flow rate (in liters per hour) requirements for the calculation of the annual DHW energy needs in the considered buildings DHW flow rate (l/h) 00:00- 07:00 07:00- 10:00 10:00- 12:00 12:00- 14:00 14:00- 18:00 18:00- 21:00 21:00- 00:00 Single-storey 0 12.5 0 12.5 0 12.5 0 Double-storey 0 21.5 0 21.5 0 21.5 0 2.2 Heating plant with the air-water heat pump The heating plant is equipped with a commercial and modulating air-water heat pump (AWHP), and the produced hot water supplies a radiant floor inside every thermal zone whose indoor temperature is regulated by a thermostat with a dead band of 1°C and a set-point of 20°C. The cut-off temperature of the heat pump is -20°C, with thermal power and the coefficient of performance trends showed in Fig. 4a as function of the outdoor air temperature, by setting the set-point water temperature to 45°C. Preliminary simulations have allowed for determining the size of the air-water heat pump, by choosing as nominal power the value with the highest frequency detected during the heating period. Thermal powers of about 4 kW and 8 kW respectively for the single and the double-storey building were determined, so two commercial air- water heat pumps, with a minimum modulation factor of 30%, were identified and simulated. Among the different climatic zones, it is worth noting that the nominal power values remain almost the same because the increase of the envelope insulation allowed for compensating the thermal losses detectable with the HDD growth, mantaining similar design loads. Radiant floors were implemented in TRNSYS with a pipe spacing of 25 cm, an internal diameter of 10 mm, embedded in a lightweight concrete layer thick 9 cm. The AWHP thermal plant was simulated by considering a vertical tank of 3 m³ as thermal storage system equipped with an electric resistance as backup, maintained at a set-point temperature of 40°C for a rational exploitation of the AWHP and to manage PV surpluses rationally (see Fig. 3). Indeed, the control system activates the heat pump also when heating loads are not required if the tank temperature is lower than the set-point and the PV electricity is available. The inlet temperature in the radiant floors was set constant to 35°C, by implementing a motorized 3-way valve that recirculate a fraction of the returning water flow rate from the same emitters. The latter was always retained supplied by the grid, in accordance with national regulation that hinders the electricity used by Joule effect to be covered by the PV production [8]. Fig. 2.2 Heating plant with the air-water heat pump 3 –Project implemented in the TRNSYS interface for the building-plant system with the AWHP It is worth noting that the TRNSYS model employed to simulate the AWHP determines the electric consumptions by considering the COP variation with the temperature sources, assuming a full load operation. Actually, the presence of the storage tank determines noticeable functioning times in part load mode, therefore the performance index is further modified when the heat pump capacity ratio (CR) is lower than minimum modulation factor (30% in accordance with datasheet). In order to consider this aspect, a further correction factor fc was introduced to determine an actual COP in part-load mode by means of the relation: 𝐶𝐶𝐶𝑓𝐶𝐶𝐶𝑡𝐶𝑖𝑖𝐶𝐶 Fig. 3 –Project implemented in the TRNSYS interface for the building-plant system with the AWHP Fig. 3 –Project implemented in the TRNSYS interface for the building-plant system with the AWHP It is worth noting that the TRNSYS model employed to simulate the AWHP determines the electric consumptions by considering the COP variation with the temperature sources, assuming a full load operation. Actually, the presence of the storage tank determines noticeable functioning times in part load mode, therefore the performance index is further modified when the heat pump capacity ratio (CR) is lower than minimum modulation factor (30% in accordance with datasheet). In order to consider this aspect, a further correction factor fc was introduced to determine an actual COP in part-load mode by means of the relation: 𝐶𝐶𝐶𝑓𝐶𝐶𝐶𝑡𝐶𝑖𝑖𝐶𝐶 𝐶𝐶𝐶𝐶𝐶𝐶𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟= 𝑓𝑓𝑐𝑐∙𝐶𝐶𝐶𝐶𝐶𝐶𝑁𝑁𝑁𝑁𝑁𝑁 (𝑡𝑡𝑜𝑜𝑜𝑜, 45°𝐶𝐶) 𝑖𝑖𝑖𝑖 𝐶𝐶𝐶𝐶< 30% 𝐶𝐶𝐶𝐶𝐶𝐶𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟= 𝑓𝑓𝑐𝑐∙𝐶𝐶𝐶𝐶𝐶𝐶𝑁𝑁𝑁𝑁𝑁𝑁 (𝑡𝑡𝑜𝑜𝑜𝑜, 45°𝐶𝐶) 𝑖𝑖𝑖𝑖 𝐶𝐶𝐶𝐶< 30% (1) 4 https://doi.org/10.1051/e3sconf/202131202014 E3S Web of Conferences 312, 02014 (2021) 76° Italian National Congress ATI in which the nominal COP was determined by TRNSYS as function of the outdoor air temperature (toa) and by the setting the supplied water flow rate at a temperature of 45 °C. The correction factor, instead, was calculated by a TRNSYS equation for every timestep t in accordance with the procedure described in the EN 14825 [25]: 𝐶𝐶𝑡 𝑓𝑓𝑐𝑐(𝑡𝑡) = 𝐶𝐶𝐶𝐶(𝑡𝑡) 1 −𝐶𝐶𝑐𝑐+ 𝐶𝐶𝑐𝑐∙𝐶𝐶𝐶𝐶(𝑡𝑡) (2) (2) in which Cc is the device degradation factor set to 0.9, as suggested by the standard when manufacturer does not provide pertinent information. 2.2 Heating plant with the air-water heat pump 4 – Trends of the thermal power and COP as function of the outdoor air temperature in nominal conditions for: a) AWH (Tcut,off=-20°C) b) WHHP (Tcut,off=-10°C) 2.2 Heating plant with the air-water heat pump Regarding the capacity ratio, it was calculated as: 𝑃 𝐶𝐶𝐶𝐶(𝑡𝑡) = 𝑃𝑃ℎ,𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟 𝑃𝑃ℎ,𝑁𝑁𝑁𝑁𝑁𝑁(𝑡𝑡𝑜𝑜𝑜𝑜, 45°𝐶𝐶) (3) (3) namely the real thermal power provided by the AHWP to the storage tank and the thermal power that the heat pump could produce in nominal condition when the outdoor air temperature value is tOA and the water flow rate temperature is 45°C. Therefore, Eq. (2) was implemented inside TRNSYS to determine the real COP in accordance to Eq. (1), and successively used to quantify the actual electric consumptions starting from the real provided thermal power. namely the real thermal power provided by the AHWP to the storage tank and the thermal power that the heat pump could produce in nominal condition when the outdoor air temperature value is tOA and the water flow rate temperature is 45°C. Therefore, Eq. (2) was implemented inside TRNSYS to determine the real COP in accordance to Eq. (1), and successively used to quantify the actual electric consumptions starting from the real provided thermal power. Regarding the DHW production, an independent water heater heat pump (WHHP) equipped with an electric booster and with performance depicted in Fig. 4b, assuming DHW at 55 °C, was simulated. Again, for the same reason discussed for the AWHP, the electric energy of the WHHP booster was considered completely absorbed from the grid. The correction of the COP described by Eq. (1) was not implemented, assuming a functioning always in full load mode. The produced renewable electricity was considered to operate the AWHP and WHHP electric demands, excluding backup and booster systems, and to drive the hydronic pumps. So, the global electric energy absorbed from the grid determined the non-renewable primary energy, by applying a conversion factor of 1.95 as indicated by Italian rules [8]. Fig. 4 – Trends of the thermal power and COP as function of the outdoor air temperature in nominal conditions for: a) AWHP (Tcut,off=-20°C) b) WHHP (Tcut,off=-10°C) 2.5 3.5 4.5 5.5 6.5 1.5 2.5 3.5 4.5 5.5 6.5 7.5 8.5 9.5 -20 -15 -10 -5 0 5 10 15 20 kW a) Tw=45 C Ph - Single-Story C 2 2.4 2.8 3.2 3.6 1 1.5 2 2.5 3 3.5 -10 -5 0 5 10 15 20 Ph COP kWth b) HWHP (TDHW=55 C) C Fig. 2.3 Heating plant with the biomass boiler Regarding the heating plant equipped with a boiler supplied by solid biomass, a commercial device with a burner working with a forced air system, supplies directly radiators by a water flow rate at a constant temperature of 80°C, in accordance with the plant scheme depicted in Fig. 5. Again, a zone thermostat controls the indoor air temperature with a dead band of 1 °C and a set-point of 20°C. The biomass boiler has a combined functioning, therefore the same device is used to provide heating and DHW, but exclusively in winter. In order to cover the DHW demand in summer and to respect the constrain to produce at least the 50% by means of a renewable system [8], the buildings were equipped with 4 m² of plane solar thermal collectors with selective surfaces, including a storage tank of 0.5 m³ operating by natural circulation. The building-plant system involves a 2 kWp of PV panels (minimum required peak power), whose electricity was used to meet auxiliary consumptions, mainly represented by the hydraulic pumps and the fan of the biomass boiler. In simulations, the energy input in the generation system was computed by considering a boiler efficiency depending on the capacity ratio, by means of the linear relation (determined by interpolating the datasheet values): 5 5 E3S Web of Conferences 312, 02014 (2021) 76° Italian National Congress ATI https://doi.org/10.1051/e3sconf/202131202014 𝜂𝜂𝑡𝑡ℎ= 0.0769 ∙𝐶𝐶𝐶𝐶+ 0.8431 (4) (4) that provides a value of 0.92 in nominal conditions (CR=1) and of 0.86 at the minimum modulation factor (CR=0.22). Regarding the fan consumptions, the absorbed electric power was determined by the formula: 𝑄𝐶𝐶𝐶𝐶 (5) 𝑄𝑄̇𝑒𝑒𝑒𝑒,𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏= −325.8 ∙𝐶𝐶𝐶𝐶2 + 730.8 ∙𝐶𝐶𝐶𝐶+ 15 (5) 𝑄𝑄̇𝑒𝑒𝑒𝑒,𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏= −325.8 ∙𝐶𝐶𝐶𝐶2 + 730.8 ∙𝐶𝐶𝐶𝐶+ 15 that produces an consumption of 420 W in nominal conditions, of 160 W at the minimum modulation factor and 15 W with a switched-off burner. The boiler capacity ratio was calculated as: 𝑃 𝐶𝐶𝐶𝐶(𝑡𝑡) = 𝑃𝑃𝑏𝑏,𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟 𝑃𝑃𝑏𝑏,𝑁𝑁𝑁𝑁𝑁𝑁 (3) (3) namely the ratio between the actual produced thermal power and that achievable in nominal condition operation. For the latter, design evaluations on the two simulated edifices have allowed to identify a nominal thermal power of 14 kW (assuming a suitable contemporary factor for heating and DHW) for the single-storey building and of 20 kW for the double-storey building. 2.3 Heating plant with the biomass boiler Finally, the non-renewable primary energy was computed by associating a conversion factor of 0.2 for the energy input of the biomass boiler and again of 1.95 for the electricity absorbed from the grid. Fig. 5 - Project implemented in the TRNSYS interface for the building-plant system with the biomass boiler Fig. 5 - Project implemented in the TRNSYS interface for the building-plant system with the biomass boiler 3. RESULTS In Fig. 6, with reference to the single-storey building (S/V=0.75), the yearly non-renewable primary energy per square meter of heated net surface (non-renewable Energy Performance index, EP [kWh/m²]) is depicted as a function of the considered HDD and, only for the heating plant equipped with the AWHP, of the PV size. It is clear that, despite the augment of the envelope insulation level with the HDD growth, the fossil share increases with the number of HDD due to the prolonged employment of the generation system and the unfavorable outdoor air temperatures. However, for the single-storey building, the performances of the AHWP assisted by the PV generator are always better than the biomass boiler, indicating that the magnitude of the electricity absorbed from the grid is not in such amount to be affected excessively by the penalizing conversion factor in fossil primary energy. Furthermore, it is worth noting that the increase of the PV size does not produce an evident limitation of the primary fossil demand. Indeed, the precise and instantaneous electric energy balance carried out by TRNSYS has highlighted that, in every climatic zone and in continuos functioning conditions, the thermal storage system and its control strategy are not sufficient to relieve the mismatching between PV production and thermal load request. In particular, greater heating loads are required prevalently at night, with a quick discharge of the storage system, and consequent activation of the AWHP that uses electricity from the grid. The slight reduction of the fossil primary energy share with the PV size growth is essentially due to the wider availability of renewable electricity during the AHWP and the WHHP operation that in some circumstances allows for limiting the electricity absorbed from the grid. Fig. 6 – Single-storey building: fossil primary energy per square meter of net heated surface as function of the HDD number and for the different heating plant configurations 0 5 10 15 20 25 30 35 772 1317 1861 2394 2909 3445 Non-renewable primary energy demands : S/V=0.75 Biomass Boiler + 2 kWp HP+2 kWp HP+3 kWp HP+4 kWp HP+5 kWp HP+6 kWp HDD [ C/year] [kWh/m²] Non-renewable primary energy demands : S/V=0.75 Fig. 2.4 Non-renewable energy share with the quasi-steady approach Currently, the Italian building energy standard requires the calculation of the heating energy requirements, which are determined at a monthly level by the quasi-steady approach that uses the concept of heat gain utilization factor and a mono-capacitive building model to take into account the transient phenomena. Successively, the primary energy requirements are calculated by applying 5 efficiency values that consider the thermal losses in the heating plant (production, storage, distribution, regulation and emission) by separating the fossil and renewable shares with an apposite conversion factor. A similar approach is used for the estimation of the primary energy demand for DHW production. When heat pumps are involved as generation system, the energy input (electric energy) is determined with the quasi- dynamic BIM method [27], whereas the thermal efficiency of the boiler at monthly level is calculated by referring to three different operation mode (full load, partial load, and null load) in accordance to the Directive 92/42/CEE procedure [28]. Finally, PV production is determined by introducing a correction coefficient to consider the module ventilation and the effects due to the cell's thermal drift, as well as BOS efficiency. Furthermore, the energy standard considers the whole PV producibility entirely absorbed by the technical plants (except for the devices operating with the sole Joule effect) by assuming the employment of an ideal electric storage system. 6 6 6 https://doi.org/10.1051/e3sconf/202131202014 E3S Web of Conferences 312, 02014 (2021) 76° Italian National Congress ATI 3. RESULTS 7 – Double-storey building: fossil primary energy per square meter of net heated surface as a function of the HDD number and for the different heating plant configurations The same evaluations conducted on the double-storey building were repeated by adopting the quasi-steady calculation, in accordance to the standard currently employed in Italy for the building energy certification (Fig. 8). The differences in results are obviously due to the different approaches used to determine the thermal and primary energy requirements, however, it is clear the evident simplification to consider the whole PV producibility completely used to satisfy heating and DHW requirements in the quasi-steady approach. Indeed, this hypothesis produces fossil primary energies that reduce linearly with the PV size and, for the location with 3445 HDD, the AWHP assisted by a 6 kWp PV generator offers an EP lower than the one detected for the biomass boiler, in contradictory with the results highlighted in Fig. 7. Therefore, considering the whole PV production instead of the actual self-consumed PV electricity produces a very different scenarios in presence of large PV surfaces that do not reflect the actual energy balance of the building-plant system. p g gy g p y The results reported in Fig. 6 and Fig. 7 give information concerning the energy performance worsening of the heat generator systems prevalently due to the outdoor temperature reduction observable with the HDD growth, whereas the same results are independent of the PV performances due to similar producibility among the considered locations. For this reason, in order to define the role of the PV producibility on the AWHP performances, further dynamic simulations have been carried out by considering two localities with different latitudes but similar HDD. In particular, the EPs were determined as a function of the PV size for the location with 3445 HDD considered in the previous evaluations (Lat. 41 °N) and another location with 3452 HDD but with a higher latitude (about 45°N). The results showed in Fig. 9 for the double-storey building demonstrate the important role of the PV production, with an EP worsening detected when moving toward higher latitudes. In particular, these results are due to the limited availability of solar radiation that determines deviances of non-renewable energy ranging between 5.7 and 8.2 kWh/m²; whereas at a seasonal level the available aerothermal energy can be considered similar due to the equivalent HDD of the two localities. 3. RESULTS 6 – Single-storey building: fossil primary energy per square meter of net heated surface as function of the HDD number and for the different heating plant configurations A different situation was detected instead for the double-storey building (S/V=0.625), which requires more thermal energy to maintain the indoor set-point temperature and for the DHW production. In this case, the employment of the biomass boiler can be suggested rather than other heating plant configurations equipped with the AHWP, as depicted in Fig. 7. In particular, it appears that the biomass boiler becomes more performant when the HDD number increases; this is due to the higher electric consumption of the heat pumps for the greater thermal requirements in colder zones [29]. Therefore, in such severe climates, it is difficult for the assisted AWHP to compete with the biomass boiler. On the contrary, from the same graph, it is possible to appreciate that larger PV peak powers allow for counterbalancing the gap with the biomass boiler when the climatic zone is favorable. Nevertheless, this aspect is not always true because the non- renewable primary energy depends strongly on the combination of weather data and building-plant features, as demonstrated by the location with 1317 HDD in which the heating plant equipped with the AWHP can be used rationally with every PV size. Nevertheless, the detected trends suggest to prefer biomass boilers especially in the locations with high HDD, whereas in more favorable climatic contexts the gap can be recovered with AWHP by properly increasing the PV size. It is worth noting that, in comparison with the single-storey building, the smaller EPs are due to the smaller building aspect ratio: in fact, the doubling of the heated volume does not correspond with the doubling of the dispersing surface. 7 7 https://doi.org/10.1051/e3sconf/202131202014 E3S Web of Conferences 312, 02014 (2021) 76° Italian National Congress ATI Fig. 7 – Double-storey building: fossil primary energy per square meter of net heated surface as a function of the HDD number and for the different heating plant configurations 0 5 10 15 20 25 30 35 772 1317 1861 2394 2909 3445 Non-renewable primary energy demands : S/V=0.625 Biomass Boiler + 2 kWp HP+2 kWp HP+3 kWp HP+4 kWp HP+5 kWp HP+6 kWp HDD [ C/year] [kWh/m²] Non-renewable primary energy demands : S/V=0.625 Fig. 3. RESULTS For the same reason, the PV cell thermal drift effects are comparable and do not contribute to the PV producibility, affected exclusively by the limited availability of solar radiation. It is worth noting that the gap tends to reduce with the PV size growth, by confirming that the differences in terms of solar radiation can be recovered by increasing the caption surface because the probability to meet PV production and thermal load request increases. 8 8 https://doi.org/10.1051/e3sconf/202131202014 E3S Web of Conferences 312, 02014 (2021) 76° Italian National Congress ATI 76° Italian National Congress ATI Fig. 8 – Double-storey building: fossil primary energy per square meter of net heated surface as a function of the HDD number and for the different heating plant configurations determined with the quasi-steady model 0 5 10 15 20 25 30 35 40 45 50 772 1317 1861 2394 2909 3445 Non-renewable primary energy demands : S/V=0.625 Biomass Boiler + 2 kWp HP+2 kWp HP+3 kWp HP+4 kWp HP+5 kWp HP+6 kWp HDD [ C/year] [kWh/m²] Non-renewable primary energy demands : S/V=0.625 Fig. 8 – Double-storey building: fossil primary energy per square meter of net heated surface as a function of the HDD number and for the different heating plant configurations determined with the quasi-steady model Fig. 9 – Double-storey building: fossil primary energy per square meter of net heated surface as a function of the PV size for two localities with similar HDD 24 26 28 30 32 34 36 38 40 2 3 4 5 6 Non-renewable primary energy detected with HP+PV (S/V=0.625) HDD_3445_Lat.41.0°N HDD_3452_Lat.44.8°N [kWh/m²] [kWp] 24 26 28 30 32 34 36 38 40 2 3 4 5 6 Non-renewable primary energy detected with HP+PV (S/V=0.625) HDD_3445_Lat.41.0°N HDD_3452_Lat.44.8°N [kWh/m²] [kWp] Non-renewable primary energy detected with HP+PV (S/V=0.625) Non-renewable primary energy detected with HP+PV (S/V=0.625) Fig. 9 – Double-storey building: fossil primary energy per square meter of net heated surface as a function of the PV size for two localities with similar HDD 9 https://doi.org/10.1051/e3sconf/202131202014 E3S Web of Conferences 312, 02014 (2021) 76° Italian National Congress ATI The analysis conducted has demonstrated that the role of the self-consumed PV electricity is decisive to attain the share of primary renewable energy employed to satisfy heating and DHW production requirements. Nevertheless, the increase of the self-consumed PV electricity share does not mean necessarily a preference for the heat pump systems. Fig. 3. RESULTS 10 shows the monthly values of self-consumed PV electricity (in kWhel per square meter of heated surface), with the correspondent percentage referred to the PV production for the single and double-storey buildings for the locality with 3445 HDD assuming a PV peak power of 6kWp. It can be noticed a greater self-consumed share for the double-storey building, especially in the coldest months, concluding that the greater operation of the heat pumps systems led to better exploitation of the PV generator. Nevertheless, in light of the results in Fig. 7, still, the renewable primary energy shares are worse than those detected for the biomass boiler, meaning that the remaining part absorbed from the grid assumes a prevalent role in the fossil primary consumptions forming. Therefore, it is confirmed that heat pump systems are preferable in presence of contained energy consumptions, even with the thermal storage system, and consequently in severe climatic zones is mandatory to limit as possible the thermal energy requirements in buildings. Moreover, it can be appreciated how the self-consumed PV electricity ranges between a maximum value of 48.6% in December and minimum values around 0.5-1% in summer, assuming a mean seasonal percentage of 9.4% and 26% for the single and the double- storey respectively. y p y In order to investigate how to increase the self-consumed PV electricity, in Fig. 11 for the single-storey building equipped with the heat pumps and for the locality with 1861 HDD, the non-renewable energy shares were determined by assuming a heating plant functioning closer to the actual conditions. In particular, in such locality, the indoor set-point temperature can be 20 °C for a maximum of 12 hours per day, as listed in the schedule of Tab.4, whereas in the remaining part of the day an attenuation operation mode was simulated by supplying the radiant floors at a temperature of 25 °C. Furthermore, a PV peak-power of 6 kWp was set. Tab. 4 – Time slots of the indoor set-point temperatures for the calculation of the primary energy requirements the considered buildings assuming a non-continuous operating regime of the heating plant Tab. 4 – Time slots of the indoor set-point temperatures for the calculation of the primary energy requirements in the considered buildings assuming a non-continuous operating regime of the heating plant Tab. 3. RESULTS 4 – Time slots of the indoor set-point temperatures for the calculation of the primary energy requirements in the considered buildings assuming a non-continuous operating regime of the heating plant Indoor set-point temperature [°C] 00:00-06:00 06:00- 10:00 10:00-12:00 12:00- 15:00 15:00-17:00 17:00- 22:00 22:00-00:00 Attenuated 20°C Attenuated 20°C Attenuated 20°C Attenuated Fig. 10 – Self-consumed PV electricity and percentage value referred to the monthly producibility for the two buildings located in the locality with 3445 HDD equipped with a PV generator of 6 kWp 20.2% 16.8% 13.3% 8.8% 1.7% 0.5% 4.9% 11.0% 22.6% 35.0% 32.8% 16.4% 6.2% 1.0% 5.0% 21.1% 48.6% 0 30 60 90 120 150 180 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec HDD 3445 Single-Story Double-Story kWhel/m² 20.2% 16.8% 13.3% 8.8% 1.7% 0.5% 4.9% 11.0% 22.6% 35.0% 32.8% 16.4% 6.2% 1.0% 5.0% 21.1% 48.6% 0 30 60 90 120 150 180 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec HDD 3445 Single-Story Double-Story kWhel/m² Fig. 10 – Self-consumed PV electricity and percentage value referred to the monthly producibility for the two buildings located in the locality with 3445 HDD equipped with a PV generator of 6 kWp It is worth noting that the new management of the heating plant, and especially of the storage system, leads to better use of the PV generator because the mean seasonal percentage of the self-consumed electric energy increases from 9.7% of the base case up to 19.9% of the actual scenario. This allows for achieving a better Energy Performance index that 10 https://doi.org/10.1051/e3sconf/202131202014 E3S Web of Conferences 312, 02014 (2021) 76° Italian National Congress ATI reduces from 14.71 kWh/m² of the base case down to 12.33 kWh/m². Nevertheless, despite the self-consumed PV electricity doubled, the non-renewable primary energy improvement is limited because it reduces of about 137 kWh. This means that the building energy performances continue to be strongly penalized by the high conversion factor in fossil primary energy despite the electricity absorbed from the grid was reduced. reduces from 14.71 kWh/m² of the base case down to 12.33 kWh/m². Nevertheless, despite the self-consumed PV electricity doubled, the non-renewable primary energy improvement is limited because it reduces of about 137 kWh. This means that the building energy performances continue to be strongly penalized by the high conversion factor in fossil primary energy despite the electricity absorbed from the grid was reduced. Fig. 3. RESULTS 11 – Self-consumed PV electricity and percentage value referred to the monthly producibility for the single-storey buildings located in the locality with 1861 HDD equipped with a PV generator of 6 kWp and assuming a continuous and an attenuation operation regime of the heating plant 10.67% 11.39% 10.52% 4.02% 0.43% 0.45% 0.36% 0.39% 0.42% 0.45% 9.24% 11.41% 24.61% 20.81% 19.63% 11.89% 20.13% 23.97% 0 30 60 90 120 150 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec HDD 1861 Continuous Operation regime Attenuated operation regime kWhel/m² 10.67% 11.39% 10.52% 4.02% 0.43% 0.45% 0.36% 0.39% 0.42% 0.45% 9.24% 11.41% 24.61% 20.81% 19.63% 11.89% 20.13% 23.97% 0 30 60 90 120 150 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec HDD 1861 Continuous Operation regime Attenuated operation regime kWhel/m² HDD 1861 Fig. 11 – Self-consumed PV electricity and percentage value referred to the monthly producibility for the single-storey buildings located in the locality with 1861 HDD equipped with a PV generator of 6 kWp and assuming a continuous and an attenuation operation regime of the heating plant CONCLUSION In this paper, the non-renewable shares of two different heat generator systems, represented by a boiler supplied by solid biomass and connected to high-temperature emitters and a PV assisted air-water heat pump supplying radiant floors, were calculated in a transient regime by considering different HDD and by varying the size of the PV generator. The non- renewable share includes also the primary energy required for the DHW production, obtained in a combined manner with the biomass boiler and with an independent water heater heat pump in presence of PV assisted heat pumps. Finally, two typologies of buildings were simulated and designed in order to meet the minimum energy requirements required by Italian legislation. The same calculation has been repeated adopting the quasi-steady model in order to highlight the deviances when the energy certification procedure is carried out The results can be summarized as follow: deviances when the energy certification procedure is carried out. The results can be summarized as follow: - assisted air-water heat pumps offer better performances in buildings with reduced thermal energy needs because the electricity from grid is limited. Consequently, the share of fossil primary energy is reduced despite the employment of the highest conversion factor among the energy carriers; gy p - assisted air-water heat pumps offer better performances in buildings with reduced thermal energy needs because the electricity from grid is limited. Consequently, the share of fossil primary energy is reduced despite the employment of the highest conversion factor among the energy carriers; - with the increase of the building thermal energy demands and with the HDD growth, biomass boilers produce better results because the electric energy from the grid becomes significant, furthermore the non-renewable share is further penalized by low outdoor air temperatures that penalize the COP increasing the electric consumptions; - whit limited HDD, the gap between biomass boilers and heat pumps can be recovered easily by increasing the PV generator size; - the results obtained with the quasi-steady approach show a different scenario because the PV electricity is considered completely absorbed from heating and DHW devices. CONCLUSION Dynamic simulations, instead, showed an evident winter mismatching between solar radiation availability and thermal load request, still difficult to solve by adopting thermal energy storage systems, even if managed by suitable control strategies; - the results obtained with the quasi-steady approach show a different scenario because the PV electricity is considered completely absorbed from heating and DHW devices. Dynamic simulations, instead, showed an evident winter mismatching between solar radiation availability and thermal load request, still difficult to solve by adopting thermal energy storage systems, even if managed by suitable control strategies; - at parity of climatic zone, the renewable shares determined with heat pumps are strongly affected by the locality latitude due to the different PV producibility. By augmenting the caption surface, a self-consumed PV electricity growth was observed because the probability to meet PV production and thermal load request increases. - at parity of climatic zone, the renewable shares determined with heat pumps are strongly affected by the locality latitude due to the different PV producibility. By augmenting the caption surface, a self-consumed PV electricity growth was observed because the probability to meet PV production and thermal load request increases. - at parity of climatic zone, the renewable shares determined with heat pumps are strongly affected by the locality latitude due to the different PV producibility. By augmenting the caption surface, a self-consumed PV electricity growth was observed because the probability to meet PV production and thermal load request increases. - by assuming continuous functioning condition and despite the priority of the PV production was given to the generation systems, it was difficult to assess PV self-consumed electricity greater than 50%. Nevertheless, these by assuming continuous functioning condition and despite the priority of the PV production was given to the generation systems, it was difficult to assess PV self-consumed electricity greater than 50%. Nevertheless, these percentages improve with the thermal energy requirements increase and with the PV size growth. by assuming continuous functioning condition and despite the priority of the PV production was given to the generation systems, it was difficult to assess PV self-consumed electricity greater than 50%. Nevertheless, these percentages improve with the thermal energy requirements increase and with the PV size growth. References 1. I. E. A. IEA, Data and Statistics, Https://Www.Iea.Org/Data-and-Statistics, Accessed on 15th June 2021 (2021) 2. European Commission, Directive 28/2009 of the European Parliament and of the Council of 23 April 2009on the Promotion of the Use of Energy from Renewable Sources and Amending and Subsequently Repealing Directives 2001/77/EC and 2003/30/EC (2009), p. L 140/16-62 3. European Commission, Directive (EU) 2018/844 of the European Parliament and of the Council of 30 May 2018 Amending Directive 2010/31/EU on the Energy Performance of Buildings and Directive 2012/27/EU on Energy Efficiency (2018), pp. 1–17 4. N. Aste, P. Caputo, C. Del Pero, G. Ferla, H. E. Huerto-Cardenas, F. Leonforte, and A. Miglioli, Energy 206, (2020) 5. N. Arcuri, R. Bruno, and C. Carpino, in 2018 IEEE Int. Conf. Environ. Electr. Eng. 2018 IEEE Ind. Commer. Power Syst. Eur. (EEEIC / I&CPS Eur. (IEEE, 2018), pp. 1–6, doi:10.1109/EEEIC.2018.8493492 7. M. Carpio, M. Zamorano, and M. Costa, Energy Build. 66, 732 (2013) 7. M. Carpio, M. Zamorano, and M. Costa, Energy Build. 66, 732 (2013) Italian iterministerial decree 26th June 2015: Application of calculation methods for energy performance an definition of minimum building requirements, Off. Gazzette Ital. Repub. N°39 15th July 2015 1 (2015) 9. R. Bruno, F. Nicoletti, G. Cuconati, S. Perrella, and D. Cirone, Energies 13, (2020) 10. T. Olkowski, S. Lipiński, and A. Olȩdzka, E3S Web Conf. 19, (2017) 11. J. A. Lozano Miralles, R. López García, J. M. Palomar Carnicero, and F. J. R. Martínez, Renew. Energy 152, 1439 (2020) 12. M. J. Stolarski, M. Krzyzaniak, K. Warmiński, and D. Niksa, Energy Convers. Manag. 121, 71 (2016) 13. A. Saari, T. Kalamees, J. Jokisalo, R. Michelsson, K. Alanne, and J. Kurnitski, Appl. Energy 92, 76 (2012) pp gy ( 14. B. Hebenstreit, R. Schnetzinger, R. Ohnmacht, E. Höftberger, J. Lundgren, W. Haslinger, and A. Biomass and Bioenergy 71, 12 (2014) 15. VV.AA., TRNSYS Libr. Vol. 4 Math. Ref. Sol. Energy Lab. Univ. Wisconsin-Madison, USA (2016) 16 Italian Unification Institution UNI TS 11300 1: Energy Performance of Buildings Part 1: Evaluation of 16. Italian Unification Institution, UNI TS 11300-1: Energy Performance of Buildings - Part 1: Evaluation of Energy Need Far Space Heating and Cooling (2014) , gy f f g f gy Need Far Space Heating and Cooling (2014) Need Far Space Heating and Cooling (2014) Italian Unification Institution, UNI 11300-2: Energy Performances of Buildings- Part 2- Evaluation of Prima 17. CONCLUSION 11 https://doi.org/10.1051/e3sconf/202131202014 E3S Web of Conferences 312, 02014 (2021) 76° Italian National Congress ATI E3S Web of Conferences 312, 02014 (2021) 76° Italian National Congress ATI - in attenuated functioning conditions, the PV self-consumed electricity increases due to a better exploitation of the thermal storage tank. Globally, in cold climates biomass are recommended to increase the shareof the renewable primary energy. PV assisted heat pumps are negatively affected by lower COP detectable in localities with high HDD, which requires a larger use of the electricity from the grid, especially at night. Since localities with high HDD numbers usually do not require cooling, the advantage to use the same heat pumps in summer fails. Conversely, climatic zones with high HDD are generally located in mountainous regions where there is a wider availability of solid biomass, making these devices able to exploit “local” fuel. The thermal storage systems managed by appropriate control strategies can improve the heat pump performance in favorable climates, however, a suitable PV size has to be installed. Nevertheless, to contrast the issues related to the aleatory nature of the renewable source and the misalignment with the building thermal loads, a great role could be assumed by the energy communities assuming the building-plant system able to rationalize the energy produced in-situ by exchanging thermal and/or electric energies with other structures, making heat pumps more competitive. References Italian Unification Institution, UNI 11300-2: Energy Performances of Buildings- Part 2- Evaluation of Primary Energy Need and of System Efficiency for Space Heating, Domestic Hot Water Production, Ventilation and Lighting for Non-Residential Buildings (2014), pp. 37–40 gy Need and of System Efficiency for Space Heating, Energy Need and of System Efficiency for Space Heating, Domestic Hot Water Production, Ventilation and Lighting for Non-Residential Buildings (2014), pp. 37–40 ghting for Non-Residential Buildings (2014), pp. 37–40 g g f g ( ) pp Italian Unification Institution, UNI TS 11300-4. Energy Performances of Building: Renewable Energy and Oth Generation Systems for Space Heating and Domestic Hot Water Production (2016) alian Unification Institution, UNI TS 11300-4. Energy Pe 18. Italian Unification Institution, UNI TS 11300-4. Energy Performances of Building: Rene Generation Systems for Space Heating and Domestic Hot Water Production (2016) Generation Systems for Space Heating and Domestic Hot Water Production (2016) 19. TRANSSOLAR & Energietechnik GmbH, TRNSYS Doc. (2016) 20. J. Szyszka, Energies 13(3), (2020) 21. B. Gil, S. Rosiek, J. Kaspersky, M. Nems, A. Nems, ECOS 2019 - Proceedings of the 32nd International Conference on Efficiency, Cost, Optimization, Simulation and Environmental Impact of Energy Systems, pp. 2559-2570, 22. G. Evola and L. Marletta, Energy and Buildings 65, pp. 448-457 (2013) 23. M. Cucumo, V. Ferraro, D. Kaliakatsos, M. Mele, Energy and Buildings 158, pp. 677-683, (2018) gy g pp 24. R. Bruno, P. Bevilacqua, L. Longo, and N. Arcuri, in Energy Procedia (2015) R. Bruno, P. Bevilacqua, L. Longo, and N. Arcuri, in E 25. P. Bevilacqua, R. Bruno, and N. Arcuri, Energy 195, 116950 (2020) 26. EN 14825:2018, Air conditioners, liquid chilling packages and heat pumps, with electrically driven compressors, for space heating and cooling - Testing and rating at part load conditions and calculation of seasonal performance 27. M. Dongellini, C. Naldi, G.L. Morini, Applied Thermal engineering 90, (2015) 27. M. Dongellini, C. Naldi, G.L. Morini, Applied Thermal engineering 90, (2015) 28. European Commission, Directive 92/42/EEC of the European Parliament and of the Council efficiencyrequirementsfornewhot-waterboilersfiredwithliquidorgaseousfuel, p. L 167/17 European Commission, Directive 92/42/EEC of the European Parliament and of the Cou efficiencyrequirementsfornewhot-waterboilersfiredwithliquidorgaseousfuel, p. L 167/17 29. S. Korol, N. Shushunova, T. Shushunova, MATEC Web of Conferences, Volume 193, Article Number 05075 29. S. Korol, N. Shushunova, T. Shushunova, MATEC Web of Conferences, Volume 193, Article Number 05075 12
https://openalex.org/W3047781482	https://veterinaryresearch.biomedcentral.com/track/pdf/10.1186/s13567-020-00825-6	English	null	C. perfringens challenge reduces matrix metalloproteinase activity in the jejunal mucosa of Eimeria-infected broiler chickens	Veterinary research	2,020	cc-by	6,861	© The Author(s) 2020. 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://creativeco mmons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Matrix metalloproteinases (MMPs) play an important role in intestinal extracellular matrix homeostasis. An overexpres- sion of MMPs results in tissue destruction and local inflammation and has been associated with multiple inflamma- tory diseases. These host proteases might also be important in tissue damage caused by infectious agents, such as in intestinal damage in Clostridium perfringens-induced avian necrotic enteritis (NE). The aim of the present study was to elucidate the effect of a C. perfringens infection on the MMP activity in the small intestine of birds with a pre-disposing coccidial infection to obtain a more thorough understanding of the pathogenesis of NE. For this purpose, the gelati- nolytic activity present in jejunal tissue of Eimeria infected birds which were challenged with either a pathogenic C. perfringens type G strain or a commensal C. perfringens type A strain was analyzed using substrate zymography. The results show that infection of broilers with Eimeria and different C. perfringens strains, independent of their patho- genicity, decreases the expression of a 40–45 kDa host collagenase in the jejunum, as compared to the expression in Eimeria-infected control birds. It was also shown that the expression of 2 MMPs with molecular weights of approxi- mately 50–60 and 60–70 kDa was significantly lower in necrotic tissue as compared to the activity in macroscopically healthy tissue adjacent to the lesion. These results indicate that host collagenases are not elicited by the C. perfringens infection for permeabilizing the host mucosa to allow penetration of the NetB toxin in Eimeria infected broilers. Keywords: broiler, Clostridium perfringens, Eimeria, gut, matrix metalloproteinases, necrotic enteritis initiates damage is hitherto not determined. It is cur- rently believed that NetB is not involved in early disease pathogenesis and targets deeper layers of the intestinal mucosa rather than superficial structures [6]. Therefore, initial breakdown or permeabilization of the mucosal layer might be required in order for NetB to exert its action. This permeabilization can be either a direct effect on the host tissues of C. perfringens virulence factors other than NetB, or it can be a host response elicited indirectly by C. perfringens. C. perfringens challenge reduces matrix metalloproteinase activity in the jejunal mucosa of Eimeria‑infected broiler chickens Lore Van Damme1 , Natasja Cox1, Chana Callens1, Freddy Haesebrouck1 , Michelle Dargatz2, Richard Ducatelle1 , Filip Van Immerseel1* and Evy Goossens1 Richard Ducatelle1 , Filip Van Immerseel1* and Evy Goossens1 Introduction Necrotic Enteritis (NE) is one of the most common and financially devastating bacterial diseases in the modern poultry industry [1, 2]. The disease is caused by netB- positive C. perfringens strains and is characterized by severe necrosis and inflammation of the small intestine [3–5]. The pore-forming toxin NetB is an essential viru- lence factor in the development of necrotic ulcers which are typical for NE in broilers [4]. Where and how NetB Overexpression of host collagenases has been associ- ated with intestinal tissue destruction in several gastro- intestinal inflammatory diseases in humans, including Crohn’s disease, ulcerative colitis and several forms of Correspondence: Filip.VanImmerseel@UGent.be 1 Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium Full list of author information is available at the end of the article Correspondence: Filip.VanImmerseel@UGent.be 1 Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium Full list of author information is available at the end of the article *Correspondence: Filip.VanImmerseel@UGent.be 1 Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium Full list of author information is available at the end of the article Van Damme et al. Vet Res (2020) 51:100 https://doi.org/10.1186/s13567-020-00825-6 Van Damme et al. Vet Res (2020) 51:100 https://doi.org/10.1186/s13567-020-00825-6 Open Access Necrotic enteritis trialh The in vivo NE model used in this trial was based on a previously described study [27]. In short, 1-day-old unvaccinated Ross 308 broilers were randomly allo- cated to 3 different treatment groups with 27 birds/pen (4 replicate pens challenged with strain CP56, 1 pen challenged with the commensal C. perfringens strain JIR4857 and 1 pen with control birds, which were not challenged with C. perfringens). All broilers were fed a wheat/rye-based (43%/7.5%) diet supplemented with soybean meal as a protein source. From day 17 on, the diet was altered with fishmeal (30%) replacing the soy bean meal as a protein source. These diets contain high levels of proteins and non-starch polysaccharides which predispose chicken to the development of NE. Mild immunosuppression was induced by adminis- tering the commercial Nobilis Gumboro D78 vaccine, containing attenuated infectious bursal disease virus, on days 4 and 9 (MSD Animal Health). On days 14 and 16, all animals received a tenfold dose of live attenuated Eimeria vaccines, respectively Hipracox (containing 5 Eimeria species: E. tenella, E. acervulina, E. maxima, E. praecox and E. mitis) (Hipra, Melle, Belgium) and Paracox-8 (containing 7 Eimeria species: E. acervulina, E.brunetti, E. maxima, E. mitis, E. necatrix, E. prae- cox and E. tenella) (MSD Animal Health, Brussels, Bel- gium), to induce a predisposing coccidial infection. On days 18, 19 and 20, birds in the first group were chal- lenged with approximately 5 × 108 CFU of netB-pos- itive C. perfringens strain 56. Birds in the second and third group received the same predisposing factors as the first group but were inoculated on day 18, 19 and 20 with respectively 5 × 108 CFU of netB-negative C. perfringens strain JIR4857 or sterile bacterial growth medium. On day 21, all animals were euthanized. NE is a multifactorial disease and outbreaks of NE are almost exclusively found to co-occur or follow upon a predisposing coccidial infection [17–21]. During coc- cidiosis, Eimeria spp. colonize the intestine and destroy epithelial cells as a consequence of the intracellular stages of their lifecycle [20]. Eimeria induces leakage of serum proteins in the lumen of the gut and increases mucus secretion. Both mucus and serum proteins are rich sources of nutrients which C. perfringens can exploit for proliferation and toxin production [22, 23]. In addition to providing nutrients for C. © The Author(s) 2020. 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://creativeco mmons.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. Van Damme et al. Vet Res (2020) 51:100 Page 2 of 8 overnight at 37 °C in brain heart infusion broth (Oxoid, Basingstoke, UK). gastro-intestinal cancer [7–11]. These host collagenases might also be important in tissue damage caused by infectious agents, such as in intestinal damage in NE. Indeed, collagen is widely distributed throughout the gastrointestinal tract and is an integral component of the connective tissue and basement membrane of the intestinal mucosal layer [12–14]. Disruption of this structural protein may result in loss of tissue integrity and allow penetration of bacterial toxins to deeper tis- sues and so contribute to subsequent tissue necrosis [15]. Furthermore, Olkowski and colleagues showed an increased MMP activity in necrotic tissue of broilers which were challenged with NE-producing C. perfrin- gens strains as compared to tissue of non-challenged birds [16]. Necrotic enteritis trialh perfringens, a predisposing Eimeria infection also has tremendous effects on the intestinal tissue itself by shortening of the villi [24], inducing inflammation [25], reducing the activity of digestive enzymes and disrupting tissue integrity [20]. In order to fully characterize the events that contribute to disease pathogenesis, even under experimental settings, this predisposing coccidial infec- tion should be taken into account.h Therefore, the aim of the present study was to focus on the host response to C. perfringens, trying to eluci- date the effect of a C. perfringens infection on the host MMP activity in the small intestine of birds in the pres- ence of a coccidial infection, known to be a crucial pre- disposing factor for NE. At necropsy, NE severity was evaluated by scoring lesions in the small intestine (duodenum, jejunum, ileum) as previously described by Keyburn et al. [28] as follows: score 0 = no lesions, score 2 = focal necro- sis or ulcerations (1–5 foci), score 3 = focal necrosis or ulcerations (6–15 foci), score 4 = focal necrosis or ulcerations (≥ 16 foci), score 5 = patches of necro- sis of 2–3 cm long, score 6 = diffuse necrosis. Birds with a lesion score of 2 or more were considered NE positive. From each scoring class, jejunal lesion tissue (except for scoring class 0) and macroscopically unaf- fected tissue, 1 cm adjacent to the lesion of 5 different animals was collected. The samples that were collected from NE-affected birds were derived from 2 different pens (5 samples were collected from pen 1, 4 samples Zymography Th l l The gelatinolytic activity in the intestinal tissue lysates collected from the NE trial was analyzed using gelatin zymography. Briefly, aliquots containing 60 µg of pro- tein of each sample were mixed with 2× loading buffer (0.5 M Tris–HCl pH 6.8, 20% glycerol, 4% SDS, a pinch of bromophenol blue) and separated on 8% SDS page containing 0.1% gelatin under non-denaturing condi- tions. After separation, the gel was incubated with renaturing buffer (2.5% Triton X-100, 30 min, room temperature) to remove SDS from the gel. This allows the separated enzymes in the gel to renature and auto- activate. Subsequently, the gel was washed with devel- oping buffer (150 mM NaCl, 5 mM CaCl2, 0.05% NaN3 and 50 mM Tris–HCl buffer pH 7.5) and incubated with fresh developing buffer under continuous shaking at 37 °C for 18 h. Afterwards, the gel was stained for 1 h with Coomassie Brilliant Blue (Sigma-Aldrich, Overi- jse, Belgium) and destained for 20 min with destaining solution [40% methanol (v/v), 10% acetic acid (v/v)]. Activity of gelatin-degrading enzymes is visible as clear colorless bands against a blue background. Gels were scanned using a GS-800 calibrated densitometer and the approximate molecular weight of the present bands and their intensities were determined using the Quan- tity One software (BioRad, Hercules, CA, USA). The gelatinolytic activity of each band was calculated as 100 OD/mm2 and is described in arbitrary units (AU). Six dif- ferent gelatinolytic bands were observed in the jejunal tissue. The lowest and highest bands were not quanti- fied due to distortion and oversaturation of the bands. j j Broilers of the control group which were not challenged with C. perfringens but received all predisposing factors including Eimeria infection did not develop NE (n = 26). Additionally, no necrotic lesions were seen in the intes- tine of birds challenged with a commensal C. perfrin- gens type A strain JIR4857 (n = 27). Only C. perfringens type G strain CP56 was able to induce necrotic lesions in Eimeria infected birds. Lesions were observed in 85.05% (91/107) of the birds challenged with this netB–positive C. perfringens strain. In NE positive chickens the lesions could be observed in all segments of the small intestine, but were most severe in the jejunum as compared to the duodenum or ileum (p < 0.0001). Zymography Th l l A significant association was observed between the small intestinal segment and the development of necrotic lesions, with the jejunum being 17.89 to 22.9 times more likely to develop necrotic lesions as compared to the duodenum or ileum, respec- tively. Moderate enteric lesions (score 3 and 4) were dominant, being present in 68.13% (62/91) of the affected birds. Lesion score distributions for each segment of the small intestine are summarized in Table 1. Bacterial strains and culture conditionsf Bacterial strains and culture conditionsf Two different C. perfringens strains were used. Strain 56 was originally isolated from the gut of a NE-affected broiler and is characterized as a toxinotype G strain. The strain is able to express NetB toxin and is routinely used to induce NE in an in vivo infection model in broilers [26]. Strain JIR4857 is characterized as a com- mensal toxinotype A strain and cannot express NetB toxin. The inoculum for the oral infection of chickens was prepared by culturing both strains anaerobically Van Damme et al. Vet Res (2020) 51:100 Van Damme et al. Vet Res (2020) 51:100 Page 3 of 8 Page 3 of 8 were collected from pen 2). The samples were stored at − 20 °C. Statistical analysisf were collected from pen 2). The samples were stored at − 20 °C. Differences in the occurrence of NE lesions between the different segments of the small intestine of C. per- fringens type G challenged broilers were evaluated by a binary logistic regression analysis using R statisti- cal software. All other analyses and calculations were performed using GraphPad Prism software (version 5.03, San Diego, CA, USA). A paired Student’s t test was used to compare gelatinase activity in necrotic and macroscopically healthy tissue derived from the same bird. For all other data, two-group experiments were analyzed with unpaired Student’s t test, whereas one-way ANOVA followed by Tukey’s test was used for comparison of more than two groups. Results are pre- sented as mean ± SEM. Analyses were performed with 95% confidence intervals and significance was deter- mined as p ≤ 0.05. Protein extraction Proteins were extracted from the intestinal tissue using mechanical lysis. Briefly, ± 27 mg of intestinal tissue was mixed with 400 µL TBS-1% NP-40 [50 mM Tris/HCl, pH 8.0, 150 mM NaCl and 1% (v/v) NP-40 supplemented with EDTA-free protease inhibitor cocktail (Complete, Roche, Mannheim, Germany)]. The mixture was homog- enized by grinding with 2.3 mm zircon/silica and 3.2 mm stainless steel beads (BioSpec Products, Bartlesville, OK, USA) in a bead beater (twice for 1.5 min, 22.5 Hz; Tis- sueLyser) with a 30 s interval between shakings. Subse- quently, samples were centrifuged for 10 min at 8000 × g and the supernatant was transferred to a new tube. Pro- tein concentration was measured using the BCA protein assay (Thermo Fisher Scientific, Merelbeke, Belgium) and samples were stored at − 20 °C until further analysis. Clostridium perfringens challenge decreases the activity of a specific host collagenase On day 18, 19 and 20, birds were challenged with either a pathogenic C. perfringens type G strains (CP56), a commensal C. perfringens type A strain (JIR4857) or sterile bacterial culture medium (control). a NE lesion scoring of the small intestine was performed as previously described by Keyburn et al. [28]. a NE lesion scoring of the small intestine was performed as previously described by Keyburn et al. [28]. b NE positive = lesion score ≥ 2. c Average lesion score of NE positive birds. Data represents mean ± SE. Average lesion score of NE positive birds. Data represents mean ± SE. d Means within the same column with different superscripts differ significantly (p < 0.05); A, B binary logistic regression analysis or C, D one-way ANOVA followed by k ’ l i l i d Means within the same column with different superscripts differ significantly (p < 0.05); A, B binary logistic regression analysis or C, D one Tukey’s multiple comparisons test. challenged with either the C. perfringens type A or type G strain. Furthermore, C. perfringens challenge of Eimeria infected birds did not affect the activity of the other gelatinolytic enzymes in the jejunal tissue (Table 2). As NE is a complex disease, not all animals are equally affected by NE in experimental trials and field outbreaks. In the current trial, a minority of the birds challenged with the pathogenic, C. perfringens type G strain did not develop macroscopic lesions of NE. When focusing on these birds that did not develop NE (i.e. birds with- out macroscopic intestinal necrosis), significantly lower gelatinase activity of a 40–45 kDa protein was meas- ured in the jejunal tissue of birds challenged with a C. perfringens type A or type G strain as compared to con- trol chickens receiving only the predisposing Eimeria infection (p-value respectively 0.0317 and 0.0098, Table 2). No difference in gelatin-degrading activity of this 40–45 kDa protein was observed between birds MMP activity is decreased within NE lesionsf In order to assess whether the difference in disease sus- ceptibility between birds within the C. perfringens type G strain challenged group might be related to an altered reaction of host MMP activity on the C. perfringens chal- lenge, the gelatinolytic activity of macroscopically healthy jejunal tissue of birds that developed NE was compared to birds that did not develop NE, but were infected with the same C. perfringens type G strain. No differences in Table 2 Gelatinolytic activity of jejunal samples of Eimeria and C. perfringens type A- or type G-infected broilers Control: Eimeria infected birds, Type A and Type G: Eimeria infected birds challenged with respectively a commensal C. perfringens type A strain (JIR4857) or pathogenic C. perfringens type G strain (CP56). NE: birds without (No) or with (Yes) necrotic enteritis. Gelatinolytic activity of the digested spots is expressed in arbitrary units = 100 OD/mm2 . Data represents mean ± SE. One-way ANOVA followed by Tukey’s multiple comparisons test was used to compare the means of the NE = No groups. Values within the same column with different superscripts differ significantly (p < 0.05). Group NE Gelatinolytic enzyme, approximate MW ~ 40–45 kDa ~ 50–60 kDa ~ 60–70 kDa ~ 75–85 kDa Control (n = 5) No 1.10 ± 0.054A 1.19 ± 0.072 1.03 ± 0.039 1.09 ± 0.077 Type A (n = 5) No 0.81 ± 0.083B 1.10 ± 0.15 1.05 ± 0.14 1.09 ± 0.18 Type G (n = 5) No 0.74 ± 0.051B 1.01 ± 0.086 0.93 ± 0.063 0.89 ± 0.087 Type G (n = 9) Yes 0.65 ± 0.030 0.98 ± 0.045 0.86 ± 0.051 0.74 ± 0.045 Table 2 Gelatinolytic activity of jejunal samples of Eimeria and C. perfringens type A- or type G-infected broilers Group NE Gelatinolytic enzyme, approximate MW 40 45 kDa 50 60 kDa 60 70 kDa 75 8 atinolytic activity of jejunal samples of Eimeria and C. perfringens type A- or type G-infected broilers Table 2 Gelatinolytic activity of jejunal samples of Eimeria and C. perfringens type A- or type G-i Control: Eimeria infected birds, Type A and Type G: Eimeria infected birds challenged with respectively a commensal C. perfringens type A strain (JIR4857) or pathogenic C. perfringens type G strain (CP56). NE: birds without (No) or with (Yes) necrotic enteritis. Gelatinolytic activity of the digested spots is expressed in arbitrary units = 100 OD/mm2 . Clostridium perfringens challenge decreases the activity of a specific host collagenase As the NE lesions occurred most frequently in the jeju- num of broilers, we focused on this section of the small intestine to study the effect of C. perfringens challenge on host MMP activity in Eimeria infected birds. The gelatinolytic activity present in jejunal tissue from broil- ers in the different treatment groups of the NE trial was assessed by gelatin zymography. Van Damme et al. Vet Res (2020) 51:100 Page 4 of 8 Table 1 Score distribution of necrotic enteritis lesions in the small intestine of 21-day old broilers All birds were challenged with a ten-fold dose of a live attenuated Eimeria vaccine at day 14 and 16 to induce a predisposing coccidiosis infection. On day 18, 19 and 20, birds were challenged with either a pathogenic C. perfringens type G strains (CP56), a commensal C. perfringens type A strain (JIR4857) or sterile bacterial culture medium (control). a NE lesion scoring of the small intestine was performed as previously described by Keyburn et al. [28]. b NE positive=lesion score≥2 Group Segment Lesion scorea NE positiveb Average lesion scorec 0 2 3 4 5 6 Control Duodenum 26 0 0 0 0 0 0% (0/26) – Jejunum 26 0 0 0 0 0 0% (0/26) – Ileum 26 0 0 0 0 0 0% (0/26) – Overall 0% (0/26) – Type A (JIR 4857) Duodenum 27 0 0 0 0 0 0% (0/27) – Jejunum 27 0 0 0 0 0 0% (0/27) – Ileum 27 0 0 0 0 0 0% (0/27) – Overall 0% (0/27) – Type G (CP56) Duodenum 85 11 6 4 1 0 20.56% (22/107)A 2.77C Jejunum 19 7 15 46 18 2 82.24% (88/107)B 3.92D Ileum 89 6 5 6 0 1 16.82% (18/107)A 3.17C Overall 85.05% (91/107) 3.91 Table 1 Score distribution of necrotic enteritis lesions in the small intestine of 21-day old broilers All birds were challenged with a ten-fold dose of a live attenuated Eimeria vaccine at day 14 and 16 to induce a predisposing coccidiosis infection. On day 18, 19 and 20, birds were challenged with either a pathogenic C. perfringens type G strains (CP56), a commensal C. perfringens type A strain (JIR4857) or sterile bacterial culture medium (control). All birds were challenged with a ten-fold dose of a live attenuated Eimeria vaccine at day 14 and 16 to induce a predisposing coccidiosis infection. MMP activity is decreased within NE lesionsf Data represents mean ± SE. One-way ANOVA followed by Tukey’s multiple comparisons test was used to compare the means of the NE = No groups. Values within the same column with different superscripts differ significantly (p < 0.05). Van Damme et al. Vet Res (2020) 51:100 Van Damme et al. Vet Res (2020) 51:100 Page 5 of 8 MMP activities were observed in macroscopically unaf- fected jejunal tissue of birds suffering from NE as com- pared to unaffected birds (Table 2). MMP activities were observed in macroscopically unaf- fected jejunal tissue of birds suffering from NE as com- pared to unaffected birds (Table 2). MMP2 which has a MW of ~ 62 kDa) was significantly lower in tissue derived from the lesions as compared to macroscopically unaffected tissue adjacent to the lesions (respectively p = 0.0107 and 0.0341). No dif- ference in activity of the 40–45 kDa or 75–85 kDa enzymes was observed (Figure 1). MMP2 which has a MW of ~ 62 kDa) was significantly lower in tissue derived from the lesions as compared to macroscopically unaffected tissue adjacent to the lesions (respectively p = 0.0107 and 0.0341). No dif- ference in activity of the 40–45 kDa or 75–85 kDa enzymes was observed (Figure 1). MMP2 which has a MW of ~ 62 kDa) was significantly lower in tissue derived from the lesions as compared to macroscopically unaffected tissue adjacent to the lesions (respectively p = 0.0107 and 0.0341). No dif- ference in activity of the 40–45 kDa or 75–85 kDa enzymes was observed (Figure 1). f Additionally, to assess the involvement of host MMPs in lesion development, the MMP activity within the necrotic lesions was compared to the activity in mac- roscopically unaffected tissue adjacent to the lesions. The activity of two MMPs with a molecular weight of approximately 50–60 and 60–70 kDa (presumably Figure 1 Gelatinolytic activity in jejunal tissue of necrotic enteritis affected birds. Lesions and macroscopically unaffected jejunal tissue 1 cm adjacent to the lesion was derived from broilers receiving a predisposing Eimeria infection, followed by a pathogenic C. perfringens type G strain (CP56) challenged. Gelatinolytic activity was analyzed by gelatin zymography and quantification of the digested spots is expressed in arbitrary units = 100 OD/mm2. = p ≤ 0.05 (Paired Student’s t-test). Figure 1 Gelatinolytic activity in jejunal tissue of necrotic enteritis affected birds. Discussion Nev- ertheless, our study indicates the importance of using an experimental NE model which resembles the reality in animal farming as close as possible as different models can lead to unexpected contradictory results. As expected, only the netB-positive C. perfringens type G strain was able to induce NE. Postmortem examina- tions of NE affected birds showed that necrotic lesions are more prevalent and severe in the jejunum compared to other segments of the small intestine. The distribu- tion of intestinal lesions in the present infection study is in accordance with older studies of both laboratory con- signments and field cases of NE [18]. Why this segment of the small intestine is more prone to develop lesions is hitherto unknown. y In conclusion, C. perfringens challenge reduced the MMP activity in the jejunal tissue of Eimeria infected broilers. These results indicate that host collagenases are not elicited by the C. perfringens infection for per- meabilizing the host mucosa to allow penetration of the NetB toxin in Eimeria infected broilers. This is in con- trast to the previous reported increase of MMP activity in the necrotic lesions of C. perfringens challenged birds in the absence of a predisposing Eimeria infection. Fur- ther studies are needed to fully characterize the effect of Eimeria challenge, as well as the combined challenge of Eimeria and C. perfringens, on specific MMP activ- ity in the broiler intestine, as compared to uninfected healthy controls. One interesting topic for future work includes the identification of the differential expressed host MMPs that were observed in this study. Clostridial challenge of Eimeria infected birds signifi- cantly reduced the gelatinolytic activity in the broiler jejunum as compared to unchallenged Eimeria infected control birds. Indeed, even when no macroscopic lesions were observed, challenge with either a non- pathogenic type A strain or a pathogenic C. perfringens type G strain both resulted in reduced activity of a spe- cific intestinal collagenase with a molecular weight of 40-45 kDa. Moreover, the activity of two larger MMPs was significantly reduced (an unknown MMP with a MW of 50-60 kDa and a 60-70 kDa MMP, presumably MMP2) in necrotic tissue as compared to the activity in macroscopically healthy tissue adjacent to the lesion. Discussion important role in the occurrence and severity of out- breaks of NE and is commonly used as a predisposing factor in experimental NE models [17]. Therefore, in the present study, a necrotic enteritis challenge model was used that includes a predisposing Eimeria infection caused by administering an overdose of commercially available coccidial vaccines which is followed by the administration of C. perfringens for 3 consecutive days.f MMPs play an important role in degrading compo- nents of the extracellular matrix and basement mem- brane [29]. In addition to ECM degradation, MMPs are involved in multiple physiological processes, like innate and adaptive immunity and inflammation, by regulat- ing the release and activation of cytokines, growth fac- tors, antibiotic peptides and other bioactive molecules [30, 31]. Expression of MMPs is tightly regulated by a complex process of enzyme synthesis, secretion, activa- tion, and inhibition, which results in low basal levels of these enzymes in healthy tissue [32]. Still, an overexpres- sion of host collagenases results in tissue destruction and local inflammation and has been associated with multi- ple inflammatory diseases and pathological processes [33]. In this study we investigated whether challenge of broiler chickens with either a pathogenic, netB-positive or a non-pathogenic, netB-negative C. perfringens strain affects the jejunal expression of host-derived MMPs using an experimental model of NE involving a predis- posing Eimeria infection. Park et al. already demonstrated a differential expres- sion of various genes involved in innate immunity when broilers were co-infected with Eimeria and C. perfrin- gens as compared to challenge with either pathogen alone, suggesting that the host inflammatory response is fundamentally different during dual infection [34]. In the current study, we assessed the effect of C. perfrin- gens challenge on host MMP activity, in the presence of a predisposing Eimeria infection. The contradictory findings between the current study and previous results from Olkowski et al. indicate that primary infection with Eimeria might increase the collagenase activity, whereas subsequent C. perfringens might dampen this response again. However, no reports are available studying the effect of Eimeria infection on the collagenase activity in the small intestine. Also in the current study no conclu- sions can be drawn on the effect of single Eimeria infec- tion on host MMP activity, as no unchallenged birds or birds infected with C. perfringens alone were used. MMP activity is decreased within NE lesionsf Lesions and macroscopically unaffected jejunal tissue 1 cm adjacent to the lesion was derived from broilers receiving a predisposing Eimeria infection, followed by a pathogenic C. perfringens type G strain (CP56) challenged Gelatinolytic activity was analyzed by gelatin zymography and quantification of the digested spots is expressed in arbitrary d bi d L d ll ff d l Figure 1 Gelatinolytic activity in jejunal tissue of necrotic enteritis affected birds. Lesions and macroscopically unaffected jejunal tissue 1 cm adjacent to the lesion was derived from broilers receiving a predisposing Eimeria infection, followed by a pathogenic C. perfringens type G strain (CP56) challenged. Gelatinolytic activity was analyzed by gelatin zymography and quantification of the digested spots is expressed in arbitrary units = 100 OD/mm2. = p ≤ 0.05 (Paired Student’s t-test). Figure 1 Gelatinolytic activity in jejunal tissue of necrotic enteritis affected birds. Lesions and macroscopically unaffected jejunal tissue 1 cm adjacent to the lesion was derived from broilers receiving a predisposing Eimeria infection, followed by a pathogenic C. perfringens type G strain (CP56) challenged. Gelatinolytic activity was analyzed by gelatin zymography and quantification of the digested spots is expressed in arbitrary units = 100 OD/mm2. = p ≤ 0.05 (Paired Student’s t-test). Van Damme et al. Vet Res (2020) 51:100 Van Damme et al. Vet Res (2020) 51:100 Page 6 of 8 Page 6 of 8 Abbreviations NE: necrotic enteritis; AU: arbitrary units; MMP: matrix metalloproteinase. Discussion This is in contrast to previous findings from Olkowski et al., where a significant increase of MMP activity was observed in necrotic tissue as compared to the activity in healthy tissue from unchallenged control birds [16]. However, it should be noted that no comparison with unaffected tissue from C. perfringens challenged birds was made. Furthermore, Olkowski et al. induced NE by two oral C. perfringens inoculations without a pre- ceding Eimeria infection [16]. Yet, coccidiosis plays an Abbreviations Van Damme et al. Vet Res (2020) 51:100 Page 7 of 8 Van Damme et al. Vet Res (2020) 51:100 Competing interestsl No potential conflict of interest was reported by the authors. 21. Williams RB, Marshall RN, La Ragione RM, Catchpole J (2003) A new method for the experimental production of necrotic enteritis and its use for studies on the relationships between necrotic enteritis, coccidiosis and anticoccidial vaccination of chickens. Parasitol Res 90:19–26 Acknowledgements 10. O’Sullivan S, Gilmer JF, Medina C (2015) Matrix metalloproteinases in inflammatory bowel disease: an update. Mediators Inflamm 2015:964131 https://doi.org/10.1155/2015/964131 g The authors are thankful for the assistance of the Ph.D. students, post-docs and scientific staff of the Department of Pathology, Bacteriology and Avian Diseases during the conduct of the necrotic enteritis in vivo trial. 11. Verma S, Kesh K, Ganguly N, Jana S, Swarnakar S (2014) Matrix metallo- proteinases and gastrointestinal cancers: impacts of dietary antioxidants. World J Biol Chem 5:355–376 Consent to publish Not applicable. Porter RE (1998) Bacterial enteritides of poultry. Poult Sci 77:1159–1 20. Williams RB (2005) Intercurrent coccidiosis and necrotic enteritis of chickens: rational, integrated disease management by maintenance of gut integrity. Avian Pathol 34:159–180 Author details 1 1 Department of Pathology, Bacteriology and Avian Diseases, Faculty of Vet- erinary Medicine, Ghent University, Merelbeke, Belgium. 2 Division Nutrition & Care‑Animal Nutrition, Evonik Operations GmbH, Halle, Westfalen 33790, Germany. 22. Collier CT, Hofacre CL, Payne AM, Anderson DB, Kaiser P, Mackie RI, Gaskins HR (2008) Coccidia-induced mucogenesis promotes the onset of necrotic enteritis by supporting Clostridium perfringens growth. Vet Immunol Immunopathol 122:104–115 Received: 4 June 2020 Accepted: 28 July 2020 23. Van Immerseel FV, De Buck J, Pasmans F, Huyghebaert G, Haesebrouck F, Ducatelle R (2004) Clostridium perfringens in poultry: an emerging threat for animal and public health. Avian Pathol 33:537–549 24. Kettunen H, Tiihonen K, Peuranen S, Saarinen MT, Remus JC (2001) Die- tary betaine accumulates in the liver and intestinal tissue and stabilizes the intestinal epithelial structure in healthy and coccidia-infected broiler chicks. Comp Biochem Physiol A: Mol Integr Physiol 130:759–769 Availability of data and materials All data are available on request. Availability of data and materials All data are available on request. All data are available on request. 16. Olkowski AA, Wojnarowicz C, Chirino-Trejo M, Laarveld B, Sawicki G (2008) Sub-clinical necrotic enteritis in broiler chickens: novel etiological consid- eration based on ultra-structural and molecular changes in the intestinal tissue. Res Vet Sci 85:543–553 Authors’ contributions 12. Frantz C, Stewart KM, Weaver VM (2010) The extracellular matrix at a glance. J Cell Sci 123:4195–4200 Study design: LVD, RD, FVI, EG; Animal experiments: LVD, NC, MD, CC, EG; in vitro experiments: LVD, NC, CC; Preparation of the manuscript: LVD, FH, RD, FVI, EG; All authors offered a critical review of the manuscript. All authors read and approved the final manuscript. 13. Graham MF, Diegelmann RF, Elson CO, Lindblad WJ, Gotschalk N, Gay S, Gay R (1988) Collagen content and types in the intestinal strictures of Crohn’s disease. Gastroenterology 94:257–265 Ethical approval and consent to participate All experimental procedures involving animals were approved by the ethical committee of the Faculties of Veterinary Medicine and Bioscience Engineering of Ghent University (EC2017/44). Consent to participate is not applicable. 17. Al-Sheikhly F, Al-Saieg A (1980) Role of coccidia in the occurrence of necrotic enteritis of chickens. Avian Dis 24:324–333 18. Long JR, Pettit JR, Barnum DA (1974) Necrotic enteritis in broiler chickens II. Pathology and proposed pathogenesis. Can J Comp Med 38:467–474 References 1. Skinner JT, Bauer S, Young V, Pauling G, Wilson J (2010) An economic analysis of the impact of subclinical (mild) necrotic enteritis in broiler chickens. Avian Dis 54:1237–1240 1. Skinner JT, Bauer S, Young V, Pauling G, Wilson J (2010) An economic analysis of the impact of subclinical (mild) necrotic enteritis in broiler chickens. Avian Dis 54:1237–1240 p y g y 25. Yun CH, Lillehoj HS, Lillehoj EP (2000) Intestinal immune responses to coccidiosis. Dev Comp Immunol 24:303–324 25. Yun CH, Lillehoj HS, Lillehoj EP (2000) Intestinal immune 25. Yun CH, Lillehoj HS, Lillehoj EP (2000) Intestina coccidiosis. Dev Comp Immunol 24:303–324 coccidiosis. Dev Comp Immunol 24:303–324 2. Wade B, Keyburn A (2015) The true cost of necrotic enteritis. World Poult 31:16–17 2. Wade B, Keyburn A (2015) The true cost of necrotic enteritis. World Poult 31:16–17 26. Timbermont L, Lanckriet A, Gholamiandehkordi AR, Pasmans F, Martel A, Haesebrouck F, Ducatelle R, Van Immerseel F (2009) Origin of Clostridium perfringens isolates determines the ability to induce necrotic enteritis in broilers. Comp Immunol Microbiol Infect Dis 32:503–512 3. Cooper KK, Songer JG (2009) Necrotic enteritis in chickens: a paradigm of enteric infection by Clostridium perfringens type A. Anaerobe 15:55–60 3. Cooper KK, Songer JG (2009) Necrotic enteritis in chickens: a paradigm of enteric infection by Clostridium perfringens type A. Anaerobe 15:55–60 4. Keyburn AL, Boyce JD, Vaz P, Bannam TL, Ford ME, Parker D, Di Rubbo A, Rood JI, Moore RJ (2008) NetB, a new toxin that is associated with avian necrotic enteritis caused by Clostridium perfringens. PLoS Pathog. https:// doi.org/10.1371/journal.ppat.0040026 27. Gholamiandehkordi AR, Timbermont L, Lanckriet A, Van Den Broeck W, Pedersen K, Dewulf J, Pasmans F, Haesebrouck F, Ducatelle R, Van Immerseel F (2007) Quantification of gut lesions in a subclinical necrotic enteritis model. Avian Pathol 36:375–382 5. Olkowski A, Wojnarowicz C, Chirino-Trejo M, Drew M (2006) Responses of broiler chickens orally challenged with Clostridium perfringens isolated from field cases of necrotic enteritis. Res Vet Sci 81:99–108 28. Keyburn AL, Sheedy SA, Ford ME, Williamson MM, Awad MM, Rood JI, Moore RJ (2006) Alpha-toxin of Clostridium perfringens is not an essential virulence factor in necrotic enteritis in chickens. Infect Immun 74:6496–6500 i 6. Parreira VR, Russell K, Athanasiadou S, Prescott JF (2016) Comparative transcriptome analysis by RNAseq of necrotic enteritis Clostridium perfrin- gens during in vivo colonization and in vitro conditions. BMC Microbiol 16:186. https://doi.org/10.1186/s12866-016-0792-6 i 6. Funding G 14. Verbeke S, Gotteland M, Fernandez M, Bremer J, Rios G, Brunser O (2002) Basement membrane and connective tissue proteins in intestinal mucosa of patients with coeliac disease. J Clin Pathol 55:440–445 g EG is supported by the Research Foundation Flanders (FWO) under Grant Number [12W8919N]. The presented work was funded by a Grant from Evonik Nutrition and Care. 15. Harrington DJ (1996) Bacterial collagenases and collagen-degrading enzymes and their potential role in human disease. Infect Immun 64:1885–1891 34. Park SS, Lillehoj HS, Allen PC, Park DW, FitzCoy S, Bautista DA, Lillehoj EP (2008) Immunopathology and cytokine responses in broiler chickens coinfected with Eimeria maxima and Clostridium perfringens with the use of an animal model of necrotic enteritis. Avian Dis 52:14–22 Van Damme et al. Vet Res (2020) 51:100 References Parreira VR, Russell K, Athanasiadou S, Prescott JF (2016) Comparative transcriptome analysis by RNAseq of necrotic enteritis Clostridium perfrin- gens during in vivo colonization and in vitro conditions. BMC Microbiol 16:186. https://doi.org/10.1186/s12866-016-0792-6 29. Fowlkes JL, Thrailkill KM, Serra DM, Suzuki K, Nagase H (1995) Matrix met- alloproteinases as insulin-like growth factor binding protein-degrading proteinases. Prog Growth Factor Res 6:255–263 7. Kirkegaard T, Hansen A, Bruun E, Brynskov J (2004) Expression and locali- sation of matrix metalloproteinases and their natural inhibitors in fistulae of patients with Crohn’s disease. Gut 53:701–709 30. Nagase H, Visse R, Murphy G (2006) Structure and function of matrix metalloproteinases and TIMPs. Cardiovasc Res 69:562–573 31. Van den Steen PE, Proost P, Wuyts A, Van Damme J, Opdenakker G (2000) Neutrophil gelatinase B potentiates interleukin-8 tenfold by aminotermi- nal processing, whereas it degrades CTAP-III, PF-4, and GRO-α and leaves RANTES and MCP-2 intact. Blood 96:2673–2681 8. Liabakk NB, Talbot I, Smith RA, Wilkinson K, Balkwill F (1996) Matrix metal- loprotease 2 (MMP-2) and matrix metalloprotease 9 (MMP-9) type IV collagenases in colorectal cancer. Cancer Res 56:190–196 8. Liabakk NB, Talbot I, Smith RA, Wilkinson K, Balkwill F (1996) Matrix metal- loprotease 2 (MMP-2) and matrix metalloprotease 9 (MMP-9) type IV collagenases in colorectal cancer. Cancer Res 56:190–196 g 9. Murray GI, Duncan ME, O’Neil P, McKay JA, Melvin WT, Fothergill JE (1998) Matrix metalloproteinase-1 is associated with poor prognosis in oesopha- geal cancer. J Pathol 185:256–261 9. Murray GI, Duncan ME, O’Neil P, McKay JA, Melvin WT, Fothergill JE (1998) Matrix metalloproteinase-1 is associated with poor prognosis in oesopha- geal cancer. J Pathol 185:256–261 32. Caley MP, Martins VL, O’Toole EA (2015) Metalloproteinases and wound healing. Adv Wound Care (New Rochelle) 4:225–234 33. Elkington PTG, O’kane CM, Friedland JS (2005) The paradox of matrix metalloproteinases in infectious disease. Clin Exp Immunol 142:12–20 Page 8 of 8 Van Damme et al. Vet Res (2020) 51:100 34. Park SS, Lillehoj HS, Allen PC, Park DW, FitzCoy S, Bautista DA, Lillehoj EP (2008) Immunopathology and cytokine responses in broiler chickens coinfected with Eimeria maxima and Clostridium perfringens with the use of an animal model of necrotic enteritis. 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https://openalex.org/W4362392858	https://figshare.com/articles/journal_contribution/Supplementary_Data_from_Gene_Expression_Profile_Correlates_with_T-Cell_Infiltration_and_Relative_Survival_in_Glioblastoma_Patients_Vaccinated_with_Dendritic_Cell_Immunotherapy/22442459/1/files/39893351.pdf	English	null	Supplementary Data from Gene Expression Profile Correlates with T-Cell Infiltration and Relative Survival in Glioblastoma Patients Vaccinated with Dendritic Cell Immunotherapy	null	2,023	cc-by	742	Prins et al., Suppl. Fig. 1 Prins et al., Suppl. Fig. 1 Initial DC Vaccination A 3000 4000 5000 Initial DC Vaccination -6] (pg/mL) 1000 10000 Booster DC Vaccinations -6] (pg/mL) A -Tx t-Tx 0 1000 2000 serum [IL Tx Tx 100 serum [IL- Supplementary Figure 1. IL-6 Responses After DC Vaccination. Serum IL-6 cytokine concentrations, measured pre- and post-vaccination, after the initial course of DC vaccination (A) or after booster DC vaccinations with either 5% imiquimod or poly ICLC Pre-T Post-T Timepoint Pre-T Post-T Timepoint vaccination (A) or after booster DC vaccinations with either 5% imiquimod or poly ICLC (B). Serum from patients enrolled on this clinical trial was thawed, labeled with cytometric bead array (CBA) antibody-coated beads, washed and subjected to analysis on a BD FacsCalibur flow cytometer together with cytokine standards. Quantitative assessment of cytokine levels was accomplished with a Microsoft Excel-based CBA software program. Initial DC Vaccination A 3000 4000 5000 Initial DC Vaccination -6] (pg/mL) A -Tx t-Tx 0 1000 2000 serum [IL Pre-T Post-T Timepoint A 1000 10000 Booster DC Vaccinations -6] (pg/mL) Tx Tx 100 serum [IL- Pre-T Post-T Timepoint P Timepoint Timepoint Supplementary Figure 1. IL-6 Responses After DC Vaccination. Serum IL-6 cytokine concentrations, measured pre- and post-vaccination, after the initial course of DC vaccination (A) or after booster DC vaccinations with either 5% imiquimod or poly ICLC vaccination (A) or after booster DC vaccinations with either 5% imiquimod or poly ICLC (B). Serum from patients enrolled on this clinical trial was thawed, labeled with cytometric bead array (CBA) antibody-coated beads, washed and subjected to analysis on a BD FacsCalibur flow cytometer together with cytokine standards. Quantitative assessment of cytokine levels was accomplished with a Microsoft Excel-based CBA software program. Prins et al., Suppl. Fig. 2 Prins et al., Suppl. Fig. 2 100 OS when vaccine 20 40 60 80 given in ND setting OS when vaccine given in rec setting Percent survival 0 500 1000 1500 2000 2500 0 Overall Survival (Days) Supplementary Figure 2: Extended survival for patients vaccinated in the newly diagnosed setting. Overall survival of y g g patients who received DC vaccinations in the newly diagnosed setting (n=15) vs. at the time of tumor recurrence (n=8). The overall survival from the date of initial surgical diagnosis of glioblastoma is depicted for all vaccinated patients. p=0.03 by Log-Rank testing. Prins et al., Suppl. Fig. 1 100 OS when vaccine 20 40 60 80 given in ND setting OS when vaccine given in rec setting Percent survival 0 500 1000 1500 2000 2500 0 Overall Survival (Days) Supplementary Figure 2: Extended survival for patients vaccinated in the newly diagnosed setting. Overall survival of y g g patients who received DC vaccinations in the newly diagnosed setting (n=15) vs. at the time of tumor recurrence (n=8). The overall survival from the date of initial surgical diagnosis of glioblastoma is depicted for all vaccinated patients. p=0.03 by Log-Rank testing. Prins et al., Suppl. Table 1 Suppl. Table 1. DC Phenotype Subset (by FACS) % Positive (Median) HLA-DR+CD86+ 95 HLA-DR+CD14- 66 CD14+ 22 CD40+ 95 CD83+ 41 % Viable 84 % Large Cells 62 Prins et al., Suppl. Table 1 Suppl. Table 1. DC Phenotype Subset (by FACS) % Positive (Median) HLA-DR+CD86+ 95 HLA-DR+CD14- 66 CD14+ 22 CD40+ 95 CD83+ 41 % Viable 84 % Large Cells 62 Prins et al., Suppl. Table 2 Table 2: Dose Escalation Survival Data Dose of DC's Tumor Avg. # Patients KPS TTP (mo ) OS (mo ) Dose of DC s Pathology Age # Patients KPS TTP (mo.) OS (mo.) 1x10e6 GBM 47.6 9 70-100 27.07 34.72 5x10e6 GBM 43.5 6 80-90 18.03 28.65 10x10e6 GBM 61.6 8 60-100 13.57 25.97 Prins et al., Suppl. Table 2 Table 2: Dose Escalation Survival Data Dose of DC's Tumor Avg. # Patients KPS TTP (mo ) OS (mo ) Dose of DC s Pathology Age # Patients KPS TTP (mo.) OS (mo.) 1x10e6 GBM 47.6 9 70-100 27.07 34.72 5x10e6 GBM 43.5 6 80-90 18.03 28.65 10x10e6 GBM 61.6 8 60-100 13.57 25.97 Prins et al., Suppl. Table 2 Prins et al., Suppl. Table 2 Table 2: Dose Escalation Survival Data Dose of DC's Tumor Avg. # Patients KPS TTP (mo ) OS (mo ) Dose of DC s Pathology Age # Patients KPS TTP (mo.) OS (mo.) 1x10e6 GBM 47.6 9 70-100 27.07 34.72 5x10e6 GBM 43.5 6 80-90 18.03 28.65 10x10e6 GBM 61.6 8 60-100 13.57 25.97
https://openalex.org/W4205125113	https://link.springer.com/content/pdf/10.1007/JHEP12(2021)070.pdf	English	null	Folding orthosymplectic quivers	The Journal of high energy physics/The journal of high energy physics	2,021	cc-by	20,879	Published for SISSA by Springer Received: August 21, 2021 Accepted: November 4, 2021 Published: December 10, 2021 Received: August 21, 2021 Accepted: November 4, 2021 Published: December 10, 2021 Open Access, c⃝The Authors. Article funded by SCOAP3. Keywords: Brane Dynamics in Gauge Theories, Extended Supersymmetry, Supersym- metric Gauge Theory Folding orthosymplectic quivers orthosymplectic 6 2.3.1 D4 aﬃne Dynkin diagram 6 2.3.2 T4 theory 8 3 Folding framed orthosymplectic quivers 9 3.1 Height two nilpotent orbits 9 3.2 Coulomb branch global symmetry 14 4 Folding unframed orthosymplectic quivers 15 4.1 En orbits 15 4.2 Z2 projection on representations 18 4.3 Magnetic quivers of 4d N = 2 19 5 Hasse diagrams 21 5.1 Maximal height 2 orbits 24 5.2 General families 25 6 Brane conﬁgurations 27 6.1 Generalisation of height two quivers 28 6.2 Kraft-Procesi transitions 29 7 Conclusion and outlook 32 A Hall Littlewood polynomials and star shaped quivers 34 Contents 1 Introduction 1 2 The Coulomb branch 3 2.1 The monopole formula 3 2.2 Hall-Littlewood computations 6 2.3 Unitary vs. orthosymplectic 6 2.3.1 D4 aﬃne Dynkin diagram 6 2.3.2 T4 theory 8 3 Folding framed orthosymplectic quivers 9 3.1 Height two nilpotent orbits 9 3.2 Coulomb branch global symmetry 14 4 Folding unframed orthosymplectic quivers 15 4.1 En orbits 15 4.2 Z2 projection on representations 18 4.3 Magnetic quivers of 4d N = 2 19 5 Hasse diagrams 21 5.1 Maximal height 2 orbits 24 5.2 General families 25 6 Brane conﬁgurations 27 6.1 Generalisation of height two quivers 28 6.2 Kraft-Procesi transitions 29 7 Conclusion and outlook 32 A Hall Littlewood polynomials and star shaped quivers 34 1 Introduction JHEP12(2021)070 4.1 En orbits 4.2 Z2 projection on representations 4.3 Magnetic quivers of 4d N = 2 5.1 Maximal height 2 orbits 6.1 Generalisation of height two quivers 6.2 Kraft-Procesi transitions 7 Conclusion and outlook A Hall Littlewood polynomials and star shaped quivers Folding orthosymplectic quivers JHEP12(2021)070 Antoine Bourget,a Julius F. Grimminger,a Amihay Hanany,a Rudolph Kalveks,a Marcus Sperlingb and Zhenghao Zhonga aTheoretical Physics Group, The Blackett Laboratory, Imperial College London, Prince Consort Road, London SW7 2AZ, U.K. bYau Mathematical Sciences Center, Tsinghua University, Haidian District, Beijing 100084, China E-mail: a.bourget@imperial.ac.uk, julius.grimminger17@imperial.ac.uk, a.hanany@imperial.ac.uk, rudolph.kalveks09@imperial.ac.uk, msperling@mail.tsinghua.edu.cn, zhenghao.zhong14@imperial.ac.uk Antoine Bourget,a Julius F. Grimminger,a Amihay Hanany,a Rudolph Kalveks,a Marcus Sperlingb and Zhenghao Zhonga msperling@mail.tsinghua.edu.cn, zhenghao.zhong14@imperial.ac.uk Abstract: Folding identical legs of a simply-laced quiver creates a quiver with a non- simply laced edge. So far, this has been explored for quivers containing unitary gauge groups. In this paper, orthosymplectic quivers are folded, giving rise to a new family of quivers. This is realised by intersecting orientifolds in the brane system. The monopole formula for these non-simply laced orthosymplectic quivers is introduced. Some of the folded quivers have Coulomb branches that are closures of minimal nilpotent orbits of ex- ceptional algebras, thus providing a new construction of these fundamental moduli spaces. Moreover, a general family of folded orthosymplectic quivers is shown to be a new magnetic quiver realisation of Higgs branches of 4d N = 2 theories. The Hasse (phase) diagrams of certain families are derived via quiver subtraction as well as Kraft-Procesi transitions in the brane system. Keywords: Brane Dynamics in Gauge Theories, Extended Supersymmetry, Supersym- metric Gauge Theory ArXiv ePrint: 2107.00754 ArXiv ePrint: 2107.00754 ArXiv ePrint: 2107.00754 Open Access, c⃝The Authors. Article funded by SCOAP3. https://doi.org/10.1007/JHEP12(2021)070 Contents 1 Introduction 1 2 The Coulomb branch 3 2.1 The monopole formula 3 2.2 Hall-Littlewood computations 6 2.3 Unitary vs. orthosymplectic 6 2.3.1 D4 aﬃne Dynkin diagram 6 2.3.2 T4 theory 8 3 Folding framed orthosymplectic quivers 9 3.1 Height two nilpotent orbits 9 3.2 Coulomb branch global symmetry 14 4 Folding unframed orthosymplectic quivers 15 4.1 En orbits 15 4.2 Z2 projection on representations 18 4.3 Magnetic quivers of 4d N = 2 19 5 Hasse diagrams 21 5.1 Maximal height 2 orbits 24 5.2 General families 25 6 Brane conﬁgurations 27 6.1 Generalisation of height two quivers 28 6.2 Kraft-Procesi transitions 29 7 Conclusion and outlook 32 A Hall Littlewood polynomials and star shaped quivers 34 Contents 1 Introduction 1 2 The Coulomb branch 3 2.1 The monopole formula 3 2.2 Hall-Littlewood computations 6 2.3 Unitary vs. 1 Introduction Amongst 3d N = 4 quiver gauge theories, a natural set of theories to consider is that of aﬃne Dynkin quivers with unitary gauge nodes. The Coulomb branches of aﬃne ADE Dynkin quivers are moduli spaces of instantons which can be identiﬁed with the closures of minimal nilpotent orbits of the An, Dn, and E6,7,8 algebras. These are examples of simply- laced quivers. The Coulomb branches of aﬃne Dynkin quivers of BCFG-type, explored in [1], are the closures of minimal nilpotent orbits of the Bn, Cn, F4, and G2 algebras. The corresponding quivers are termed non-simply laced quivers. – 1 – The concept of folding, in the sense that identical legs of simply-laced quivers are folded into a quiver with a non-simply laced edge, has been studied recently in the context of the Coulomb branch Hilbert series of 3d N = 4 quivers [2–5]. In particular, the non- simply laced edge within the quiver implies that the theory has no obvious path integral formulation. Nonetheless, the Coulomb branch Hilbert series can be readily computed via the monopole formula [1, 6]. This allows one to study non-simply laced quivers that are more general than aﬃne Dynkin type quivers. One purpose of this paper is to demonstrate that these quivers can provide new magnetic quiver constructions of known moduli spaces and in many cases lead to new interesting moduli spaces. Other approaches to folding include [7–10]. JHEP12(2021)070 So far, works on non-simply laced quivers have focused solely on quivers with unitary gauge groups. In light of the recent understanding of orthosymplectic quivers [11–17], and the applicability of the monopole formula to framed and unframed orthosymplectic quivers [18], one is ﬁnally able to extend this program to non-simply laced orthosymplectic quivers. Amongst framed/ﬂavoured orthosymplectic quivers, one natural set to fold is that of magnetic quivers for nilpotent orbit closures of so(2n). To be more precise, framed orthosymplectic quivers whose Coulomb branches are height 2 nilpotent orbit closures of so(2n) carry a natural Z2 symmetry that allows us to fold the identical legs. The Coulomb branches of the resulting non-simply laced framed orthosymplectic quivers turn out to be height 2 nilpotent orbit closures of sl(n). Furthermore, for framed non-simply laced orthosymplectic quivers there exist corresponding brane conﬁgurations with D3-D5-NS5 branes in the presence of O3, O5 and ON orientifold planes. 1 Introduction For unframed/unﬂavoured orthosymplectic quivers, there is a nice set of En quivers which are studied in detail in [15, 18]. Upon folding their identical legs, one obtains the following key results: • First, folding orthosymplectic quivers, whose Coulomb branches are closures of En minimal nilpotent orbits for 4 ≤n ≤8, leads to non-simply laced orthosymplec- tic quivers, whose Coulomb branches are also closures of minimal nilpotent orbits. Folding the E8, E7, E6, E5 ∼= D5, E4 ∼= A4 quivers, leads to non-simply laced or- thosymplectic quivers, whose Coulomb branches are closures of minimal orbits of E7, E6, D5, D4, D3 respectively. This can be depicted as follows: • First, folding orthosymplectic quivers, whose Coulomb branches are closures of En minimal nilpotent orbits for 4 ≤n ≤8, leads to non-simply laced orthosymplec- tic quivers, whose Coulomb branches are also closures of minimal nilpotent orbits. Folding the E8, E7, E6, E5 ∼= D5, E4 ∼= A4 quivers, leads to non-simply laced or- thosymplectic quivers, whose Coulomb branches are closures of minimal orbits of E7, E6, D5, D4, D3 respectively. This can be depicted as follows: E8 E7 E6 D5 A4 · · · E7 E6 D5 D4 A3 · · · (1.1) E8 E7 E6 D5 A4 · · · E7 E6 D5 D4 A3 · · · (1.1) (1.1) The red arrows denote orthosymplectic folding. Note that the top line corresponds to the standard exceptional sequence while the bottom line corresponds to a chain of inclusions of associated aﬃne Weyl groups studied in [19, 20]. • Second, each member of the En family of orthosymplectic quivers can be generalized to an inﬁnite sequence of quivers, as shown in [18]. These quivers are magnetic quivers for 5d N = 1 SQCD theories. Each of these families of quivers can be folded, – 2 – producing inﬁnite sequences of non-simply laced orthosymplectic quivers. Some of these families are magnetic quivers for 4d N = 2 theories. The outline of the paper is as follows: section 2 provides a brief recap of magnetic quivers and the monopole formula for non-simply laced orthosymplectic quivers. The alternative method of calculating such Coulomb branches via Hall-Littlewood polynomials and their related functions is also summarised. Thereafter, orthosymplectic quivers with a known unitary quiver counterpart are considered in section 2.3. 1 Introduction A comparison from folding both types of quivers, namely the orthosymplectic as well the unitary realisation, demonstrates that the non-simply laced orthosymplectic quiver produces results consistent with the expectation from folding. Section 3 details the folding of framed orthosymplectic quivers, i.e. quivers that contain ﬂavour nodes. Section 4 investigates non-simply laced orthosymplectic quivers whose Coulomb branches are closures of E5,6,7 minimal nilpotent orbits. Section 4.3 presents the non-simply laced orthosymplectic quivers that are new magnetic quiver constructions for certain 4d N = 2 theories. Having derived a new class of magnetic quivers, section 5 details the construction of their Hasse diagrams by extending the quiver subtraction algorithm to non-simply laced orthosymplectic quivers. Section 6 provides brane realisations for ﬂavoured non-simply laced orthosymplectic quivers. Lastly, section 7 concludes and provides an outlook. JHEP12(2021)070 2 The Coulomb branch The notion of a magnetic quiver was recently introduced and studied in [14, 15, 21–27]. A given hyper-Kähler moduli space X is said to have a magnetic quiver construction if there exist ﬁnitely many quivers Qi such that X = [ i C3d(Qi) (2.1) (2.1) holds as equality of moduli spaces, where the intersections are lower dimensional and also admit magnetic quiver constructions. In other words, each magnetic quiver is taken as input data for a 3d N = 4 Coulomb branch C3d(Qi) and each of them is a symplectic singularity [28] itself; in contrast, X might be a union of hyper-Kähler cones. Note that 3d N = 4 Coulomb branches are used only as a black box to construct moduli spaces of theories that do not need to be three dimensional. In many physically motivated examples, X is taken as a Higgs branch of a theory with 8 supercharges in space-time dimensions d = 3, 4, 5, 6. It is important to note that the magnetic quiver construction is not unique, as there are several known examples for which diﬀerent magnetic quivers describe the same space X. For example, in section 4 we consider the exceptional En families introduced in [15] or the diﬀerent representations of the minimal nilpotent orbit of E6 discussed in [18]. 2.1 The monopole formula Given a magnetic quiver, the associated Coulomb branch moduli space can be studied via various techniques such as abelianisation [29, 30], Coulomb branch quantisation [31, 32], – 3 – Hilbert series [6], or more mathematical approaches [33–35]. For this work, the central tool is the monopole formula which allows one to evaluate the Coulomb branch Hilbert series by counting dressed monopole operators. The monopole formula for simply-laced unitary quivers and simply-laced orthosymplectic quivers was introduced in [6]. To brieﬂy review, for a given 3d N = 4 gauge theory with gauge group G and matter content transforming in some representation R of G, the unreﬁned monopole formula takes the form HS(t) = X m∈Λ/W P(t; m) t2∆(m) 2∆(m) = X ρ∈R \|ρ(m)\| −2 X α∈Φ+ \|α(m)\| (2.2) (2.2) JHEP12(2021)070 with Φ+ the set of positive roots of G. The magnetic lattice Λ is the weight lattice of the GNO dual group G∨[36], which has Weyl group W. The classical factor P(t, m) originates from dressings by gauge invariant combinations of the residual massless degrees of freedom in the monopole background labelled by m. The reader is referred to [6] for details. For an unframed orthosymplectic quiver with gauge nodes {gi} a possible choice of gauge group is G′ = Q i gi. If there is a subgroup H ⊂G′ acting trivially on the matter content, one may choose a diﬀerent global form of the gauge group: G = G′/H. This aﬀects the magnetic lattice in a non-trivial way [36], as discussed in detail in [18]. In this paper, the discrete subgroup for the unframed simply-laced orthosymplectic quiver is always chosen to be H = Z2. The magnetic lattice Λ of G = G′/Z2 can be divided into two parts, Λ = Λ1 + Λ2, where Λ1 ∼= Zr is the magnetic lattice of G′, and Λ2 ∼= Z + 1 2 r, r being the rank of G. The full Hilbert series is HSΛ(t) = HSΛ1(t) + HSΛ2(t) . (2.3) (2.3) HSΛ(t) = HSΛ1(t) + HSΛ2(t) . (2.3) Besides the magnetic lattice, another ingredient for the monopole formula is the confor- mal dimension. For non-simply laced unitary quivers, the conformal dimension is proposed in [1]. For framed non-simply laced orthosymplectic quivers, one may propose a similar set of amendments such that the conformal dimension is modiﬁed as summarised in ﬁgure 1 to accommodate for the non-simply laced edge. 2.1 The monopole formula For unframed non-simply laced orthosymplectic quivers, one needs to take into con- sideration both the changes to the magnetic lattice due to H as well as the change to the conformal dimension due to the non-simply laced edge. One can divide the nodes of non-simply laced quivers long and short nodes (in the sense of the long and short nodes of Dynkin diagrams). Denote by ΛL the magnetic lattice of the long nodes/gauge groups and by ΛS the magnetic lattice of the short nodes/gauge groups. A vector of magnetic charges m ∈Λ is represented as a pair m ∈(mL, mS) ∈ΛL × ΛS. Let rL denote the sum of the ranks of all long nodes and rS the sum of the ranks of all short nodes. If the non-simply laced edge is even (i.e. with double, quadruple bond etc.), then the magnetic lattice to be summed over is as follows: ZrS+rL ⊕ Z + 1 2 rS × ZrL . (2.4) (2.4) – 4 – JHEP12(2021)070 ∆edge = 1 2 kP i=1 lP j=1 \|b m1,i −m2,j\| U(k) U(l) ∆edge = 1 2 kP i=1 lP j=1 (\|b m1,i −m2,j\| + \|b m1,i + m2,j\|) USp(2k) SO(2l) b b USp(2l) SO(2k) b USp(2k) SO(2l + 1) b USp(2l) SO(2k + 1) b ∆edge = 1 2 kP i=1 lP j=1 (\|b m1,i −m2,j\| + \|b m1,i + m2,j\|) ∆edge = 1 2 kP i=1 lP j=1 (\|b m1,i −m2,j\| + \|b m1,i + m2,j\|) + 1 2 kP i=1 \|b m1,i\| ∆edge = 1 2 kP i=1 lP j=1 (\|b m1,i −m2,j\| + \|b m1,i + m2,j\|) + 1 2 lP j=1 \|m2,j\| Figure 1. The contribution of the edges to the conformal dimension ∆edge is given for the two- U(k) U(l) b ∆edge = 1 2 kP i=1 lP j=1 \|b m1,i −m2,j\| ∆edge = 1 2 kP i=1 lP j=1 (\|b m1,i −m2,j\| + \|b m1,i + m2,j\|) JHEP12(2021)070 b ∆edge = 1 2 kP i=1 lP j=1 (\|b m1,i −m2,j\| + \|b m1,i + m2,j\|) ∆edge = 1 2 kP i=1 lP j=1 (\|b m1,i −m2,j\| + \|b m1,i + m2,j\|) + 1 2 kP i=1 \|b m1,i\| USp(2l) SO(2k + 1) b ∆edge = 1 2 kP i=1 lP j=1 (\|b m1,i −m2,j\| + \|b m1,i + m2,j\|) + 1 2 lP j=1 \|m2,j\| Figure 1. 2.1 The monopole formula Furthermore, when a unitary magnetic quiver construction is known for the same geometric space, the reﬁned Hilbert series can be given in the form of a highest weight generating function (HWG) [37] for characters of the global symmetry group. In certain cases, these HWGs can be generalised to arbitrary rank. The monopole formula results for the orthosymplectic magnetic quivers in this paper have been tested against the unreﬁned Hilbert series for unitary magnetic quivers, calculated either directly, or by expanding and unreﬁning their HWGs. JHEP12(2021)070 2.3 Unitary vs. orthosymplectic In this section, the folding of simply-laced orthosymplectic quivers is demonstrated on a set of examples. These examples are chosen such that the orthosymplectic quivers have unitary counterparts. The resulting non-simply laced quivers are analysed and are found to be consistent with each other. 2.2 Hall-Littlewood computations The relationship between the monopole formula with background charges and Hall-Little- wood polynomials was explored in [38]. This relationship permits an alternative method of calculating Coulomb branches that is applicable to many star shaped quivers, both unitary and/or orthosymplectic, with a central node of type G. The approach is to identify the Coulomb branches of the linear quiver legs as framed Slodowy slices [39], and to compose these by summing over the weight lattice of the GNO dual G∨, while incorporating sym- metry factors, all as described in appendix A. This method permits the exact calculation of reﬁned (or in some cases, partially reﬁned) Hilbert series and HWGs, thereby providing many consistency checks on the results herein. 2.1 The monopole formula The contribution of the edges to the conformal dimension ∆edge is given for the two- node quivers on the left. The magnetic charges for the left nodes are denoted by {m1,i} and for the right node by {m2,j}. The non-simply laced edge has multiplicity b, which then appears as a multiplicative factor for the m1,i magnetic charges. The contribution of the vector multiplets is not aﬀected by non-simply laced edges. In contrast, if the non-simply laced edge is odd (i.e. with triple, quintuple bond etc.), then the magnetic lattice is: + ZrS+rL ⊕ Z + 1 2 rS+rL . (2.5) (2.5) If the non-simply laced orthosymplectic quiver is framed, then the Hilbert series sum is evaluated only over the integer-valued magnetic charges, because the discrete group H is trivial, see [18]. In this paper, moduli spaces are identiﬁed and compared by Hilbert series computa- tions. These can be computed for the Coulomb branches of magnetic quivers by alternative methods. The central method used in this paper is the monopole formula (2.2). This yields – 5 – Hilbert series, which can be computed exactly for small rank quivers and perturbatively to high orders for larger rank quivers. For unitary quivers, the Hilbert series can be reﬁned, but there is no known prescription for obtaining the reﬁned Hilbert series of an orthosym- plectic quiver via the monopole formula. Nonetheless, such unreﬁned Hilbert series can be compared with the Hilbert series of known moduli spaces. Furthermore, when a unitary magnetic quiver construction is known for the same geometric space, the reﬁned Hilbert series can be given in the form of a highest weight generating function (HWG) [37] for characters of the global symmetry group. In certain cases, these HWGs can be generalised to arbitrary rank. The monopole formula results for the orthosymplectic magnetic quivers in this paper have been tested against the unreﬁned Hilbert series for unitary magnetic quivers, calculated either directly, or by expanding and unreﬁning their HWGs. Hilbert series, which can be computed exactly for small rank quivers and perturbatively to high orders for larger rank quivers. For unitary quivers, the Hilbert series can be reﬁned, but there is no known prescription for obtaining the reﬁned Hilbert series of an orthosym- plectic quiver via the monopole formula. Nonetheless, such unreﬁned Hilbert series can be compared with the Hilbert series of known moduli spaces. 2.3.1 D4 aﬃne Dynkin diagram Due to the isomorphisms so(2) ∼= u(1) and sl(2) ∼= usp(2), one can construct two quivers with equivalent Coulomb branches: 2 2 2 2 2 1 1 2 1 1 (2.6) 2 2 2 2 2 1 1 2 1 1 (2.6) (2.6) where white nodes with label n denote U(n) gauge groups, red nodes with label n denote SO(n) gauge groups, and blue nodes with label 2n denote USp(2n) gauge groups. Note that the central node has rank 2 on the left hand side and rank 1 on the right hand side. As a reminder, for unframed unitary quivers, there is always a diagonal U(1) that where white nodes with label n denote U(n) gauge groups, red nodes with label n denote SO(n) gauge groups, and blue nodes with label 2n denote USp(2n) gauge groups. Note that the central node has rank 2 on the left hand side and rank 1 on the right hand side. As a reminder, for unframed unitary quivers, there is always a diagonal U(1) that – 6 – one needs to ungauge. Whereas for the unframed orthosymplectic quiver, one chooses H = Z2 ⊂USp(2) × SO(2)4 as the diagonal subgroup to be ungauged. As shown in [18], the Coulomb branch Hilbert series of both quivers are the same. Since the two quivers have the same shape, one can fold identical legs and check if the non-simply laced quivers reproduce the same results. Using the monopole formula, one can verify that the following foldings reproduce the same Coulomb branch Hilbert series. Folding two identical legs. First one folds two of the four identical legs in each quiver such that one obtains 2 1 2 2 1 2 1 1 2 2 (2.7) 2 2 1 2 1 1 2 2 (2.7) JHEP12(2021)070 (2.7) For non-simply laced quivers, the node where one ungauges the U(1) is important and diﬀerent nodes can yield diﬀerent Coulomb branches [40]. In this paper, the choice taken is to ungauge on a long node (ungaugings on long nodes all give the same moduli space). The Coulomb branch of the unitary quiver is known be Oso(7) min as it is the aﬃne Dynkin diagram of B3 [41]. 2.3.2 T4 theory Next, let us turn our attention to a more involved example. The theory known as T4 is constructed by gluing together quivers whose Coulomb branches are closures of maximal nilpotent orbits of sl(4). Due to the isomorphism sl(4) ∼= so(6), the following quivers have equivalent moduli spaces: 1 2 3 4 3 2 1 3 2 1 6 4 4 4 2 2 4 2 2 4 4 2 2 (2.13) JHEP12(2021)070 (2.13) Computation of the Coulomb branch Hilbert series of the orthosymplectic quiver is given in [18, ﬁgure 39] and is consistent with the unitary counterpart. Computation of the Coulomb branch Hilbert series of the orthosymplectic quiver is given in [18, ﬁgure 39] and is consistent with the unitary counterpart. Computation of the Coulomb branch Hilbert series of the orthosymplectic quiver is given in [18, ﬁgure 39] and is consistent with the unitary counterpart. As a ﬁrst step, one can fold two of the quiver legs which yields: 4 3 2 1 3 2 1 6 4 4 4 2 2 4 2 2 4 3 2 1 3 2 1 6 4 4 4 2 2 4 2 2 (2.14) Computation of the Coulomb branch Hilbert series of both quivers yields: HSU(t) = HSOSp(t) =            1+21t2+68t3+341t4+1300t5+4936t6+15988t7 +50242t8+142812t9+384411t10+960772t11+2270650t12 +5038840t13+10601001t14+21083004t15+39862377t16 +71590384t17+122553812t18+199944220t19+311642452t20 +464078612t21+661421665t22+902317920t23+1179751147t24 +1478423752t25+1777451140t26+2050065624t27 +2269933494t28+2412458048t29+2462182956t30 +palindromic+···+21t58+t60            (1 −t2)9 (1 −t3)12 (1 −t4)9 . (2.15) HSU(t) = HSOSp(t) =            1+21t2+68t3+341t4+1300t5+4936t6+15988t7 +50242t8+142812t9+384411t10+960772t11+2270650t12 +5038840t13+10601001t14+21083004t15+39862377t16 +71590384t17+122553812t18+199944220t19+311642452t20 +464078612t21+661421665t22+902317920t23+1179751147t24 +1478423752t25+1777451140t26+2050065624t27 +2269933494t28+2412458048t29+2462182956t30 +palindromic+···+21t58+t60            (1 −t2)9 (1 −t3)12 (1 −t4)9 . (2.15) HSU(t) = HSOSp(t) =            1+21t2+68t3+341t4+1300t5+4936t6+15988t7 +50242t8+142812t9+384411t10+960772t11+2270650t12 +5038840t13+10601001t14+21083004t15+39862377t16 +71590384t17+122553812t18+199944220t19+311642452t20 +464078612t21+661421665t22+902317920t23+1179751147t24 +1478423752t25+1777451140t26+2050065624t27 +2269933494t28+2412458048t29+2462182956t30 +palindromic+···+21t58+t60            (1 −t2)9 (1 −t3)12 (1 −t4)9 . (2.15) (2.15) As a next step, one folds all three identical legs which yields As a next step, one folds all three identical legs which yields 4 4 6 3 2 1 4 2 2 (2.16) (2.16) The unitary quiver in (2.16) is a known member of the generalised rank 1 4d N = 2 sequence studied in [42]. 2.3.1 D4 aﬃne Dynkin diagram An explicit Coulomb branch Hilbert series computation shows that the folded orthosymplectic quiver is consistent with having the same Coulomb branch: HSU(t) = HSOSp(t) = 1 + 13t2 + 28t4 + 13t6 + t8 (1 −t2)8 (2.8) (2.8) where HSU(t) and HSOSp(t) are the Hilbert series of the unitary and orthosymplectic quivers, respectively. where HSU(t) and HSOSp(t) are the Hilbert series of the unitary and orthosymplectic quivers, respectively. Folding three identical legs. Next, one proceeds to fold three of the identical legs: 2 2 1 1 2 2 (2.9) (2.9) The unitary quiver is the aﬃne Dynkin diagram of G2 and, hence, the Coulomb branch is Og2 min. An explicit computation of the Coulomb branch Hilbert series of the orthosymplectic quiver HSU(t) = HSOSp(t) = (1 + t2)(1 + 7t2 + t4) (1 −t2)6 (2.10) (2.10) conﬁrms the equality of the moduli spaces in terms of Hilbert series. Folding four identical legs. Finally, one folds all the identical legs and obtains th following quivers Folding four identical legs. Finally, one folds all the identical legs and obtains the following quivers 2 1 2 2 (2.11) (2.11) The unitary non-simply laced quiver is investigated in [42] and the Coulomb branch is Osl3 min. An explicit computation of the Coulomb branch Hilbert series of the orthosymplectic quiver HSU(t) = HSOSp(t) = 1 + 4t2 + t4 (1 −t2)4 (2.12) (2.12) – 7 – shows that both magnetic quiver constructions agree in the unreﬁned Hilbert series. shows that both magnetic quiver constructions agree in the unreﬁned Hilbert series. 2.3.2 T4 theory An explicit computation of the Coulomb branch Hilbert series of the both quivers in (2.16) yields HSU(t) = HSOSp(t) =    1−t+10t2+23t3+67t4+190t5+525t6+1053t7 +2292t8+4167t9+7299t10+11494t11+17114t12+23080t13 +29925t14+35107t15+39221t16+40320t17 +palindromic+···+10t32−t33+t34    (1 −t)(1 −t2)5(1 −t3)7(1 −t4)5 . (2.17) (2.17) – 8 – – 8 – As a reminder, in all calculations in this article that involve non-simply laced unitary quivers which lack explicit framing, the overall U(1) framing is applied on a long node, such as the central node, in order to obtain consistent results. The above examples reinforce the conjecture that folding orthosymplectic quivers yields valid results, so one may proceed to fold quivers where the resulting Coulomb branches cannot easily be determined from accidental isomorphisms. 3 Folding framed orthosymplectic quivers As a next step, examine the folding of certain families of orthosymplectic quivers treated in [11, 13], whose Coulomb branches are closures of nilpotent orbits. To be concrete, the focus is placed on so-called height two orbits, which are orbits of elements x ∈g such that ad(x)2 ̸= 0 and ad(x)3 = 0 [43]. These are the lowest dimensional non-trivial nilpotent orbits and yield a few clear candidates with the necessary symmetry for folding. In particular, height two orbits of type D are considered. JHEP12(2021)070 The monopole formula for orthosymplectic quivers only returns unreﬁned Hilbert se- ries [38]. However, these are often suﬃcient to identify known moduli spaces (such as nilpotent orbit closures). For these, the encoding of reﬁned Hilbert series into HWGs is often straightforward. Indeed, for each of the orthosymplectic quivers in the follow- ing sections, the Coulomb branches turn out to be well-known moduli spaces, for which HWGs provide a concise description. Furthermore, as is shown below, one can ﬁnd natural projection maps between the HWGs for orthosymplectic quivers before and after folding. 3.1 Height two nilpotent orbits Even D-type. For Oso(4n) [22n] , the orbit is the union of two identical cones [44]. One of these cones has the magnetic quiver: . . . . . . 2 2n 2n−2 2n−2 4 2 2 4 2 2 (3.1) The reﬁned Coulomb branch Hilbert series can be encoded as the HWG (3.1) The reﬁned Coulomb branch Hilbert series can be encoded as the HWG HWG(3.1) = PE "n−1 X i=1 ρ2it2i + ρ2 2nt2n # , (3.2) (3.2) where ρi for i = 1, . . . , 2n are the highest weight fugacities of so(4n). Note, the fugacity for the ρ2n spinor is present in the expression [45]. For the second cone, the quiver is the same as (3.1), but the other spinor ρ2n−1 is used in the HWG. The orbit is the union of the two cones and includes both spinors (whereas the intersection contains neither). where ρi for i = 1, . . . , 2n are the highest weight fugacities of so(4n). Note, the fugacity for the ρ2n spinor is present in the expression [45]. For the second cone, the quiver is the same as (3.1), but the other spinor ρ2n−1 is used in the HWG. The orbit is the union of the two cones and includes both spinors (whereas the intersection contains neither). Folding (3.1) results in the following quiver: Folding (3.1) results in the following quiver: . . . 2n−2 4 2 2 2n 2 (3.3) (3.3) – 9 – Explicit computation shows the Coulomb branch of (3.3) to be the moduli space Osl(2n) [2n] . The unitary quiver with the same Coulomb branch is well-known and takes the form: Explicit computation shows the Coulomb branch of (3.3) to be the moduli space Osl(2n) [2n] . The unitary quiver with the same Coulomb branch is well-known and takes the form: 2 n n−1 n−1 . . . . . . 2 1 2 1 (3.4) (3.4) mb branches of these quivers share the HWG: The Coulomb branches of these quivers share the HWG: HWG(3.3) = HWG(3.4) = PE " n X i=1 µiµ2n−it2i # , (3.5) (3.5) JHEP12(2021)070 where µi for i = 1, . . . , 2n −1 are the highest weight fugacities of sl(2n). where µi for i = 1, . . . , 2n −1 are the highest weight fugacities of sl(2n). 3.1 Height two nilpotent orbits When folding quivers, one observes that the creation of a non-simply laced edge leads to a change in the global symmetry. For unitary quivers this is obvious as the Dynkin diagram changes from simply laced to non-simply laced. For these unitary quivers, the action of folding also results in a mapping of the highest weight fugacities [5, 40]. As a result, in this class of examples, the HWG of the Coulomb branch of the folded quiver can be inferred if the HWG for the original quiver is known, see also section 6. By studying (3.2) and (3.5) one observes that the global symmetry changes from SO(4n) to SU(2n). Furthermore, for this family, the action of folding results in the following mapping of highest weight monomials for even D-type: For i = 1, . . . , n −1 , (ρ2i)so(4n) →(µiµ2n−i)sl(2n), (3.6a) (ρ2n−1)so(4n), (ρ2n)so(4n) →(µn)sl(2n), (3.6b) (ρ2 2n−1)so(4n), (ρ2n−1ρ2n)so(4n), (ρ2 2n)so(4n) →(µ2 n)sl(2n). (3.6c) (3.6c) One can repeat this procedure for the remaining so(4n) height 2 orbits: Oso(4n) [22k,14n−4k], where n ≥k ≥1. These geometric spaces are given by the Coulomb branches of ≥ ≥ g p g y . . . . . . 2 2 4 1 1 . . . 2k 2k 2k 2k 2k + 1 2k + 1 4n −4k −1 nodes 4 2 2 . . . 2k 2k + 1 2k (3.7) and their HWGs are given by: HWG(3.7) = PE " k X i=1 ρ2it2i # . (3.8) After folding of (3.7), one obtains the quiver . . . 2k 2 2 2k + 1 2k + 1 . . . 2k 2k + 1 2k 1 2k 2n −2k nodes (3.9) . . . . . . 2 2 4 1 1 . . . 2k 2k 2k 2k 2k + 1 2k + 1 4n −4k −1 nodes 4 2 2 . . . 2k 2k + 1 2k (3.7) and their HWGs are given by: . . . . . . 2 2 4 1 1 . . . 2k 2k 2k 2k 2k + 1 2k + 1 4n −4k −1 nodes 4 2 2 . . . 2k 2k + 1 2k (3.7) and their HWGs are given by: (3.7) and their HWGs are given by: HWG(3.7) = PE " k X i=1 ρ2it2i # . (3.8) (3.8) HWG(3.7) = PE "X i=1 ρ2it2i # . 3.1 Height two nilpotent orbits (3.8) After folding of (3.7), one obtains the quiver . . . 2k 2 2 2k + 1 2k + 1 . . . 2k 2k + 1 2k 1 2k 2n −2k nodes (3.9) After folding of (3.7), one obtains the quiver . . . 2k 2 2 2k + 1 2k + 1 . . . 2k 2k + 1 2k 1 2k 2n −2k nodes (3.9) (3.9) k nodes – 10 – whose Coulomb branch, after computing its Hilbert series, is found to be the closure of the sl(2n) orbit Osl(2n) [2k,12n−2k]. The unitary quiver with the same Coulomb branch is: whose Coulomb branch, after computing its Hilbert series, is found to be the closure of the sl(2n) orbit Osl(2n) [2k,12n−2k]. The unitary quiver with the same Coulomb branch is: . . . . . . 1 2 1 2 1 1 . . . k −1 k k k k k −1 (3.10) (3.10) 2n −2k + 1 nodes 2n −2k + 1 nodes WG of (3.9) is therefore: The HWG of (3.9) is therefore: The HWG of (3.9) is therefore: JHEP12(2021)070 HWG(3.9) = HWG(3.10) = PE " k X i=1 µiµ2n−it2i # . (3.11) (3.11) With the absence of spinors in the HWG, one can get from (3.8) to (3.11) by the map- ping (3.6). The folding of a quiver whose Coulomb branch has so(4n) global symmetry into a quiver whose Coulomb branch is sl(2n) global symmetry is not surprising, as in the k = 1 case it reduces to the following simple observation. One can see this by folding the aﬃne Dynkin quiver of D2n along its vertical symmetry axis 2 2 . . . 1 1 2 2 2 2 1 1 . . . . . . 1 1 2 2 2 2n −3 nodes n −1 nodes 1 1 . . . 2 3 2 2 3 3 4n −5 nodes . . . 2 3 2 2 3 3 . . . 2 3 2 1 2 2n −2 nodes (3.12) where the diagram on the top right is the twisted aﬃne Dynkin quiver of A(2) 2n−1,1 whose Coulomb branch is the minimal nilpotent orbit closure of sl(2n). 1We follow the labelling of Kac [46] for twisted aﬃne algebras, as this predicts the Coulomb branch of the balanced quiver. 3.1 Height two nilpotent orbits 2 2n 2n 2n 4 2 2 4 2 2 (3.13) with the following HWG: (3.13) with the following HWG: HWG(3.13) = PE "n−1 X i=1 ρ2it2i + ρ2nρ2n+1t2n # . (3.14) (3.14) JHEP12(2021)070 Folding the quiver (3.13) gives: . . . 2n 2 2 2n 2 (3.15) (3.15) The Coulomb branch of (3.15) turns out to be Osl(2n+1) [2n,1] . The unitary quiver counterpart with the same Coulomb branch takes the form: . . . . . . 1 2 1 2 1 1 n n n −1 n −1 (3.16) (3.16) The HWG of the folded quiver is: The HWG of the folded quiver is: HWG(3.15) = HWG(3.16) = PE " n X i=1 µiµ2n+1−it2i # . (3.17) (3.17) By comparing (3.14) and (3.17), one observes that there is again a mapping of the highest weight monomials of so(4n + 2) to sl(2n + 1): By comparing (3.14) and (3.17), one observes that there is again a mapping of the highest weight monomials of so(4n + 2) to sl(2n + 1): (ρ2i)so(4n+2) →(µiµ2n+1−i)sl(2n+1) , for i = 1, . . . , n −1 (3.18a) (ρ2n)so(4n+2) →(µn)sl(2n+1) (3.18b) (ρ2n+1)so(4n+2) →(µn+1)sl(2n+1) (3.18c) (ρ2nρ2n+1)so(4n+2) →(µnµn+1)sl(2n+1) (3.18d) 3.1 Height two nilpotent orbits In this case, one observes that folding either a unitary quiver or an orthosymplectic quiver, whose Coulomb branch is the closure of the minimal so(4n) orbit, produces a quiver whose Coulomb branch is the minimal orbit closure of sl(2n). The diﬃculty in reproducing this procedure for other unitary quivers whose Coulomb branch are height 2 nilpotent orbits of so(4n) is that they do not have identical legs to fold. 2 2 . . . 1 1 2 2 n −1 nodes n −1 nodes 4n −5 nodes 4n −5 nodes 2n −2 nodes (3.12) ( ) where the diagram on the top right is the twisted aﬃne Dynkin quiver of A(2) 2n−1,1 whose Coulomb branch is the minimal nilpotent orbit closure of sl(2n). In this case, one observes that folding either a unitary quiver or an orthosymplectic quiver, whose Coulomb branch is the closure of the minimal so(4n) orbit, produces a quiver whose Coulomb branch is the minimal orbit closure of sl(2n). The diﬃculty in reproducing this procedure for other unitary quivers whose Coulomb branch are height 2 nilpotent orbits of so(4n) is that they do not have identical legs to fold. ( ) where the diagram on the top right is the twisted aﬃne Dynkin quiver of A(2) 2n−1,1 whose Coulomb branch is the minimal nilpotent orbit closure of sl(2n). In this case, one observes that folding either a unitary quiver or an orthosymplectic quiver, whose Coulomb branch is the closure of the minimal so(4n) orbit, produces a quiver whose Coulomb branch is the minimal orbit closure of sl(2n). The diﬃculty in reproducing this procedure for other unitary quivers whose Coulomb branch are height 2 nilpotent orbits of so(4n) is that they do not have identical legs to fold. – 11 – Odd D-type. For Oso(4n+2) [22n,12] , there is a single cone. The moduli space is given by the Coulomb branch of: . . . . . . 2 2n 2n 2n 4 2 2 4 2 2 (3.13) with the following HWG: Odd D-type. For Oso(4n+2) [22n,12] , there is a single cone. The moduli space is given by the Coulomb branch of: . . . . . . 2 2n 2n 2n 4 2 2 4 2 2 (3.13) owing HWG: . . . . . . for odd D-type. As with the even D-type case, one can repeat the same folding procedure for the remaining height 2 orbits Oso(4n+2) [22k,14n−4k+2], for n ≥k ≥1. This moduli space is given by the Coulomb branch of Coulomb branch of . . . . . . 2 2 4 1 1 . . . 2k 2k 2k 2k 2k + 1 2k + 1 4n + 1 −4k nodes 4 2 2 . . . 2k + 1 2k 2k + 1 (3.19) . . . . . . 2 2 4 1 1 . . . 2k 2k 2k 2k 2k + 1 2k + 1 4n + 1 −4k nodes 4 2 2 . . . 2k + 1 2k 2k + 1 (3.19) . . . . . . 2 2 4 1 1 . . . 2k 2k 2k 2k 2k + 1 2k + 1 4n + 1 −4k nodes 4 2 2 . . . 2k + 1 2k 2k + 1 (3.19) (3.19) 4n + 1 −4k nodes – 12 – with the HWG being the same as (3.8). After folding of (3.19), one obtains with the HWG being the same as (3.8). After folding of (3.19), one obtains . . . 2k + 1 2 2 2k 2k . . . 2k 2k + 1 2k 1 2k 2n + 1 −2k nodes (3.20) (3.20) 2n + 1 −2k nodes whose Coulomb branch is the moduli space Osl(2n+1) [2k,12n+1−2k]. The unitary quiver with the same Coulomb branch is therefore: JHEP12(2021)070 . . . . . . 1 2 1 2 1 1 . . . k −1 k k k k k −1 2n −2k + 2 nodes (3.21) (3.21) with the HWG with the HWG with the HWG HWG(3.20) = HWG(3.21) = PE " k X i=1 µiµ2n+1−it2i # . (3.22) (3.22) Comment. For orthosymplectic quivers whose Coulomb branch are closures of nilpotent orbits of classical algebras, only the height 2 orbits of so(2n) have symmetric quivers that can be folded. For orthosymplectic quivers whose Coulomb branch are closures of height 2 orbits of so(2n+1), the quivers have orthogonal gauge group(s) on one of the legs, making the quiver asymmetric. For closures of height 2 orbits of usp(2n), the quiver legs contain ‘bad’ nodes in the sense of [47] that cause the monopole formula to diverge. 3.2 Coulomb branch global symmetry For quivers composed of unitary gauge groups, it is easy to read oﬀ(in most cases2) the algebra gglobal of the global symmetry group by studying the balance of the gauge groups. A U(k) gauge group is balanced if the ﬂavour from the neighboring nodes is Nf = 2k. The balanced nodes form the Dynkin diagram of h which is a subalgebra of gglobal. In most cases where all the gauge nodes are balanced with n nodes overbalanced, one ﬁnds that gglobal = Q i hi × u(1)n which considers all balanced subset of nodes that are connected and form Dynkin diagrams hi. If the unitary quiver is unframed, then an overall u(1) factor needs to be removed from the global symmetry. JHEP12(2021)070 The same idea can be carried on for orthosymplectic quivers. In [47], the balance conditions for (special) orthogonal and symplectic gauge groups with Nf fundamental hypermultiplets (i.e. 2Nf half-hypermultiplets) are as follows: SO(2k) : Nf = 2k −1, SO(2k + 1) : Nf = 2k, USp(2k) : Nf = 2k + 1. (3.23) (3.23) It has been shown in [47] that a linear chain of n balanced orthosymplectic gauge nodes gives a global symmetry of so(n + 1). To read this full global symmetry, it may be necessary to add balanced USp(0) nodes, but these are omitted in the drawings as they do not contribute to the Coulomb branch computations. The above is true regardless of the gauge groups being O or SO, noting that USp(0) nodes should not be attached to O(2) nodes. Building on the investigation of non-simply laced orthosymplectic quivers, the following subsets of balanced nodes: . . . . . . . . . n −1 nodes (3.24) . . . . . . . . . n −1 nodes (3.25) both contribute an sl(n) factor to the global symmetry. Here, red nodes are SO and blue nodes are USp. (3.24) (3.25) n −1 nodes n −1 nodes both contribute an sl(n) factor to the global symmetry. Here, red nodes are SO and blue nodes are USp. both contribute an sl(n) factor to the global symmetry. Here, red nodes are SO and blue nodes are USp. Presence of SO(2). As highlighted in [47], when an SO(2) gauge group is present, the global symmetry from a chain of n balanced orthosymplectic gauge nodes is enhanced. This is due to the accidental isomorphism SO(2) ∼= U(1). 2One does observe, however, that more complicated quivers such as moduli space of k-instantons [1] and some non-simply laced unitary quivers [48] have factors in gglobal which cannot be read oﬀfrom the balance of gauge groups. In such cases, the best way to obtain the global symmetry group is an explicit computation of the Hilbert series to order t2 which reveals the dimension of the global symmetry group. for odd D-type. Therefore, this section provides an exhaustive list of orthosymplectic quivers that are closures of nilpotent orbits, which can both be folded and have their Hilbert series computed with the monopole formula. – 13 – 3.2 Coulomb branch global symmetry However, this can be understood from an alternative point of view using D3-D5-NS5 brane conﬁgurations with O3 planes. – 14 – As shown in [49], whenever a balanced SO(2) gauge node is present, it is implied that a USp(0) gauge group is connected to it. Therefore, the following two quivers are identical: As shown in [49], whenever a balanced SO(2) gauge node is present, it is implied that a USp(0) gauge group is connected to it. Therefore, the following two quivers are identical: . . . . . . n −1 nodes 2 . . . . . . n nodes 2 = 0 (3.26) . . . . . . n −1 nodes 2 . . . . . . n nodes 2 = 0 (3.26) . . . n nodes 2 0 (3.26) . . . . . . n −1 nodes 2 (3.26) n −1 nodes n −1 nodes Both quivers contribute an sl(n) factor to the global symmetry group. The quiver on the right hand side is in a more convenient form as it allows us to apply the same rule of reading oﬀthe global symmetry based on the number of balanced nodes. Throughout the paper, it is implicit that whenever there is a balanced SO(2) gauge group, there is always a balanced USp(0) gauge node connected to it. JHEP12(2021)070 These rules for reading oﬀthe global symmetry based on balanced of gauge groups work for all framed non-simply laced orthosymplectic quiver. However, there can be rare cases among unframed orthosymplectic quivers where the global symmetry is enhanced. This is already observed in some cases for unframed simply-laced orthosymplectic in [18]. In the next section it is shown how the global symmetry of unframed non-simply laced orthosymplectic quivers can become enhanced to exceptional en algebras. 4 Folding unframed orthosymplectic quivers In this section, unframed quivers are considered, i.e. quivers without ﬂavour nodes. Un- framed orthosymplectic quivers have been investigated recently in [15, 18]. The simplest of these theories are magnetic quivers of 5d N = 1 SQCD theories. To begin with, the magnetic quivers corresponding to rank 1 5d SQCD theories are considered; their Coulomb branches are closures of the minimal orbits of exceptional algebras En. Thereafter, one focuses on the generalisation of these families of [15] and folds them into new families of non-simply laced unframed orthosymplectic quivers. Some of these fall into the category of star shaped quivers whose Coulomb branches can also be evaluated using the Slodowy slice approach of appendix A. 4.1 En orbits To begin with, consider the folding of orthosymplectic quivers whose Coulomb branches are closures of En minimal nilpotent orbits: Oen min for n = 4, 5, 6, 7, 8. Since the quivers are all unframed, there is a non-trivial choice of the discrete group H ⊂Z2 that one can ungauge. For all the quivers in this section, the Coulomb branches are deﬁned by the choice H = Z2, see [18] for more details. The results are summarised in table 1 along with the identiﬁcation of the Coulomb branch. Below, some observations for the individual cases are discussed in turn and how they are compared with folding their unitary quiver counterparts. E8 orbit. The unitary quiver whose Coulomb branch is the closure of the minimal E8 orbit takes the form of the aﬃne Dynkin diagram of E8. The unitary quiver does not have any identical legs and, therefore, cannot be folded. In contrast, the orthosymplectic quiver with the same Coulomb branch is given in the ﬁrst row of table 1 and has two identical legs – 15 – The orthosymplectic quivers on the left have Coulomb branches that are closures of exceptional algebras En for n = 8, 7, 6, 5, 4. Red an index k denote SO(k) groups while blue nodes with index 2k denote USp(2k) groups. Folding these quivers along the identical legs on-simply laced orthosymplectic quivers on the right. The Coulomb branches of these theories are given as well. In all the quivers here, ll Z hi h i d [18] f d t il Table 1. The orthosymplectic quivers on the left have Coulomb branches that are closures of exceptional algebras En for n = 8, 7, 6, 5, 4. Red nodes with an index k denote SO(k) groups while blue nodes with index 2k denote USp(2k) groups. Folding these quivers along the identical legs gives the non-simply laced orthosymplectic quivers on the right. The Coulomb branches of these theories are given as well. In all the quivers here, there is an overall Z2 which is ungauged, see [18] for more details. 4.1 En orbits Before folding After folding Orthosymplectic quiver Coulomb branch Orthosymplectic quiver Coulomb branch 4 6 6 8 4 2 2 6 2 2 4 4 6 2 Oe8 min = e8 2 8 6 6 4 4 2 2 Oe7 min = e7 4 6 4 4 4 2 2 2 2 2 1 Oe7 min = e7 2 6 4 4 2 2 1 Oe6 min = e6 4 4 2 2 4 2 2 1 Oe6 min = e6 4 4 2 2 1 Oso(10) min = d5 2 4 2 2 2 1 Oso(10) min = d5 4 2 2 1 Oso(8) min = d4 2 2 2 1 1 Osl(5) min = a4 2 2 1 1 Osl(4) min = a3 Table 1. The orthosymplectic quivers on the left have Coulomb branches that are closures of exceptional algebras En for n = 8, 7, 6, 5, 4. Red nodes with an index k denote SO(k) groups while blue nodes with index 2k denote USp(2k) groups. Folding these quivers along the identical legs gives the non-simply laced orthosymplectic quivers on the right. The Coulomb branches of these theories are given as well. In all the quivers here, there is an overall Z2 which is ungauged, see [18] for more details. JHEP12(2021)070 – 16 – that one can fold. Folding these identical legs gives a non-simply laced quiver, see table 1, whose Coulomb branch is the closure of the minimal E7 orbit Oe7 min. E7 orbit. The unitary quiver whose Coulomb branch is the closure of the minimal E7 orbit takes the form of the aﬃne Dynkin diagram of E7 and, hence, has two identical legs one can fold. Folding them yields the non-simply laced unitary quiver whose Coulomb branch is Oe6 min. The orthosymplectic quiver of E7 is provided in the second row of table 1 and has two identical legs. Folding these two legs also gives the non-simply laced orthosymplectic quiver whose Coulomb branch is Oe6 min. JHEP12(2021)070 E6 orbit. The unitary quiver is the aﬃne E6 Dynkin diagram which has three identical legs. When two of the legs are folded, the resulting Coulomb branch is Of4 min [40].3 The orthosymplectic quiver of E6 is listed in the third row of table 1 and has only two identical legs. Folding them results in the non-simply laced quiver whose Coulomb branch is Oe5 min ∼= Oso(10) min . 3Folding all three identical legs gives the minimal nilpotent orbit of so(8) [42]. 4.1 En orbits The discrepancy is not necessarily a surprise since there are several diﬀerent embeddings of Z2 in E6. To summarise, one reaches the surprising statement that folding orthosymplectic quiv- ers whose Coulomb branch are closures of the minimal E8, E7, E6 nilpotent orbits gives non-simply laced quivers whose Coulomb branches are closures of the E7, E6, E5 ∼= D5 orbits respectively. E5 orbit. The unitary quiver is the aﬃne D5 Dynkin diagram. Folding the pairs of identical nodes on the two sides of the diagram produces a quiver with two non-simply laced edges 1 1 1 1 2 2 1 2 2 1 (4.1) (4.1) The Coulomb branches of the quivers on the right are the minimal orbits of Oso(8) min [42]. The orthosymplectic quiver of D5 is listed in the fourth row of table 1 (which is reproduced here): The Coulomb branches of the quivers on the right are the minimal orbits of Oso(8) min [42]. The orthosymplectic quiver of D5 is listed in the fourth row of table 1 (which is reproduced here): 1 4 2 2 2 2 4 2 2 1 (4.2) 1 4 2 2 2 2 4 2 2 1 (4.2) (4.2) One can verify that the Coulomb branch of the folded orthosymplectic quiver is also Oso(8) min . One can verify that the Coulomb branch of the folded orthosymplectic quiver is also Oso(8) min . ne can verify that the Coulomb branch of the folded orthosymplectic quiver is also Oso(8) min . As a comment, (4.1) has the D5 Dynkin diagram on the left and the twisted aﬃne D(2) 4 Dynkin diagram on the right [50]. This pattern generalises to any n, meaning that the aﬃne Dn Dynkin quiver, whose Coulomb branch is Oso(2n) min , can be folded to the twisted aﬃne D(2) n−1 Dynkin quiver, whose Coulomb branch is Oso(2n−2) min . – 17 – E4 orbit. The unitary quiver is the aﬃne A4 Dynkin diagram, which after framing does not have identical legs attached to a pivot node, and hence cannot be folded in the common way. The orthosymplectic quiver with the same Coulomb branch is listed in the ﬁfth row of table 1, which does have two identical legs that one can fold. The wiggly line denotes a charge 2 hypermultiplet, see [15] for more details. The Coulomb branch of the folded non-simply laced orthosymplectic quiver is Osl(4) min . 4.1 En orbits A feature of orthosymplectic quivers whose Coulomb branches are closures of excep- tional algebras is that they always have two identical legs one can fold. This reﬂection symmetry is not always present in the unitary quiver counterparts. JHEP12(2021)070 4To be more precise, it gives the algebra of the global symmetry group. 4To be more precise, it gives the algebra of the global symmetry group. 4.2 ZZZ2 projection on representations For the E8 quiver, one natural branching to subgroups is to identify the identical legs: 4 6 6 8 4 2 2 6 2 2 4 4 6 2 Balance gives SO(8)B global symmetry Z2 2 8 6 6 4 4 2 2 Balance gives SO(8)diag global symmetry Oe7 min: Oe8 min: Balance gives SO(8)A global symmetry (4.7) (4.7) JHEP12(2021)070 Balance gives SO(8)diag global symmetry For a unitary magnetic quiver of E8, the quiver takes the form of the E8 aﬃne Dynkin diagram which does not have a natural SO(8) × SO(8) subgroup one can identify and fold. This is an advantage of the En orthosymplectic quivers in general which always has a natural Z2 symmetry one can fold. One can repeat this procedure for the remaining En families and the result reproduce the global symmetry of the folded quivers. 4.2 ZZZ2 projection on representations The results can be explained using representation theory. In [51], 5d N = 1 theories are compactiﬁed on a circle with Z2 twist. First, one seeks to ﬁnd a subgroup H5d of the global symmetry group G5d of the 5d SCFT such that H5d ∼= H1 × H1 × H2. In other words, H5d must contain two identical groups. Next, consider the Z2 invariant part of H1 × H1. This way, during the compactiﬁcation, the Z2 acts diagonally and only representations invariant under this action remain. Consider the E8 quiver. E8 contains the following subgroup: Consider the E8 quiver. E8 contains the following subgroup: E8 ⊃SO(8)A × SO(8)B . (4.3) (4.3) The adjoint representation of E8 decomposes as: The adjoint representation of E8 decomposes as: The adjoint representation of E8 decomposes as: presentation of E8 decomposes as: (µ8)E8 →(µ1)SO(8)A(µ1)SO(8)B + (µ2)SO(8)A + (µ2)SO(8)B + (µ3)SO(8)A(µ4)SO(8)B + (µ4)SO(8)A(µ3)SO(8)B (4.4) (4.4) where (µi)E8, (µi)A, (µi)B are the highest weight fugacities of E8, SO(8)A, and SO(8)B respectively. The Z2 group acts on the adjoint representation as follows: where (µi)E8, (µi)A, (µi)B are the highest weight fugacities of E8, SO(8)A, and SO(8)B respectively. The Z2 group acts on the adjoint representation as follows: (µ2)SO(8)A + (µ2)SO(8)B −→(µ2)SO(8)diag (µ1)SO(8)A(µ1)SO(8)B −→(µ2 1)SO(8)diag (µ3)SO(8)A(µ4)SO(8)B + (µ4)SO(8)A(µ3)SO(8)B −→(µ2 3)SO(8)diag + (µ2 4)SO(8)diag . (4.5) (4.5) Since SO(8)diag ⊂SU(8) ⊂E7, the irreducible representation after the projection precisely gives the adjoint representation for E7: Since SO(8)diag ⊂SU(8) ⊂E7, the irreducible representation after the projection precisely gives the adjoint representation for E7: (ρ1)E7 = (κ1κ7)SU(8) + (κ4)SU(8) = (µ2)SO(8) + (µ2 1)SO(8) + (µ2 3)SO(8) + (µ2 4)SO(8) (4.6) (4.6) where ρi, κi are highest weight fugacities of E7 and SU(8) respectively. here ρi, κi are highest weight fugacities of E7 and SU(8) respectively. This process is beautifully encoded in the folding procedure. When given a quiver, the balance of the gauge nodes determines the global symmetry group.4 If one singles out – 18 – a subset of balanced nodes, then this subquiver gives a subgroup of the global symmetry. 4.3 Magnetic quivers of 4d N = 2 The orthosymplectic quivers before folding are magnetic quivers tain 5d N = 1 SQCD theories at inﬁnite gauge coupling. The subscript next to the gauge group is the Chern-Simons level. For the E3−2l , the Coulomb branch of the magnetic quiver is only one of the two cones in the Higgs branch of the 5d theory. The global symmetry is given > 1 and k > l + 1, it enhances for k = 1 or k = l + 1 as shown in Table 1. JHEP12(2021)070 Before folding After folding Family Magnetic quiver (inﬁnite coupling) 5d quiver Global Symmetry Magnetic quiver Global Symmetry E8 2 2 2k + 6 2k + 4 2 2 · · · · · · 2 2k + 4 SU(k + 1)± 1 2 2k + 5 so(4k + 12) . . . 2k + 4 4 2 2 2k + 6 2 su(2k + 6) E7 2 2 2k + 4 2k + 2 2 2 · · · · · · 2 2k + 2 1 SU(k + 1)±1 2k + 4 so(4k + 8) ⊕su(2) . . . 2k + 2 4 2 2 2k + 4 2 1 su(2k + 4) ⊕su(2) E6 2 2 2k + 2 2k + 2 2 2 · · · · · · 2k + 2 1 SU(k + 1)± 3 2 2k + 3 so(4k + 6) ⊕u(1) . . . 2k + 2 4 2 2 2k + 2 1 u(2k + 3) E5 2 2 2k + 2 2k 2 2 · · · · · · 2k 1 SU(k + 1)±2 2k + 2 so(4k + 4) ⊕u(1) . . . 2k 4 2 2 2k + 2 1 u(2k + 2) E4−2l 2 2 2k −2l 2k −2l 2k −2l 2 2 1 l + 1 · · · · · · SU(k + 1)±( 5 2 +l) 2k −2l + 1 so(4k −4l + 2) ⊕u(1) . . . 2k −2l 4 2 2 2k −2l 1 l + 1 u(2k −2l + 1) E3−2l 2 2 2k −2l −2 2k −2l 2k −2l −2 2 2 1 l + 1 · · · · · · SU(k + 1)±(3+l) 2k −2l so(4k −4l) ⊕u(1) . . . 2k −2l −2 4 2 2 2k −2l 1 l + 1 u(2k −2l) Table 2. 4.3 Magnetic quivers of 4d N = 2 In [42], a class of unitary magnetic quivers of 5d N = 1 SQCD has been folded to produce general sequences whose limiting cases are 4d N = 2 rank 1 theories. In cases where the folding involves two identical legs, this procedure produces the Higgs branches of 5d theories compactiﬁed on a circle with a Z2 twist [51]. However, note that folding magnetic quivers of 5d N = 1 theories does not always give rise to magnetic quivers of 4d N = 2 theories. As seen in the above subsection, some unitary magnetic quivers, which do not have identical legs, have orthosymplectic counterparts that do have identical legs. The orthosymplectic quivers studied here are examples like that where the unitary counterparts (tabulated in [15, table 1]) lack this symmetry. In this section, the generalised families of orthosymplectic quivers are considered and folded. The results are summarised in table 2. Like the unitary quivers, one conjectures that some of the folded orthosymplectic quiv- ers are magnetic quivers of known 4d N = 2 theories. In other words, the Coulomb branch of these folded orthosymplectic quivers are the Higgs branch of 4d N = 2 theories. To be concrete, focus on the rank 1 cases in table 1. After folding the E8, E7, D5 orthosymplectic quivers, the resulting Coulomb branches are minimal nilpotent orbit closures of E7, E6, D4 respectively. These are Higgs branches of known 4d N = 2 rank 1 theories. On the other hand, folding the E6, A4 orthosymplectic quivers give Coulomb branches that are minimal nilpotent orbit closures of D5, A3 respectively which are not the Higgs branches – 19 – JHEP12(2021)070 able 2. Generalised families of En orthosymplectic quivers of those in Table 1. The orthosymplectic quivers before folding are magnetic quivers certain 5d N = 1 SQCD theories at inﬁnite gauge coupling. The subscript next to the gauge group is the Chern-Simons level. For the E3−2l mily, the Coulomb branch of the magnetic quiver is only one of the two cones in the Higgs branch of the 5d theory. The global symmetry is given r k > 1 and k > l + 1, it enhances for k = 1 or k = l + 1 as shown in Table 1. 2. Generalised families of En orthosymplectic quivers of those in Table 1. 4.3 Magnetic quivers of 4d N = 2 Generalised families of En orthosymplectic quivers of those in Table 1. The orthosymplectic quivers before folding are magnetic quivers of certain 5d N = 1 SQCD theories at inﬁnite gauge coupling. The subscript next to the gauge group is the Chern-Simons level. For the E3−2l family, the Coulomb branch of the magnetic quiver is only one of the two cones in the Higgs branch of the 5d theory. The global symmetry is given for k > 1 and k > l + 1, it enhances for k = 1 or k = l + 1 as shown in Table 1. JHEP12(2021)070 Before folding Before foldin Family Magnetic quiver (inﬁnite coupling) 5 E8 2 2 2k + 6 2k + 4 2 2 · · · · · · 2 2k + 4 S E7 2 2 2k + 4 2k + 2 2 2 · · · · · · 2 2k + 2 1 E6 2 2 2k + 2 2k + 2 2 2 · · · · · · 2k + 2 1 S E5 2 2 2k + 2 2k 2 2 · · · · · · 2k 1 E4−2l 2 2 2k −2l 2k −2l 2k −2l 2 2 1 l + 1 · · · · · · SU E3−2l 2 2 2k −2l −2 2k −2l 2k −2l −2 2 2 1 l + 1 · · · · · · SU Table 2. Generalised families of En orthosymplectic q of certain 5d N = 1 SQCD theories at inﬁnite gauge family, the Coulomb branch of the magnetic quiver is o for k > 1 and k > l + 1, it enhances for k = 1 or k = l – 20 – of known rank 1 4d theories [52]. It has been shown in [53], via anomaly matching on the Higgs branch, that D5, A3 minimal nilpotent orbit closures (or equivalently, one-instanton moduli spaces) are excluded as Higgs branches of rank 1 4d N = 2 theories. This shows only a subset of the folded orthosymplectic quivers are actually magnetic quivers for 4d N = 2 theories. Following this argument, one can generalise each En non-simply laced orthosymplectic quiver to inﬁnite families as in table 2. The families obtained from folding the E8, E7 and E5 ∼= D5 families give rise to known 4d N = 2 theories. 4.3 Magnetic quivers of 4d N = 2 These are all class S theories. For E8 and E7 folded families, these are Sicilian theories with A-type punctures (A-type 6d N = (2, 0) theories compactiﬁed on a sphere with 3 punctures) as studied in [54]. Using the parameterisation given in table 3, the folded E8 family gives the [k+3], [k+3], [22, 1k−1] Sicilian theory where punctures are labeled by their partition data. The folded E7 family gives [k + 2], [k + 2], [3, 1k−1] Sicilian theory. Finally, the E5 ∼= D5 folded family is the magnetic quiver for the 4d N = 2 SCFT of SU(k + 1) with 2k + 2 ﬂavours. JHEP12(2021)070 For the remaining three families in table 3, the theories do not resemble magnetic quivers of known 4d N = 2 theories. Nevertheless, they are magnetic quivers for 5d N = 1 theories. For the E6 folded family, the corresponding 5d theory is SU(k + 1)±1 with 2k + 2 ﬂavours at inﬁnite gauge coupling. The E4−2l folded family is a magnetic quiver of one of the two cones of the Higgs branch of the 5d SU(k+1)± 1 2 with 2k−2l+1 ﬂavours at inﬁnite gauge coupling. The E3−2l folded family is a magnetic quiver of one of the two cones of the Higgs branch of the 5d SU(k + 1)0 with 2k −2l + 1 ﬂavours at inﬁnite gauge coupling. The HWGs in table 3 can be obtained by taking the Coulomb branch HWG of the magnetic quiver before folding, see [18], and applying the projection (3.6)/(3.18), for the global symmetries so(4n)/so(4n + 2) respectively. The HWGs of the folded quivers are already computed in [23, 55–57]. 5 Hasse diagrams Hasse diagrams are useful tools for understanding the geometry of Higgs branches of various supersymmetric gauge theories. In particular, for theories with 8 supercharges in 3, 4, 5, 6 dimensions, this is explored in detail in recent works [14, 15, 42, 49, 58–60]. Instead of studying the Higgs branch of an electric theory directly, one can equivalently study the 3d N = 4 Coulomb branch of the corresponding magnetic quiver(s). This allows one to obtain the Hasse diagram using quiver subtraction [61]. Starting with a magnetic quiver, one can systematically subtract quivers to ﬁnd the diﬀerent transverse slices and symplectic leaves of the moduli space. The Coulomb branches of quivers one can subtract are elementary slices. The most up to date list of such quivers can be found in [62, table 1] for unitary quivers and in [15] for simply-laced orthosymplectic quivers. Here, the list is extended by non-simply laced orthosymplectic quivers whose Coulomb branches are elementary slices. These are summarised in table 4, for framed quivers giving closures of nilpotent orbits of type A, and table 1, for unframed quivers. – 21 – Table 3. The non-simply laced orthosymplectic quiver families and the unitary quiver family which have the same Coulomb branches. The highest weight generating function (HWG) is presented in the form of a plethystic logarithm (PL). The fugacities correspond to the global symmetry given in the last column of Table 2, with q denoting a u(1) factor and ν denoting an su(2) factor when present. Table 3. The non-simply laced orthosymplectic quiver families and the unitary quiver family which have the same Coulomb branches. The highest weight generating function (HWG) is presented in the form of a plethystic logarithm (PL). The fugacities correspond to the global symmetry given in the last column of Table 2, with q denoting a u(1) factor and ν denoting an su(2) factor when present. ( ) Magnetic quiver (orthosymplectic) Magnetic quiver (unitary) PL[HWG] . . . 2k + 4 4 2 2 2k + 6 2 2 k + 3 k + 2 k + 2 . . . . . . 2 1 2 1 Pk+2 i=1 µiµ2k+6−it2i + t4 + µk+3(tk+1 + tk+3) . . . 2k + 2 4 2 2 2k + 4 2 1 2 k + 2 k + 1 k + 1 . . . . . . 5 Hasse diagrams 2 1 2 1 1 Pk+2 i=1 µiµ2k+4−it2i + ν2t2 + t4 +νµk+2(tk+1 + tk+3) −ν2µ2 k+2t2k+6 . . . 2k + 2 4 2 2 2k + 2 1 k + 1 k k . . . . . . 2 1 2 1 k + 1 1 1 Pk i=1 µiµ2k+3−it2i + t2 + (µk+1q + µk+2/q)tk+1 . . . 2k 4 2 2 2k + 2 1 1 k + 1 k k . . . . . . 2 1 2 1 1 Pk i=1 µiµ2k+2−it2i + t2 + µk+1(q + 1/q)tk+1 . . . 2k −2l 4 2 2 2k −2l 1 l + 1 k −l k −l −1 k −l −1 . . . . . . 2 1 2 1 k −l 1 1 l + 1 Pk−l i=1 µiµ2k−2l+1−it2i + t2 +(µk−lq)tk+1 + (µk−l+1/q)tk+1 −µk−lµk−l+1t2k+2 . . . 2k −2l −2 4 2 2 2k −2l 1 l + 1 1 k −l k −l −1 k −l −1 . . . . . . 2 1 2 1 1 l + 1 Pk−l i=1 µiµ2k−2l−it2i + t2 +µk−l(q + 1/q)tk+1 −µ2 k−lt2k+2 Table 3. The non-simply laced orthosymplectic quiver families and the unitary quiver family which have the same Coulomb branches. The highest weight generating function (HWG) is presented in the form of a plethystic logarithm (PL). The fugacities correspond to the global symmetry given in the last column of Table 2, with q denoting a u(1) factor and ν denoting an su(2) factor when present. JHEP12(2021)070 – 22 – e 4. The orthosymplectic quivers on the left have Coulomb branches that are closures of nilpotent orbits of Dn algebras. Red nodes with dex k denote SO(k) groups while blue nodes with index 2k denote USp(2k) groups. Folding these quivers along the identical legs gives the imply laced orthosymplectic quivers on the right. The Coulomb branches of these theories are given as well. Note that the central node for uivers on the left are orthogonal on the ﬁrst row and symplectic on the second row. Before folding After folding Orthosymplectic quiver Coulomb branch Orthosymplectic quiver Coulomb branch 1 1 . . . 2 2 2 2 3 3 4n −3 nodes . . . 2 3 2 Oso(4n) min = d2n 2 2 for n = 1 2 3 3 . . . 5For general orthosymplectic quivers, while the rules for electric quiver subtractions are straightforward, there are many complications when dealing with magnetic quivers, such as the choice of SO/O gauge groups, and shifts between SO(odd) vs. SO(even) gauge nodes to obtain nodes of the desired dimensions and rank, all of which make the recipe for subtraction quite involved. 5 Hasse diagrams 2 3 2 1 2 2n −1 nodes for n > 1 Osl(2n) min = a2n−1 1 1 . . . 2 2 2 2 3 3 4n −1 nodes . . . 3 2 3 Oso(4n+2) min = d2n+1 2 2 2 for n = 1 3 2 2 . . . 2 3 2 1 2 2n nodes for n > 1 Osl(2n+1) min = a2n Table 4. The orthosymplectic quivers on the left have Coulomb branches that are closures of nilpotent orbits of Dn algebras. Red nodes with an index k denote SO(k) groups while blue nodes with index 2k denote USp(2k) groups. Folding these quivers along the identical legs gives the non-simply laced orthosymplectic quivers on the right. The Coulomb branches of these theories are given as well. Note that the central node for the quivers on the left are orthogonal on the ﬁrst row and symplectic on the second row. JHEP12(2021)070 2n −1 nodes – 23 – 6 8 2 6 4 4 2 2 6 7 6 4 4 2 2 1 4 5 5 4 4 2 2 1 2 3 3 2 3 2 2 1 2 3 3 2 3 2 2 1 2 2 2 3 1 2 3 3 2 1 2 2 {1} a1 a3 a5 a7 Figure 2. Hasse diagram of O sl(8) (24) . 6 8 2 6 4 4 2 2 6 7 6 4 4 2 2 1 4 5 5 4 4 2 2 1 2 3 3 2 3 2 2 1 2 3 3 2 3 2 2 1 2 2 2 3 1 2 3 3 2 1 2 2 {1} a1 a3 a5 a7 Figure 2. Hasse diagram of O sl(8) (24) . JHEP12(2021)070 {1} Figure 2. Hasse diagram of O sl(8) (24) . 5.1 Maximal height 2 orbits Quiver subtraction for orthosymplectic quivers. The rules for quiver subtraction between unitary quivers are given in [61]. The general idea is to align two quivers and subtract the respective gauge nodes Gi −G′ i →G′′ i such that rank(Gi) −rank(G′ i) = rank(G′′ i ). The resulting gauge groups need to be rebalanced by adding ﬂavours. For orthosymplectic quivers, rules are given in [39]. From a brane perspective, subtracting orthosymplectic quivers for so(2n) orbits corre- sponds to Kraft-Procesi transitions [49].5 It turns out that all one needs for a Kraft-Procesi decomposition of the families of orthosymplectic quivers treated in this paper are the fol- lowing subtraction guides [14], which apply to subtraction of special minimal nilpotent orbits: SO(2k) −SO(2r) →SO(2k −2r + 1), (5.1a) SO(2k + 1) −SO(2r + 1) →SO(2k −2r + 1), (5.1b) USp(2k) −USp(2r) →USp(2k −2r). (5.1c) (5.1c) This means that one can subtract minimal nilpotent orbit closures of type an, dn, e6,7,8. To begin with, focus on a framed orthosymplectic quiver explored in section 3. Ta ing (3.3) with n = 4, one obtains the Hasse diagram shown in ﬁgure 2. The quivers on th – 24 – 6 6 2 4 4 2 2 4 3 2 2 3 2 2 {1} 5 4 4 2 2 1 1 6 6 2 4 4 2 2 4 3 2 2 3 2 2 {1} 5 4 4 2 2 2 2 2 1 2 3 2 1 2 1 3 2 2 3 2 2 1 a2 a4 a6 Figure 3. Hasse diagram for O sl(7) (23,1). JHEP12(2021)070 {1} Figure 3. Hasse diagram for O sl(7) (23,1). right have Coulomb branches that are closures of the symplectic leaves denoted by . The quivers on the left are the subtracted quivers, where the labeling an−1 means the Coulomb branch is the closure of the minimal nilpotent orbit of sl(n). The result is consistent with the Hasse diagram of the Osl(8) (24) [63]. Considering (3.15), instead, for the case n = 3, the Hasse diagram is shown in ﬁgure 3. This is the Hasse diagram for Osl(7) (23,1) [63]. right have Coulomb branches that are closures of the symplectic leaves denoted by . The quivers on the left are the subtracted quivers, where the labeling an−1 means the Coulomb branch is the closure of the minimal nilpotent orbit of sl(n). 5.1 Maximal height 2 orbits The result is consistent with the Hasse diagram of the Osl(8) (24) [63]. Considering (3.15), instead, for the case n = 3, the Hasse diagram is shown in ﬁgure 3. This is the Hasse diagram for Osl(7) (23,1) [63]. 5.2 General families Now, one performs quiver subtraction to obtain Hasse diagrams for the general families tabulated in table 2. The only diﬀerence here is that the orthosymplectic quiver is un- framed. The ﬁrst non-simply laced quiver one subtracts are those in table 1 whose Coulomb branches are also closures of minimal nilpotent orbits. The subtraction rules for unframed orthosymplectic magnetic quivers are incomplete. However, the unframed orthosymplectic quivers (before folding) are studied in [14, 15, 18, 25] where the Hasse diagrams are ob- tained through transitions on brane webs. By observing those transitions, one realises that the rules for subtracting the nodes for these families are the same as (5.1). The resulting Hasse diagrams are again what one expects through quiver subtraction on their unitary quiver counterparts. Below, the Hasse diagram is drawn for one example from each of the families as the general Hasse diagram is already known from the unitary quivers and is given in [59]. The examples for k = 3 are shown in ﬁgures 4, 5, 6, 7, 8, 9. In fact, for the entire E4−2l and E3−2l families (l ≥1), the Coulomb branch of the ﬁrst quiver to subtract is always a Al = C2/Zl+1 Kleinian singularity given by a U(1) with l + 1 charge 2 hypermultiplets. This identiﬁcation follows from the rules of quiver subtraction established in [39, 59, 61]. We remark that the moduli spaces for the folded E6 and E5 families (third and fourth rows in table 3) coincide with the classical Higgs branches of SQCD theories, speciﬁcally – 25 – 10 12 2 10 4 5 5 4 5 4 5 2 3 3 2 3 2 3 {1} 2 4 2 3 2 2 2 2 1 6 6 4 4 2 2 8 8 6 8 2 6 2 2 4 4 1 2 3 3 2 3 2 3 2 2 1 2 3 3 2 3 2 3 2 3 2 2 1 e7 a9 a11 Figure 4. Hasse diagram of the folded E8 family for k = 3. 10 12 2 10 4 5 5 4 5 4 5 2 3 3 2 3 2 3 {1} 2 4 2 3 2 2 2 2 1 6 6 4 4 2 2 8 8 1 e7 a9 11 JHEP12(2021)070 {1} Figure 4. Hasse diagram of the folded E8 family for k = 3. 5.2 General families 4 4 2 2 4 {1} 4 2 2 1 2 3 2 2 2 2 1 A1 a2 a4 4 4 2 2 4 {1} 4 2 2 1 2 3 2 2 2 1 2 2 2 2 2 1 2 3 2 2 1 A1 a2 a4 Figure 8. Hasse diagram of the folded E4−2l family for k = 3 and l = 1. {1} Figure 8. Hasse diagram of the folded E4−2l family for k = 3 and l = 1. SU(k +1) gauge theories with respectively 2k +3 and 2k +2 fundamental hypermultiplets. Therefore these can be realized as hyper-Kähler quotients. SU(k +1) gauge theories with respectively 2k +3 and 2k +2 fundamental hypermultiplets. Therefore these can be realized as hyper-Kähler quotients. 5.2 General families 2 6 2 2 4 4 2 2 3 3 2 3 2 2 1 2 3 3 2 3 2 3 2 2 1 e6 a7 a9 2 10 4 5 5 4 5 4 4 {1} 2 2 6 6 4 4 8 8 2 2 2 1 2 3 3 2 3 2 3 2 2 1 2 10 4 5 5 4 5 4 4 {1} 2 2 6 6 4 4 8 8 2 2 2 2 6 2 2 4 4 2 1 2 3 3 2 3 2 2 1 2 3 3 2 3 2 3 2 2 1 2 3 3 2 3 2 3 2 2 1 e6 a7 a9 Figure 5. Hasse diagram of the folded E7 family for k = 3. {1} Figure 5. Hasse diagram of the folded E7 family for k = 3. 4 4 2 2 1 1 3 2 2 3 2 2 2 3 2 3 2 3 2 2 1 8 8 6 6 4 4 4 {1} 5 4 5 4 4 1 2 2 4 4 2 2 1 2 2 1 1 3 2 2 3 2 2 2 3 2 3 2 3 2 2 1 2 3 2 3 2 3 2 2 1 d5 a6 a8 Figure 6. Hasse diagram of the folded E6 family for k = 3. 8 8 6 6 4 4 4 {1} 5 4 5 4 4 1 2 2 2 2 1 2 3 2 3 2 3 2 2 1 d5 a6 a8 {1} Figure 6. Hasse diagram of the folded E6 family for k = 3. – 26 – 1 8 4 5 5 4 4 2 2 {1} 6 6 4 4 2 2 2 3 3 2 3 2 2 1 4 2 2 1 2 3 3 2 2 1 1 2 3 3 2 3 2 2 1 d4 a5 a7 Figure 7. Hasse diagram of the folded E5 family for k = 3. 1 4 2 2 2 3 3 2 2 1 2 3 3 2 3 2 2 1 1 8 4 5 5 4 4 2 2 {1} 6 6 4 4 2 2 2 3 3 2 3 2 2 1 1 JHEP12(2021)070 {1} Figure 7. Hasse diagram of the folded E5 family for k = 3. 6One can also insert O5−plane which will give a B-type non-simply laced orthosymplectic quiver. Such quivers will be studied in detail somewhere else. 6 Brane conﬁgurations Inspired by the results above, one can study the respective brane conﬁgurations that give rise to the non-simply laced orthosymplectic quivers. This section focuses only on the framed orthosymplectic quivers of section 3. The quiver gauge theories are 3d N = 4 eﬀective ﬁeld theories living on D3 branes that are stretched between 5-branes in the presence of orientifold planes. To begin with, consider the magnetic quiver in ﬁgure 10, whose Coulomb branch is the next-to-minimal nilpotent orbit of sl(2n). The brane conﬁguration is presented on the right. By inserting O3 planes, one produces an orthosymplectic quiver that is the minimal – 27 – 1 4 2 4 2 {1} 2 2 2 3 2 2 2 2 2 1 2 3 2 1 1 2 A1 a1 a3 Figure 9. Hasse diagram of the folded E3−2l family for k = 3 and l = 1. 1 4 2 4 2 {1} 2 2 2 3 2 2 2 1 JHEP12(2021)070 {1} Figure 9. Hasse diagram of the folded E3−2l family for k = 3 and l = 1. nilpotent orbit of so(2n). One can insert an O5+ plane in the centre in combination with the presence of the O3 planes.6 An ON+ plane then sits at the intersection of the two orientifolds [64]. The proposal is that the resulting brane conﬁguration is the folded orthosymplectic quiver whose Coulomb branch is the minimal nilpotent orbit of sl(n), which is expected as the gauge theory on 2k D3 branes between two NS5 branes in the presence of this intersection of orientifolds is U(k) [65, 66]. This shows the insertion of O3 and O5 branes is a commutative process. Similarly, one can start with the sl(2n) unitary quiver and insert an O5+ plane in the middle. This creates a non-simply laced unitary quiver [1] whose Coulomb branch is the next-to-minimal orbit of usp(2n). Inserting O3 planes after that reproduces the same folded orthosymplectic quiver as above. 6.1 Generalisation of height two quivers Electric (left) and magnetic (below) quivers for the brane systems are provided. The magnetic quivers are most conveniently read after suitable Hanany-Witten transitions. The O5+ insertion on the left is the brane realisation of what is called folding in [5]. reproduces (3.9) whose Coulomb branch is the closure of (2k, 1n−2k) orbit of sl(n). This process is demonstrated in ﬁgure 12. n odd. For sl(2n) and n odd, height 2 orbits are identiﬁed by the partition (22k+1, 12n−4k−2) for n ≥2k+1. Putting O3 planes reproduces (3.13) for n = 2k+1 and (3.19) for n > 2k+1 whose Coulomb branches are closures of (2n−1, 12) and (22k−1, 12n−4k+2) nilpotent orbits of so(2n) respectively. Adding an O5+ plane gives the folded orthosymplectic quiver in (3.15) and (3.20) whose Coulomb branches are the closure of the 2 n−1 2 , 1 and (2k, 1n−2k) orbit of sl(n) respectively. This process is demonstrated in ﬁgure 13. 6.1 Generalisation of height two quivers The above procedure can be generalised by starting with a unitary quiver whose Coulomb branch is the closure of a general height 2 sl(2n) nilpotent orbit. n even. For the maximal height 2 orbit of sl(2n) the partition is (2n). Adding O3 planes to the brane conﬁguration produces the orthosymplectic quiver in (3.1) whose Coulomb branch is (one of the two identical cones) of the (2n) orbit of so(2n). Adding O5 planes then yield the folded orthosymplectic quiver (3.3) whose Coulomb branch is the 2 n 2 orbit of sl(n). This process is demonstrated in ﬁgure 11. The remaining height 2 orbits of sl(2n) are identiﬁed by the partition (2k+1, 12n−4k−2) with n > 2k. Adding an orientifold produces the orthosymplectic quiver in (3.7) whose Coulomb branch is the closure of (22k, 12n−4k) orbit of so(2n). Adding an O5+ plane – 28 – 1 2 2 2 2 2 2 2 1 1 1 2 10 2 2 2 2 1 1 O(2) USp(10) 2 2 3 2 3 2 2 1 1 USp(2) SO(10) 2 3 2 2 1 1 5 O5+ O5+ O3+ O3+ Figure 10. The commutative diamond represents the construction of the non-simply laced or- thosymplectic quiver. Vertical lines depict D5 branes, circles with crosses NS5 branes, and horizon- tal lines D3 branes. The vertical green line depicts an O5+ plane, the horizontal dotted line an O3+ plane, the horizontal dashed line an eO3+ plane, and the orange circle with a cross an ON+ plane. Electric (left) and magnetic (below) quivers for the brane systems are provided. The magnetic quivers are most conveniently read after suitable Hanany-Witten transitions. The O5+ insertion on the left is the brane realisation of what is called folding in [5]. 1 2 2 2 2 2 2 2 1 1 1 2 10 2 2 2 2 1 1 O(2) USp(10) 2 2 3 2 3 2 2 1 1 USp(2) SO(10) 2 3 2 2 1 1 5 O5+ O5+ O3+ O3+ JHEP12(2021)070 Figure 10. The commutative diamond represents the construction of the non-simply laced or- thosymplectic quiver. Vertical lines depict D5 branes, circles with crosses NS5 branes, and horizon- tal lines D3 branes. The vertical green line depicts an O5+ plane, the horizontal dotted line an O3+ plane, the horizontal dashed line an eO3+ plane, and the orange circle with a cross an ON+ plane. 6.2 Kraft-Procesi transitions In section 5, the diﬀerent phases of a quiver theory are studied via the Hasse diagram. The phase diagram can be explicitly derived via Kraft-Procesi transitions [49] in a brane conﬁguration. For concreteness, consider the brane conﬁguration at the top of ﬁgure 14, which displays a Higgs branch phase of the D3-D5-NS5 system. The corresponding mag- – 29 – 2 . . . . . . 2 1 2 1 . . . . . . 2 n n−2 n−2 4 2 2 4 2 2 O3+ O5+ . . . n−2 4 2 2 n 2 2 n 2 n 2 −1 n 2 −1 . . . . . . 2 1 2 1 n−1 n n−1 = Figure 11. The even n case. The Coulomb branch of the top quiver is the sl(2n) nilpotent orbit closure with partition (2n). With the addition of O3+ planes, the following orthosymplectic quiver is the so(2n) nilpotent orbit closure with partition (2n). Finally, adding O5+ planes, the resulting non-simply laced orthosymplectic quiver is obtained. This quiver has a unitary counterpart on the right whose Coulomb branch is the sl(n) nilpotent orbit closure of partition 2 n 2 . JHEP12(2021)070 2 n 2 n 2 −1 n 2 −1 . . . . . . 2 1 2 1 = Figure 11. The even n case. The Coulomb branch of the top quiver is the sl(2n) nilpotent orbit closure with partition (2n). With the addition of O3+ planes, the following orthosymplectic quiver is the so(2n) nilpotent orbit closure with partition (2n). Finally, adding O5+ planes, the resulting non-simply laced orthosymplectic quiver is obtained. This quiver has a unitary counterpart on the right whose Coulomb branch is the sl(n) nilpotent orbit closure of partition 2 n 2 . netic quiver for this Higgs branch is displayed on the left and describes the closure of the maximal height 2 orbit of sl(6). A Kraft-Procesi transition is realised by moving a full D3 brane onto the O3 plane such that the D3 can split on the NS5 branes. The D3 seg- ment, which is solely suspended between NS5 branes, gives rise to an electric theory that characterises the KP transition via its Higgs branch. In other words, this is the transverse slice and the associated magnetic quiver is depicted next to the arrow in ﬁgure 14. 6.2 Kraft-Procesi transitions Moving this D3 brane segment along the NS5 branes implies that this modulus has left the Higgs branch and entered the Coulomb branch. The remaining brane conﬁguration, displayed at the centre of ﬁgure 14, has an associated magnetic quiver which accounts for the sym- plectic leaf below the top. The next KP transition proceeds as before, segments of full D3 branes are aligned and moved onto the O3 plane such that the resulting D3 can split on the NS5 brane. The Higgs branch of the electric theory characterises the transverse slice and ﬁgure 14 displays the associated magnetic quiver. After moving this D3 onto the Coulomb branch, the remaining parts of the Hasse diagram are obtained by repeating KP transition until all D3 branes are moved onto the Coulomb branch. Since there are no Higgs branch moduli left, the magnetic quiver is trivial. This is the trivial symplectic leaf of the Hasse diagram. The Kraft-Procesi transitions are almost identical to those in [49] and, therefore, are – 30 – JHEP12(2021)070 O3+ O5+ = · · · · · · 2 2 4 1 1 . . . 2k 2k 2k 2k 2k+1 2k+1 2n−4k−1 nodes 4 2 2 . . . 2k 2k+1 2k . . . 2k 2 2 2k+1 2k + 1 . . . 2k 2k+1 2k 1 2k n−2k nodes . . . . . . 1 2 1 2 1 1 . . . k−1 k k k k k−1 n−2k+1 nodes . . . . . . 1 2 1 2 1 1 . . . 2k 2k + 1 2k + 1 2k + 1 2k + 1 2k 2n−4k−1 nodes Figure 12. The even n case. The Coulomb branch of the top quiver is the sl(2n) nilpotent orbit closure with partition (22k+1, 12n−4k−2). With the addition of O3+ planes, the following orthosymplectic quiver is the so(2n) nilpotent orbit closure with partition (22k, 12n−4k). Finally, adding O5+ planes, the resulting non-simply laced orthosymplectic quiver is obtained. This quiver has a unitary counterpart on the right whose Coulomb branch is the sl(n) nilpotent orbit closure of partition (2k, 1n−2k). O3+ O5+ = · · · · · · 2 2 4 1 1 . . . 2k 2k 2k 2k 2k+1 2k+1 2n−4k−1 nodes 4 2 2 . . . 2k 2k+1 2k . . . 2k 2 2 2k+1 2k + 1 . 6.2 Kraft-Procesi transitions . . 2k 2k+1 2k 1 2k n−2k nodes . . . . . . 1 2 1 2 1 1 . . . k−1 k k k k k−1 n−2k+1 nodes . . . . . . 1 2 1 2 1 1 . . . 2k 2k + 1 2k + 1 2k + 1 2k + 1 2k 2n−4k−1 nodes 2n−4k−1 nodes JHEP12(2021)070 2n−4k−1 nodes O5+ = . . . 2k 2 2 2k+1 2k + 1 . . . 2k 2k+1 2k 1 2k n−2k nodes . . . . . . 1 2 1 2 1 1 . . . k−1 k k k k k−1 n−2k+1 nodes n−2k+1 nodes n−2k+1 nodes n−2k+1 nodes Figure 12. The even n case. The Coulomb branch of the top quiver is the sl(2n) nilpotent orbit closure with partition (22k+1, 12n−4k−2). With the addition of O3+ planes, the following orthosymplectic quiver is the so(2n) nilpotent orbit closure with partition (22k, 12n−4k). Finally, adding O5+ planes, the resulting non-simply laced orthosymplectic quiver is obtained. This quiver has a unitary counterpart on the right whose Coulomb branch is the sl(n) nilpotent orbit closure of partition (2k, 1n−2k). Figure 12. The even n case. The Coulomb branch of the top quiver is the sl(2n) nilpotent orbit closure with partition (22k+1, 12n−4k−2). With the addition of O3+ planes, the following orthosymplectic quiver is the so(2n) nilpotent orbit closure with partition (22k, 12n−4k). Finally, adding O5+ planes, the resulting non-simply laced orthosymplectic quiver is obtained. This quiver has a unitary counterpart on the right whose Coulomb branch is the sl(n) nilpotent orbit closure of partition (2k, 1n−2k). – 31 – JHEP12(2021)070 O3+ O5+ = . . . . . . 1 2 1 2 1 1 . . . k−1 k k k k k−1 n−2k+1 nodes . . . . . . 1 2 1 2 1 1 . . . 2k 2k+1 2k+1 2k+1 2k+1 k 2n−4k−1 nodes . . . . . . 2 2 4 1 1 . . . 2k 2k 2k 2k 2k+1 2k+1 2n−4k−1 nodes 4 2 2 . . . 2k+1 2k 2k+1 . . . 2k+1 2 2 2k 2k . . . 2k 2k+1 2k 1 2k n−k−1 nodes Figure 13. The odd n case. The Coulomb branch of the top quiver is the sl(2n) nilpotent orbit closure with partition (22k+1, 12n−4k−2). 6.2 Kraft-Procesi transitions With the addition of O3+ planes, the following orthosym- plectic quiver is the so(2n) nilpotent orbit closure with partition (22k−1, 12n−4k+2). Finally, adding O5+ planes, the resulting non-simply laced orthosymplectic quiver is obtained. This quiver has a unitary counterpart on the right whose Coulomb branch is the sl(n) nilpotent orbit closure of partition (2k, 1n−2k). only shown for one example. The only complication is that the presence of both O3 and O5 planes ﬁxes the ON plane. only shown for one example. The only complication is that the presence of both O3 and O5 planes ﬁxes the ON plane. 6.2 Kraft-Procesi transitions With the addition of O3+ planes, the following orthosym- plectic quiver is the so(2n) nilpotent orbit closure with partition (22k−1, 12n−4k+2). Finally, adding O5+ planes, the resulting non-simply laced orthosymplectic quiver is obtained. This quiver has a unitary counterpart on the right whose Coulomb branch is the sl(n) nilpotent orbit closure of partition (2k, 1n−2k). O3+ O5+ = . . . . . . 1 2 1 2 1 1 . . . k−1 k k k k k−1 n−2k+1 nodes . . . . . . 1 2 1 2 1 1 . . . 2k 2k+1 2k+1 2k+1 2k+1 k 2n−4k−1 nodes . . . . . . 2 2 4 1 1 . . . 2k 2k 2k 2k 2k+1 2k+1 2n−4k−1 nodes 4 2 2 . . . 2k+1 2k 2k+1 . . . 2k+1 2 2 2k 2k . . . 2k 2k+1 2k 1 2k n−k−1 nodes Figure 13. The odd n case. The Coulomb branch of the top quiver is the sl(2n) nilpotent orbit 2n−4k−1 nodes O3 O5+ = . . . . . . 1 2 1 2 1 1 . . . k−1 k k k k k−1 n−2k+1 nodes . . . . . . 2 4 1 1 . . . 2k 2k 2k 2k 2k+1 2k+1 2n−4k−1 nodes 4 2 . . . 2k+1 2k 2k+1 . . . 2k+1 2 2 2k 2k . . . 2k 2k+1 2k 1 2k n−k−1 nodes JHEP12(2021)070 2n−4k−1 nodes O5+ = . . . . . . 1 2 1 2 1 1 . . . k−1 k k k k k−1 n−2k+1 nodes . . . 2k+1 2 2 2k 2k . . . 2k 2k+1 2k 1 2k n−k−1 nodes n−2k+1 nodes n−2k+1 nodes Figure 13. The odd n case. The Coulomb branch of the top quiver is the sl(2n) nilpotent orbit closure with partition (22k+1, 12n−4k−2). With the addition of O3+ planes, the following orthosym- plectic quiver is the so(2n) nilpotent orbit closure with partition (22k−1, 12n−4k+2). Finally, adding O5+ planes, the resulting non-simply laced orthosymplectic quiver is obtained. This quiver has a unitary counterpart on the right whose Coulomb branch is the sl(n) nilpotent orbit closure of partition (2k, 1n−2k). Figure 13. The odd n case. The Coulomb branch of the top quiver is the sl(2n) nilpotent orbit closure with partition (22k+1, 12n−4k−2). Acknowledgments We would like to thank Satoshi Nawata, Dan Xie and Gabi Zafrir for helpful discussions. This work is supported by STFC grants ST/P000762/1 and ST/T000791/1. M.S. is sup- ported by the National Natural Science Foundation of China (grant no. 11950410497), and the China Postdoctoral Science Foundation (grant no. 2019M650616). Z.Z. is thank- ful for the kind hospitality of YMSC and Tsinghua University where part of this work is completed. 7 Conclusion and outlook The notation for twisted Aodd punctures is adapted from [71, 72]. Type J′ J J∨ Dynkin Labels of J′ Standard Gr∨ Gr Gr∨ [n1, . . . , nr] Twisted AI odd A2r−1 Dr Dr [n1, . . . , M, A, M, . . . , n1] Twisted AII odd A2r−1 Cr Br [n1, . . . , nr−1, nr, nr−1, . . . , n1] Twisted Aeven A2r Br Cr [n1, . . . , nr, nr, . . . , n1] Twisted D Dr+1 Br Cr [n1, . . . , nr, nr] Table 5. Types of Twisted Puncture. The maps express the Dynkin labels [n′] of J′ in terms of the Dynkin labels [n] of J∨. Here M ≡Min[nr−1, nr] and A ≡Abs[nr−1 −nr]. The notation for twisted Aodd punctures is adapted from [71, 72]. Table 5. Types of Twisted Puncture. The maps express the Dynkin labels [n′] of J′ in terms the Dynkin labels [n] of J∨. Here M ≡Min[nr−1, nr] and A ≡Abs[nr−1 −nr]. The notation f twisted Aodd punctures is adapted from [71, 72]. Table 5. Types of Twisted Puncture. The maps express the Dynkin labels [n′] of J′ in terms of the Dynkin labels [n] of J∨. Here M ≡Min[nr−1, nr] and A ≡Abs[nr−1 −nr]. The notation for twisted Aodd punctures is adapted from [71, 72]. JHEP12(2021)070 branches are sl(n) nilpotent orbit closures of height 2. Amongst unframed orthosymplectic quivers, the magnetic quivers of the 5d En families discussed in [15] have been treated. The Coulomb branches of the folded quivers have known unitary quiver counterparts, and in some cases are magnetic quivers for known 5d N = 1 or 4d N = 2 theories. The brane systems studied alternatively allow for f O5 + and O5−orientifold planes, giving rise to orthosymplectic quivers of B and D type. We leave the study of these quivers for future work. It would furthermore be interesting to study ﬁve-brane webs including O5 and O7+ orientifold planes, which are expected to yield the unframed non-simply laced orthosymplectic magnetic quivers studied in this work. 7 Conclusion and outlook In this article, the folding of orthosymplectic quivers, both framed and unframed, has been studied. In the case of framed orthosymplectic quivers, whose Coulomb branches are so(2n) nilpotent orbit closures of height 2, folding gives non-simply laced quivers whose Coulomb – 32 – JHEP12(2021)070 4 6 2 4 2 2 4 5 4 2 2 1 2 3 3 2 2 1 2 2 2 3 1 2 2 3 3 2 2 1 {1} a1 a3 a5 Figure 14. Deriving the Hasse diagram via quiver subtraction and Kraft-Procesi transitions. The left-hand side details the quiver subtraction algorithm; while the right-hand side shows the corresponding Kraft-Procesi transitions in the brane conﬁgurations. The notation for the branes and O3 planes follows [49]. The branes colored in magenta correspond to the subtracted ﬁgures on the left side. JHEP12(2021)070 2 2 2 1 2 1 a1 a3 JHEP12(2021)070 Hasse diagram via quiver subtraction and Kraft-Procesi transitions. he quiver subtraction algorithm; while the right-hand side shows the transitions in the brane conﬁgurations. The notation for the branes The branes colored in magenta correspond to the subtracted ﬁgures on 4 6 2 4 2 2 4 5 4 2 2 1 2 3 3 2 2 1 2 2 2 3 1 2 2 3 3 2 2 1 {1} a1 a3 a5 JHEP12(2021)070 {1} Figure 14. Deriving the Hasse diagram via quiver subtraction and Kraft-Procesi transitions. The left-hand side details the quiver subtraction algorithm; while the right-hand side shows the corresponding Kraft-Procesi transitions in the brane conﬁgurations. The notation for the branes and O3 planes follows [49]. The branes colored in magenta correspond to the subtracted ﬁgures on the left side. – 33 – Type J′ J J∨ Dynkin Labels of J′ Standard Gr∨ Gr Gr∨ [n1, . . . , nr] Twisted AI odd A2r−1 Dr Dr [n1, . . . , M, A, M, . . . , n1] Twisted AII odd A2r−1 Cr Br [n1, . . . , nr−1, nr, nr−1, . . . , n1] Twisted Aeven A2r Br Cr [n1, . . . , nr, nr, . . . , n1] Twisted D Dr+1 Br Cr [n1, . . . , nr, nr] Table 5. Types of Twisted Puncture. The maps express the Dynkin labels [n′] of J′ in terms of the Dynkin labels [n] of J∨. Here M ≡Min[nr−1, nr] and A ≡Abs[nr−1 −nr]. A Hall Littlewood polynomials and star shaped quivers We can calculate the Coulomb branch Hilbert series of many 3d N = 4 unframed unitary and/or orthosymplectic star shaped quivers Q(J), that are characterised by a central node with gauge group J, by gluing resolved Slodowy slices. The procedure depends upon being able to identify the linear quivers that form the legs of the star shaped quiver as Slodowy slices; these may be transverse to orbits in the GNO dual group J∨, or in a twist related simply laced dual J′, as discussed later, see table 5. This correspondence between linear quivers and Slodowy slices draws on the Barbasch-Vogan map and related dualities, as elaborated in [39, 67]. The pairings for low rank Classical groups are tabulated in [67], and can in principle be extended to higher rank. Recall that, for any Lie group G, nilpotent orbits are labeled by homomorphisms ρ : SU(2) →G, which correspond to partitions. The Slodowy slice SG N,ρ transverse to the closure of the nilpotent orbit Oρ deﬁnes a decomposition G →F(ρ) ⊗SU(2), where – 34 – F(ρ) is the centralizer of the image of ρ in G. This partition ρ thus determines a fugacity map x →(y, t), where the x and y are Cartan subalgebra (CSA) fugacities for G and the subgroup F(ρ) ⊆G, respectively, and t is a CSA fugacity for the SU(2). This fugacity map is unique up to Weyl group transformations. The Slodowy slice transforms under F(ρ). The Hilbert series for a Slodowy slice can be found from the branching of the adjoint representation of G under ρ: χG [adjoint](x) = M [n],[m] an,m χSU(2) [n] ⊗χF [m](y) , (A.1) (A.1) where the an,m are branching coeﬃcients. The HS is obtained from (A.1) by (i) replacing the SU(2) characters by highest weight fugacities [37], χSU(2) [n] →tn, (ii) symmetrising the representations of F(ρ) under a grading by t2, and (iii) taking a quotient by the Casimirs of G. This leads to the reﬁned and unreﬁned HS: JHEP12(2021)070 g SG N ,ρ HS (y, t) = PE  M [n],[m] an,m χF [m](y)tn+2 − r X i=1 t2di  , g SG N ,ρ HS (1, t) = PE "X n antn+2 − r X i=1 t2di # , (A.2) (A.2) where the di are the degrees of the Casimirs of G. Further details on the construction of slices can be found in [67]. A Hall Littlewood polynomials and star shaped quivers Developing the methods of [38], we now introduce resolved Slodowy slices, SG N,ρ,[n], which carry background charges [n] parameterised by the Dynkin labels of G, as described in [39, appendix B]. These are related to uncharged slices (which eﬀectively carry singlet charges) by a quotient of Hall Littlewood polynomials [68] under the fugacity map for ρ: g SG N ,ρ,[n] HS (y, t) ≡g SG N ,ρ HS (y, t) HLG [n](x, t) HLG [0](x, t) ρ: x→(y,t) . (A.3) (A.3) The above considerations are quite general and apply to any Lie group G. Returning to our star shaped quiver Q(J), let us assume that we can identify its k legs with a set of Slodowy slices SJ∨ N,ρ(i), where i ∈{1, . . . , k}. The partitions ρ(i) determine fugacity maps x →(y(i), t). Given such a set of slices, we can apply charges [n] to these slices and carry out gluing [38] by summation over the weight lattice of J∨. We obtain the following Hilbert series: gQ(J) HS (y(1), . . . , y(k), t) = X [n]∈ΓJ∨/W ∨ P J∨ [n] t2∆[[n]] \| {z } central term k Y i=1 Tρ(i),[n](J). (A.4) (A.4) Here, the symmetry factors P J∨ [n] match the P(t; m) terms that appear in the monopole formula (2.2), being related by the map between the magnetic weight lattice charges m in the orthogonal basis of J∨, and the weight lattice charges [n] in the Dynkin label or ω-basis [69, 70]. The term ∆[[n]] equals the conformal dimension contribution ∆vec(m), – 35 – as in ﬁgure 1.7 Each leg, described by the remaining terms, corresponds to the Coulomb branch of a theory of type T(J) carrying external charges, expressed as a Slodowy slice: Tρ(i),[n](J) = t−∆[[n]] g SJ∨ N ,ρ(i),[n] HS (y(i), t) \| {z } slices . (A.5) (A.5) The Hilbert series gQ(J) HS transforms in the product group ⊗ i F(ρ(i)) (before possible symme- try enhancement) and matches that of the Coulomb branch of the star shaped quiver Q(J). Selection rule. Not every collection of slices yields a well formed Hilbert series wherein all the ﬁelds (other than the singlet at its origin) have positive conformal dimension, ∀n ̸= [0] : ∆[[n]] > 0. 7Alternatively, ∆[[n]] can be found directly, either from the weight map [41] associated to the nilcone of J∨, as ∆[[n]] = −[n]·ω(N), or from the Cartan matrix A∨and Weyl vector 1, as ∆[[n]] = −2[n]·A∨−1 ·1. A Hall Littlewood polynomials and star shaped quivers The selection rule can be formulated in terms of the weights asso- ciated with each slice by the partitions ρ(i) and their contribution to the overall charges carried by t. Thus, each fugacity map x →(y(i), t) incorporates a weight map ω(i) that assigns R-charges to the CSA fugacities x, viz ω(i) : {x1, . . . , xr} →{tω1(i), . . . , tωr(i)} [67]. Collecting terms that contribute to conformal dimension (via exponents of t) from (A.4) and (A.5) we ﬁnd a selection rule that requires: JHEP12(2021)070 ω(Q) ≡−2 ω(reg.) + k X i=1 (ω(reg.) −ω(i)) > strict 0, (A.6) (A.6) where ω(reg.) is the weight map associated with the zero dimensional regular slice, and the inequality requires that all the entries of the weight vector ω(Q) should be greater than zero. For example, consider the quiver with E7 global symmetry in table 2. We can read from [18, ﬁgure 25] that this comprises slices to orbits with D-partitions ρ = (16), (16) and (3, 13). Using the nilpotent orbit data in [41, appendix B], we ﬁnd that these correspond to weights [0, 0, 0], [0, 0, 0] and [2, 1, 1], and that the weight map for the regular slice is [4, 3, 3]. We obtain, ω(Q) = −2[4, 3, 3] + 2([4, 3, 3] −[0, 0, 0]) + ([4, 3, 3] −[2, 1, 1]) = [2, 2, 2]. (A.7) (A.7) This is strictly greater than [0, 0, 0], so ω(Q) is the weight vector of a good quiver. Integer and fractional integer lattices. The regular summation over resolved slices in (A.4) is carried out over the entire weight or Dynkin label lattice [n] ∈ΓJ∨/W ∨of the GNO dual group J∨of the central quiver node. This corresponds to the combination of integer and half integer lattices as in [18]. When J∨is special orthogonal, the summation can be restricted to correspond to the integer lattice by the simple expedient of restricting the summation over the weight lattice to exclude irreps from spinor lattices, i.e. to exclude those [n] where the sum of spinor Dynkin labels is odd. – 36 – Non-simply laced ﬁxtures. A Hall Littlewood polynomials and star shaped quivers The above formulae (A.4) to (A.6) can be extended to calculate reﬁned Coulomb branch Hilbert series from quivers with one or more non-simply laced legs by the expedient of applying a multiple [m(i)n] of the Dynkin label charges [n] to each leg, according to the multiplicity m(i) of the non-simply laced link i in the quiver diagram [68]. Thus, (A.5) becomes: Tρ(i),[m(i)n](J) = t−∆[[m(i)n]] g SJ∨ N ,ρ(i),[m(i)n] HS (y(i), t), (A.8) (A.8) and the selection rule (A.6) is modiﬁed to reﬂect the higher Dynkin label charges on the non-simply laced quiver legs: JHEP12(2021)070 ω(Q) ≡−2 ω(reg.) + k X i=1 m(i) (ω(reg.) −ω(i)) > strict 0. (A.9) (A.9) In a sense, the folding of m magnetic quiver legs Tρ,[n](J) corresponds to the creation of a single leg Tρ,[mn](J) carrying charges in the m-th symmetrisation of the charges carried by the original quiver leg. In a sense, the folding of m magnetic quiver legs Tρ,[n](J) corresponds to the creation of a single leg Tρ,[mn](J) carrying charges in the m-th symmetrisation of the charges carried by the original quiver leg. Twisted ﬁxtures. The foregoing applies to magnetic quivers for standard ﬁxtures, where all the slices are transverse to orbits from the same symmetry group J∨. Quivers can also be constructed for “twisted” ﬁxtures, where some of the punctures are represented by slices from a diﬀerent symmetry group J′ (being always simply laced and generally of diﬀerent rank), which are “twisted” to ﬁt the weight lattice of the J∨symmetry group. Compatibility requirements between the lattices of J∨and J′ mean that only certain pairs of groups can be related by such twists. To accommodate twisted quivers, equations (A.4) and (A.5) require modiﬁcation. We set the terms for a twisted puncture to contain a conformal dimension contribution and slice drawn from J′: Tρ(i),[n′](J′) = t−∆′[[n′]] g SJ′ N ,ρ(i),[n′] HS (y(i), t). (A.10) (A.10) 1. We identify the partition ρ(i) from amongst the orbits of J′, based on the quiver diagram, so that the resolved slices SJ′ N,ρ(i),[n′] belong to the twisted group. 1. We identify the partition ρ(i) from amongst the orbits of J′, based on the quiver diagram, so that the resolved slices SJ′ N,ρ(i),[n′] belong to the twisted group. 2. A Hall Littlewood polynomials and star shaped quivers We also need to identify the “twisted” map [n] →[n′] between the Dynkin labels of J∨and those of J′, and use this to express the ∆′[[n′]] contribution to conformal dimension in terms of [n]. 2. We also need to identify the “twisted” map [n] →[n′] between the Dynkin labels of J∨and those of J′, and use this to express the ∆′[[n′]] contribution to conformal dimension in terms of [n]. Various types of twist are encountered when dealing with mixed unitary and orthosym- plectic quivers. These are tabulated in table 5, along with the maps between the Dynkin labels of J′ and J∨that are necessary in order to carry out the summation over the J∨ lattice. The twisted A2r−1 ﬁxtures analysed in sections 2.3 and 4 contain a J′ = A2r−1 linear quiver aﬃxed to a star shaped quiver with J = Cr and J∨= Br. For example, consider the quiver in table 1 whose Coulomb branch is the minimal orbit of E6. We can read from [18, – 37 – table 20] that this comprises two slices to orbits with the B-partitions ρ = (15) and a slice to the orbit with A3 partition (3, 1). Using the nilpotent orbit data in [41, appendix B], we ﬁnd these correspond to B2 weights [0, 0] and A3 weights [2, 2, 2], respectively. The weight map for the regular slice of B2 is [4, 3] and that for A3 is [3, 4, 3]. So, ω(Q) = −2[4, 3] + 2([4, 3] −[0, 0]) \| {z } Dynkin Labels [n1,n2] + ([3, 4, 3] −[2, 2, 2]) \| {z } Dynkin Labels [n1,n2,n1] = [1, 2, 1] \| {z } Dynkin Labels [n1,n2,n1] = [2, 2] \| {z } Dynkin Labels [n1,n2] . (A.11) ω(Q) = −2[4, 3] + 2([4, 3] −[0, 0]) \| {z } Dynkin Labels [n1,n2] + ([3, 4, 3] −[2, 2, 2]) \| {z } Dynkin Labels [n1,n2,n1] ω(Q) = −2[4, 3] + 2([4, 3] −[0, 0]) \| {z } Dynkin Labels [n1,n2] + ([3, 4, 3] −[2, 2, 2]) \| {z } Dynkin Labels [n1,n2,n1] = [1, 2, 1] \| {z } Dynkin Labels [n1,n2,n1] = [2, 2] \| {z } Dynkin Labels [n1,n2] . (A.11) (A.11) JHEP12(2021)070 Application of the selection rule shows ω(Q) is strictly positive and therefore the weight vector is that of a good star shaped quiver. A Hall Littlewood polynomials and star shaped quivers The summation is carried out over the B2 Dynkin label lattice [n1, n2]. Only A3 slices with Dynkin labels of the form [n1, n2, n1] contribute to the summation. Alternatively, consider the quiver in table 1 whose Coulomb branch is the minimal orbit of D5. This comprises two slices to orbits with the D-partitions ρ = (14) and a slice to the orbit with A3 partition (3, 1). Using the nilpotent orbit data in appendix B of [41], we ﬁnd these correspond to D2 weights [0, 0] and A3 weights [2, 2, 2], respectively. The weight map for the regular slice of D2 is [1, 1] and that for A3 is [3, 4, 3]. So, ω(Q) = −2[1, 1] + 2([1, 1] −[0, 0]) \| {z } Dynkin Labels [n1,n2] + ([3, 4, 3] −[2, 2, 2]) \| {z } Dynkin Labels [M,A,M] = [1, 2, 1] \| {z } Dynkin Labels [M,A,M] . (A.12) (A.12) The contribution to conformal dimension from ω(Q) is positive for [n1, n2] ̸= [0, 0], and so application of the selection rule shows that the weight vector is that of a good star shaped quiver. The summation is carried out over the D2 weight lattice [n1, n2]. Only A3 slices with Dynkin labels of the form [M, A, M] are involved, where M = Min[n1, n2] and A = Abs[n1 −n2]. It is clearly possible to construct ﬁxtures that combine features of twists, non-simply laced legs and/or sub-lattices. Further discussion of such ﬁxtures is left for future work. Open Access. This article is distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits any use, distribution and reproduction in any medium, provided the original author(s) and source are credited. [2] A. Hanany, N. Mekareeya and S.S. Razamat, Hilbert Series for Moduli Spaces of Two Instantons, JHEP 01 (2013) 070 [arXiv:1205.4741] [INSPIRE]. [1] S. Cremonesi, G. Ferlito, A. Hanany and N. Mekareeya, Coulomb Branch and The Moduli Space of Instantons, JHEP 12 (2014) 103 [arXiv:1408.6835] [INSPIRE]. [1] S. Cremonesi, G. Ferlito, A. Hanany and N. Mekareeya, Coulomb Branch and The Moduli Space of Instantons, JHEP 12 (2014) 103 [arXiv:1408.6835] [INSPIRE]. [2] A. Hanany, N. Mekareeya and S.S. Razamat, Hilbert Series for Moduli Spaces of Two Instantons, JHEP 01 (2013) 070 [arXiv:1205.4741] [INSPIRE]. References [2] A. Hanany, N. Mekareeya and S.S. Razamat, Hilbert Series for Moduli Spaces of Two Instantons, JHEP 01 (2013) 070 [arXiv:1205.4741] [INSPIRE]. – 38 – [3] A. Dey, A. Hanany, P. Koroteev and N. Mekareeya, On Three-Dimensional Quiver Gauge Theories of Type B, JHEP 09 (2017) 067 [arXiv:1612.00810] [INSPIRE]. [4] H. Nakajima and A. Weekes, Coulomb branches of quiver gauge theories with symmetrizers, arXiv:1907.06552 [INSPIRE]. [5] A. Bourget, A. Hanany and D. Miketa, Quiver origami: discrete gauging and folding, JHEP 01 (2021) 086 [arXiv:2005.05273] [INSPIRE]. [6] S. Cremonesi, A. Hanany and A. Zaﬀaroni, Monopole operators and Hilbert series of Coulomb branches of 3d N = 4 gauge theories, JHEP 01 (2014) 005 [arXiv:1309.2657] [INSPIRE]. [7] S. Cecotti and M. Del Zotto, 4d N = 2 Gauge Theories and Quivers: the Non-Simply Laced Case, JHEP 10 (2012) 190 [arXiv:1207.7205] [INSPIRE]. JHEP12(2021)070 JHEP12(2021)070 [8] T. Kimura and V. Pestun, Fractional quiver W-algebras, Lett. Math. Phys. 108 (2018) 2425 [arXiv:1705.04410] [INSPIRE]. [9] N. Haouzi and C. Kozçaz, The ABCDEFG of little strings, JHEP 06 (2021) 092 [arXiv:1711.11065] [INSPIRE]. [10] H.-Y. Chen and T. Kimura, Quantum integrability from non-simply laced quiver gauge theory, JHEP 06 (2018) 165 [arXiv:1805.01308] [INSPIRE]. [11] S. Cremonesi, A. Hanany, N. Mekareeya and A. Zaﬀaroni, T σ ρ (G) theories and their Hilbert series, JHEP 01 (2015) 150 [arXiv:1410.1548] [INSPIRE]. [12] S. Cremonesi, A. Hanany, N. Mekareeya and A. Zaﬀaroni, Coulomb branch Hilbert series and Three Dimensional Sicilian Theories, JHEP 09 (2014) 185 [arXiv:1403.2384] [INSPIRE]. [13] S. Cabrera, A. Hanany and Z. Zhong, Nilpotent orbits and the Coulomb branch of T σ(G) theories: special orthogonal vs. orthogonal gauge group factors, JHEP 11 (2017) 079 [arXiv:1707.06941] [INSPIRE]. [14] S. Cabrera, A. Hanany and M. Sperling, Magnetic quivers, Higgs branches, and 6d N = (1, 0) theories — orthogonal and symplectic gauge groups, JHEP 02 (2020) 184 [arXiv:1912.02773] [INSPIRE]. [15] A. Bourget, J.F. Grimminger, A. Hanany, M. Sperling and Z. Zhong, Magnetic Quivers from Brane Webs with O5 Planes, JHEP 07 (2020) 204 [arXiv:2004.04082] [INSPIRE]. [16] M. Akhond, F. Carta, S. Dwivedi, H. Hayashi, S.-S. Kim and F. Yagi, Five-brane webs, Higgs branches and unitary/orthosymplectic magnetic quivers, JHEP 12 (2020) 164 [arXiv:2008.01027] [INSPIRE]. [17] M. Akhond, F. Carta, S. Dwivedi, H. Hayashi, S.-S. Kim and F. Yagi, Factorised 3d N = 4 orthosymplectic quivers, JHEP 05 (2021) 269 [arXiv:2101.12235] [INSPIRE]. [18] A. Bourget, J.F. Grimminger, A. Hanany, R. Kalveks, M. References Sperling and Z. Zhong, Magnetic Lattices for Orthosymplectic Quivers, JHEP 12 (2020) 092 [arXiv:2007.04667] [INSPIRE]. [19] H. Sakai, Rational surfaces associated with aﬃne root systems and geometry of the Painlevé equations, Commun. Math. Phys. 220 (2001) 165. [20] P. Boalch, Quivers and Diﬀerence Painlevé Equations, in Groups and Symmetries: From Neolithic Scots to John McKay 47, American Mathematical Society, Providence RI U.S.A. (2009), p. 25. [21] S. Cremonesi, G. Ferlito, A. Hanany and N. Mekareeya, Instanton Operators and the Higgs Branch at Inﬁnite Coupling, JHEP 04 (2017) 042 [arXiv:1505.06302] [INSPIRE]. [22] N. Mekareeya, K. Ohmori, Y. Tachikawa and G. Zafrir, E8 instantons on type-A ALE spaces and supersymmetric ﬁeld theories, JHEP 09 (2017) 144 [arXiv:1707.04370] [INSPIRE]. – 39 – [23] G. Ferlito, A. Hanany, N. Mekareeya and G. Zafrir, 3d Coulomb branch and 5d Higgs branch at inﬁnite coupling, JHEP 07 (2018) 061 [arXiv:1712.06604] [INSPIRE]. [24] A. Hanany and G. Zafrir, Discrete Gauging in Six Dimensions, JHEP 07 (2018) 168 [arXiv:1804.08857] [INSPIRE]. [25] A. Hanany and N. Mekareeya, The small E8 instanton and the Kraft Procesi transition, JHEP 07 (2018) 098 [arXiv:1801.01129] [INSPIRE]. [26] S. Cabrera, A. Hanany and F. Yagi, Tropical Geometry and Five Dimensional Higgs Branches at Inﬁnite Coupling, JHEP 01 (2019) 068 [arXiv:1810.01379] [INSPIRE]. [27] S. Cabrera, A. Hanany and M. Sperling, Magnetic quivers, Higgs branches, and 6d N = (1, 0) theories, JHEP 06 (2019) 071 [Erratum JHEP 07 (2019) 137] [arXiv:1904.12293] [INSPIRE]. JHEP12(2021)070 JHEP12(2021)070 [28] A. Beauville, Symplectic singularities, Invent. Math. 139 (2000) 541 [math/9903070]. [29] M. Bullimore, T. Dimofte and D. Gaiotto, The Coulomb Branch of 3d N = 4 Theories, Commun. Math. Phys. 354 (2017) 671 [arXiv:1503.04817] [INSPIRE]. [30] M. Bullimore, T. Dimofte, D. Gaiotto, J. Hilburn and H.-C. Kim, Vortices and Vermas, Ad Theor. Math. Phys. 22 (2018) 803 [arXiv:1609.04406] [INSPIRE]. [31] M. Dedushenko, Y. Fan, S.S. Pufu and R. Yacoby, Coulomb Branch Operators and Mirror Symmetry in Three Dimensions, JHEP 04 (2018) 037 [arXiv:1712.09384] [INSPIRE]. [32] M. Dedushenko, Y. Fan, S.S. Pufu and R. Yacoby, Coulomb Branch Quantization and Abelianized Monopole Bubbling, JHEP 10 (2019) 179 [arXiv:1812.08788] [INSPIRE]. [33] H. Nakajima, Questions on provisional Coulomb branches of 3-dimensional N = 4 gauge theories, arXiv:1510.03908 [INSPIRE]. [34] H. Nakajima, Towards a mathematical deﬁnition of Coulomb branches of 3-dimensional N = 4 gauge theories, I, Adv. Theor. Math. Phys. 20 (2016) 595 [arXiv:1503.03676] [INSPIRE]. [35] A. Braverman, M. Finkelberg and H. References Nakajima, Towards a mathematical deﬁnition of Coulomb branches of 3-dimensional N = 4 gauge theories, II, Adv. Theor. Math. Phys. 22 (2018) 1071 [arXiv:1601.03586] [INSPIRE]. [36] P. Goddard, J. Nuyts and D.I. Olive, Gauge Theories and Magnetic Charge, Nucl. Phys. B 125 (1977) 1 [INSPIRE]. [37] A. Hanany and R. Kalveks, Highest Weight Generating Functions for Hilbert Series, JHEP 10 (2014) 152 [arXiv:1408.4690] [INSPIRE]. [38] S. Cremonesi, A. Hanany, N. Mekareeya and A. Zaﬀaroni, Coulomb branch Hilbert series and Hall-Littlewood polynomials, JHEP 09 (2014) 178 [arXiv:1403.0585] [INSPIRE]. [39] A. Hanany and R. Kalveks, Quiver Theories and Hilbert Series of Classical Slodowy Intersections, Nucl. Phys. B 952 (2020) 114939 [arXiv:1909.12793] [INSPIRE]. [40] A. Hanany and A. Zajac, Ungauging Schemes and Coulomb Branches of Non-simply Laced Quiver Theories, JHEP 09 (2020) 193 [arXiv:2002.05716] [INSPIRE]. [41] A. Hanany and R. Kalveks, Quiver Theories for Moduli Spaces of Classical Group Nilpotent Orbits, JHEP 06 (2016) 130 [arXiv:1601.04020] [INSPIRE]. [42] A. Bourget, J.F. Grimminger, A. Hanany, M. Sperling, G. Zafrir and Z. Zhong, Magnetic quivers for rank 1 theories, JHEP 09 (2020) 189 [arXiv:2006.16994] [INSPIRE]. [42] A. Bourget, J.F. Grimminger, A. Hanany, M. Sperling, G. Zafrir and Z. Zhong, Magnetic quivers for rank 1 theories, JHEP 09 (2020) 189 [arXiv:2006.16994] [INSPIRE]. [43] D.I. Panyushev, Complexity and nilpotent orbits, Manuscripta Math. 83 (1994) 223. [43] D.I. Panyushev, Complexity and nilpotent orbits, Manuscripta Math. 83 (1994) 223. – 40 – [44] D.H. Collingwood and W.M. McGovern, Nilpotent Orbits In Semisimple Lie Algebra: An Introduction, CRC Press (1993). [45] G. Ferlito and A. Hanany, A tale of two cones: the Higgs Branch of Sp(n) theories with 2n ﬂavours, arXiv:1609.06724 [INSPIRE]. [46] V. Kac, Inﬁnite-Dimensional Lie Algebras, in Progress in Mathematics, Cambridge University Press, Cambridge U.K. (1994). [47] D. Gaiotto and E. Witten, S-duality of Boundary Conditions In N = 4 Super Yang-Mills Theory, Adv. Theor. Math. Phys. 13 (2009) 721 [arXiv:0807.3720] [INSPIRE]. [48] A. Bourget, S. Giacomelli, J.F. Grimminger, A. Hanany, M. Sperling and Z. Zhong, S-fold magnetic quivers, JHEP 02 (2021) 054 [arXiv:2010.05889] [INSPIRE]. JHEP12(2021)070 JHEP12(2021)070 [49] S. Cabrera and A. Hanany, Branes and the Kraft-Procesi transition: classical case, JHEP 04 (2018) 127 [arXiv:1711.02378] [INSPIRE]. [50] J. Fuchs and C. Schweigert, Symmetries, Lie Algebras and Representations: A Graduate Course for Physicists, in Cambridge Monographs on Mathematical Physics, Cambridge University Press, Cambridge U.K. (2003). [51] G. References Zafrir, Compactiﬁcations of 5d SCFTs with a twist, JHEP 01 (2017) 097 [arXiv:1605.08337] [INSPIRE]. [52] P.C. Argyres, M. Lotito, Y. Lü and M. Martone, Expanding the landscape of N = 2 rank 1 SCFTs, JHEP 05 (2016) 088 [arXiv:1602.02764] [INSPIRE]. [53] H. Shimizu, Y. Tachikawa and G. Zafrir, Anomaly matching on the Higgs branch, JHEP 12 (2017) 127 [arXiv:1703.01013] [INSPIRE]. [54] O. Chacaltana and J. Distler, Tinkertoys for Gaiotto Duality, JHEP 11 (2010) 099 [arXiv:1008.5203] [INSPIRE]. [55] S. Benvenuti, A. Hanany and N. Mekareeya, The Hilbert Series of the One Instanton Mod Space, JHEP 06 (2010) 100 [arXiv:1005.3026] [INSPIRE]. [56] A. Hanany and A. Pini, HWG for Coulomb branch of 3d Sicilian theory mirrors, arXiv:1707.09784 [INSPIRE]. [57] A. Bourget, S. Cabrera, J.F. Grimminger, A. Hanany and Z. Zhong, Brane Webs and Magnetic Quivers for SQCD, JHEP 03 (2020) 176 [arXiv:1909.00667] [INSPIRE]. [58] S. Cabrera and A. Hanany, Branes and the Kraft-Procesi Transition, JHEP 11 (2016) 175 [arXiv:1609.07798] [INSPIRE]. [59] A. Bourget et al., The Higgs mechanism — Hasse diagrams for symplectic singularities, JHEP 01 (2020) 157 [arXiv:1908.04245] [INSPIRE]. [60] J.F. Grimminger and A. Hanany, Hasse diagrams for 3d N = 4 quiver gauge theories — Inversion and the full moduli space, JHEP 09 (2020) 159 [arXiv:2004.01675] [INSPIRE]. [61] S. Cabrera and A. Hanany, Quiver Subtractions, JHEP 09 (2018) 008 [arXiv:1803.11205] [INSPIRE]. [62] A. Bourget, J.F. Grimminger, A. Hanany, M. Sperling and Z. Zhong, Branes, Quivers, and the Aﬃne Grassmannian, arXiv:2102.06190 [INSPIRE]. [63] H. Kraft and C. Procesi, On the geometry of conjugacy classes in classical groups, Comment. Math. Helv. 57 (1982) 539. [64] D. Kutasov, Orbifolds and solitons, Phys. Lett. B 383 (1996) 48 [hep-th/9512145] [INSPIRE]. – 41 – [65] A. Hanany and A. Zaﬀaroni, Branes and six-dimensional supersymmetric theories, Nucl. Phys. B 529 (1998) 180 [hep-th/9712145] [INSPIRE]. [66] D.R. Gulotta, C.P. Herzog and T. Nishioka, The ABCDEF’s of Matrix Models for Supersymmetric Chern-Simons Theories, JHEP 04 (2012) 138 [arXiv:1201.6360] [INSPIRE] [67] S. Cabrera, A. Hanany and R. Kalveks, Quiver Theories and Formulae for Slodowy Slices of Classical Algebras, Nucl. Phys. B 939 (2019) 308 [arXiv:1807.02521] [INSPIRE]. [68] A. Hanany and R. Kalveks, Construction and Deconstruction of Single Instanton Hilbert Series, JHEP 12 (2015) 118 [arXiv:1509.01294] [INSPIRE]. [69] R. Feger and T.W. Kephart, LieART — A Mathematica application for Lie algebras and representation theory, Comput. Phys. Commun. 192 (2015) 166 [arXiv:1206.6379] [INSPIRE]. JHEP12(2021)070 [70] R. Feger, T.W. Kephart and R.J. References Saskowski, LieART 2.0 — A Mathematica application for Lie Algebras and Representation Theory, Comput. Phys. Commun. 257 (2020) 107490 [arXiv:1912.10969] [INSPIRE]. [71] A.L. Onishchik, Lectures on real semisimple Lie algebras and their representations. Volume 1, European Mathematical Society (2004). [72] D. Bump, Lie groups, Springer (2004). [72] D. Bump, Lie groups, Springer (2004). – 42 –
W4206546647.txt	https://www.frontiersin.org/articles/10.3389/fonc.2021.801269/pdf	en		The Role of Circulating Biomarkers in Lung Cancer	Frontiers in oncology	2,022	cc-by	10,538	REVIEW published: 21 January 2022 doi: 10.3389/fonc.2021.801269 Edited by: Pasquale Pisapia, University of Naples Federico II, Italy Reviewed by: Simon Chang-Hao Tsao, The University of Melbourne, Australia Sandra V. Fernandez, Fox Chase Cancer Center, United States Correspondence: Arutha Kulasinghe arutha.kulasinghe@uq.edu.au Specialty section: This article was submitted to Thoracic Oncology, a section of the journal Frontiers in Oncology Received: 25 October 2021 Accepted: 21 December 2021 Published: 21 January 2022 Citation: Herath S, Sadeghi Rad S, Radfar P, Ladwa R, Warkiani M, O’Byrne K and Kulasinghe A (2022) The Role of Circulating Biomarkers in Lung Cancer. Front. Oncol. 11:801269. doi: 10.3389/fonc.2021.801269 Frontiers in Oncology \| www.frontiersin.org The Role of Circulating Biomarkers in Lung Cancer Sayuri Herath 1, Habib Sadeghi Rad 2, Payar Radfar 3, Rahul Ladwa 4, Majid Warkiani 3, Ken O’Byrne 2,4 and Arutha Kulasinghe 5 1 Department of Medical Laboratory Sciences, Faculty of Health Sciences, The Open University of Sri Lanka, Nugegoda, Sri Lanka, 2 Centre for Genomics and Personalised Health, Faculty of Health, School of Biomedical Sciences, Queensland University of Technology, Brisbane, QLD, Australia, 3 Faculty of Engineering and IT, School of Biomedical Engineering, University of Technology Sydney, Sydney, NSW, Australia, 4 Princess Alexandra Hospital, Cancer Care Services, Woolloongabba, QLD, Australia, 5 The University of Queensland Diamantina Institute, The University of Queensland, Brisbane, QLD, Australia Lung cancer is the leading cause of cancer morbidity and mortality worldwide and early diagnosis is crucial for the management and treatment of this disease. Non-invasive means of determining tumour information is an appealing diagnostic approach for lung cancers as often accessing and removing tumour tissue can be a limiting factor. In recent years, liquid biopsies have been developed to explore potential circulating tumour biomarkers which are considered reliable surrogates for understanding tumour biology in a non-invasive manner. Most common components assessed in liquid biopsy include circulating tumour cells (CTCs), cell-free DNA (cfDNA), circulating tumour DNA (ctDNA), microRNA and exosomes. This review explores the clinical use of circulating tumour biomarkers found in liquid biopsy for screening, early diagnosis and prognostication of lung cancer patients. Keywords: Circulating tumour cells, cell-free DNA, circulating tumour DNA, microRNA and exosomes, lung cancer, liquid biopsy 1 January 2022 \| Volume 11 \| Article 801269 Herath et al. Circulating Biomarkers in Lung Cancer GRAPHICAL ABSTRACT \| The identiﬁcation of multi-marker analytes in liquid biopsy samples enables a personalised medicine approach to the management of lung cancer. However, one of the main challenges for managing and treating lung cancer patients is the lack of sensitive early diagnostic methods (10). To date, low-dose spiral computed tomography (LDCT) is the most commonly used approach for lung cancer screening with more than four times higher sensitivity compared to X-ray imaging (3, 11, 12). However, high false-positive results in the early stages of lung cancers and radiation exposure often limits the usage of LDCT (10). Tumour biopsy is the gold standard for lung cancer diagnosis due to the potential of investigating targeted biomarkers including carcinoembryonic antigen (CEA), fragments of cytokeratin ‐19 (CYFRA21‐1), squamous cell carcinoma antigen (SCC) and neuron‐speciﬁc enolase (NSE) (13, 14). However, tumour biopsy is an invasive approach, requiring specialist medical expertise and cannot be routinely performed outside of a hospital setting. Single site biopsy may have a sampling bias by not being representative of the whole tumour (15). Moreover, tumour biopsies represent a single time point snapshot of the tumour, where therapy-induced changes cannot be determined over the course of treatment. Therefore, alternative, more dynamic biomarkers which can be assessed serially over the course of therapy are desirable (14). Liquid biopsy have emerged as a promising tool to detect tumour biomarkers (Figure 1) in body ﬂuids in a non-invasive manner. They have shown to play a crucial role in lung cancer screening, early diagnosis, monitoring and determining patient prognosis (16). The minimal invasiveness, ease of use and ability to have repeat measurements over time make it a useful companion diagnostic tool. To date, liquid biopsies has been developed as a novel diagnostic approach to explore potential circulating tumour biomarkers and is considered as a reliable way to understand dynamically changing tumour biology under the stressors of treatment. Liquid biopsy biomarkers include cell-free DNA (cfDNA), circulating tumour DNA (ctDNA), microRNA INTRODUCTION Lung Cancer Lung cancer is the leading cause of cancer related deaths and accounted for over 2.1 million new cases and 1.8 million deaths in 2018 (1). If the cancer is diagnosed in the early stages (i.e., Stage I – II), the ﬁve-year survival rate is estimated to be around 56% (2). However, only 16% of lung cancer cases are diagnosed early, which causes the overall ﬁve-year survival rate of lung cancer patients to be less than 20% with adverse clinical outcomes (3, 4). Lung cancer is mainly categorized into two histological groups: non-small cell carcinoma (NSCLC) and small cell lung carcinoma (SCLC) (5). NSCLC is the most prevalent lung cancer type that accounts for 80% to 85% of all lung cancer cases and is further divided into three histological subtypes of adenocarcinoma, squamous cell carcinoma, and large cell (undifferentiated) carcinoma (6). Adenocarcinoma accounts for 40% of all lung cancers and is frequently found in peripheral bronchi (7, 8). Squamous cell carcinoma comprises 25%-30% of all lung cancer cases which arises from the main bronchi and disseminates into the carina. Large cell (undifferentiated) carcinoma represents about 10% and may originate from different part of the lung. SCLC accounts for 10-15% of all lung cancers and it is the most aggressive type with the lowest overall survival (OS) (5, 6). LIQUID BIOPSY Lung cancer patients often suffer from progression of their disease with adverse clinical outcomes, complications and recurrence (9). Therefore, early diagnosis is vital for effective disease management and preventing the advancement of cancer. Frontiers in Oncology \| www.frontiersin.org 2 January 2022 \| Volume 11 \| Article 801269 Herath et al. Circulating Biomarkers in Lung Cancer FIGURE 1 \| The process of cancer metastasis. Metastasis occurs via the vascular or lymph circulatory systems, where cancer cells from the primary tumour, intravasate into the blood/lymphatics systems and travel through the body, and extravasate at local/distant sites/organs. Adapted from “Overview of Metastatic Cascade”, by BioRender.com (2021). Retrieved from https://app.biorender.com/biorender-templates. cfDNA and ctDNA (miRNA), exosomes and circulating tumour cells (CTCs) (3, 12, 17–19). This review provides an overview of these circulating tumour biomarkers and their clinical signiﬁcance in screening, early diagnosis and prognostication of lung cancer (Figure 2). Cell free DNA (cfDNA) is derived from dividing and apoptotic cells as a result of the normal physiological process of tissue remodeling (21–23). In healthy subjects, the amount of cfDNA is FIGURE 2 \| Multi-marker analytes in a liquid biopsy sample: CTCs, cfDNA and exosomes can be analysed at different time points during clinical progression/ treatment. Adapted from Ref (20) and created with BioRender.com. Frontiers in Oncology \| www.frontiersin.org 3 January 2022 \| Volume 11 \| Article 801269 Herath et al. Circulating Biomarkers in Lung Cancer ensure the EGFR mutation does not exist in the patient. In addition to detection of EGFR mutation, ALK rearrangements have been assessed in ctDNA of lung cancer patients (43–45). ALK is a membrane-bound tyrosine kinase receptor encoded by the ALK gene (46, 47) and rearrangement of ALK can be seen in 2-7% of NSCLC patients. Hence, ALK rearrangement has emerged as the second most studied targetable mutation in order to develop a novel treatment approach to lung cancer (48). In addition to single-gene assays, higher through-put NGS multi-gene readout assays have been developed to identify single-nucleotide variants (SNVs), copy number alterations (CNAs), inserts/deletions and or fusions. These assays include the FoundationOne Liquid CDx, Guardant 360 CDx, MSKACCESS, OncoDNA and Archer Reveal. These assays have the advantage of broad genotyping and utility in clinical trials (49). The blood TMB (bTMB) assays have shown concordance with the tumour TMB assays in advanced stage NSCLC, in particular for predicting clinical outcomes to immunotherapy (50, 51). Usually, at the time of sample collection, tumour-derived DNA is found highly fragmented and mixed with non-tumour DNA/cfDNA. Separation of ctDNA from cfDNA can be pursued using ultrasensitive analytical assays (52). Several testing approaches have been identiﬁed; including emulsions, beads, ampliﬁcation, magnetics (BEAMing), droplet digital PCR (ddPCR) and next generation sequencing (NGS), which can detect down to a few copies of ctDNA (53–55). In contrast, the low amount of ctDNA in plasma has been challenging the clinical applicability of this technique. Therefore, evaluation of ctDNA in other body ﬂuids than plasma may potentially provide a solution to this. The concentration of cfDNA in body ﬂuids is higher near the proximal tumour sites (37). It is worth mentioning that the current guidelines emphasize plasma as a preferred specimen than serum for the detection of ctDNA, as leukocyte lysis takes place during clotting which thereby leads to high contamination rate of germinal DNA than in plasma (56). Furthermore, the specimen should be processed within 6 hours of collection in order to prevent release of DNA from normal blood cell lysis and leukocyte stabilization reagents would be useful in these circumstances (57). quite low and it has been estimated as 5–10 ng/mL in body ﬂuids (24). The half-life of cfDNA is approximately 2 hours (25). The proportion of cell free DNA derived from tumour cells is known as ctDNA (18). The concentration of ctDNA in plasma varies from 0.01% to 90% of total cfDNA (25). The amount of ctDNA present in plasma correlates with the tumour burden, progression free survival (PFS) and OS (10), which is a useful biomarker to monitor NSCLC patients in different stages (26). A study conducted by Newman et al. has been found that a 100% detection rate of ctDNA in stage II–IV NCSLC patients and 50% in early-stage patients (26). Owing to the short half-life of ctDNA, treatment efﬁcacy of patients can be rapidly detected, earlier than radiological changes (25). On the other hand, in order to detect ctDNA in the peripheral circulation, blood samples should be collected within a certain time period to avoid degradation (24). ctDNA can identify speciﬁc molecular changes present in the original tumour. These include mutations in oncogenes/tumour suppressor genes and gene ampliﬁcations or epigenetic changes (19). Therefore, cfDNA/ctDNA has been used as a prognostic marker for the diagnosis of different types of cancers including lung cancer (17, 27, 28). Furthermore, ctDNA provides a molecular picture of the residual disease which helps in decision making for the commencement of adjuvant chemotherapy after the surgery (29). The concentration of cfDNA is found to be higher in early stage NSCLC patients, recurrent and advanced stage NSCLC patients compared to healthy subjects (26, 29). A study by Pohomaryova et al., reported that the plasma concentration of cfDNA in lung cancer patients is eight times higher than normal healthy adults (30). Several studies have been reported the presence of high cfDNA concentrations has a signiﬁcant association with the worse clinical outcome (31–34). Furthermore, cfDNA plays an important role for identifying blood tumour mutational burden (bTMB) in NSCLC, indicating the number of somatic mutations in the genome coding regions (35). It has been found that sensitivity and speciﬁcity of bTMB assay were 93.9%, 93.9% and 100.0%, compared to tissue TMB (36). cfDNA mutation analysis is also important to identify speciﬁc mutations and molecular targets for personalized therapies (37). This can be beneﬁcial to select patients for immunotherapy and pursue a clinically meaningful improvement in terms of survival (38, 39). The United States Food and Drug Administration (U.S. FDA) approved ctDNA as the ﬁrst liquid biopsy test for the detection of NSCLC patients with EGFR mutations who were suitable for personalized therapy (Roche Cobas EGFR mutation test v2) (40). This assay can detect multiple mutations in exons 18, 19, 20 and 21 in NSCLC including L858R, T790M, G719X, S7681, and L861Q (41). Using ctDNA, 62.5% of patients were identiﬁed with EGFR mutations (exon 19 deletion, exon 20 T790 M insertion and exon 21 L858R mutation) at the baseline, while the rate of EGFR mutation positivity was higher among patients with metastatic disease (42). ctDNA, as a speciﬁc tumour marker, has a high speciﬁcity of 80-95% for the detection of EGFR mutations, which can inform on the use of tyrosine kinase targeted therapies. However, the sensitivity of this approach is comparatively lower – about 60-85%, which cannot be used to Frontiers in Oncology \| www.frontiersin.org Exosomes Exosomes are small extracellular vesicles derived by endocytosis, with 40–100 nm in size (58), which can be found in all the body ﬂuids and capable of transporting DNA, mRNAs, noncoding miRNAs, proteins and lipids (59). It consists of a lipid bilayer that prevents degradation by enzymes such as ribonuclease and high pH conditions. Hence, exosomes play an important role in cell mediated communication in normal healthy cells and disease cells including tumour cells (60). All cell types, including normal, disease or tumour cells can release exosomes into the extracellular space (61). Recent studies suggest a higher number of exosomes in patients with cancer than in healthy adults (62, 63). There is emerging evidence that exosomes have a crucial role in carcinogenesis, cancer progression and metastasis of several tumours, including NSCLC (14). Exosomes derived from tumour cells can promote carcinogenesis by transferring oncogenic factors and thereby 4 January 2022 \| Volume 11 \| Article 801269 Herath et al. Circulating Biomarkers in Lung Cancer according to the density, blood and other components are separated with different centrifugal speeds and exosomes are separated in the ﬁnal ultracentrifugation step. Ultraﬁltration is another physical method that utilizes the exosome size for separation of these extracellular vesicles (59). Furthermore, Polymeric-based precipitation is one of the promising techniques that used the chemical properties of exosomes by using beads coated with speciﬁc antibodies coated. induce malignant transformation (64). Acquiring the oncogenic factors from by non-cancerous cells leads to change of cellular behaviour and share comparable characteristics that the tumour poses (65). Tumour derived exosomes also have the potential of inﬂuencing epithelial-mesenchymal transition (EMT) and facilitate metastasis by transferring migratory and metastatic capacity to the non-cancerous cells (66). Moreover, these extracellular vesicles cause the expression of vimentin in normal cells, stimulating EMT and increasing the metastasis ability. This can further facilitate neoangiogenesis by releasing proteins such as ﬁbroblast growth factor (FGF), vascular endothelial growth factor (VEGF), IL-6, and IL-8, and stimulating vascular endothelial cells via miRNAs (67, 68). The analysis of nucleic acids in exosomes has shown to be a more reliable and sensitive mutation detection approach than using cfDNA/ctDNA. Thakur et al. showed tumour-derived exosomes carry double-stranded DNA which represents the whole genome and the mutation proﬁle of the primary tumour cells (69). Exosomes hold great promise as biomarkers for the early diagnosis and treatment via analysis of nucleic acids and other markers to ﬁnd clues on primary tumour and metastasis condition of patients. Different exosomal proteins and miRNA have been currently employed for the diagnostic and prognostic utility. Some surface proteins offer promising potential as tumour markers including CD91, CD317 and EGFR (70). Exosomal microRNA-96 (miRNA-96) fosters lung cancer progression by suppressing the activity of Lim domain 7 (LMO7) protein. LMO7 is a ﬁbrous actin-binding protein functions in the formation and maintenance of actin cytoskeleton. In lung cancers, it functions as a tumour suppressor molecule while a shortage of LMO7 confers a genetic predisposition to lung cancer (67). Studies have been documented that the amount of exosomal miRNA is different between healthy individuals and lung cancer patients at different stages (71, 72). Therefore, exosomal miRNA is imperative as a promising and effective noninvasive candidate biomarker for early diagnosis, tumour proﬁling and enabling a timely treatment plan in NSCLC (10). It has been found that EGFR protein can be transferred from tumour cells to non-cancerous cells via exosomes which leading to downregulation of the VEGF pathway (73). Similar to cfDNA, exosomal RNA could be utilized to detect EGFR mutations. It is worth mentioning that exosomal RNA has been found to be more efﬁcient in analyzing EGFR mutations than cfDNA (74). Although exosomes have provided prospective clinical evidence on cancer, still several constraints limit their clinical utility. One of the major drawbacks is difﬁculty in puriﬁcation due to its’ smaller size. Further, few drawbacks are associated with the difﬁculty to adopt exosome isolation techniques in clinical environment. In this context, several isolation techniques have been employed based on the physical, chemical and biological properties of exosomes (75). Physical methods include ultracentrifugation, ultraﬁltration, density gradient separation and size exclusion chromatography (75). All these aforementioned methods are based on the molecular size and density of the exosomes. In the ultracentrifugation, Frontiers in Oncology \| www.frontiersin.org CTCs CTCs were ﬁrst described by an Australian physician, Thomas Ashworth in 1869, where he observed cells similar to the primary tumour were found in the blood of a patient with metastatic disease. Accordingly, metastasis is thought to be facilitated by metastatic precursor cells, known as CTCs, which are tumour cells that detach from a tumour into the vasculature (76). They are extremely rare and approximately 1-10 CTCs can be present per milliliter of whole blood (77). Emerging evidence has demonstrated that the presence of CTCs in the vascular system associate with worse clinical outcomes in terms of OS of cancers (5). It has been shown that patients with NSCLC have ten times higher concentration of CTCs in their blood circulation than in other cancer patients (78–80). CTCs are considered to play a pivotal role in metastasis of cancer and during which they go through a process called EMT (81, 82). EMT is a complex biological process that results in the gradual lowering of epithelial features of the tumour cells and obtains mesenchymal properties to acquire migratory and invasive metastatic characteristics (83). In this process, tumour cells enter the circulation and reach a distant site where a metastatic deposit is initiated. Here, CTCs regain epithelial characteristics allowing further proliferation and formation of metastases deposits at the distant site (Figure 1) (84). A sensitive and speciﬁc isolation method is therefore imperative to provide sufﬁcient and puriﬁed CTCs to analyse (85, 86). Since CTCs are extremely rare, their isolation is greatly constrained by technological implications (87, 88). With the advancement of novel technologies for CTC enrichment, in particular that of microﬂuidics, CTC capture is becoming increasingly efﬁcient. The current CTC enrichment methods are based either on the biological or physical properties of CTCs. The CellSearch (Menarini Silicon Biosystems) platform is the only FDAapproved CTC enumeration method which target epithelial cell adhesion molecule (EpCAM), a transmembrane glycoprotein involved in cell to cell adhesion (89). This platform is approved for breast, colon and prostate cancers. However, this platform has a number of limitations, including that is only captured epithelial CTCs and does not capture mesenchymal shifted CTCs with low or absent EpCAM expression (90, 91). Furthermore, isolation and enrichment of CTCs have been extremely challenging in NSCLC due to the downregulation of the epithelial tumour markers during EMT, which consequently masks the true number of CTCs (10). CTCs often have partialEMT or hybrid states, which can downregulated markers such as EpCAM (92–94). This is further confounded by the presence of CTC clusters or microemboli which have a higher metastatic 5 January 2022 \| Volume 11 \| Article 801269 Herath et al. Circulating Biomarkers in Lung Cancer released into the circulation via protein-miRNA complexes, exosomes, tumor-educated platelets (TEP), and apoptotic bodies (122, 123). Accordingly, evidence suggests that cancer therapeutic approaches such as chemotherapy and radiotherapy may affect circulating miRNA levels, implying that miRNAs can serve as biomarkers for response or resistance to therapy (120, 121, 124, 125). In lung cancer, miRNAs have been found to distinguish healthy individuals from cancer patients (126–128). miRNAs may also serve as biomarkers of therapeutic success in NSCLC patients, allowing for better patient management decisions (129). As a result, studies on lung cancer patients revealed that, when compared to LDCT, miRNAs had signiﬁcantly lower false-positive results when it came to detecting lung cancer patients in their early stages (129). Furthermore, a meta-analysis study from 65 studies (6919 patients with lung cancer and 7064 healthy volunteers), discovered a panel of four miRNAs, including miR-21-5p, miR-126-3p, miR-155-5p, and miR-223-3p, which can be used as a potential biomarker in lung cancer screening (130). Moreover, a different study conducted on early NSCLC patients showed that a number of miRNAs were differentially regulated between short-term survivors and long-term survivors. These miRNAs, which included miR-1, miR-30d, miR-486, and miR-499, were also linked to the OS (127). In line with these ﬁndings, Li et al. studied miR-486 and miR-150 on plasma samples from lung cancer patients to explore their early diagnostic value (131). As a result, these miRNAs were discovered to have greater than 80% speciﬁcity and sensitivity in discriminating healthy individuals from lung cancer patients (131). Another study on patients with metastatic lung cancer discovered that miR-18a, miR-28-3p, miR-191, miR-145, and miR-328 were associated with 3-year survival (132). In studies on NSCLC tumors, researchers discovered six serum miRNAs from patients that had signiﬁcantly different expressions when compared to healthy people. Accordingly, miR-15b-5p was found to be overexpressed, while miR-19-3p, miR-92-3p, miR16-5p, miR-17b-5p, and miR-20a-5p were found to have downregulation (133). Among these miRNAs, miR-16-5p, miR-15b-5p, and miR-20a-5p had the highest sensitivity and speciﬁcity values (133). However, despite these promising ﬁndings, there are some drawbacks to using miRNAs as diagnostic biomarkers, which are listed in Table 1. capacity compared to single CTCs, including immune evasion strategies by the inclusion of leukocytes (83, 95–97). To tackle these drawbacks, label-free CTC isolation technologies have been established to isolate CTCs based on size, density and deformability (87, 98). Still, considerable constraints of label-free technologies are inevitable and the usage is limited due to their throughput (99). To overcome this, recently microﬂuidic technologies have been developed including electrophoresis, hydrodynamic and cross-ﬂow ﬁltration, micropore and micropost trapping, deterministic lateral displacement and inertial focusing systems to capture CTCs (90, 99–103). This has added values through effective cell sorting without need for puriﬁcation, high system throughputs and ability to analyze functions of CTCs in vitro (90, 99, 104–106). Isolated CTCs can be analysed through different approaches including molecular and proteomic studies. For instance, captured CTCs can be used for EGFR mutation analysis which will provide a better understanding of the tumour genetic proﬁle similar to ctDNA (10). Studies emphasized that detection of EGFR mutations in CTC could assist in determining prospective therapeutic decisions which ultimately would lead to the advancement of precision medicine and personalized oncology (5, 107). Other than EGFR mutations, programmed death ligand-1 (PD-L1) expression on CTC has also been studied; however, authors have not been established a clear relationship between its expression and cancer progression and prognosis in patients with NSCLC (108, 109). The isolation of a sufﬁcient amount of CTCs in the blood of NSCLC patients and identify its various biomarkers can aid in early detection of the NSCLC and will also provide real-time monitoring of cancer progression, treatment efﬁcacy and prognosis. Circulating miRNAs miRNAs are a type of gene expression regulator that works at the post-translational level with a multi-protein complex known as the RNA-induced silencing complex (miRISC), exerting their function at the 3’-untranslated region (3’UTR) of target complementary messenger RNA (mRNA) sequences (110, 111). miRNAs have been discovered to be single-stranded RNA molecules with a length of 19 to 22 nucleotides (14, 112). It has been well established that each miRNA can regulate and act as a target for multiple mRNAs and that each mRNA, resulting in a cascade of gene regulation (113, 114). Evidence suggests that any dysregulation of miRNAs could have an impact on a variety of diseases, most notably cancer (115). In the context of cancer, miRNA dysregulation has been linked to tumor initiation, growth, and progression, with evidence pointing to miRNAs acting as tumor suppressors and oncogenes (115). In addition, it was found that miRNAs are packed into extracellular vesicles before being released into the extracellular space. These vesicles could be exosome and microvesicles (116–118). Several studies have been conducted to explore the role and application of miRNAs in cancer (75, 119–121). The serum or plasma level of miRNAs can serve as a predictive marker in cancer patients, indicating signs of the disease stage (75, 119). miRNAs are Frontiers in Oncology \| www.frontiersin.org Tumor Educated Platelets Platelets are enucleated cells that participate in coagulation. Platelet activation has also been found in inﬂammatory diseases like eczema and asthma (140). Evidence suggests that platelets may play an important role in tumorigenesis by helping tumor evasion, angiogenesis, and metastasis (141, 142). Activated platelets secrete a-granules to release transforming growth factor-beta (TGFb) and adenosine triphosphate (ATP), leading to epithelial mesenchymal transition (EMT) and metastasis (143). On the other hand, tumour cells have been found to induce thrombocytosis by producing growth factors and cytokines such as granulocyte colony-stimulating factor (GCSF), granulocyte-macrophage colony-stimulating factor (GMCSF), interleukin (IL)-1 and IL-6 (144). Given this information, 6 January 2022 \| Volume 11 \| Article 801269 Herath et al. Circulating Biomarkers in Lung Cancer TABLE 1 \| Comparison of different liquid biopsy markers including cfDNA/ctDNA, exosome, CTC, cmiRNA and TEP. Marker cfDNA/ ctDNA Exosomes CTC Sample type • • • • • • • Serum Plasma CSF Ascites Pleural effusion Strength • • • • Nearly all body • ﬂuids Peripheral blood • • • • cmiRNA • • Serum Plasma • • TEP • Peripheral blood • • • Reﬂective of tumor molecular alterations/mutations Stable up to 2 days in blood samples Reﬂective of tumor heterogeneity Highly sensitive assays (NGS, PCR) Stable source of tumor genetic material (DNA, RNA, protein, miRNA) Commercial kits available Assessment of tumor markers (PD-L1) during treatment Demonstration of signal colocalization Cell morphology and functional studies Different proﬁle among early-stage cancer patients Distinguishable between cancer patients and healthy individuals Weakness • • • • • • • • • • • • • • TEP-RNA is reﬂective of • tumor transcriptome • Abundant Dynamic mRNA repertoire because of short life-span Contamination of germinal cfDNA Cannot reﬂect every gene mutation Low amount in plasma Undetectable in many patients with early-stage cancer Less stable than non-tumor DNA Clinical application in oncology • Elevated in cancer patients compared to healthy individuals Increases with tumor size and stage (14, 49, 134, 135) Elevated in cancer patients compared to healthy individuals Exosome size positively correlates with unfavorable outcomes Predictive of early relapse after primary treatment CTC number correlates with progressionfree survival and overall survival (5, 110, 136) • cmi-RNA expression correlates with tumor development, progression and metastasis (14, 110, 122) • Distinguishable between healthy individuals and cancer patients Distinguishable between patients with early-stage cancer and patients with advanced-stage cancer (138, 139) • required standardization for extraction • and detection Unreliable isolation procedures • Not predictive of therapeutic beneﬁt in metastatic setting Undetectable in most patients with early-stage cancer Rare to capture in the bloodstream Loss of epithelial speciﬁc markers during epithelial mesenchymal transition (EMT) High variability Lack of standardization Unspeciﬁc for a cancer type Reproducibility Lack of validated assay Ref. • • • (109, 122, 137) The sample type, strength, weakness and clinical applications of each marker is discussed. found 38 cases had an EML4-ALK-rearrangement in platelets with 65% sensitivity and 100% speciﬁcity (157). In addition, it was reported that the EML4-ALK rearrangement in platelets had a correlation with PFS and OS in patients who received crizotinib. Accordingly, patients with EML4-ALK+platelets had 3.7 month PFS, whereas patients with EML4-ALK- platelets had 16 month PFS (157). When compared to patients with noncancerous inﬂammatory diseases, patients with cancer have a hyperactive state of TEPs, according to functional analysis (158). In addition, using RNA sequencing, the platelet RNAs were investigated in patients with early stage NSCLC and healthy individuals. It was demonstrated that integrin alpha-IIb (ITGA2B) was expressed more in NSCLC patients than healthy individuals (159). As a result of their ﬁndings, the researchers concluded that TEP ITGA2B could be a promising marker for detecting patients with early-stage NSCLC (159). the interaction between tumour, tumour microenvironment (TME), and platelets account for tumor-educated platelets (TEPs) (145, 146). Accordingly, numerous studies explored the use of TEPs as a liquid biopsy in cancer initiation and progression (147–150). It has been discovered that growth factors released and produced by platelets and tumor cells, such as vascular endothelial growth factor (VEGF), contribute to changes in mRNA expression within platelets, resulting in a speciﬁc spliced mRNA signature (151, 152). The mRNA signature could be used as a biomarker to distinguish cancer patients from healthy individuals. According to multiple studies, patients with cancer onset and progression have a highly dynamic mRNA repertoire (138, 153). Therefore, analyzing mRNA proﬁles may be useful in detecting primary tumors, metastasis, and cancer staging (45, 138). A study that compared patients with localized and metastatic tumors to healthy people discovered that platelet mRNA proﬁles could distinguish cancer cases from healthy ones with 96% accuracy, 97% sensitivity, and 94% speciﬁcity (153). In the context of lung cancer, Geraci and colleagues investigated platelet mRNA proﬁles in the context of lung cancer using a lung cancer model. As a result, they discovered distinct platelet mRNA gene expression between metastatic and control groups (154). Furthermore, research has revealed that some patients with lung adenocarcinoma have ALK rearrangement, resulting in the EML4–ALK fusion gene product (155, 156). Using reverse transcription-polymerase chain reaction (RT-PCR), Nelson et al. examined blood platelets from 77 NSCLC patients and Frontiers in Oncology \| www.frontiersin.org DISCUSSION Liquid biopsy has shown promise for the early diagnosis and management of lung cancer due to its high sensitivity, speciﬁcity, non-invasive sampling and low-risk proﬁle. cfDNA/ctDNA, CTCs, miRNAs and exosomes are considered as potentially actionable biomarkers which are used in liquid biopsy. However, robust characterization of each marker is needed for the comprehensive understanding of their role in NSCLC disease 7 January 2022 \| Volume 11 \| Article 801269 Herath et al. Circulating Biomarkers in Lung Cancer FIGURE 3 \| Importance of liquid biopsy at different stages of cancer propagation, indicating the biomarker purpose, evolution of clones, and treatment effects. Adapted from Ref (160) and created with BioRender.com. panels have come onto the marker, cross comparisons of blood and tumour studies will be warranted to determine their utility for targeted therapies. Moreover, large prospective clinical trials are needed to provide a better understanding of the clinical utility of liquid biopsy assays. progression, prognostication and determining a tailored treatment regimen. It is clear that certain liquid biopsy analytes would be useful over the course of disease progression. For example, ctDNA and exosomes may be informative early in disease onset, and could be used to identify speciﬁc actionable mutations/and early stage disease which cannot be determined using LDCT. Whereas CTCs may be used to identify the risk of developing metastatic disease, and the types of clones which may develop treatment resistance (Figure 3). ctDNA has also shown utility over the course of therapy where the variant allele frequencies can be monitored over time/therapy to determine increases or decreases of tumour speciﬁc mutations in response to treatment. ctDNA has been the most studied marker in the ﬁeld of lung cancer with clinical utility for a number of gene mutations. The detection of tumour speciﬁc mutations post therapy may also present a window of therapeutic opportunity where the patient has minimal residual disease (MRD), prior to clinically detectable disease progression with radiological evidence. Whilst larger multi-marker NGS AUTHOR CONTRIBUTIONS All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication. FUNDING The authors are supported by project grants and fellowships for AK from NHMRC (1157741), Cure Cancer (1182179), and the PA Research Foundation (KOB). 5. Kapeleris J, Kulasinghe A, Warkiani ME, Vela I, Kenny L, O’Byrne K, et al. The Prognostic Role of Circulating Tumor Cells (Ctcs) in Lung Cancer. Front Oncol (2018) 8:311. doi: 10.3389/fonc.2018.00311 6. Lemjabbar-Alaoui H, Hassan OU, Yang Y-W, Buchanan P. Lung Cancer: Biology and Treatment Options. Biochim Biophys Acta (BBA)-Reviews Cancer (2015) 1856(2):189–210. doi: 10.1016/j.bbcan. 2015.08.002 7. Rubin P, Hansen JT. TNM Staging Atlas With Oncoanatomy. Lippincott Williams & Wilkins (2013). 8. Travis WD, Brambilla E, Riely GJ. New Pathologic Classiﬁcation of Lung Cancer: Relevance for Clinical Practice and Clinical Trials. J Clin Oncol (2013) 31(8):992–1001. doi: 10.1200/JCO.2012.46.9270 REFERENCES 1. Boloker G, Wang C, Zhang J. Updated Statistics of Lung and Bronchus Cancer in United States (2018). J Thorac Dis (2018) 10(3):1158. doi: 10.21037/jtd.2018.03.15 2. Siegel RL, Miller KD, Jemal A. Cancer Statistics, 2019. CA: Cancer J Clin (2019) 69(1):7–34. doi: 10.3322/caac.21551 3. Patz EFJr., Campa MJ, Gottlin EB, Kusmartseva I, Guan XR, Herndon JE. Panel of Serum Biomarkers for the Diagnosis of Lung Cancer. J Clin Oncol (2007) 25(35):5578–83. doi: 10.1200/JCO.2007.13.5392 4. Howlader N, Noone AM, Krapcho M, Miller D, Brest A, Yu M, et al. SEER Cancer Statistics Review, 1975-2017. Bethesda, MD: National Cancer Institute (2020) https://seer.cancer.gov/csr/1975_2017/ Frontiers in Oncology \| www.frontiersin.org 8 January 2022 \| Volume 11 \| Article 801269 Herath et al. Circulating Biomarkers in Lung Cancer 30. Ponomaryova AA, Rykova EY, Cherdyntseva NV, Skvortsova TE, Dobrodeev AY, Zav’yalov AA, et al. Potentialities of Aberrantly Methylated Circulating DNA for Diagnostics and Post-Treatment FollowUp of Lung Cancer Patients. Lung Cancer (2013) 81(3):397–403. doi: 10.1016/j.lungcan.2013.05.016 31. Hyun MH, Sung JS, Kang EJ, Choi YJ, Park KH, Shin SW, et al. Quantiﬁcation of Circulating Cell-Free DNA to Predict Patient Survival in non-Small-Cell Lung Cancer. Oncotarget (2017) 8(55):94417. doi: 10.18632/ oncotarget.21769 32. Alama A, Coco S, Genova C, Rossi G, Fontana V, Tagliamento M, et al. Prognostic Relevance of Circulating Tumor Cells and Circulating Cell-Free DNA Association in Metastatic Non-Small Cell Lung Cancer Treated With Nivolumab. J Clin Med (2019) 8(7):1011. doi: 10.3390/jcm8071011 33. Nygaard A, Holdgaard P, Spindler KG, Pallisgaard N, Jakobsen A. The Correlation Between Cell-Free DNA and Tumour Burden was Estimated by PET/CT in Patients With Advanced NSCLC. Br J Cancer (2014) 110(2):363– 8. doi: 10.1038/bjc.2013.705 34. Lee Y, Park S, Kim WS, Lee JC, Jang SJ, Choi J, et al. Correlation Between Progression-Free Survival, Tumor Burden, and Circulating Tumor DNA in the Initial Diagnosis of Advanced-Stage EGFR-Mutated non-Small Cell Lung Cancer. Thorac Cancer (2018) 9(9):1104–10. doi: 10.1111/17597714.12793 35. Ou S-HI, Nagasaka M, Zhu VW. Liquid Biopsy to Identify Actionable Genomic Alterations. Am Soc Clin Oncol Educ Book (2018) 38:978–97. doi: 10.1200/EDBK_199765 36. Fabrizio D, Malboeuf C, Lieber D, Zhong S, He J, White E, et al. Analytic Validation of a Next Generation Sequencing Assay to Identify Tumor Mutational Burden From Blood (Btmb) to Support Investigation of an AntiPD-L1 Agent, Atezolizumab, in a First Line non-Small Cell Lung Cancer Trial (BFAST). Ann Oncol (2017) 28:v27. doi: 10.1093/annonc/mdx363.018 37. Villatoro S, Mayo-de-las-Casas C, Jordana-Ariza N, Viteri-Ramı́rez S, Garzó n-Ibañez M, Moya-Horno I, et al. Prospective Detection of Mutations in Cerebrospinal Fluid, Pleural Effusion, and Ascites of Advanced Cancer Patients to Guide Treatment Decisions. Mol Oncol (2019) 13(12):2633–45. doi: 10.1002/1878-0261.12574 38. Wang Z, Duan J, Cai S, Han M, Dong H, Zhao J, et al. Assessment of Blood Tumor Mutational Burden as a Potential Biomarker for Immunotherapy in Patients With non–Small Cell Lung Cancer With Use of a Next-Generation Sequencing Cancer Gene Panel. JAMA Oncol (2019) 5(5):696–702. doi: 10.1001/jamaoncol.2018.7098 39. Gandara DR, Paul SM, Kowanetz M, Schleifman E, Zou W, Li Y, et al. BloodBased Tumor Mutational Burden as a Predictor of Clinical Beneﬁt in nonSmall-Cell Lung Cancer Patients Treated With Atezolizumab. Nat Med (2018) 24(9):1441–8. doi: 10.1038/s41591-018-0134-3 40. Keppens C, Palma JF, Das PM, Scudder S, Wen W, Normanno N, et al. Detection of EGFR Variants in Plasma: A Multilaboratory Comparison of a Real-Time PCR EGFR Mutation Test in Europe. J Mol Diagn (2018) 20 (4):483–94. doi: 10.1016/j.jmoldx.2018.03.006 41. Yang J, Hui Y, Zhang Y, Zhang M, Ji B, Tian G, et al. Application of Circulating Tumor DNA as a Biomarker for non-Small Cell Lung Cancer. Front. Oncol (2021) 11:725938. doi: 10.3389/fonc.2021.725938 42. Akamatsu H, Koh Y, Okamoto I, Fujimoto D, Bessho A, Azuma K, et al. Clinical Signiﬁcance of Monitoring EGFR Mutation in Plasma Using Multiplexed Digital PCR in EGFR Mutated Patients Treated With Afatinib (West Japan Oncology Group 8114LTR Study). Lung Cancer (2019) 131:128–33. doi: 10.1016/j.lungcan.2019.03.021 43. Cui S, Zhang W, Xiong L, Pan F, Niu Y, Chu T, et al. Use of Capture-Based Next-Generation Sequencing to Detect ALK Fusion in Plasma Cell-Free DNA of Patients With Non-Small-Cell Lung Cancer. Oncotarget (2017) 8 (2):2771. doi: 10.18632/oncotarget.13741 44. Bordi P, Tiseo M, Roﬁ E, Petrini I, Restante G, Danesi R, et al. Detection of ALK and KRAS Mutations in Circulating Tumor DNA of Patients With Advanced ALK-Positive NSCLC With Disease Progression During Crizotinib Treatment. Clin Lung Cancer (2017) 18(6):692–7. doi: 10.1016/j.cllc.2017.04.013 45. McCoach CE, Blakely CM, Banks KC, Levy B, Chue BM, Raymond VM, et al. Clinical Utility of Cell-Free DNA for the Detection of ALK Fusions and Genomic Mechanisms of ALK Inhibitor Resistance in Non–Small Cell Lung 9. Polanski J, Jankowska-Polanska B, Rosinczuk J, Chabowski M, SzymanskaChabowska A. Quality of Life of Patients With Lung Cancer. OncoTargets Ther (2016) 9:1023. doi: 10.2147/OTT.S100685 10. Revelo AE, Martin A, Velasquez R, Kulandaisamy PC, Bustamante J, Keshishyan S, et al. Liquid Biopsy for Lung Cancers: An Update on Recent Developments. Ann Trans Med (2019) 7(15):349. doi: 10.21037/ atm.2019.03.28 11. Team NLSTR. Reduced Lung-Cancer Mortality With Low-Dose Computed Tomographic Screening. N Engl J Med (2011) 365(5):395–409. doi: 10.1056/ NEJMoa1102873 12. Bauml J, Levy B. Clonal Hematopoiesis: A New Layer in the Liquid Biopsy Story in Lung Cancer. Clin Cancer Res (2018) 24(18):4352–4. doi: 10.1158/ 1078-0432.CCR-18-0969 13. Chu X-Y, Hou X-B, Song W-A, Xue Z-Q, Wang B, Zhang L-B. Diagnostic Values of SCC, CEA, Cyfra21-1 and NSE for Lung Cancer in Patients With Suspicious Pulmonary Masses: A Single Center Analysis. Cancer Biol Ther (2011) 11(12):995–1000. doi: 10.4161/cbt.11.12.15526 14. Rijavec E, Coco S, Genova C, Rossi G, Longo L, Grossi F. Liquid Biopsy in non-Small Cell Lung Cancer: Highlights and Challenges. Cancers (2020) 12 (1):17. doi: 10.3390/cancers12010017 15. Bailey C, Black JR, Reading JL, Litchﬁeld K, Turajlic S, McGranahan N, et al. Tracking Cancer Evolution Through the Disease Course. Cancer Discov (2021) 11(4):916–32. doi: 10.1158/2159-8290.CD-20-1559 16. Lim M, Kim C-J, Sunkara V, Kim M-H, Cho Y-K. Liquid Biopsy in Lung Cancer: Clinical Applications of Circulating Biomarkers (Ctcs and Ctdna). Micromachines (2018) 9(3):100. doi: 10.3390/mi9030100 17. Bettegowda C, Sausen M, Leary RJ, Kinde I, Wang Y, Agrawal N, et al. Detection of Circulating Tumor DNA in Early-and Late-Stage Human Malignancies. Sci Trans Med (2014) 6(224):224ra24–ra24. doi: 10.1158/ 1538-7445.AM2014-5606 18. Sozzi G, Conte D, Leon M, Cirincione R, Roz L, Ratcliffe C, et al. Quantiﬁcation of Free Circulating DNA as a Diagnostic Marker in Lung Cancer. J Clin Oncol (2003) 21(21):3902–8. doi: 10.1200/JCO.2003.02.006 19. Santarpia M, Liguori A, D’Aveni A, Karachaliou N, Gonzalez-Cao M, Dafﬁnà MG, et al. Liquid Biopsy for Lung Cancer Early Detection. J Thorac Dis (2018) 10 (Suppl 7):S882. doi: 10.21037/jtd.2018.03.81 20. De Rubis G, Rajeev Krishnan S, Bebawy M. Liquid Biopsies in Cancer Diagnosis, Monitoring, and Prognosis. Trends Pharmacol Sci (2019) 40 (3):172–86. doi: 10.1016/j.tips.2019.01.006 21. Fernandez-Garcia D, Hills A, Page K, Hastings RK, Toghill B, Goddard KS, et al. Plasma Cell-Free DNA (Cfdna) as a Predictive and Prognostic Marker in Patients With Metastatic Breast Cancer. Breast Cancer Res (2019) 21(1):1– 13. doi: 10.1186/s13058-019-1235-8 22. Kustanovich A, Schwartz R, Peretz T, Grinshpun A. Life and Death of Circulating Cell-Free DNA. Cancer Biol Ther (2019) 20(8):1057–67. doi: 10.1080/15384047.2019.1598759 23. Zhu G, Guo YA, Ho D, Poon P, Poh ZW, Wong PM, et al. Tissue-Speciﬁc Cell-Free DNA Degradation Quantiﬁes Circulating Tumor DNA Burden. Nat Commun (2021) 12(1):1–11. doi: 10.1038/s41467-021-22463-y 24. Giacona MB, Ruben GC, Iczkowski KA, Roos TB, Porter DM, Sorenson GD. Cell-Free DNA in Human Blood Plasma: Length Measurements in Patients With Pancreatic Cancer and Healthy Controls. Pancreas (1998) 17(1):89–97. doi: 10.1097/00006676-199807000-00012 25. Diehl F, Schmidt K, Choti MA, Romans K, Goodman S, Li M, et al. Circulating Mutant DNA to Assess Tumor Dynamics. Nat Med (2008) 14 (9):985–90. doi: 10.1038/nm.1789 26. Newman AM, Bratman SV, To J, Wynne JF, Eclov NC, Modlin LA, et al. An Ultrasensitive Method for Quantitating Circulating Tumor DNA With Broad Patient Coverage. Nat Med (2014) 20(5):548–54. doi: 10.1038/ nm.3519 27. Dawson S-J, Tsui DW, Murtaza M, Biggs H, Rueda OM, Chin S-F, et al. Analysis of Circulating Tumor DNA to Monitor Metastatic Breast Cancer. N Engl J Med (2013) 368(13):1199–209. doi: 10.1056/NEJMoa1213261 28. Spellman PT, Gray JW. Detecting Cancer by Monitoring Circulating Tumor DNA. Nat Med (2014) 20(5):474–5. doi: 10.1038/nm.3564 29. Diaz LAJr., Bardelli A. Liquid Biopsies: Genotyping Circulating Tumor DNA. J Clin Oncol (2014) 32(6):579. doi: 10.1200/JCO.2012.45.2011 Frontiers in Oncology \| www.frontiersin.org 9 January 2022 \| Volume 11 \| Article 801269 Herath et al. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. Circulating Biomarkers in Lung Cancer 64. Kharaziha P, Ceder S, Li Q, Panaretakis T. Tumor Cell-Derived Exosomes: A Message in a Bottle. Biochim Biophys Acta (BBA)-Reviews Cancer (2012) 1826(1):103–11. doi: 10.1016/j.bbcan.2012.03.006 65. Zomer A, Maynard C, Verweij FJ, Kamermans A, Schäfer R, Beerling E, et al. In Vivo Imaging Reveals Extracellular Vesicle-Mediated Phenocopying of Metastatic Behavior. Cell (2015) 161(5):1046–57. doi: 10.1016/ j.cell.2015.04.042 66. Kahlert C, Kalluri R. Exosomes in Tumor Microenvironment Inﬂuence Cancer Progression and Metastasis. J Mol Med (2013) 91(4):431–7. doi: 10.1007/s00109-013-1020-6 67. Wu H, Zhou J, Mei S, Wu D, Mu Z, Chen B, et al. Circulating Exosomal Microrna-96 Promotes Cell Proliferation, Migration and Drug Resistance by Targeting LMO7. J Cell Mol Med (2017) 21(6):1228–36. doi: 10.1111/ jcmm.13056 68. Kholia S, Ranghino A, Garnieri P, Lopatina T, Deregibus MC, Rispoli P, et al. Extracellular Vesicles as New Players in Angiogenesis. Vasc Pharmacol (2016) 86:64–70. doi: 10.1016/j.vph.2016.03.005 69. Thakur BK, Zhang H, Becker A, Matei I, Huang Y, Costa-Silva B, et al. Double-Stranded DNA in Exosomes: A Novel Biomarker in Cancer Detection. Cell Res (2014) 24(6):766–9. doi: 10.1038/cr.2014.44 70. Yamashita T, Kamada H, Kanasaki S, Maeda Y, Nagano K, Abe Y, et al. Epidermal Growth Factor Receptor Localized to Exosome Membranes as a Possible Biomarker for Lung Cancer Diagnosis. Die Pharmazie-An Int J Pharm Sci (2013) 68(12):969–73. 71. Fortunato O, Gasparini P, Boeri M, Sozzi G. Exo-Mirnas as a New Tool for Liquid Biopsy in Lung Cancer. Cancers (2019) 11(6):888. doi: 10.3390/ cancers11060888 72. Wu J, Shen Z. Exosomal Mirnas as Biomarkers for Diagnostic and Prognostic in Lung Cancer. Cancer Med (2020) 9(19):6909–22. doi: 10.1002/cam4.3379 73. Al-Nedawi K, Meehan B, Kerbel RS, Allison AC, Rak J. Endothelial Expression of Autocrine VEGF Upon the Uptake of Tumor-Derived Microvesicles Containing Oncogenic EGFR. Proc Natl Acad Sci (2009) 106 (10):3794–9. doi: 10.1073/pnas.0804543106 74. Fujita Y, Suda K, Kimura H, Matsumoto K, Arao T, Nagai T, et al. Highly Sensitive Detection of EGFR T790M Mutation Using Colony Hybridization Predicts Favorable Prognosis of Patients With Lung Cancer Harboring Activating EGFR Mutation. J Thorac Oncol (2012) 7(11):1640–4. doi: 10.1097/JTO.0b013e3182653d7f 75. Zhang Y, Roth JA, Yu H, Ye Y, Xie K, Zhao H, et al. A 5-Microrna Signature Identiﬁed From Serum Microrna Proﬁling Predicts Survival in Patients With Advanced Stage non-Small Cell Lung Cancer. Carcinogenesis (2019) 40 (5):643–50. doi: 10.1093/carcin/bgy132 76. Jin H, Varner J. Integrins: Roles in Cancer Development and as Treatment Targets. Br J Cancer (2004) 90(3):561–5. doi: 10.1038/sj.bjc.6601576 77. Nagrath S, Sequist LV, Maheswaran S, Bell DW, Irimia D, Ulkus L, et al. Isolation of Rare Circulating Tumour Cells in Cancer Patients by Microchip Technology. Nature (2007) 450(7173):1235–9. doi: 10.1038/nature06385 78. Tanaka F, Yoneda K, Hasegawa S. Circulating Tumor Cells (Ctcs) in Lung Cancer: Current Status and Future Perspectives. Lung Cancer: Targets Ther (2010) 1:77. doi: 10.2147/LCTT.S6828 79. Krebs MG, Sloane R, Priest L, Lancashire L, Hou J-M, Greystoke A, et al. Evaluation and Prognostic Signiﬁcance of Circulating Tumor Cells in Patients With non–Small-Cell Lung Cancer. J Clin Oncol (2011) 29 (12):1556–63. doi: 10.1200/JCO.2010.28.7045 80. Alix-Panabières C, Pantel K. Characterization of Single Circulating Tumor Cells. FEBS Lett (2017) 591(15):2241–50. doi: 10.1002/1873-3468.12662 81. Lee JM, Dedhar S, Kalluri R, Thompson EW. The Epithelial–Mesenchymal Transition: New Insights in Signaling, Development, and Disease. J Cell Biol (2006) 172(7):973–81. doi: 10.1083/jcb.200601018 82. Zheng Q-M, Chen X-Y, Bao Q-F, Yu J, Chen L-H. ILK Enhances Migration and Invasion Abilities of Human Endometrial Stromal Cells by Facilitating the Epithelial–Mesenchymal Transition. Gynecol Endocrinol (2018) 34 (12):1091–6. doi: 10.1080/09513590.2018.1498477 83. Herath S, Razavi Bazaz S, Monkman J, Ebrahimi Warkiani M, Richard D, O’Byrne K, et al. Circulating Tumor Cell Clusters: Insights Into Tumour Dissemination and Metastasis. Expert Rev Mol Diagn (2020) 20(11):1139–47. doi: 10.1080/14737159.2020.1846523 Cancer. Clin Cancer Res (2018) 24(12):2758–70. doi: 10.1158/10780432.CCR-17-2588 Ivan D, Prieto VG. Use of Immunohistochemistry in the Diagnosis of Melanocytic Lesions: Applications and Pitfalls. Future Oncol (2010) 6 (7):1163–75. doi: 10.2217/fon.10.81 Huang H. Anaplastic Lymphoma Kinase (ALK) Receptor Tyrosine Kinase: A Catalytic Receptor With Many Faces. Int J Mol Sci (2018) 19(11):3448. doi: 10.3390/ijms19113448 Rosas G, Ruiz R, Araujo JM, Pinto JA, Mas L. ALK Rearrangements: Biology, Detection and Opportunities of Therapy in non-Small Cell Lung Cancer. Crit Rev Oncol/Hematol (2019) 136:48–55. doi: 10.1016/j.critrevonc. 2019.02.006 Ignatiadis M, Sledge GW, Jeffrey SS. Liquid Biopsy Enters the Clinic — Implementation Issues and Future Challenges. Nat Rev Clin Oncol (2021) 18 (5):297–312. doi: 10.1038/s41571-020-00457-x Wang Z, Duan J, Cai S, Han M, Dong H, Zhao J, et al. Assessment of Blood Tumor Mutational Burden as a Potential Biomarker for Immunotherapy in Patients With Non-Small Cell Lung Cancer With Use of a Next-Generation Sequencing Cancer Gene Panel. JAMA Oncol (2019) 5(5):696–702. doi: 10.1001/jamaoncol.2018.7098 Jia Q, Chiu L, Wu S, Bai J, Peng L, Zheng L, et al. Tracking Neoantigens by Personalized Circulating Tumor DNA Sequencing During Checkpoint Blockade Immunotherapy in Non-Small Cell Lung Cancer. Adv Sci (Weinheim Baden-Wurttemberg Germany) (2020) 7(9):1903410. doi: 10.1002/advs.201903410 Eslami-S Z, Corté s-Herná ndez LE, Alix-Panabières C. The Metastatic Cascade as the Basis for Liquid Biopsy Development. Front Oncol (2020) 10:1055. doi: 10.3389/fonc.2020.01055 Esposito Abate R, Pasquale R, Fenizia F, Rachiglio AM, Roma C, Bergantino F, et al. The Role of Circulating Free DNA in the Management of NSCLC. Expert Rev Anticancer Ther (2019) 19(1):19–28. doi: 10.1080/14737140. 2019.1548938 Vanni I, Coco S, Truini A, Rusmini M, Dal Bello MG, Alama A, et al. NextGeneration Sequencing Workﬂow for NSCLC Critical Samples Using a Targeted Sequencing Approach by Ion Torrent PGM™ Platform. Int J Mol Sci (2015) 16(12):28765–82. doi: 10.3390/ijms161226129 Dono M, De Luca G, Lastraioli S, Anselmi G, Dal Bello MG, Coco S, et al. TagBased Next Generation Sequencing: A Feasible and Reliable Assay for EGFR T790M Mutation Detection in Circulating Tumor DNA of non Small Cell Lung Cancer Patients. Mol Med (2019) 25(1):1–13. doi: 10.1186/s10020-019-0082-5 Normanno N, Denis MG, Thress KS, Ratcliffe M, Reck M. Guide to Detecting Epidermal Growth Factor Receptor (EGFR) Mutations in Ctdna of Patients With Advanced non-Small-Cell Lung Cancer. Oncotarget (2017) 8(7):12501. doi: 10.18632/oncotarget.13915 Kang Q, Henry NL, Paoletti C, Jiang H, Vats P, Chinnaiyan AM, et al. Comparative Analysis of Circulating Tumor DNA Stability in K3EDTA, Streck, and Cellsave Blood Collection Tubes. Clin Biochem (2016) 49 (18):1354–60. doi: 10.1016/j.clinbiochem.2016.03.012 Zheng H, Zhan Y, Liu S, Lu J, Luo J, Feng J, et al. The Roles of TumorDerived Exosomes in non-Small Cell Lung Cancer and Their Clinical Implications. J Exp Clin Cancer Res (2018) 37(1):1–11. doi: 10.1186/ s13046-018-0901-5 Vanni I, Alama A, Grossi F, Dal Bello MG, Coco S. Exosomes: A New Horizon in Lung Cancer. Drug Discov Today (2017) 22(6):927–36. doi: 10.1016/j.drudis.2017.03.004 Valadi H, Ekström K, Bossios A, Sjöstrand M, Lee JJ, Lötvall JO. ExosomeMediated Transfer of Mrnas and Micrornas is a Novel Mechanism of Genetic Exchange Between Cells. Nat Cell Biol (2007) 9(6):654–9. doi: 10.1038/ncb1596 Masaoutis C, Mihailidou C, Tsourouﬂis G, Theocharis S. Exosomes in Lung Cancer Diagnosis and Treatment. From translating Res into Future Clin Practice Biochimie (2018) 151:27–36. doi: 10.1016/j.biochi.2018.05.014 Rabinowits G, Gerç el-Taylor C, Day JM, Taylor DD, Kloecker GH. Exosomal Microrna: A Diagnostic Marker for Lung Cancer. Clin Lung Cancer (2009) 10(1):42–6. doi: 10.3816/CLC.2009.n.006 Logozzi M, Mizzoni D, Di Raimo R, Fais S. Exosomes: A Source for New and Old Biomarkers in Cancer. Cancers (2020) 12(9):2566. doi: 10.3390/ cancers12092566 Frontiers in Oncology \| www.frontiersin.org 10 January 2022 \| Volume 11 \| Article 801269 Herath et al. Circulating Biomarkers in Lung Cancer 104. Khoo BL, Warkiani ME, Tan DS-W, Bhagat AAS, Irwin D, Lau DP, et al. Clinical Validation of an Ultra High-Throughput Spiral Microﬂuidics for the Detection and Enrichment of Viable Circulating Tumor Cells. PLoS One (2014) 9(7):e99409. doi: 10.1371/journal.pone.0099409 105. Fachin F, Spuhler P, Martel-Foley JM, Edd JF, Barber TA, Walsh J, et al. Monolithic Chip for High-Throughput Blood Cell Depletion to Sort Rare Circulating Tumor Cells. Sci Rep (2017) 7(1):1–11. doi: 10.1038/s41598-01711119-x 106. Zhou J, Kulasinghe A, Bogseth A, O’Byrne K, Punyadeera C, Papautsky I. Isolation of Circulating Tumor Cells in non-Small-Cell-Lung-Cancer Patients Using a Multi-Flow Microﬂuidic Channel. Microsystems Nanoeng (2019) 5(1):1–12. doi: 10.1038/s41378-019-0045-6 107. Wang C, Mu Z, Chervoneva I, Austin L, Ye Z, Rossi G, et al. Longitudinally Collected Ctcs and CTC-Clusters and Clinical Outcomes of Metastatic Breast Cancer. Breast Cancer Res Treat (2017) 161(1):83–94. doi: 10.1007/s10549016-4026-2 108. Kulasinghe A, Perry C, Kenny L, Warkiani ME, Nelson C, Punyadeera C. PD-L1 Expressing Circulating Tumour Cells in Head and Neck Cancers. BMC Cancer (2017) 17(1):1–6. doi: 10.1186/s12885-017-3316-3 109. Mazel M, Jacot W, Pantel K, Bartkowiak K, Topart D, Cayrefourcq L, et al. Frequent Expression of PD-L1 on Circulating Breast Cancer Cells. Mol Oncol (2015) 9(9):1773–82. doi: 10.1016/j.molonc.2015.05.009 110. Freitas C, Sousa C, Machado F, Serino M, Santos V, Cruz-Martins N, et al. The Role of Liquid Biopsy in Early Diagnosis of Lung Cancer. Front Oncol (2021) 11:1130. doi: 10.3389/fonc.2021.634316 111. Esquela K, Slack J. Oncomirs Mirnas With a Role in Cancer. Nat Rev Cancer (2006) 6:259–569. doi: 10.1038/nrc1840 112. Ambros V. The Functions of Animal Micrornas. Nature (2004) 431 (7006):350–5. doi: 10.1038/nature02871 113. Lim L, Lau NC, Garrett-Engele P, Grimson A, Schelter JM, Castle J, et al. Microarray Analysis Shows That Some Micrornas Downregulate Large Numbers of Target Mrnas. Nature (2005) 433:769–73. doi: 10.1038/ nature03315 114. Krek A, Grü n D, Poy MN, Wolf R, Rosenberg L, Epstein EJ, et al. Combinatorial Microrna Target Predictions. Nat Genet (2005) 37:495–500. doi: 10.1038/ng1536 115. Zhang B, Pan X, Cobb GP, Anderson TA. Micrornas as Oncogenes and Tumor Suppressors. Dev Biol (2007) 302(1):1–12. doi: 10.1016/ j.ydbio.2006.08.028 116. Turchinovich A, Weiz L, Burwinkel B. Extracellular Mirnas: The Mystery of Their Origin and Function. Trends Biochem Sci (2012) 37(11):460–5. doi: 10.1016/j.tibs.2012.08.003 117. Arroyo JD, Chevillet JR, Kroh EM, Ruf IK, Pritchard CC, Gibson DF, et al. Argonaute2 Complexes Carry a Population of Circulating Micrornas Independent of Vesicles in Human Plasma. Proc Natl Acad Sci (2011) 108 (12):5003–8. doi: 10.1073/pnas.1019055108 118. Vickers KC, Palmisano BT, Shoucri BM, Shamburek RD, Remaley AT. Micrornas are Transported in Plasma and Delivered to Recipient Cells by High-Density Lipoproteins. Nat Cell Biol (2011) 13(4):423–33. doi: 10.1038/ ncb2210 119. Hu Z, Chen X, Zhao Y, Tian T, Jin G, Shu Y, et al. Serum Microrna Signatures Identiﬁed in a Genome-Wide Serum Microrna Expression Proﬁling Predict Survival of non-Small-Cell Lung Cancer. J Clin Oncol (2010) 28(10):1721–6. doi: 10.1200/JCO.2009.24.9342 120. Pigati L, Yaddanapudi SC, Iyengar R, Kim D-J, Hearn SA, Danforth D, et al. Selective Release of Microrna Species From Normal and Malignant Mammary Epithelial Cells. PLoS One (2010) 5(10):e13515. doi: 10.1371/ journal.pone.0013515 121. Okada H, Kohanbash G, Lotze MT. Micrornas in Immune Regulation— Opportunities for Cancer Immunotherapy. Int J Biochem Cell Biol (2010) 42 (8):1256–61. doi: 10.1016/j.biocel.2010.02.002 122. Siravegna G, Marsoni S, Siena S, Bardelli A. Integrating Liquid Biopsies Into the Management of Cancer. Nat Rev Clin Oncol (2017) 14(9):531–48. doi: 10.1038/nrclinonc.2017.14 123. Schwarzenbach H, Nishida N, Calin GA, Pantel K. Clinical Relevance of Circulating Cell-Free Micrornas in Cancer. Nat Rev Clin Oncol (2014) 11 (3):145–56. doi: 10.1038/nrclinonc.2014.5 84. Thiery JP, Acloque H, Huang RY, Nieto MA. Epithelial-Mesenchymal Transitions in Development and Disease. Cell (2009) 139(5):871–90. doi: 10.1016/j.cell.2009.11.007 85. Alix-Panabières C, Pantel K. Circulating Tumor Cells: Liquid Biopsy of Cancer. Clin Chem (2013) 59(1):110–8. doi: 10.1373/clinchem.2012.194258 86. Alix-Panabières C, Pantel K. Challenges in Circulating Tumour Cell Research. Nat Rev Cancer (2014) 14(9):623–31. doi: 10.1038/nrc3820 87. Alix-Panabières C. EPISPOT Assay: Detection of Viable Dtcs/Ctcs in Solid Tumor Patients. Minimal Residual Dis Circulating Tumor Cells Breast Cancer (2012) 195:69–76. doi: 10.1007/978-3-642-28160-0_6 88. Yu M, Stott S, Toner M, Maheswaran S, Haber DA. Circulating Tumor Cells: Approaches to Isolation and Characterization. J Cell Biol (2011) 192(3):373– 82. doi: 10.1083/jcb.201010021 89. Königsberg R, Obermayr E, Bises G, Pfeiler G, Gneist M, Wrba F, et al. Detection of Epcam Positive and Negative Circulating Tumor Cells in Metastatic Breast Cancer Patients. Acta Oncol (2011) 50(5):700–10. doi: 10.3109/0284186X.2010.549151 90. Kulasinghe A, Zhou J, Kenny L, Papautsky I, Punyadeera C. Capture of Circulating Tumour Cell Clusters Using Straight Microﬂuidic Chips. Cancers (2019) 11(1):89. doi: 10.3390/cancers11010089 91. Pantel K, Alix-Panabières C. Circulating Tumour Cells in Cancer Patients: Challenges and Perspectives. Trends Mol Med (2010) 16(9):398–406. doi: 10.1016/j.molmed.2010.07.001 92. Jolly MK, Boareto M, Huang B, Jia D, Lu M, Ben-Jacob E, et al. Implications of the Hybrid Epithelial/Mesenchymal Phenotype in Metastasis. Front. Oncol (2015) 5:155. doi: 10.3389/fonc.2015.00155 93. Semaan A, Bernard V, Kim DU, Lee JJ, Huang J, Kamyabi N, et al. Characterisation of Circulating Tumour Cell Phenotypes Identiﬁes a Partial-EMT Sub-Population for Clinical Stratiﬁcation of Pancreatic Cancer. Br J Cancer (2021) 124(12):1970–7. doi: 10.1038/s41416-02101350-9 94. Kulasinghe A, Perry C, Jovanovic L, Nelson C, Punyadeera C. Circulating Tumour Cells in Metastatic Head and Neck Cancers. Int J Cancer (2015) 136 (11):2515–23. doi: 10.1002/ijc.29108 95. Au SH, Storey BD, Moore JC, Tang Q, Chen YL, Javaid S, et al. Clusters of Circulating Tumor Cells Traverse Capillary-Sized Vessels. Proc Natl Acad Sci U S A (2016) 113(18):4947–52. doi: 10.1073/pnas.1524448113 96. Kulasinghe A, Kapeleris J, Kimberley R, Mattarollo SR, Thompson EW, Thiery JP, et al. The Prognostic Signiﬁcance of Circulating Tumor Cells in Head and Neck and Non-Small-Cell Lung Cancer. Cancer Med (2018) 7 (12):5910–9. doi: 10.1002/cam4.1832 97. Szczerba BM, Castro-Giner F, Vetter M, Krol I, Gkountela S, Landin J, et al. Neutrophils Escort Circulating Tumour Cells to Enable Cell Cycle Progression. Nature (2019) 566(7745):553–7. doi: 10.1038/s41586-0190915-y 98. Ding L, Radfar P, Rezaei M, Warkiani ME. An Easy-to-Operate Method for Single-Cell Isolation and Retrieval Using a Microﬂuidic Static Droplet Array. Microchimica Acta (2021) 188(8):242. doi: 10.1007/s00604-021-04897-9 99. Zeinali M, Lee M, Nadhan A, Mathur A, Hedman C, Lin E, et al. HighThroughput Label-Free Isolation of Heterogeneous Circulating Tumor Cells and CTC Clusters From Non-Small-Cell Lung Cancer Patients. Cancers (2020) 12(1):127. doi: 10.3390/cancers12010127 100. Rostami P, Kashaninejad N, Moshksayan K, Saidi MS, Firoozabadi B, Nguyen N-T. Novel Approaches in Cancer Management With Circulating Tumor Cell Clusters. J Sci: Adv Mater Dev (2019) 4(1):1–18. doi: 10.1016/ j.jsamd.2019.01.006 101. Alshareef M, Metrakos N, Juarez Perez E, Azer F, Yang F, Yang X, et al. Separation of Tumor Cells With Dielectrophoresis-Based Microﬂuidic Chip. Biomicroﬂuidics (2013) 7(1):011803. doi: 10.1063/1.4774312 102. Tang Y, Shi J, Li S, Wang L, Cayre YE, Chen Y. Microﬂuidic Device With Integrated Microﬁlter of Conical-Shaped Holes for High Efﬁciency and High Purity Capture of Circulating Tumor Cells. Sci Rep (2014) 4(1):1–7. doi: 10.1038/srep06052 103. Wan S, Kim TH, Smith KJ, Delaney R, Park G-S, Guo H, et al. New Labyrinth Microﬂuidic Device Detects Circulating Tumor Cells Expressing Cancer Stem Cell Marker and Circulating Tumor Microemboli in Hepatocellular Carcinoma. Sci Rep (2019) 9(1):1–11. doi: 10.1038/s41598-019-54960-y Frontiers in Oncology \| www.frontiersin.org 11 January 2022 \| Volume 11 \| Article 801269 Herath et al. Circulating Biomarkers in Lung Cancer 124. Nagasaka M, Uddin MH, Al-Hallak MN, Rahman S, Balasubramanian S, Sukari A, et al. Liquid Biopsy for Therapy Monitoring in Early-Stage nonSmall Cell Lung Cancer. Mol Cancer (2021) 20(1):1–16. doi: 10.1186/s12943021-01371-1 125. Cui M, Wang H, Yao X, Zhang D, Xie Y, Cui R, et al. Circulating Micrornas in Cancer: Potential and Challenge. Front Genet (2019) 10:626. doi: 10.3389/ fgene.2019.00626 126. Wang P, Yang D, Zhang H, Wei X, Ma T, Cheng Z, et al. Early Detection of Lung Cancer in Serum by a Panel of Microrna Biomarkers. Clin Lung Cancer (2015) 16(4):313–9.e1. doi: 10.1016/j.cllc.2014.12.006 127. Montani F, Marzi MJ, Dezi F, Dama E, Carletti RM, Bonizzi G, et al. MirTest: A Blood Test for Lung Cancer Early Detection. JNCI: J Natl Cancer Institute (2015) 107(6):djv063. doi: 10.1093/jnci/djv063 128. Wozniak MB, Scelo G, Muller DC, Mukeria A, Zaridze D, Brennan P. Circulating Micrornas as Non-Invasive Biomarkers for Early Detection of Non-Small-Cell Lung Cancer. PLoS One (2015) 10(5):e0125026. doi: 10.1371/journal.pone.0125026 129. Umu SU, Langseth H, Bucher-Johannessen C, Fromm B, Keller A, Meese E, et al. A Comprehensive Proﬁle of Circulating Rnas in Human Serum. RNA Biol (2018) 15(2):242–50. doi: 10.1080/15476286.2017.1403003 130. Yang Y, Hu Z, Zhou Y, Zhao G, Lei Y, Li G, et al. The Clinical Use of Circulating Micrornas as non-Invasive Diagnostic Biomarkers for Lung Cancers. Oncotarget (2017) 8(52):90197. doi: 10.18632/oncotarget.21644 131. Li W, Wang Y, Zhang Q, Tang L, Liu X, Dai Y, et al. Microrna-486 as a Biomarker for Early Diagnosis and Recurrence of Non-Small Cell Lung Cancer. PLoS One (2015) 10(8):e0134220. doi: 10.1371/journal.pone. 0134220 132. Wang Y, Gu J, Roth JA, Hildebrandt MA, Lippman SM, Ye Y, et al. PathwayBased Serum Microrna Proﬁling and Survival in Patients With Advanced Stage Non–Small Cell Lung Cancer. Cancer Res (2013) 73(15):4801–9. doi: 10.1158/0008-5472.CAN-12-3273 133. Fan L, Qi H, Teng J, Su B, Chen H, Wang C, et al. Identiﬁcation of Serum Mirnas by Nano-Quantum Dots Microarray as Diagnostic Biomarkers for Early Detection of Non-Small Cell Lung Cancer. Tumor Biol (2016) 37 (6):7777–84. doi: 10.1007/s13277-015-4608-3 134. Marques JF, Junqueira-Neto S, Pinheiro J, Machado JC, Costa JL. Induction of Apoptosis Increases Sensitivity to Detect Cancer Mutations in Plasma. Eur J Cancer (2020) 127:130–8. doi: 10.1016/j.ejca.2019.12.023 135. Abbosh C, Birkbak NJ, Swanton C. Early Stage NSCLC—Challenges to Implementing Ctdna-Based Screening and MRD Detection. Nat Rev Clin Oncol (2018) 15(9):577–86. doi: 10.1038/s41571-018-0058-3 136. Chemi F, Rothwell DG, McGranahan N, Gulati S, Abbosh C, Pearce SP, et al. Pulmonary Venous Circulating Tumor Cell Dissemination Before Tumor Resection and Disease Relapse. Nat Med (2019) 25(10):1534–9. doi: 10.1038/ s41591-019-0593-1 137. Zhang L, Ridgway LD, Wetzel MD, Ngo J, Yin W, Kumar D, et al. The Identiﬁcation and Characterization of Breast Cancer Ctcs Competent for Brain Metastasis. Sci Trans Med (2013) 5(180):180ra48–ra48. doi: 10.1126/ scitranslmed.3005109 138. Lomnytska M, Pinto R, Becker S, Engström U, Gustafsson S, Björklund C, et al. Platelet Protein Biomarker Panel for Ovarian Cancer Diagnosis. Biomark Res (2018) 6(1):1–14. doi: 10.1186/s40364-018-0118-y 139. Chen R, Xu X, Qian Z, Zhang C, Niu Y, Wang Z, et al. The Biological Functions and Clinical Applications of Exosomes in Lung Cancer. Cell Mol Life Sci (2019) 76(23):4613–33. doi: 10.1007/s00018-019-03233-y 140. Page C, Pitchford S. Platelets and Allergic Inﬂammation. Clin Exp Allergy (2014) 44(7):901–13. doi: 10.1111/cea.12322 141. Wojtukiewicz MZ, Sierko E, Hempel D, Tucker SC, Honn KV. Platelets and Cancer Angiogenesis Nexus. Cancer Metastasis Rev (2017) 36(2):249–62. doi: 10.1007/s10555-017-9673-1 142. Heeke S, Mograbi B, Alix-Panabières C, Hofman P. Never Travel Alone: The Crosstalk of Circulating Tumor Cells and the Blood Microenvironment. Cells (2019) 8(7):714. doi: 10.3390/cells8070714 143. Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y, et al. Clinical Features of Patients Infected With 2019 Novel Coronavirus in Wuhan, China. Lancet (2020) 395(10223):497–506. doi: 10.1016/S0140-6736(20)30183-5 144. Suzuki A, Takahashi T, Nakamura K, Tsuyuoka R, Okuno Y, Enomoto T, et al. Thrombocytosis in Patients With Tumors Producing Colony-Stimulating Frontiers in Oncology \| www.frontiersin.org 145. 146. 147. 148. 149. 150. 151. 152. 153. 154. 155. 156. 157. 158. 159. 160. Factor. Blood (1992) 80(8):2052–9. doi: 10.1182/blood.V80.8.2052. bloodjournal8082052 Sadeghi Rad H, Monkman J, Warkiani ME, Ladwa R, O’Byrne K, Rezaei N, et al. Understanding the Tumor Microenvironment for Effective Immunotherapy. Med Res Rev (2021) 41(3):1474–98. doi: 10.1002/ med.21765 Kalita-de Croft P, Sadeghi Rad H, Gasper H, O’Byrne K, Lakhani SR, Kulasinghe A. Spatial Proﬁling Technologies and Applications for Brain Cancers. Expert Rev Mol Diagn (2021) 21(3):323–32. doi: 10.1080/ 14737159.2021.1900735 Li D, Yang W, Zhang Y, Yang JY, Guan R, Xu D, et al. Genomic Analyses Based on Pulmonary Adenocarcinoma in Situ Reveal Early Lung Cancer Signature. BMC Med Genomics (2018) 11(5):89–97. doi: 10.1186/s12920018-0413-3 Liefaard M, Lips E, Best M, Sol N, Rookus M, Sonke G, et al. RNA Signatures From Tumor-Educated Platelets (TEP) Enable Detection of Early-Stage Breast Cancer. Ann Oncol (2019) 30:iii13. doi: 10.1093/annonc/mdz095.036 Xue L, Xie L, Song X, Song X. Identiﬁcation of Potential Tumor-Educated Platelets RNA Biomarkers in non-Small-Cell Lung Cancer by Integrated Bioinformatical Analysis. J Clin Lab Anal (2018) 32(7):e22450. doi: 10.1002/ jcla.22450 Sheng M, Dong Z, Xie Y. Identiﬁcation of Tumor-Educated Platelet Biomarkers of non-Small-Cell Lung Cancer. OncoTargets Ther (2018) 11:8143. doi: 10.2147/OTT.S177384 Sabrkhany S, Kuijpers MJ, van Kuijk SM, Sanders L, Pineda S, Damink SWO, et al. A Combination of Platelet Features Allows Detection of Early-Stage Cancer. Eur J Cancer (2017) 80:5–13. doi: 10.1016/j.ejca.2017.04.010 Plantureux L, Mège D, Crescence L, Dignat-George F, Dubois C, PanicotDubois L. Impacts of Cancer on Platelet Production, Activation and Education and Mechanisms of Cancer-Associated Thrombosis. Cancers (2018) 10(11):441. doi: 10.3390/cancers10110441 Best M, Sol N, Kooi I, Tannous B, Wesseling P. RNA-Seq of TumorEducated Platelets Enables Article RNA-Seq of Tumor-Educated Platelets Enables. Cancer Cell (2015) 28:666–76. doi: 10.1016/j.ccell.2015.09.018 Calverley DC, Phang TL, Choudhury QG, Gao B, Oton AB, Weyant MJ, et al. Signiﬁcant Downregulation of Platelet Gene Expression in Metastatic Lung Cancer. Clin Trans Sci (2010) 3(5):227–32. doi: 10.1111/j.17528062.2010.00226.x Rodig SJ, Mino-Kenudson M, Dacic S, Yeap BY, Shaw A, Barletta JA, et al. Unique Clinicopathologic Features Characterize ALK-Rearranged Lung Adenocarcinoma in the Western Population. Clin Cancer Res (2009) 15 (16):5216–23. doi: 10.1158/1078-0432.CCR-09-0802 Dacic S. Molecular Genetic Testing for Lung Adenocarcinomas: A Practical Approach to Clinically Relevant Mutations and Translocations. J Clin Pathol (2013) 66(10):870–4. doi: 10.1136/jclinpath-2012-201336 Nilsson RJA, Karachaliou N, Berenguer J, Gimenez-Capitan A, Schellen P, Teixido C, et al. Rearranged EML4-ALK Fusion Transcripts Sequester in Circulating Blood Platelets and Enable Blood-Based Crizotinib Response Monitoring in non-Small-Cell Lung Cancer. Oncotarget (2016) 7(1):1066. doi: 10.18632/oncotarget.6279 Sol N, Wurdinger T. Platelet RNA Signatures for the Detection of Cancer. Cancer Metastasis Rev (2017) 36(2):263–72. doi: 10.1007/s10555-017-9674-0 Xing S, Zeng T, Xue N, He Y, Y-z L, Li H-l, et al. Development and Validation of Tumor-Educated Blood Platelets Integrin Alpha 2b (ITGA2B) RNA for Diagnosis and Prognosis of Non-Small-Cell Lung Cancer Through RNA-Seq. Int J Biol Sci (2019) 15(9):1977. doi: 10.7150/ijbs.36284 Søreide K, Primavesi F, Labori KJ, Watson MM, Stättner S. Molecular Biology in Pancreatic Ductal Adenocarcinoma: Implications for Future Diagnostics and Therapy. Eur Surg (2019) 51(3):126–34. doi: 10.1007/ s10353-019-0575-z 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. 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 12 January 2022 \| Volume 11 \| Article 801269 Herath et al. Circulating Biomarkers in Lung Cancer this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Copyright © 2022 Herath, Sadeghi Rad, Radfar, Ladwa, Warkiani, O’Byrne and Kulasinghe. This is an open-access article distributed under the terms of the Creative Frontiers in Oncology \| www.frontiersin.org 13 January 2022 \| Volume 11 \| Article 801269
https://openalex.org/W866462402	https://pressto.amu.edu.pl/index.php/rpeis/article/download/15901/15681	Polish	null	„Interes publiczny’’, ,,interes społeczny’’ i ,,interes społecznie uzasadniony’’. Pro´ba dookres´- lenia poje˛c´	Ruch Prawniczy, Ekonomiczny i Socjologiczny	2,018	cc-by	7,845	1 Nazwa ,,klauzula generalna’’ pochodzi od J. W. Hedemanna (z pracy Die Flucht in die General- klauseln. Eine Gefahr für Recht und Staat, Tübingen 1933. Podaje˛ za: A. Szpunar, Naduz˙ycie prawa podmiotowego, Krako´w 1947, s. 63, przyp. 1). Klauzule generalne sa˛ konstrukcja˛ prawna˛ dos´c´ kontrowersyjna˛; spory dotycza˛juz˙ samej ich definicji, nie mo´wia˛c o kwestiach bardziej szczego´łowych. Ich geneza wia˛z˙e sie˛ z szeroko rozumiana˛ cywilistyka˛, choc´ obecnie ro´wniez˙ i w prawie publicznym sa˛ nierzadkie. Chodzi jednak nie o to, gdzie klauzule sa˛stosowane, ale jak sa˛stosowane. Praktyka pokazuje, z˙e – jako zwroty niedookres´lone – dostarczaja˛ one wielu problemo´w interpretacyjnych, dlatego pojawiaja˛ sie˛ pogla˛dy, z˙e stosowanie klauzul generalnych jest w pewnym sensie kle˛ska˛ ustawodawcy, kto´ry przez ich nadmierne wprowadzanie do systemu prawnego sam pozbawia sie˛ kontroli nad prawidłowos´cia˛ procesu stosowania prawa (zob. M. Wyrzykowski, Poje˛cie interesu społecznego w prawie administra- cyjnym, Warszawa 1986, s. 199). Postuluje sie˛ wie˛c niekiedy zasta˛pienie tych zwroto´w jednoznacznie brzmia˛cymi przepisami prawnymi, co oczywis´cie nie jest moz˙liwe ani poz˙a˛dane, maja˛ one bowiem sprawic´, by prawo było bardziej elastyczne, a dzie˛ki temu lepiej dostosowywało sie˛ do zmieniaja˛cej sie˛ rzeczywistos´ci (zob. tez˙: M. Zielin´ ski, Wykładnia prawa. Zasady. Reguły. Wskazo´wki, Warszawa 2002, s. 163-171). 5 Zob. E. Modlin´ ski, Poje˛cie interesu publicznego w prawie administracyjnem, Warszawa 1932, s. 3 i n. Por. M. Wyrzykowski, op. cit., s. 9. 3 Zob. art. 1 ustawy z 16 lutego 2007 r. o ochronie konkurencji i konsumento´w (Dz. U. 2007, Nr oz. 331 ze zm.). 4 2 Zob. art. 17 ust. 1, 22, 63, 213 ust. 1. 4 Zob. np. art. 21 § 2 Kodeksu poste˛powania karnego. RUCH PRAWNICZY, EKONOMICZNY I SOCJOLOGICZNY Rok LXXV – zeszyt 2 – 2013 RUCH PRAWNICZY, EKONOMICZNY I SOCJOLOGICZNY Rok LXXV – zeszyt 2 – 2013 ur Zurawik ,,INTERES PUBLICZNY’’, ,,INTERES SPOŁECZNY’’ I ,,INTERES SPOŁECZNIE UZASADNIONY’’ PRO´ BA DOOKRES´LENIA POJE˛C´ Jedna˛ z najwaz˙niejszych klauzul generalnych 1 w prawodawstwie polskim jest klauzula interesu publicznego, ma bowiem charakter konstytucyjny 2, choc´ i ustawodawca zwykły cze˛sto do niej sie˛ga. Czasem klauzula ta stanowi podstawe˛ ograniczania praw i wolnos´ci, a czasem jest przesłanka˛ wdraz˙ania mechanizmo´w słuz˙a˛cych ochronie wybranych wartos´ci3. Ma wie˛c zro´z˙nicowany charakter. Zwia˛zana jest przede wszystkim z prawem publicznym: gło´wnie administracyjnym, w mniejszym stopniu karnym procesowym 4. Zasadniczo jest ona dyrektywa˛ kierowana˛ do organo´w publicznych, kto´re swoje zadania wypeł- niaja˛, a przynajmniej powinny, w interesie publicznym. Ta kluczowa dla prawa publicznego klauzula była i jest przedmiotem refleksji i bardzo skrajnych ocen – uznaje sie˛ ja˛ za kategorie˛ normatywna˛ ba˛dz´ traktuje jako kategorie˛ polityczna˛, socjologiczna˛, czy wszelka˛ inna˛, byle nie normatywna˛, jako niedaja˛ca˛ sie˛ prawnie kategoryzowac´ 5. Zwia˛zki pomie˛dzy prawem a z˙yciem społecznym sa˛ szerokie, to zas´ ma swe odzwierciedlenie w refleksji teoretycznoprawnej innych nauk społecznych. Prawo, w szczego´lnos´ci publiczne, jest wynikiem realizacji okres´lonej polityki, ale ro´wniez˙ nos´nikiem i odzwierciedleniem waz˙nych społecznie wartos´ci. To zas´ wywołuje potrzebe˛ analizy i odkrywania normatywnej zawartos´ci przywołanej © Wydział Prawa i Administracji UAM, Poznań 2013 58 Artur Z˙urawik klauzuli oraz klauzul podobnych. Do tych ostatnich nalez˙a˛ m.in. klauzule ,,interes społeczny’’ oraz ,,interes społecznie uzasadniony’’. Co wie˛cej, bardzo cze˛sto klauzule˛ interesu publicznego wre˛cz utoz˙samia sie˛ z klauzula˛ interesu społecznego i pogla˛dy dotycza˛ce jednej z nich automatycznie odnosi do drugiej6. Na tej samej zasadzie czasem traktuje sie˛ tak samo klauzule˛ interesu społecznego oraz interesu społecznie uzasadnionego. klauzuli oraz klauzul podobnych. Do tych ostatnich nalez˙a˛ m.in. klauzule ,,interes społeczny’’ oraz ,,interes społecznie uzasadniony’’. Co wie˛cej, bardzo cze˛sto klauzule˛ interesu publicznego wre˛cz utoz˙samia sie˛ z klauzula˛ interesu społecznego i pogla˛dy dotycza˛ce jednej z nich automatycznie odnosi do drugiej6. Na tej samej zasadzie czasem traktuje sie˛ tak samo klauzule˛ interesu społecznego oraz interesu społecznie uzasadnionego. W tym konteks´cie warto zastanowic´ sie˛, czy klauzule te rzeczywis´cie znacza˛ to samo, czy moz˙e maja˛ jednak ro´z˙norodny charakter7. Jes´li traktowac´ je normatywnie, a nie jak słowa bez pokrycia, to pro´ba dookres´lenia ich tres´ci wydaje sie˛ oczywista. M. Wyrzykowski, pisza˛c o klauzuli interesu społecznego, nota bene stosował to poje˛cie zamiennie z klauzula˛ interesu publicznego, stwierdził: ,,Wzgle˛dny charakter tres´ci interesu publicznego nie oznacza jej dowolnos´ci. Dlatego wszelkie pro´by, nawet te, kto´re zmierzaja˛ do okres´lenia minimum pewnos´ci znaczenia poje˛cia, musza˛ byc´ powitane z uznaniem. Sa˛ to bowiem pro´by okres´lenia nienaruszalnych, w danych warunkach społecznych i ustrojowych, granic interesu publicznego jako dobra wspo´lnego’’ 8. 6 Np. A. Duda stwierdził: ,,W obecnej sytuacji polityczno-ustrojowej – tj. w pan´ stwie prawnym – poje˛cia »interesu społecznego« i »interesu publicznego« sa˛ znaczeniowo toz˙same i nie sa˛ ro´wnoznaczne z poje˛ciem »interesu pan´ stwa«’’ (A. Duda, Interes prawny w polskim prawie administracyjnym, Warszawa 2008, s. 24). ur Zurawik ,,INTERES PUBLICZNY’’, ,,INTERES SPOŁECZNY’’ I ,,INTERES SPOŁECZNIE UZASADNIONY’’ PRO´ BA DOOKRES´LENIA POJE˛C´ Słowa te nich be˛da˛ mys´la˛ przewodnia˛ niniejszego opracowania9. Dalsze rozwaz˙ania poprzedze˛ kro´tkimi uwagami dotycza˛cymi pojmowania terminu ,,interes’’, kto´ry to termin stanowi podstawe˛ wszystkich analizowanych klauzul. 6 Np. A. Duda stwierdził: ,,W obecnej sytuacji polityczno-ustrojowej – tj. w pan´ stwie prawnym – poje˛cia »interesu społecznego« i »interesu publicznego« sa˛ znaczeniowo toz˙same i nie sa˛ ro´wnoznaczne z poje˛ciem »interesu pan´ stwa«’’ (A. Duda, Interes prawny w polskim prawie administracyjnym, Warszawa 2008, s. 24). y y , p , 9 Dodam, z˙e konstrukcje˛ klauzuli generalnej pojmuje˛ podobnie jak L. Leszczyn´ ski. Autor ten swoja˛ koncepcje˛ wywodzi z poje˛cia odesłania i wyro´z˙nia generalne klauzule odsyłaja˛ce od klauzul generalnych potocznie rozumianych, kto´re nie odsyłaja˛ do kryterio´w pozaprawnych. Odesłaniem jest wg niego ,,[...] be˛da˛ce wyraz´na˛ konstrukcja˛ prawodawcza˛ (elementem przepisu prawnego) i sformułowane w je˛zyku prawnym, upowaz˙nienie dla organu stosuja˛cego prawo do wyznaczenia zachowania adresata decyzji lub ustalenia podstaw kwalifikacji zachowan´ adresata normy, na podstawie kryterio´w (norm, ocen) wyraz˙o- nych (nazwanych) w teks´cie prawnym, ale nieinkorporowanych (z ro´z˙nych powodo´w) do systemu przepiso´w prawnych’’. Klauzula generalna odsyłaja˛ca – jego zdaniem – odpowiada wszystkim elementom odesłania, z tym z˙e kryteria i oceny pozaprawne, do kto´rych prawodawca odwołuje sie˛ przez klauzule generalne, maja˛ charakter ukierunkowany i ,,systemowy’’. Omawiane klauzule maja˛ niewa˛tpliwie charakter klauzul odsyłaja˛cych (L. Leszczyn´ ski, Tworzenie generalnych klauzul odsyłaja˛cych, Lublin 2000, s. 17 i 23). W pis´miennictwie wyro´z˙nia sie˛ takz˙e zwroty ocenne, kto´re nie sa˛ toz˙same z klauzulami odsyłaja˛cymi. Zwrot ,,ocenny’’ jest bowiem normatywnym wyraz˙eniem złoz˙onym, zawieraja˛cym nazwe˛ abstrakcyjnie uje˛tych fakto´w oraz wskazanie rodzaju wypowiedzi oceniaja˛cej, upowaz˙niaja˛cej do oszacowania (swoistej kwalifikacji) skali wysta˛pienia tych fakto´w. W odro´z˙nieniu od generalnej klauzuli odsyłaja˛cej nie ma tu odesłania do ,,systemu’’ wartos´ci czy norm, lecz do jednostkowej czynnos´ci szaco- wania (oceniania poro´wnawczego). Typowe zwroty ocenne to np. ,,waz˙ne powody’’, ,,szczego´lne okolicz- nos´ci’’ czy ,,waz˙ny interes’’. Pierwszy składnik omawianych wyraz˙en´ wskazuje na szacowanie, a drugi – na przedmiot szacowania, czyli na okres´lone fakty, kto´re musza˛ zostac´ ustalone (L. Leszczyn´ ski, Wykładania generalnych klauzul odsyłaja˛cych prawa administracyjnego, w: System prawa administra- cyjnego, t. 4: Wykładnia w prawie administracyjnym, red. R. Hauser, Z. Niewiadomski, A. Wro´bel, Warszawa 2012, s. 340). Zob. tez˙ szerzej o klauzulach generalnych np.: L. Leszczyn´ ski, Stosowanie generalnych klauzul odsyłaja˛cych, Krako´w 2001, s. 21 i n.; Z. Ziembin´ ski, Stan dyskusji nad pro- blematyka˛ klauzul generalnych, ,,Pan´ stwo i Prawo’’ 1989, z. 3, s. 14 i n.; K. Wo´jcik, Teoretyczna konstrukcja klauzuli generalnej, ,,Studia Prawno-Ekonomiczne’’, t. 44, 1990, s. 7 Szerzej zob. A. Z˙urawik, Interes publiczny w prawie gospodarczym, Warszawa 2013, passim. 8 M. Wyrzykowski, op. cit., s. 47. 8 M. Wyrzykowski, op. cit., s. 47. 6 Np. A. Duda stwierdził: ,,W obecnej sytuacji polityczno-ustrojowej – tj. w pan´ stwie prawnym – poje˛cia »interesu społecznego« i »interesu publicznego« sa˛ znaczeniowo toz˙same i nie sa˛ ro´wnoznaczne z poje˛ciem »interesu pan´ stwa«’’ (A. Duda, Interes prawny w polskim prawie administracyjnym, Warszawa 2008, s. 24). 7 Szerzej zob. A. Z˙urawik, Interes publiczny w prawie gospodarczym, Warszawa 2013, passim. 8 M. Wyrzykowski, op. cit., s. 47. 9 Dodam, z˙e konstrukcje˛ klauzuli generalnej pojmuje˛ podobnie jak L. Leszczyn´ ski. Autor ten swoja˛ koncepcje˛ wywodzi z poje˛cia odesłania i wyro´z˙nia generalne klauzule odsyłaja˛ce od klauzul generalnych potocznie rozumianych, kto´re nie odsyłaja˛ do kryterio´w pozaprawnych. Odesłaniem jest wg niego ,,[...] be˛da˛ce wyraz´na˛ konstrukcja˛ prawodawcza˛ (elementem przepisu prawnego) i sformułowane w je˛zyku prawnym, upowaz˙nienie dla organu stosuja˛cego prawo do wyznaczenia zachowania adresata decyzji lub ustalenia podstaw kwalifikacji zachowan´ adresata normy, na podstawie kryterio´w (norm, ocen) wyraz˙o- nych (nazwanych) w teks´cie prawnym, ale nieinkorporowanych (z ro´z˙nych powodo´w) do systemu przepiso´w prawnych’’. Klauzula generalna odsyłaja˛ca – jego zdaniem – odpowiada wszystkim elementom odesłania, z tym z˙e kryteria i oceny pozaprawne, do kto´rych prawodawca odwołuje sie˛ przez klauzule generalne, maja˛ charakter ukierunkowany i ,,systemowy’’. Omawiane klauzule maja˛ niewa˛tpliwie charakter klauzul odsyłaja˛cych (L. Leszczyn´ ski, Tworzenie generalnych klauzul odsyłaja˛cych, Lublin 2000, s. 17 i 23). W pis´miennictwie wyro´z˙nia sie˛ takz˙e zwroty ocenne, kto´re nie sa˛ toz˙same z klauzulami odsyłaja˛cymi. Zwrot ,,ocenny’’ jest bowiem normatywnym wyraz˙eniem złoz˙onym, zawieraja˛cym nazwe˛ abstrakcyjnie uje˛tych fakto´w oraz wskazanie rodzaju wypowiedzi oceniaja˛cej, upowaz˙niaja˛cej do oszacowania (swoistej kwalifikacji) skali wysta˛pienia tych fakto´w. W odro´z˙nieniu od generalnej klauzuli odsyłaja˛cej nie ma tu odesłania do ,,systemu’’ wartos´ci czy norm, lecz do jednostkowej czynnos´ci szaco- wania (oceniania poro´wnawczego). Typowe zwroty ocenne to np. ,,waz˙ne powody’’, ,,szczego´lne okolicz- nos´ci’’ czy ,,waz˙ny interes’’. Pierwszy składnik omawianych wyraz˙en´ wskazuje na szacowanie, a drugi – na przedmiot szacowania, czyli na okres´lone fakty, kto´re musza˛ zostac´ ustalone (L. Leszczyn´ ski, Wykładania generalnych klauzul odsyłaja˛cych prawa administracyjnego, w: System prawa administra- cyjnego, t. 4: Wykładnia w prawie administracyjnym, red. R. Hauser, Z. Niewiadomski, A. Wro´bel, Warszawa 2012, s. 340). Zob. tez˙ szerzej o klauzulach generalnych np.: L. Leszczyn´ ski, Stosowanie generalnych klauzul odsyłaja˛cych, Krako´w 2001, s. 21 i n.; Z. Ziembin´ ski, Stan dyskusji nad pro- blematyka˛ klauzul generalnych, ,,Pan´ stwo i Prawo’’ 1989, z. 3, s. 14 i n.; K. Wo´jcik, Teoretyczna konstrukcja klauzuli generalnej, ,,Studia Prawno-Ekonomiczne’’, t. 44, 1990, s. 47 i n.; Z. Radwan´ ski, M. Zielin´ ski, Uwagi de lege ferenda o klauzulach generalnych w prawie prywatnym, ,,Przegla˛d Legislacyjny’’ 2001, nr 2, s. 11 i n. oraz podana w ww. pozycjach literatura. ur Zurawik ,,INTERES PUBLICZNY’’, ,,INTERES SPOŁECZNY’’ I ,,INTERES SPOŁECZNIE UZASADNIONY’’ PRO´ BA DOOKRES´LENIA POJE˛C´ 47 i n.; Z. Radwan´ ski, M. Zielin´ ski, Uwagi de lege ferenda o klauzulach generalnych w prawie prywatnym, ,,Przegla˛d Legislacyjny’’ 2001, nr 2, s. 11 i n. oraz podana w ww. pozycjach literatura. ,,Interes publiczny’’, ,,interes społeczny’’ i ,,interes społecznie uzasadniony’’ 59 10 W. Wołodkiewicz (red.), Prawo rzymskie. Słownik encyklopedyczny, Warszawa 1986, s. 77; J. Dra˛z˙kiewicz, Interesy a struktura społeczna, Warszawa 1982, s. 5. 11 Zob. np. J. Dra˛z˙kiewicz, Interesy a struktura społeczna, Warszawa 1982, s. 5 i n.; P. J. Suwaj, Konflikt intereso´w w administracji publicznej, Warszawa 2009, s. 19 i n.; A. S. Duda, op. cit., s. 1 i n. 12 V. Van Dyke, Values and Interests, ,,The American Political Science Review’’ 3, 1962, s. 567 i n. 13 Por. R. Flathman, The Public Interest. An Essay Concerning the Normative Discourse of Politics, New York-London-Sydney 1966, s. 15-16; J. Dra˛z˙kiewicz, op. cit., s. 20 i n.; V. Held, The Public Interest and Individual Interests, New York-London 1970, np. s. 51. 10 W. Wołodkiewicz (red.), Prawo rzymskie. Słownik encyklopedyczny, Warszawa 1986, s. 77; J. Dra˛z˙kiewicz, Interesy a struktura społeczna, Warszawa 1982, s. 5. 11 Zob. np. J. Dra˛z˙kiewicz, Interesy a struktura społeczna, Warszawa 1982, s. 5 i n.; P. J. Suwaj, Konflikt intereso´w w administracji publicznej, Warszawa 2009, s. 19 i n.; A. S. Duda, op. cit., s. 1 i n. 12 V. Van Dyke, Values and Interests, ,,The American Political Science Review’’ 3, 1962, s. 567 i n. 13 Por. R. Flathman, The Public Interest. An Essay Concerning the Normative Discourse of Politics, New York-London-Sydney 1966, s. 15-16; J. Dra˛z˙kiewicz, op. cit., s. 20 i n.; V. Held, The Public Interest and Individual Interests, New York-London 1970, np. s. 51. 14 J. Mucha, Konfliktowe modele społeczen´ stwa we wspo´łczesnej socjologii niemarksistowskiej – pro´ba typologii, ,,Studia Socjologiczne’’ 1975, nr 1; Podaje˛ za: J. Dra˛z˙kiewicz, op. cit., s. 28. 15 Zob. tez˙ A. Z˙urawik, Klauzula interesu publicznego w prawie gospodarczym krajowym i unijnym, ,,Europejski Przegla˛d Sa˛dowy’’ 2012, nr 12, s. 25. 15 Zob. tez˙ A. Z˙urawik, Klauzula interesu publicznego w prawie gospodarczym krajowym i unijnym, ,,Europejski Przegla˛d Sa˛dowy’’ 2012, nr 12, s. 25. 14 J. Mucha, Konfliktowe modele społeczen´ stwa we wspo´łczesnej socjologii niemarksistowskiej – pro´ba typologii, ,,Studia Socjologiczne’’ 1975, nr 1; Podaje˛ za: J. Dra˛z˙kiewicz, op. cit., s. 28. I. POJMOWANIE ,,INTERESU’’ Z´ro´dłosłowu poje˛cia ,,interes’’ nalez˙y doszukiwac´ sie˛ w łacin´ skim słowie interesse, kto´re oznacza ,,byc´ w czyms´’’, ,,znajdowac´ sie˛ przy czyms´’’, ,,byc´ po- mie˛dzy’’, ,,brac´ udział’’, ,,byc´ obecnym’’ 10. W doktrynie podejmowano ro´z˙norodne pro´by zdefiniowania omawianego terminu, nie ma jednak potrzeby ich tu przywoływac´ 11. Istotne be˛dzie zwro´cenie uwagi na pewne grupy pogla˛do´w, kto´re moz˙na wyodre˛bnic´. Przedstawiane w pis´miennictwie pogla˛dy na ten temat moz˙na sprowadzic´ do trzech podstawowych typo´w. Za podstawe˛ interesu uwaz˙a sie˛ bowiem, po pierwsze, pewien rodzaj w a r t o s´ c i – x jest interesem A ze wzgle˛du na wartos´c´ y. Pewien stan lub przedmiot zostaje uznany za interes danego podmiotu, poniewaz˙ jest on wartos´ciowy, korzystny, dla niego. Jes´li układem odniesienia sa˛ tu wartos´ci lub systemy wartos´ci, to taka˛ podstawe˛ interesu nazwac´ moz˙na aksjologiczna˛. Skrajne stanowisko aksjologiczne zajmował V. Van Dyke, kto´ry poje˛cia ,,interes’’ oraz ,,wartos´c´’’ traktował synonimicznie12. Po drugie, pod- stawa˛ dookres´lania interesu moga˛ byc´ p o t r z e b y l u b z b i o´ r p o t r z e b – x be˛dzie tu interesem A ze wzgle˛du na potrzebe˛ y. Cze˛s´c´ autoro´w przy tym wre˛cz utoz˙samia interes z potrzeba˛, twierdza˛c np., z˙e interesy to społecznie zde- terminowane potrzeby, a cze˛s´c´ je wyraz´nie oddziela, z tym z˙e sama potrzeba moz˙e byc´ przez nich rozumiana dwojako: albo subiektywistycznie (jako wy- raz˙one z˙yczenie, s´wiadoma skłonnos´c´, odczuwane pragnienie), albo obiektywnie (gdy potrzeba wyste˛puja˛ca w podstawie interesu jest ujmowana niezalez˙nie od wyobraz˙en´ i subiektywnych przekonan´ poszczego´lnych oso´b)13. I po trzecie, podstawa˛ interesu moz˙e byc´ tez˙ k a t e g o r i a c e l u, gdy x jest interesem podmiotu A ze wzgle˛du na cel y, stoja˛cy przed podmiotem. Zwolennikiem traktowania celo´w jako podstawy interesu jest m.in. J. Mucha. Jego zdaniem, relatywizacja interesu pewnego podmiotu w stosunku do celo´w tego podmiotu jest waz˙niejsza niz˙ relatywizacja w stosunku do ,,udziału w dobrach (warto- s´ciach)’’, poniewaz˙ zwie˛kszaja˛cy sie˛ udział podmiotu (grupy) w dobrach nie zawsze jest korzystny z punktu widzenia jego dalszych, na ogo´ł waz˙niejszych, celo´w14. Tego rodzaju podstawe˛ interesu nazywa sie˛ prakseologiczna˛15. Jak natomiast rozumie sie˛ interes publiczny? Jak natomiast rozumie sie˛ interes publiczny? 60 Artur Z˙urawik 16 M. Wyrzykowski, op. cit., s. 30-31. Zob. tez˙ A. Wilczyn´ ska, Interes publiczny w prawie stanowio- nym i orzecznictwie Trybunału Konstytucyjnego, ,,Przegla˛d Prawa Handlowego’’ 2009, czerwiec, s. 50-51. 17 M. Wyrzykowski, op. cit., s. 31. Zob. tez˙ R. Flathman, op. cit., s. 21 i przywołane tam pogla˛dy Benthama. 18 J. Nawrot, s.v. Interes publiczny, w: A. Powałowski (red.), Leksykon prawa gospodarczego publicznego, Warszawa 2009, s. 67-68. 19 Wyrok TK z 20 marca 2006 r., K 1705, OTK-A 2006, nr 3, poz. 30; wyrok SN z 18 listopada 1993 r., III ARN 4993; L. Leszczyn´ ski, Kategoria interesu w stosowaniu prawa administracyjnego. Przykład art. 7 KPA, w: A. Korybski, M. W. Kostyckij, L. Leszczyn´ ski (red.), Poje˛cie interesu w naukach prawnych, prawie stanowionym i orzecznictwie sa˛dowym Polski i Ukrainy, Lublin 2006, s. 81; M. Wyrzykowski, op. cit., passim. 18 J. Nawrot, s.v. Interes publiczny, w: A. Powałowski (red.), Leksykon prawa gospodarczego icznego, Warszawa 2009, s. 67-68. 19 Wyrok TK z 20 marca 2006 r., K 1705, OTK-A 2006, nr 3, poz. 30; wyrok SN z 18 listopada 1993 r., III ARN 4993; L. Leszczyn´ ski, Kategoria interesu w stosowaniu prawa administracyjnego. Przykład art. 7 KPA, w: A. Korybski, M. W. Kostyckij, L. Leszczyn´ ski (red.), Poje˛cie interesu w naukach prawnych, prawie stanowionym i orzecznictwie sa˛dowym Polski i Ukrainy, Lublin 2006, s. 81; M. Wyrzykowski, op. cit., passim. 16 M. Wyrzykowski, op. cit., s. 30-31. Zob. tez˙ A. Wilczyn´ ska, Interes publiczny w prawie stanowio- nym i orzecznictwie Trybunału Konstytucyjnego, ,,Przegla˛d Prawa Handlowego’’ 2009, czerwiec, s. 50-51. 17 M. Wyrzykowski, op. cit., s. 31. Zob. tez˙ R. Flathman, op. cit., s. 21 i przywołane tam pogla˛dy Benthama. 18 16 M. Wyrzykowski, op. cit., s. 30-31. Zob. tez˙ A. Wilczyn´ ska, Interes publiczny w prawie stanowio- nym i orzecznictwie Trybunału Konstytucyjnego, ,,Przegla˛d Prawa Handlowego’’ 2009, czerwiec, s. 50-51. 17 M. Wyrzykowski, op. cit., s. 31. Zob. tez˙ R. Flathman, op. cit., s. 21 i przywołane tam pogla˛dy Benthama. 18 J. Nawrot, s.v. Interes publiczny, w: A. Powałowski (red.), Leksykon prawa gospodarczego publicznego, Warszawa 2009, s. 67-68. 19 Wyrok TK z 20 marca 2006 r., K 1705, OTK-A 2006, nr 3, poz. 30; wyrok SN z 18 listopada 1993 r., III ARN 4993; L. Leszczyn´ ski, Kategoria interesu w stosowaniu prawa administracyjnego. Przykład 20 Zob. tez˙ M. Wyrzykowski, op. cit., s. 31; A. Banaszkiewicz, Cel publiczny w gospodarce nieruchomos´ciami, Acta Universitatis Wratislaviensis, Przegla˛d Prawa i Administracji LXV, Wrocław 2005, s. 90. W. Jakimowicz zauwaz˙a, z˙e w uje˛ciu indywidualistycznym interes publiczny interpretuje sie˛ z punktu widzenia interesu indywidualnego, kto´ry jest punktem wyjs´cia, a zasadnos´c´ interesu indywidualnego niekoniecznie musi korespondowac´ z interesami ogo´łu. W uje˛ciu uniwersalistycznym interes publiczny ocenia sie˛ z punktu widzenia dobra wspo´lnego, dobra ogo´łu, a działanie jednostki jest oceniane w aspekcie zgodnos´ci z interesami innych podmioto´w, zwłaszcza z interesami całego społe- czen´ stwa. ,,Charakterystyczny dla uje˛cia uniwersalistycznego był pogla˛d, wyraz˙ony w jednym z pierw- szych orzeczen´ NSA, z˙e ochrona interesu indywidualnego i jej zakres sie˛gaja˛do granic kolizji z interesem społecznym – jako wartos´cia˛ nadrze˛dna˛ w pan´ stwie socjalistycznym. W okresie PRL oparte na doktrynie komunistycznej załoz˙enie, z˙e cecha˛ pan´ stwa kapitalistycznego jest sprzecznos´c´ intereso´w ogo´lnego i indywidualnego, kto´ra nie moz˙e miec´ miejsca w pan´ stwie socjalistycznym, utwierdzało o´wczesna˛ nauke˛ w przekonaniu o słusznos´ci konstruowania jednej kategorii wspo´lnego interesu. Na tej zasadzie we wzajemnych relacjach intereso´w indywidualnych i intereso´w ogo´lnych pogla˛d o niesprzecznos´ci tych intereso´w i koniecznos´ci ustalania kaz˙dorazowo słusznego interesu w konkretnym wypadku zaste˛powano zasada˛ dominacji interesu społecznego, zgodnie z kto´ra˛ sposo´b interpretacji i realizacji prawa powinien byc´ podporza˛dkowany interesowi społecznemu, a podporza˛dkowanie to powinno wynikac´ z samej istoty prawa’’ (W. Jakimowicz, Wykładania w prawie administracyjnym, Warszawa 2006, s. 118). II. POJMOWANIE ,,INTERESU PUBLICZNEGO’’ Koncepcji interesu publicznego pojawiło sie˛ wiele. Poszczego´lni autorzy zwracali uwage˛ na ro´z˙ne jego aspekty. Pierwotne koncepcje opierały sie˛ na zestawieniu intereso´w jednostki i interesu publicznego. Interes publiczny, jako poje˛ciowo przeciwstawny interesowi indywidualnemu, przeciwstawiany był mu ro´wniez˙ w sferze normatywnej i politycznej, nie tylko je˛zykowej. Dopiero dok- tryna liberalnego pan´ stwa prawnego oraz koncepcja publicznych praw podmio- towych stworzyły podstawe˛ do analizy pozycji jednostki w pan´ stwie i charak- teru jej intereso´w. Zauwaz˙ono tez˙, z˙e w praktyce wyste˛puja˛ sytuacje nie tyle przeciwstawiania, ile ro´wnoległos´ci, a nawet ła˛czenia interesu indywidualnego i interesu publicznego 16. ,,Interes publiczny zacza˛ł sie˛ jawic´ jako kategoria skomplikowana, złoz˙ona z elemento´w uprzednio odrzucanych jako jej cze˛s´ci składowe. Straciła swoich zwolenniko´w koncepcja »matematyczna«, zakłada- ja˛ca, z˙e interes publiczny jest suma˛ intereso´w prywatnych’’ 17. W tym konteks´cie moz˙na mo´wic´ o trzech uje˛ciach zagadnienia relacji pomie˛dzy interesem publicznym a interesem jednostki. W pierwszym ze wspom- nianych modeli (tzw. teoria nadrze˛dnos´ci interesu publicznego) interes publiczny jest nadrze˛dny w stosunku do interesu jednostki. Drugi model, nazywany w literaturze przedmiotu teoria˛ wspo´lnego interesu (common in- terest), ro´wniez˙ odwołuje sie˛ do intereso´w jednostek. Od teorii nadrze˛dnos´ci interesu publicznego ro´z˙ni go to, z˙e zakłada sumowanie wszystkich intereso´w indywidualnych przy jednoczesnym uwzgle˛dnieniu intereso´w mniejszos´ci, podczas gdy tzw. teoria nadrze˛dnos´ci dopuszcza moz˙liwos´c´ eliminacji intereso´w mniejszos´ci w procesie definiowania dobra wspo´lnego. Ostatni zas´ ze wspom- nianych modeli, czyli teoria jednos´ci (unitary conception), zakłada, z˙e poje˛cie interesu publicznego opiera sie˛ na rywalizacji konkurencyjnych roszczen´ , bazuja˛cych jednak na pewnych wspo´lnych wartos´ciach uznawanych w społe- czen´ stwie i stanowia˛cych podstawe˛ rozstrzygnie˛c´ władzy publicznej18. Obecnie nie budzi wie˛kszych wa˛tpliwos´ci to, z˙e nie moz˙na arbitralnie przypisywac´ wyz˙szos´ci interesu publicznego nad indywidualnym, oba bowiem interesy musza˛ byc´ wywaz˙ane w kaz˙dej sytuacji. Czasem pierwszen´ stwo be˛dzie nalez˙ec´ do interesu publicznego, a czasem be˛dzie on musiał usta˛pic´ przed interesem indywidualnym. Na tym stanowisku stoja˛ Sa˛d Najwyz˙szy, Trybunał Konstytucyjny, a takz˙e zdecydowana wie˛kszos´c´ doktryny 19. Warto tez˙ dodac´, z˙e dyskusje na temat interesu publicznego determinowane były przez naste˛puja˛ce uje˛cia: 61 ,,Interes publiczny’’, ,,interes społeczny’’ i ,,interes społecznie uzasadniony’’ 1) z punktu widzenia dobra ogo´łu, dobra wspo´lnego, uwzgle˛dniaja˛ce przede wszystkim fakt, z˙e człowiek nie z˙yje w s´wiecie sam, lecz musi dostosowac´ sie˛ do intereso´w innych oso´b, grupy, w kto´rej z˙yje; 2) indywidualistyczne, zakładaja˛ce przewage˛ interesu indywidualnego, kłada˛ce nacisk na rozwo´j jednostki, realizacje˛ jej ambicji i osobistych celo´w20. 2) indywidualistyczne, zakładaja˛ce przewage˛ interesu indywidualnego, kłada˛ce nacisk na rozwo´j jednostki, realizacje˛ jej ambicji i osobistych celo´w20. 24 Zob. J. Nawrot, op. cit., s. 66-72. W szczego´lnos´ci chodzi o wartos´ci wymienione w art. 31 ust. 3 Konstytucji, kto´re sa˛podstawa˛interesu publicznego. Do wartos´ci w swoich koncepcjach nawia˛zuja˛takz˙e: A. Wro´bel, Interes publiczny w poste˛powaniu administracyjnym, w: Administracja publiczna u progu XXI wieku. Prace dedykowane prof. zw. dr. hab. Janowi Szreniawskiemu z okazji Jubileuszu 45-lecia pracy naukowej, Przemys´l 2000, s. 701-702; M. Szaraniec, Klauzula interesu publicznego i okres´lenia nieostre – pro´ba wyodre˛bnienia tych poje˛c´ na gruncie ustawy o działalnos´ci ubezpieczeniowej, w: B. Gnela (red.), Ubezpieczenia gospodarcze. Wybrane zagadnienia prawne, Warszawa 2011, s. 247; wyrok TK z 25 lutego 1999 r., K 2398. 25 Zob. M. Wyrzykowski, op. cit. s. 37 i przywołane tam pogla˛dy. 22 E. Bodenheimer, Prolegomena to a Theory of the Public Interest, w: C. J. Friedrich (red.), The Public Interest, New York 1962, s. 207. 23 M. Wyrzykowski, op. cit., s. 33 i podana tam literatura. 21 Zob. tez˙ M. Wyrzykowski, op. cit., s. 31. II. POJMOWANIE ,,INTERESU PUBLICZNEGO’’ Te dwa uje˛cia co do zasady pozostaja˛ – choc´ cze˛sto w zmodyfikowanej postaci – aktualne i determinuja˛ w zasadniczy sposo´b rozumienie pozycji jednostki w społeczen´ stwie i pan´ stwie21. Osobis´cie zgadzam sie˛ z pogla˛dem, z˙e elementem interesu publicznego jest ochrona jednostki i ,,[...] bez jakos´ciowego wartos´ciowania (oceny) intereso´w indywidualnych włas´ciwa determinacja dobra publicznego moz˙e sprawic´ powaz˙ne trudnos´ci’’ 22. Zagadnienie to znalazło ro´wniez˙ wyraz w słowach: ,,Powszechnie akceptowane jest stanowisko przyjmuja˛ce, z˙e konstytucyjna ogo´lna koncepcja dobra ogo´lnego czy interesu ogo´lnego nie moz˙e byc´ formuło- wana w sposo´b jednostronny, poprzez elementy tradycyjnej »racji stanu«, lecz z˙e w zakres dobra ogo´łu wła˛czona jest ochrona jednostki, z˙e nie moz˙e istniec´ dobro ogo´łu, w skład kto´rego nie wchodziłaby, jako istotny element, wolnos´c´ jednostki’’23. W pogla˛dach odnosza˛cych sie˛ do rozumienia interesu publicznego widac´ tez˙ dylemat, czy interesowi publicznemu nadawac´ podstawe˛ aksjologiczna˛, wia˛z˙a˛c go z wartos´ciami24, czy prakseologiczna˛, wia˛z˙a˛ca˛go z celami25, czy moz˙e jeszcze 62 Artur Z˙urawik inna˛, według kto´rej to potrzeby stanowic´ maja˛ jego podstawe˛ 26. Prezentowane sa˛ tez˙ stanowiska oparte na koncepcji hybrydowej, wia˛z˙a˛cej interes publiczny z ro´z˙nymi podstawami27. Sam ustawodawca nie jest zdecydowany, kto´ra˛ z koncepcji wybrac´. Czasem wia˛z˙e on interes publiczny z wartos´ciami, wre˛cz utoz˙samia obie kategorie, a innym razem opiera sie˛ na koncepcji prakseolo- gicznej i odwołuje sie˛ dodatkowo do ,,zobiektywizowanych potrzeb’’28. Skoro w definicjach legalnych znajdziemy ro´z˙ne koncepcje interesu publicznego, to błe˛dem metodologicznym byłoby, moim zdaniem, budowanie jednej doktry- nalnej koncepcji interesu publicznego, tak jak to sie˛ czasem pro´buje czynic´. Bardzo łatwo moz˙na wykazac´ istnienie przypadku wynikaja˛cego wprost z przepiso´w, kto´ry jest wyrazem innej koncepcji i nie przystaje do stworzonej definicji. Przepisy z kolei musza˛ dla prawnika stanowic´ punkt wyjs´cia. Pozostaje wie˛c najogo´lniej przyja˛c´, z˙e interes publiczny moz˙e wia˛zac´ sie˛ z okres´lonymi wartos´ciami, celami lub z potrzebami, przy czym podstawy te moga˛ byc´ i czasem sa˛ ła˛czone29. , p ; y y , p , p 28 Jedna˛ z definicji znajdziemy w ustawie z 27 marca 2003 r. o planowaniu i zagospodarowaniu przestrzennym (t.jedn.: Dz. U. 2012, Nr 647), w kto´rej ustawodawca w art. 2 pkt 4 przez interes publiczny rozumie ,,uogo´lniony cel da˛z˙en´ i działan´ , uwzgle˛dniaja˛cych zobiektywizowane potrzeby ogo´łu społeczen´ - stwa lub lokalnych społecznos´ci, zwia˛zanych z zagospodarowaniem przestrzennym’’. Odnosi sie˛ wie˛c i do celo´w, i do potrzeb. Inna˛ definicje˛ legalna˛ zawiera ustawa z 4 marca 2010 r. o s´wiadczeniu usług na terytorium Rzeczypospolitej Polskiej (Dz. U. 2010, Nr 47, poz. 278 ze zm.), kto´ra ro´wniez˙ jest stosowana na gruncie ustawy z 2 lipca 2004 r. o swobodzie działalnos´ci gospodarczej (t.jedn.: Dz. U. 2010, Nr 220, poz. 1447 ze zm.), a to na zasadzie odesłania zawartego tam w art. 5 pkt 7. Art. 2 ust. 1 pkt 7 ustawy o s´wiadczeniu usług na terytorium Rzeczypospolitej Polskiej stanowi, z˙e nadrze˛dny interes publiczny to wartos´c´ podlegaja˛ca ochronie, w szczego´lnos´ci porza˛dek publiczny, bezpieczen´ stwo publiczne, zdrowie publiczne itd. Ustawodawca w tym przypadku preferuje koncepcje˛ aksjologiczna˛, i to w skrajnej postaci, utoz˙samiaja˛c ten rodzaj interesu publicznego z wartos´ciami. Jest to wie˛c koncepcja, o kto´rej pisał V. Van Dyke. 29 Uzupełniaja˛co zob. A. Z˙urawik, op. cit., s. 24-30. 26 F. Longchamps, Osobiste s´wiadczenia wojenne, Lwo´w 1936, s. 33-34. Zob. tez˙ A. Duda, op. cit. 6-27; J. Lang, Struktura prawna skargi w prawie administracyjnym, Wrocław 1972, s. 96. 26 F. Longchamps, Osobiste s´wiadczenia wojenne, Lwo´w 1936, s. 33-34. Zob. tez˙ A. Duda, op. cit., s. 26-27; J. Lang, Struktura prawna skargi w prawie administracyjnym, Wrocław 1972, s. 96. 27 Koncepcje te preferuja˛: E. Modlin´ ski, Poje˛cie interesu publicznego w prawie administracyjnem, Warszawa 1932, passim; M. Wyrzykowski, op. cit., passim. 28 Jedna˛ z definicji znajdziemy w ustawie z 27 marca 2003 r. o planowaniu i zagospodarowaniu przestrzennym (t.jedn.: Dz. U. 2012, Nr 647), w kto´rej ustawodawca w art. 2 pkt 4 przez interes publiczny rozumie ,,uogo´lniony cel da˛z˙en´ i działan´ , uwzgle˛dniaja˛cych zobiektywizowane potrzeby ogo´łu społeczen´ - stwa lub lokalnych społecznos´ci, zwia˛zanych z zagospodarowaniem przestrzennym’’. Odnosi sie˛ wie˛c i do celo´w, i do potrzeb. Inna˛ definicje˛ legalna˛ zawiera ustawa z 4 marca 2010 r. o s´wiadczeniu usług na terytorium Rzeczypospolitej Polskiej (Dz. U. 2010, Nr 47, poz. 278 ze zm.), kto´ra ro´wniez˙ jest stosowana na gruncie ustawy z 2 lipca 2004 r. o swobodzie działalnos´ci gospodarczej (t.jedn.: Dz. U. 2010, Nr 220, poz. 1447 ze zm.), a to na zasadzie odesłania zawartego tam w art. 5 pkt 7. Art. 2 ust. 1 pkt 7 ustawy o s´wiadczeniu usług na terytorium Rzeczypospolitej Polskiej stanowi, z˙e nadrze˛dny interes publiczny to wartos´c´ podlegaja˛ca ochronie, w szczego´lnos´ci porza˛dek publiczny, bezpieczen´ stwo publiczne, zdrowie publiczne itd. Ustawodawca w tym przypadku preferuje koncepcje˛ aksjologiczna˛, i to w skrajnej postaci, utoz˙samiaja˛c ten rodzaj interesu publicznego z wartos´ciami. Jest to wie˛c koncepcja, o kto´rej pisał V. Van Dyke. 29 Uzupełniaja˛co zob. A. Z˙urawik, op. cit., s. 24-30. 27 Koncepcje te preferuja˛: E. Modlin´ ski, Poje˛cie interesu publicznego w prawie administracyjnem szawa 1932, passim; M. Wyrzykowski, op. cit., passim. 26 F. Longchamps, Osobiste s´wiadczenia wojenne, Lwo´w 1936, s. 33-34. Zob. tez˙ A. Duda, op. cit., s. 26-27; J. Lang, Struktura prawna skargi w prawie administracyjnym, Wrocław 1972, s. 96. 27 Koncepcje te preferuja˛: E. Modlin´ ski, Poje˛cie interesu publicznego w prawie administracyjnem, III. ,,INTERES PUBLICZNY’’ A ,,INTERES SPOŁECZNY’’ Omo´wiona wyz˙ej klauzula interesu publicznego jest konstrukcja˛ prawna˛ maja˛ca˛bogatsza˛ historie˛ niz˙ klauzula interesu społecznego, jednakz˙e w okresie dominacji doktryny socjalistycznej ta ostatnia zyskała wie˛ksze znaczenie. Według M. Wyrzykowskiego w pan´ stwie burz˙uazyjnym, jako ,,trzecia siła’’, obok pan´ stwa i jednostki działaja˛cej w swych prywatnych sprawach, funkcjo- nował samorza˛d realizuja˛cy zadania publiczne i dlatego termin ,,publiczny’’ był rozumiany szerzej aniz˙eli ,,pan´ stwowy’’. Poniewaz˙ samorza˛d ten w okresie socjalizmu zlikwidowano jako element struktury pan´ stwa realizuja˛cy zadania pan´ stwowe i przekazano dotychczasowe zadania samorza˛du organom władzy i administracji pan´ stwowej, pojawiła sie˛ wie˛c koniecznos´c´ zmiany nazwy klauzuli interesu publicznego. Wychodza˛c z ideologiczno-ustrojowego załoz˙enia uspołecznienia pan´ stwa, a przede wszystkim uspołecznienia administracji ,,Interes publiczny’’, ,,interes społeczny’’ i ,,interes społecznie uzasadniony’’ 63 pan´ stwowej, przyje˛to poje˛cie ,,interesu społecznego’’; zakładano, z˙e pan´ stwo jest dobrem społecznym, a wie˛c jest ,,uspołecznione’’ w wyniku udziału obywateli w sprawowaniu władzy30. W pis´miennictwie po 1989 r. ro´z˙nie podchodzono do obu klauzul. A. Duda pisał: ,,W obecnej sytuacji polityczno-ustrojowej – tj. w pan´ stwie prawnym – poje˛cia »interesu społecznego« i »interesu publicznego« sa˛znaczeniowo toz˙same i nie sa˛ ro´wnoznaczne z poje˛ciem »interesu pan´ stwa«’’31. W podobnym duchu, utoz˙samiaja˛c oba interesy, wypowiada sie˛ J. Zimmer- mann. Odnosza˛c sie˛ do art. 7 k.p.a., pisał, z˙e ,,[...] organy administracji publicznej w swojej działalnos´ci maja˛ miec´ na wzgle˛dzie interes społeczny (jest to okres´lenie interesu publicznego uz˙ywane w dawniejszym ustawodawstwie PRL – dzis´ nie ma przeszko´d, z˙eby utoz˙samiac´ te poje˛cia i posługiwac´ sie˛ poje˛ciem interesu publicznego) i (słuszny) interes obywateli (interes indywi- dualny)’’ 32. L. Leszczyn´ ski zauwaz˙a natomiast, z˙e wyła˛czaja˛c pewne interesy zinsty- tucjonalizowane, zwia˛zane z interesem pan´ stwa jako organizacji, wyraz˙ane pełniej przez kryteria interesu publicznego, moz˙na stwierdzic´, z˙e co do is- toty tres´c´ obu intereso´w moz˙e byc´ zbiez˙na, zwłaszcza w demokratycznych systemach politycznych, w kto´rych rozbiez˙nos´c´ interesu publicznego ze spo- łecznym byłaby trudno uzasadniana. Na tej podstawie uznaje, z˙e kryterium interesu publicznego stanowi pewien aspekt interesu społecznego, rozumianego w wymiarze ogo´lnospołecznym, i z˙e praktyka administracyjna w ten sposo´b ujmuje jego istote˛ 33. Problem omawianej relacji rozstrzygany był zatem ro´z˙norodnie. Uwaz˙am, z˙e moz˙na odro´z˙niac´ obie klauzule, maja˛c na uwadze moz˙liwos´c´ odniesienia cze˛s´ci intereso´w bezpos´rednio do pewnych podmioto´w publicznych, w tym np. jedno- stek samorza˛du terytorialnego, kiedy to interes samego społeczen´ stwa, w tym przypadku lokalnego, realizowany jest jedynie pos´rednio. Tego rodzaju pod- mioty publiczne maja˛ samodzielnos´c´ wynikaja˛ca˛ z przyznanej im osobowos´ci prawnej, co dotyczy np. Skarbu Pan´ stwa, gmin, powiato´w czy wojewo´dztw. 33 L. Leszczyn´ ski, Wykładania generalnych klauzul odsyłaja˛cych prawa administracyjnego, w: System prawa administracyjnego, t. 4, s. 319-320; zob. tez˙. L. Leszczyn´ ski, Kategoria interesu w sto- sowaniu prawa administracyjnego..., s. 81. 32 J. Zimmermann, Prawo administracyjne, Warszawa 2012, s. 276. Zob. tez˙ J. Wyporska- -Frankiewicz, Publicznoprawne formy działania administracji o charakterze dwustronnym, Warszawa 2009, s. 28; W. Sokolewicz, Komentarz do art. 213 Konstytucji, w: L. Garlicki, Konstytucja Rzeczy- pospolitej Polskiej. Komentarz, t. 3, Warszawa 2003, s. 14-15. 30 M. Wyrzykowski, op. cit., s. 26. 31 A. Duda, op. cit., s. 24. 32 J. Zimmermann, Prawo administracyjne, Warszawa 2012, s. 276. Zob. tez˙ J. Wyporska- -Frankiewicz, Publicznoprawne formy działania administracji o charakterze dwustronnym, Warszawa 2009, s. 28; W. Sokolewicz, Komentarz do art. 213 Konstytucji, w: L. Garlicki, Konstytucja Rzeczy- pospolitej Polskiej. Komentarz, t. 3, Warszawa 2003, s. 14-15. 33 L. Leszczyn´ ski, Wykładania generalnych klauzul odsyłaja˛cych prawa administracyjnego, w: System prawa administracyjnego, t. 4, s. 319-320; zob. tez˙. L. Leszczyn´ ski, Kategoria interesu w sto- sowaniu prawa administracyjnego..., s. 81. 31 A. Duda, op. cit., s. 24. 30 M. Wyrzykowski, op. cit., s. 26. 34 Zob. np. wyroki TK z 25 listopada 2003 r., K 3702; z 30 czerwca 2003 r., K 802; z 4 listopada 2003 r., K 103; A. Wilczyn´ ska, op. cit., s. 48-55. 35 Dz. U. 2010, Nr 234, poz. 1536 ze zm. 36 Zob. np. J. Raglewski, Komentarz do art. 212 Kodeksu karnego, w: A. Zoll (red.), Kodeks karny. Cze˛s´c´ szczego´lna. Komentarz, t. 2, Krako´w 2006. III. ,,INTERES PUBLICZNY’’ A ,,INTERES SPOŁECZNY’’ Prawnie stanowia˛ odre˛bne byty, a co za tym idzie, realizuja˛ tez˙ swoje interesy, kto´re oczywis´cie co do zasady powinny byc´ zgodne z interesami poszczego´lnych społecznos´ci. Nie zawsze jednak tak jest, skoro media niekiedy donosza˛, z˙e jakas´ społecznos´c´ lokalna sprzeciwia sie˛ władzom i podejmuje kroki prawne, by je odwołac´. Zreszta˛ najlepsza˛ weryfikacja˛ sa˛ kolejne wybory, w kto´rych to dotychczasowe władze moga˛ nie uzyskac´ mandatu na dalsze sprawowanie władzy. To z kolei oznacza, z˙e ich postrzeganie interesu społecznego musiało znacznie ro´z˙nic´ sie˛ od postrzegania go przez samo społeczen´ stwo. 64 Artur Z˙urawik Co wie˛cej, tak rozumiane interesy publiczne moga˛ nawet mie˛dzy soba˛ kolidowac´, np. interesy dwo´ch ro´z˙nych gmin ba˛dz´ interesy Skarbu Pan´ stwa z interesami jednostki samorza˛du terytorialnego34. Skoro wymienione samo- dzielne podmioty sa˛ bez wa˛tpienia podmiotami prawa publicznego, to tym samym realizuja˛ interesy ,,publiczne’’, a interesy społecznos´ci (,,społeczne’’) sa˛ tu realizowane jedynie w sposo´b pos´redni. Rozro´z˙nianie obu intereso´w moz˙na tez˙ uzasadnic´ faktem, z˙e ustawodawca czasem posługuje sie˛ obydwoma klauzulami w ramach jednego uregulowania prawnego i wre˛cz tego samego przepisu. Przykładem jest ustawa z 24 kwietnia 2003 r. o działalnos´ci poz˙ytku publicznego i o wolontariacie35. W art. 11b stwierdzono, z˙e Prezes Rady Ministro´w, jez˙eli jest to niezbe˛dne ze wzgle˛du na ochrone˛ z˙ycia lub zdrowia ludzkiego albo ze wzgle˛du na waz˙ny interes społeczny lub waz˙ny interes publiczny, moz˙e zlecac´ organizacjom pozarza˛dowym oraz podmiotom wymienionym w art. 3 ust. 3 realizacje˛ zadan´ publicznych z pomi- nie˛ciem otwartego konkursu ofert. Zatem gdyby ustawodawca traktował obie klauzule synonimicznie, posłuz˙yłby sie˛ jedna˛ z nich, a tymczasem rozro´z˙nia je i wia˛z˙e okres´lone skutki prawne zaro´wno z jedna˛ klauzula˛, jak i z druga˛. IV. KLAUZULA ,,INTERESU SPOŁECZNIE UZASADNIONEGO’’ W PRAWIE KARNYM Jeszcze inna˛ klauzula˛ jest klauzula ,,interesu społecznie uzasadnionego’’, choc´ moz˙e sie˛ wydawac´, z˙e jest toz˙sama z poprzednimi. Przykładem przepisu zawieraja˛cego odesłanie do interesuja˛cej mnie kategorii jest art. 213 § 2 k.k., kto´ry mo´wi, z˙e nie popełnia przeste˛pstwa okres´lonego w art. 212 § 1 lub 2 ten, kto publicznie podnosi lub rozgłasza prawdziwy zarzut: 1) dotycza˛cy poste˛powania osoby pełnia˛cej funkcje˛ publiczna˛ lub 2) słuz˙a˛cy obronie społecznie uzasadnionego interesu. Artykuł 212 k.k. opisuje znamiona typo´w czyno´w karalnych okres´lanych jako ,,zniesławienie’’ ba˛dz´ ,,pomo´wienie’’. Ich istota sprowadza sie˛ do zachowania, przez kto´re sprawca przypisuje innemu podmiotowi negatywnie oceniane włas´ciwos´ci lub poste˛powanie, co moz˙e doprowadzic´ do poniz˙enia go w opinii publicznej ba˛dz´ tez˙ narazic´ na utrate˛ zaufania potrzebnego do zajmowania danego stanowiska, wykonywania zawodu lub prowadzenia okres´lonego rodzaju działalnos´ci36. Realizacja prawa do zachowania dobrego imienia moz˙e w wielu wypadkach kolidowac´ z inna˛ wartos´cia˛, kto´ra˛ jest wolnos´c´ wypowiedzi innych oso´b. Moz˙liwos´c´ takiej kolizji przewiduje art. 10 ust. 2 Europejskiej konwencji o ochronie praw człowieka i podstawowych wolnos´ci. Wynika z niego, z˙e korzystanie z wolnos´ci wypowiedzi moz˙e podlegac´ takim wymogom formalnym, ,,Interes publiczny’’, ,,interes społeczny’’ i ,,interes społecznie uzasadniony’’ 65 warunkom, ograniczeniom i sankcjom, kto´re sa˛ przewidziane przez ustawe˛ i niezbe˛dne w społeczen´ stwie demokratycznym m.in. z uwagi na ochrone˛ dobrego imienia i praw innych oso´b. Podobny zapis zawiera art. 19 ust. 3 Mie˛dzynarodowego paktu praw obywatelskich i politycznych z 16 grudnia 1966 r. Zadaniem ustawodawcy jest wie˛c wypracowanie takich rozwia˛zan´ legislacyjnych, kto´re umoz˙liwia˛ zachowanie włas´ciwych relacji pomie˛dzy koliduja˛cymi tu wartos´ciami. Z tres´ci art. 213 § 2 k.k. wycia˛gna˛c´ moz˙na wniosek, z˙e sam fakt praw- dziwos´ci sformułowanego przez sprawce˛ publicznie zarzutu zniesławiaja˛cego nie prowadzi jeszcze do wyła˛czenia bezprawnos´ci pomo´wienia. Konieczne jest dodatkowo, by podniesiony lub rozgłoszony prawdziwy zarzut słuz˙ył obronie społecznie uzasadnionego interesu. W jednym z orzeczen´ Sa˛d Najwyz˙szy wskazał, z˙e ,,Społecznie uzasadniony interes nie moz˙e byc´ rozumiany w sposo´b abstrakcyjny, jest bowiem poje˛ciem konkretnym i musi wynikac´ z okres´lonej sytuacji, wymagaja˛cej obrony tego interesu nawet z naruszeniem dobrego imienia innej osoby, grupy oso´b lub instytucji. Nie kaz˙de działanie jest działaniem w obronie społecznie uzasad- nionego interesu, lecz tylko takie, kto´re faktycznie temu interesowi słuz˙y’’ 37. g , y , y y Trybunał Konstytucyjny w wyroku z 12 maja 2008 r. (SK 4305) zauwaz˙a natomiast trudnos´ci z dookres´leniem, czym jest społecznie uzasadniony interes i pojawiaja˛ce sie˛ w doktrynie rozbiez˙nos´ci. Zwraca uwage˛, z˙e jest to rodzaj odesłania pozaprawnego, gdyz˙ jego tres´c´ nie jest wyznaczona przez przepis prawny. 37 Wyrok z 25 wrzes´nia 1973 r., V KRN 35873, OSNKW 1974, nr 2, poz. 27. IV. KLAUZULA ,,INTERESU SPOŁECZNIE UZASADNIONEGO’’ W PRAWIE KARNYM Znaczenie normatywne wynikaja˛ce z tego przepisu konkretyzowane be˛dzie a casu ad casum, z uwzgle˛dnieniem dowiedzionego w danym poste˛po- waniu karnym stanu faktycznego oraz argumentacji aksjologicznej. Zaznacza, z˙e interpretacja kategorii ,,społecznie uzasadnionego interesu’’ moz˙e i powinna nawia˛zywac´ do aksjologii przyje˛tej w obowia˛zuja˛cym prawie, w tym zwłaszcza w Konstytucji. Niejednoznacznos´c´ tego odesłania polega ro´wniez˙ na tym, jak twierdzi Trybunał, z˙e nie pozwala ono w sposo´b pewny i bezdyskusyjny ustalic´ rozległos´ci odniesienia społecznego, o kto´rym mowa, a wie˛c to od uznania organu oceniaja˛cego dany czyn z punktu widzenia znamion kontratypu wymienionych w art. 213 § 2 k.k. zalez˙ec´ be˛dzie, czy uwzgle˛dniony be˛dzie interes całego społeczen´ stwa (jeden zgeneralizowany zakres wspo´lny dla obywateli), czy np. jedynie interes jakiejs´ grupy s´rodowiskowej lub zawodowej. Trybunał orzekł w tym zakresie, z˙e ustawodawca, daja˛c sa˛dom moz˙liwos´c´ doprecyzowania na tle konkretnego stanu faktycznego poje˛cia ,,społecznie uzasadnionego interesu’’, nie naruszył standardu okres´lonos´ci przepiso´w praw- nych w stopniu przekraczaja˛cym dopuszczalne konstytucyjnie granice. W pis´miennictwie z kolei wskazuje sie˛, z˙e z obrona˛ omawianego interesu mamy do czynienia zwłaszcza wtedy, gdy publicznie postawione zarzuty moga˛ słuz˙yc´ ostrzez˙eniu innych oso´b przed nagannym zachowaniem pomo´wionego i moz˙e to prowadzic´ do skorygowania nagannego zachowania, np. w sferze kierowania procesami gospodarczymi lub społecznymi, polityki kultural- nej, działalnos´ci partii politycznych, stowarzyszen´ , a takz˙e w razie krytyki 37 Wyrok z 25 wrzes´nia 1973 r., V KRN 35873, OSNKW 1974, nr 2, poz. 27. 66 Artur Z˙urawik indywidualnej działalnos´ci jednostki, jez˙eli ta działalnos´c´ ma społeczne znaczenie38. J. Raglewski pisze w tym konteks´cie, z˙e kryterium obrony społecznie uzasadnionego interesu nalez˙y rozmiec´ w sposo´b zobiektywizowany. Nie chodzi tu jedynie o subiektywne przekonanie sprawcy, z˙e stawiane przez niego zarzuty słuz˙a˛obronie społecznie uzasadnionego interesu. Dodaje, z˙e na gruncie art. 213 § 2 k.k. chodzi o ,,interes społecznie uzasadniony’’, a nie o ,,interes społeczny’’. Zakres pierwszego z wymienionych zwroto´w normatywnych jest szerszy: spo- łecznie uzasadnione moga˛ byc´ bowiem ro´wniez˙ działania podejmowane w interesie jednostki39. Jest to włas´nie podstawowa cecha odro´z˙niaja˛ca obie klauzule40. 38 J. Raglewski, Komentarz do art. 213 Kodeksu karnego, w: A. Zoll (red.), op. cit., t. 2. 39 Ibidem. Zob. tez˙ postanowienie SN z 27 wrzes´nia 2001 r., III KKN 74098. 40 Zob. tez˙ art. 44 ust. 2 ustawy z 26 stycznia 1984 r. – Prawo prasowe (Dz. U. 1984, Nr 5, poz. 24). 41 A. Pa˛zik, Obrona interesu społecznego jako przesłanka wyła˛czaja˛ca bezprawnos´c´ naruszenia dobra osobistego, ,,Prace z Prawa Własnos´ci Intelektualnej’’ 2010, z. 109, s. 117. Autor przywołuje tu: S. Grzybowski, Ochrona do´br osobistych w przepisach ogo´lnych prawa cywilnego, Warszawa 1957, s. 118. 42 Zob. A. Pa˛zik, op. cit. s. 110 i podana tam literatura; P. Ksie˛z˙ak, Komentarz do art. 24 Kodeksu cywilnego, wersja elektroniczna, Lexel. 2012; J. Dobosz, Działanie w obronie uzasadnionego interesu jako okolicznos´c´ wyła˛czaja˛ca bezprawnos´c´ naruszenia do´br osobistych, w: J. S. Pia˛towski (red.), Dobra osobiste i ich ochrona w polskim prawie cywilnym. Zagadnienia wybrane, Wrocław-Warszawa-Krako´w- 38 J. Raglewski, Komentarz do art. 213 Kodeksu karnego, w: A. Zoll (red.), op. cit., t. 2. 39 Ibid Z b t t i i SN 27 ´ i 2001 III KKN 74098 osobistego, ,,Prace z Prawa Własnosci Intelektualnej 2010, z. 109, s. 117. Autor przywołuje tu: S. Grzybowski, Ochrona do´br osobistych w przepisach ogo´lnych prawa cywilnego, Warszawa 1957, s. 118. 42 Zob. A. Pa˛zik, op. cit. s. 110 i podana tam literatura; P. Ksie˛z˙ak, Komentarz do art. 24 Kodeksu cywilnego, wersja elektroniczna, Lexel. 2012; J. Dobosz, Działanie w obronie uzasadnionego interesu jako okolicznos´c´ wyła˛czaja˛ca bezprawnos´c´ naruszenia do´br osobistych, w: J. S. Pia˛towski (red.), Dobra osobiste i ich ochrona w polskim prawie cywilnym. Zagadnienia wybrane, Wrocław-Warszawa-Krako´w- 38 J. Raglewski, Komentarz do art. 213 Kodeksu karnego, w: A. Zoll (red.), op. cit., t. 2. V. ,,INTERES SPOŁECZNIE UZASADNIONY’’ W PRAWIE CYWILNYM Klauzula ,,interesu społecznie uzasadnionego’’ stosowana jest takz˙e na gruncie prawa cywilnego, w zakresie przepiso´w dotycza˛cych ochrony do´br osobistych. Poje˛cie ,,obrona społecznie uzasadnionego interesu’’ wprowadził do doktryny prawa cywilnego S. Grzybowski w latach pie˛c´dziesia˛tych XX w. – uznał ja˛ za jedna˛ z przesłanek wyła˛czaja˛cych bezprawnos´c´ naruszenia dobra osobistego41. Ochrona do´br osobistych w polskim porza˛dku prawnym odbywa sie˛ przede wszystkim na podstawie art. 23 k.c., zawieraja˛cego otwarty katalog wartos´ci uznanych przez ustawodawce˛ za dobra osobiste. Artykuł 24 § 1 k.c. wprowadza zdaniem cze˛s´ci doktryny tzw. domniemanie bezprawnos´ci naruszenia dobra osobistego. Wynika z niego, z˙e kaz˙de naruszenie takiego dobra jest bezprawne, z wyja˛tkiem sytuacji, w kto´rych naruszycielowi uda sie˛ wykazac´, iz˙ jego poste˛powanie nie miało cech bezprawnos´ci. Z kolei o bezprawnos´ci moz˙emy mo´wic´ w wypadku sprzecznos´ci danego zachowania z przepisami prawa ba˛dz´ z zasadami wspo´łz˙ycia społecznego. Przy tym kaz˙da ingerencja w sfere˛ chro- nionych przez prawo wartos´ci zwia˛zanych z osobowos´cia˛ człowieka obarczona jest cecha˛ bezprawnos´ci, chyba z˙e zachodzi jedna z okolicznos´ci ja˛ wyła˛czaja˛- cych. Okolicznos´ci te to: 1) zgoda pokrzywdzonego (nie we wszystkich sytuacjach), 2) działanie na podstawie przepisu prawa, 3) wykonywanie prawa podmiotowego i w kon´ cu 4) działanie w obronie społecznie uzasadnionego interesu lub interesu prywatnego 42. 67 ,,Interes publiczny’’, ,,interes społeczny’’ i ,,interes społecznie uzasadniony’’ W uchwale 7 se˛dzio´w SN z 18 lutego 2005 r.43 w tym konteks´cie wskazano: ,,Wykazanie przez dziennikarza, z˙e przy zbieraniu i wykorzystaniu materiało´w prasowych działał w obronie społecznie uzasadnionego interesu oraz wypełnił obowia˛zek zachowania szczego´lnej starannos´ci i rzetelnos´ci, uchyla bez- prawnos´c´ działania dziennikarza. Jez˙eli zarzut okaz˙e sie˛ nieprawdziwy, dziennikarz zobowia˛zany jest do jego odwołania’’. Pogla˛d ten nie był jednak powszechnie podzielany44. Problematyki tej nie be˛de˛ jednak tu rozstrzygał, gdyz˙ nie jest to przedmiot niniejszego opracowania. W kaz˙dym razie A. Pa˛zik stwierdza, z˙e w czasie analizy przesłanki interesu społecznie uzasadnionego, jako okolicznos´ci legalizuja˛cej naruszenie do´br osobistych na podstawie art. 23 i 24 k.c., dorobek karnistyczny moz˙e miec´ zastosowanie jedynie pomocnicze. Wynika to z faktu, z˙e prawo cywilne i prawo karne to dwa samodzielne działy prawa, a cywilistyczna ochrona do´br osobistych ma swoja˛ specyfike˛. W prawie karnym konieczne jest bowiem wyka- zanie przez oskarz˙yciela, z˙e działanie sprawcy spełnia wszystkie znamiona typu czynu zabronionego, co wynika z zasady domniemania niewinnos´ci. Z kolei w art. 24 k.c. -Gdan´ sk-Ło´dz´ 1986, s. 289-290; Z. Bidzin´ ski, J. Serda, Cywilnoprawna ochrona do´br osobistych w prak- tyce sa˛dowej, w: J. S. Pia˛tkowski (red.), op. cit., s. 12 i n. 43 III CZP 5304, OSP 2005, z. 9, poz. 110 z glosa˛ Z. Radwan´ skiego. 44 Zob. szczego´łowo P. Ksie˛z˙ak, op. cit. i podana tam literatura. 45 A. Pa˛zik, op. cit., s. 113. 46 Ibidem, s. 111-114 i podana tam literatura. 47 Ibidem, s. 115. 46 Ibidem, s. 111-114 i podana tam literatura. y j, , p , 43 III CZP 5304, OSP 2005, z. 9, poz. 110 z glosa˛ Z. Radwan´ skiego. 45 A. Pa˛zik, op. cit., s. 113. an´ sk-Ło´dz´ 1986, s. 289-290; Z. Bidzin´ ski, J. Serda, Cywilnoprawna ochrona do´br osobistych w prak sa˛dowej, w: J. S. Pia˛tkowski (red.), op. cit., s. 12 i n. 48 W tym zakresie w 1990 r. znowelizowano art. 14 ust. 6. Zob. Z. Radwan´ ski, Prawo cywilne – cze˛s´c´ ogo´lna, Warszawa 2009, s. 171 i 172. Podobnie stwierdza M. Pazdan: ,,Najwie˛cej sporo´w dotyczy pytania, czy za samodzielna˛ okolicznos´c´ wyła˛czaja˛ca˛ bezprawnos´c´ zagroz˙enia lub naruszenia dobra osobistego moz˙e byc´ uznane działanie w obronie uzasadnionego interesu społecznego lub prywatnego. Na pytanie to udzielane sa˛ zaro´wno odpowiedzi twierdza˛ce, jak i przecza˛ce. Racje˛ przyznac´ nalez˙y zwolennikom odpowiedzi przecza˛cej. Wprowadzenie tej okolicznos´ci wyła˛czaja˛cej bezprawnos´c´ znacznie ograniczałoby ochrone˛ do´br osobistych. Prawo polskie »uzasadnionego interesu« nie uznaje za ogo´lna˛ przesłanke˛ wytyczaja˛ca˛ granice kaz˙dego prawa podmiotowego. Klauzula ta moz˙e wie˛c odgrywac´ jaka˛s´ role˛ tylko w razie odwołania sie˛ do niej przepisu szczego´lnego’’ (M. Pazdan, Dobra osobiste i ich ochrona, w: System prawa prywatnego, t. 1: Prawo cywilne – cze˛s´c´ ogo´lna, red. M. Safjan, Warszawa 2007, s. 1160). 49 Ksie˛ga Pierwsza Kodeksu Cywilnego. Projekt wraz z uzasadnieniem, Warszawa 2008. Projekt opublikowany na: http:bip.ms.gov.plpldzialalnosckomisje-kodyfikacyjnekomisja-kodyfikacyjna- prawa-cywilnego (doste˛p: 22.08.2012). V. ,,INTERES SPOŁECZNIE UZASADNIONY’’ W PRAWIE CYWILNYM ustawodawca, jego zdaniem, zdecydował sie˛ na wprowadzenie domniemania bezprawnos´ci, co oznacza sytuacje˛ odwrotna˛: powo´d nie jest zobowia˛zany do udowodnienia przed sa˛dem bezprawnos´ci działania pozwanego, lecz to na pozwanym cia˛z˙y obowia˛zek wykazania, z˙e jego działanie w istocie nie nosiło cech bezprawnos´ci. Inaczej jest ro´wniez˙ definiowana sama bez- prawnos´c´ 45. Autor ten dodatkowo utoz˙samia poje˛cia ,,interesu społecznie uzasadnionego’’ oraz ,,interesu społecznego’’, zauwaz˙aja˛c przy tym, z˙e działanie w ramach społecznie uzasadnionego interesu nie zostało uje˛te w z˙adnym przepisie prawa cywilnego, brakuje ro´wniez˙ powszechnie uznawanej definicji tej klauzuli. Zgadza sie˛ z J. Barta˛ i R. Markiewiczem, z˙e wszystkie pro´by definicyjne w istocie niewiele wyjas´niaja˛, poniewaz˙ jedno poje˛cie nieostre zaste˛puja˛ innym46. Uwaz˙a, z˙e koncepcja interesu społecznego na gruncie ochrony do´br osobistych ma pełnic´ funkcje˛ ,,wagi’’, czyli instrumentu prawnego umoz˙liwia- ja˛cego wywaz˙anie koliduja˛cych z soba˛intereso´w. Wartos´ciami, kto´re kładziemy na jej szalach, sa˛ z jednej strony dobra osobiste pokrzywdzonego, a z drugiej – jedna z akceptowanych wartos´ci, reprezentowana przez naruszyciela, np. swo- boda wypowiedzi, swoboda działalnos´ci artystycznej, doste˛p społeczen´ stwa do informacji, wolnos´c´ badan´ naukowych itd.47 Przeciwnikiem wyro´z˙niania interesu społecznie uzasadnionego jako ogo´lnej okolicznos´ci wyła˛czaja˛cej bezprawnos´c´ był w swym pis´miennictwie Z. Rad- wan´ ski. Argumentował to tym, z˙e o´w interes nie został wprost wyraz˙ony przez ustawodawce˛ w z˙adnym akcie prawnym dotycza˛cym ochrony do´br osobistych. 68 Artur Z˙urawik Co wie˛cej, w ramach jednej z kolejnych nowelizacji prawa prasowego wre˛cz został z ustawy usunie˛ty 48. Na marginesie moz˙na dodac´, z˙e w projekcie ksie˛gi pierwszej Kodeksu cywilnego, przedstawionym przez Komisje˛ Kodyfikacyjna˛ Prawa Cywilnego, artykuł 22 § 2 stanowi, z˙e: ,,Nie jest bezprawne działanie podje˛te na podstawie przepiso´w ustawy lub za zgoda˛ uprawnionego. Ujawnienie informacji dotycza˛- cych jego osoby nie jest działaniem bezprawnym, jez˙eli informacje te sa˛ prawdziwe, a za ich ujawnieniem przemawia waz˙ny interes publiczny lub prywatny’’ 49. Posłuz˙enie sie˛ tu klauzula˛ dos´c´ dobrze znana˛ ustawodawstwu polskiemu jest, moim zdaniem, w pełni uzasadnione. VI. WNIOSKI Klauzule interesu publicznego, interesu społecznego oraz interesu spo- łecznie uzasadnionego sa˛ zatem ro´z˙nymi klauzulami, choc´ cze˛sto sie˛ je utoz˙samia, w szczego´lnos´ci dwie pierwsze. Ro´wniez˙ i trzecia ma swoisty charakter, inny niz˙ poprzednie. Jest on dwojakiego rodzaju: 1) ustawowy – w omo´wionych wyz˙ej karnych przepisach Kodeku karnego oraz Prawa prasowego; 1) ustawowy – w omo´wionych wyz˙ej karnych przepisach Kodeku karnego oraz Prawa prasowego; 2) pozaustawowy – na gruncie przepiso´w cywilnych, choc´ nie jest wyklu- czone, z˙e w przyszłos´ci ustawodawca zwia˛z˙e okres´lone skutki prawne z klauzula˛ interesu publicznego. W prawie karnym przyjmowane jest stanowisko, z˙e zakres poje˛cia ,,interes społecznie uzasadniony’’ jest szerszy niz˙ poje˛cia ,,interes społeczny’’; społecznie uzasadnione moga˛ byc´ bowiem ro´wniez˙ działania podejmowane w interesie jednostki. Natomiast w prawie cywilnym poje˛cie interesu społecznie uza- sadnionego budzi szczego´lne kontrowersje, a co wie˛cej, czasem utoz˙samia sie˛ je z interesem społecznym. Uwaz˙am, z˙e nie ma powodu, by obie klauzule utoz˙samiac´, powoduje to bowiem niepotrzebny zame˛t terminologiczny. Skoro w praktyce doktryna cywilistyczna i orzecznictwo w zdecydowanej wie˛kszos´ci wypadko´w posługuja˛ sie˛ kategoria˛ interesu społecznie uzasadnionego, to nie ma racjonalnych przesłanek, by pisza˛c o tej samej przesłance, nazywac´ ja˛ ,,Interes publiczny’’, ,,interes społeczny’’ i ,,interes społecznie uzasadniony’’ 69 ,,interesem społecznym’’. Dokonanie takiej zmiany wymagałoby pogłe˛bionych rozwaz˙an´ , dlaczego dotychczasowe nazewnictwo nie odpowiada głoszonej koncepcji. dr Artur Z˙urawik Uniwersytet Jagiellon´ ski w Krakowie S u m m a r y One of the most important general clauses in Polish legislation is, because of its constitutive character, a clause of public interest. This clause is also frequently used by ordinary legislators. The clause of public interest constitutes first and foremost the basis for limiting the rights and freedoms of citizens. In some cases, however, it is a premise for implementation of certain mechanisms for the protection of selected values (in, e.g. competition law or consumer protection law). Therefore, it has a diverse character. Connections between the law and the social life are wide, as is manifested by the theoretical and legal reflection, and a reflection of other social sciences. The law, in particular public law, results inter alia, from the realisation of a specific policy and is also a carrier and reflection of socially important values. This, in turn, triggers a need for a normative analysis and exploration of the normative content of the above clause and similar ones which include clauses of ‘social interest’ and ‘socially justified interest.’ These clauses are sometimes used interchangeably in the doctrine, but such an approach is not justified. This paper aims to analyse these clauses in detail to prove that they should not be treated synonymously. Copyright of Journal of Law, Economics and Sociology is the property of Faculty of Law and Administration of Adam Mickiewicz University in Poznan and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder’s express written permission. However, users may print, download, or email articles for individual use. Właścicielem praw autorskich do „Ruchu Prawniczego, Ekonomicznego i Socjologicznego” jest Wydział Prawa i Administracji Uniwersytetu im. Adama Mickiewicza w Poznaniu. Zawartość czasopisma nie może być kopiowana, przesyłana do innych stron internetowych bądź zamieszczana na blogach bez pisemnej zgody wydawcy. Niemniej artykuły można drukować, kopiować lub przesyłać w formie elektronicznej na własny użytek.
https://openalex.org/W3214360623	https://europepmc.org/articles/pmc8564353?pdf=render	English	null	Exosome CTLA-4 Regulates PTEN/CD44 Signal Pathway in Spleen Deficiency Internal Environment to Promote Invasion and Metastasis of Hepatocellular Carcinoma	Frontiers in pharmacology	2,021	cc-by	11,374	ORIGINAL RESEARCH published: 20 October 2021 doi: 10.3389/fphar.2021.757194 Reviewed by: Currently, there is no effective treatment. In traditional Chinese medicine, a large proportion of patients with HCC have been diagnosed with spleen deﬁciency (SD) syndrome and treated with tonifying traditional Chinese medicine, which has signiﬁcant clinical efﬁcacy. However, the role and molecular mechanism of SD in HCC remain unclear. In this study, 40 mice were randomly divided into four groups: control, SD, HCC, and SD-HCC groups. The liver cancer model of SD was established by reserpine induction and orthotopic transplantation. The effects of SD on the proliferation, apoptosis, invasion, and metastasis of HCC cells were studied by cell proliferation, cell apoptosis, cell scratch, and transwell assay. We found that compared with the HCC group, the protein expressions of cytotoxic T lymphocyte antigen 4 (CTLA- 4), programmed cell death protein 1 (PD-1), phosphatase and tensin homolog (PTEN), and AKT (also known as protein kinase B or PKB) in the exosomes of the SD-HCC group were upregulated. In addition, the metastases and self-renewal of exosomes in the SD-HCC group were more aggressive than those in the HCC group, which could be partially reversed with the addition of CTLA-4 inhibitors. Further studies showed that in the internal environment of SD, CTLA-4 promoted tumor invasion and metastasis by regulating the PTEN/CD44 pathway. In conclusion, our ﬁndings suggest that during SD in the internal environment, exosome CTLA-4 regulates the PTEN/CD44 signal pathway to promote the proliferation, self-renewal, and metastasis of liver cancer. Correspondence: Xifeng Liu xifengliu@126.com Shijun Zhang zhshjun@mail.sysu.edu.cn Ling Yu 994242527@qq.com †These authors have contributed equally to this work Specialty section: This article was submitted to Pharmacology of Anti-Cancer Drugs, a section of the journal Frontiers in Pharmacology Received: 11 August 2021 Accepted: 09 September 2021 Published: 20 October 2021 Reviewed by: Reviewed by: Tongkai Chen, Guangzhou University of Chinese Medicine, China Xianglong Hu, South China Normal University, China Yi Cao, Xiangtan University, China Kunyi Yu, The Afﬁliated Hospital of Southern Medical University, China Zhigang He, Zhejiang University, China Yongdan Wang 1†, Pan Li 1†, Shuai Mao 2,3†, Zhuomao Mo 1, Zhirui Cao 1, Jin Luo 1, Meiling Zhou 1, Xifeng Liu 4, Shijun Zhang 1* and Ling Yu 2,3* 1Department of Traditional Chinese Medicine, The First Afﬁliated Hospital, Sun Yat-sen University, Guangzhou, China, 2Second Clinical College, Guangzhou University of Chinese Medicine, Guangzhou, China, 3AMI Key Laboratory of Chinese Medicine in Guangzhou, Guangdong Provincial Hospital of Chinese Medicine, Guangzhou, China, 4School of Life Sciences, Xiangya Medical College, Central South University, Changsha, China Hepatocellular carcinoma (HCC) is one of the most common primary cancers, and its pathogenesis is complicated and difﬁcult to screen. Currently, there is no effective treatment. In traditional Chinese medicine, a large proportion of patients with HCC have been diagnosed with spleen deﬁciency (SD) syndrome and treated with tonifying traditional Chinese medicine, which has signiﬁcant clinical efﬁcacy. However, the role and molecular mechanism of SD in HCC remain unclear. In this study, 40 mice were randomly divided into four groups: control, SD, HCC, and SD-HCC groups. The liver cancer model of SD was established by reserpine induction and orthotopic transplantation. The effects of SD on the proliferation, apoptosis, invasion, and metastasis of HCC cells were studied by cell proliferation, cell apoptosis, cell scratch, and transwell assay. We found that compared with the HCC group, the protein expressions of cytotoxic T lymphocyte antigen 4 (CTLA- 4), programmed cell death protein 1 (PD-1), phosphatase and tensin homolog (PTEN), and AKT (also known as protein kinase B or PKB) in the exosomes of the SD-HCC group were upregulated. In addition, the metastases and self-renewal of exosomes in the SD-HCC group were more aggressive than those in the HCC group, which could be partially reversed with the addition of CTLA-4 inhibitors. Further studies showed that in the internal environment of SD, CTLA-4 promoted tumor invasion and metastasis by regulating the PTEN/CD44 pathway. In conclusion, our ﬁndings suggest that during SD in the internal environment, exosome CTLA-4 regulates the PTEN/CD44 signal pathway to promote the proliferation, self-renewal, and metastasis of liver cancer. Hepatocellular carcinoma (HCC) is one of the most common primary cancers, and its pathogenesis is complicated and difﬁcult to screen. Keywords: hepatocellular carcinoma, spleen deﬁciency, exosome, CTLA-4, PTEN Edited by: Jianxun Ding, Changchun Institute of Applied Chemistry (CAS), China ORIGINAL RESEARCH published: 20 October 2021 doi: 10.3389/fphar.2021.757194 INTRODUCTION apoptosis through the Caspase-3/PARP signaling pathway (Yu et al., 2021). The above studies suggest the importance of the internal environment of SD in the occurrence and development of immune recognition disorders of HCC. Hepatocellular carcinoma (HCC) is a highly prevalent and lethal cancer, ranking the ﬁfth most commonly diagnosed cancer and the third leading cause of cancer-associated deaths worldwide (Chen et al., 2016). The incidence of HCC has increased signiﬁcantly recently, with about 840,000 new cases each year (Bray et al., 2018). It has been reported that the incidence and mortality of HCC in China account for 50% of HCC in the world (Feng et al., 2019). Hepatitis B virus (HBV) infection is the main cause of HCC in East Asia, especially in China (Bray et al., 2018). The rapid development and high recurrence are major challenges in the treatment of HCC due to its highly metastatic nature (Gish et al., 2007; Hu et al., 2020). Therefore, it is urgent to seek valuable biomarkers and potential therapeutic targets to improve the clinical curative effect on HCC patients. The barrier to successful cancer immunotherapy is the capability of tumors to escape from the host’s immune system (Whiteside et al., 2011). Exosomes, small membranous sacs of endocytic origin (30–150 nm), are considered as intercellular messengers that can carry a large number of macromolecular cargos, including proteins, mRNA, lipids, and miRNA (Wen et al., 2016; Bach et al., 2017; Ruivo et al., 2017; Su et al., 2021). Studies have found that exosomes derived from tumors carry immunosuppressive proteins, including PD-1, CTLA-4, FasL, TRAIL, CD39, and CD73, which induce apoptosis and depletion of T lymphocytes to achieve tumor immune escape (Whiteside, 2013; Ukrainskaya et al., 2019; Benecke et al., 2021). Cytotoxic T lymphocyte antigen 4 (CTLA-4), a member of the immune protein superfamily, competes with the T cell co- stimulator CD28 for binding to CD80 and CD86 with a higher afﬁnity to antigen-presenting cells during the priming phase in the lymph nodes and conveys inhibitory signals within the T cells (Van Coillie et al., 2020; Goenka et al., 2021). Clinical evidence has shown that anti–CTLA-4 therapy can enhance the activation of effector T cells (Maker et al., 2005), increase the ratio of effector T cells to Treg (Quezada et al., 2006; Curran et al., 2010; Ou et al., 2018), and promote the transport of activated T cells to tumor tissues (Quezada et al., 2006). Citation: Wang Y, Li P, Mao S, Mo Z, Cao Z, Luo J, Zhou M, Liu X, Zhang S and Yu L (2021) Exosome CTLA-4 Regulates PTEN/CD44 Signal Pathway in Spleen Deﬁciency Internal Environment to Promote Invasion and Metastasis of Hepatocellular Carcinoma. Front. Pharmacol. 12:757194. doi: 10.3389/fphar.2021.757194 Keywords: hepatocellular carcinoma, spleen deﬁciency, exosome, CTLA-4, PTEN October 2021 \| Volume 12 \| Article 757194 1 Frontiers in Pharmacology \| www.frontiersin.org Exosome Regulates Spleen Deﬁciency HCC Wang et al. Cell Line and Cell Culture Mouse HCC cell line hep1-6 was obtained from the Institute of Biochemistry and Cell Biology (Shanghai, China). The cell line was cultured in RPMI-1640 medium (Hy Clone, Los Angeles, CA, United States), which contained 10% fetal bovine serum (FBS) and 5% carbon dioxide. The ambient temperature was controlled at about 37°C. HCC cell lines were identiﬁed by microsatellite analysis, in which the cells were cultured for no more than 2 months. The Establishment of Spleen Deﬁciency Model Based on the above studies, we proposed the hypothesis that the change of CTLA-4 expression in exosomes of SD internal environment is related to the PTEN/CD44 signaling pathway, as demonstrated in Scheme 1. Exosome CTLA-4 is an important factor in HCC. Therefore, we aimed to explore the underlying mechanism of exosome CTLA-4 in HCC progression. We have tried our best to seek a new therapeutic target for patients with liver cancer. A mouse model of SD was established by reserpine. Mice in the SD group and SD liver cancer group were subcutaneously injected with reserpine solution B, one time per day at a dose of 0.1 ml/10 g. At the same time, the normal and liver cancer groups were subcutaneously injected with sodium chloride once per day at the same dose of 0.1 ml/10 g for 14 consecutive days. The weight of all the mice was measured daily, and the amount of food they ate in each cage was recorded. At the same time, the odor, mental state, body cold and heat, respiratory status, hair color, food intake, and stool of all mice in the four groups were observed. At the end of the 14th day, the severity of SD in mice was evaluated by using the SD rating scale (Table 1). INTRODUCTION October 2021 \| Volume 12 \| Article 757194 Frontiers in Pharmacology \| www.frontiersin.org 2 Exosome Regulates Spleen Deﬁciency HCC Wang et al. from GemPharmatech Co. Ltd. (Jiangsu, China). Laboratory animal quality certiﬁcate no: SCXK(SU)2018-0008. The animals were kept in a speciﬁc pathogen-free environment with a temperature of 25°C, relative humidity of 60–80%, free eating, and drinking water. All experiments were conducted in the SPF laboratory of the animal center of the ﬁrst afﬁliated hospital, Sun Yat-Sen University (Guangzhou, China). The license number was SYXK (Guangdong) 2020-0108. Experimental animals were used in accordance with the 3R principle for humane care. The animal experiments were approved by the clinical research and animals ethics committee of the ﬁrst afﬁliated hospital, Sun Yat-Sen University. In the SPF barrier environment, 40 C57BL/6 male mice were randomly divided into the normal group, SD group, liver cancer group, and SD liver cancer group. There were 10 mice in each group and 40 mice were labeled. transformation, progression, chemotherapy response, and survival of a variety of cancers (Wang et al., 2018a; Raffone et al., 2019). PTEN regulates PI3K/Akt/mTOR by its phosphatase activity, which is one of the most important signaling pathways for cell growth and survival in cancer (Su et al., 2016; Lee et al., 2018; Luongo et al., 2019). It has been reported that CD44 is one of the biomarkers and a key regulator of cancer stem cells (CSCs), including self-renewal, tumor initiation, and metastasis (Wang et al., 2018b). PTEN plays a critical role in the immobility and regulation of the cell cycle entry of transforming stem cells (SCs), and the loss of PTEN tends to promote cancerous phenotypes (Luongo et al., 2019). It has been found that miR-21, a gene known to target PTEN, signiﬁcantly regulates the growth and/or differentiation of CSCs in colon cancer (Yu et al., 2015). The imbalance of the PTEN/CD44 signaling pathway plays a vital role in maintaining the characteristics of CSC (Luongo et al., 2019; Cetintas and Batada, 2020). Nevertheless, the link between CTLA-4 and PTEN/CD44 signaling pathway has not been reported. INTRODUCTION Our previous study found that traditional Chinese medicine could improve the tumor microenvironment of patients to treat liver cancer and speculated that the immune checkpoints CTLA-4, LAG-3, and BIRC5 are the key targets for activating immune cells to treat liver cancer (Mo et al., 2020). However, only a minority of people can respond to immunotherapy (Mahoney et al., 2015). Consequently, it is still a critical challenge to explore the molecular mechanisms of immunotherapy for HCC. The imbalance of the internal environment is closely related to the occurrence and development of tumors (Craig et al., 2020; Llovet et al., 2021). Generally, only about 5% of tumors could be explained by genetic factors alone, whereas most tumors are the result of a combination of environmental and genetic factors (Bhattacharjee et al., 2013). In the TCM (traditional Chinese medicine) therapy systems, “Bian Zheng Lun Zhi” is the core, that is, a treatment based on syndrome differentiation, which prioritizes to adjust the internal environment (Zhang et al., 2020). TCM syndromes are the characteristics of all syndromes in a patient’s clinical manifestations essentially and help guide the design of individualized treatments (Ji et al., 2016). In TCM theory, symptoms such as loose stools, abdominal distension after meals, loss of appetite, sallow complexion, weight loss, general weakness, and/or low disease resistance could be summarized as spleen deﬁciency (SD) (Luo et al., 2017). Interestingly, the majority of patients with cancer cachexia have been diagnosed with SD in China (Sun et al., 2016a; Zhang et al., 2020). According to the literature, SD may lead to dysfunction in T cell recognition by disordering the expression of TCRVβ in rats (Chen et al., 2014). In addition, another study reported that immunoregulatory traditional Chinese medicine is beneﬁcial to liver cancer and induces cell PTEN is known as a tumor suppressor protein in most tumors, which is located in Chromosome 10 (Su et al., 2016; Luongo et al., 2019). The loss of PTEN function due to epigenetic silencing or genetic aberration has been associated with malignant SCHEME 1 \| In the internal environment of SD, exosome CTLA-4 regulate the PTEN/CD44 signal pathway to promote the proliferation, self-renewal, and metastasis of liver cancer. SCHEME 1 \| In the internal environment of SD, exosome CTLA-4 regulate the PTEN/CD44 signal pathway to promote the proliferation, self-renewal, and metastasis of liver cancer. Chemicals and Materials Chemicals and Materials Reserpine powder (R817202), purity greater than 98.0%, was purchased from Jiusuo Technology Co. Ltd. (Guangzhou, China). Glacial acetic acid (B018) was purchased from Feng Wei Technology Co. Ltd. (Guangzhou, China). The preparation process of the reserpine solution is as follows: 10 mg of reserpine powder was added with 10 ml acetic acid to obtain 1 mg/ml reserpine solution A and stored at 4°C. Before each experiment, 0.1 ml reserpine solution A and 9.9 ml sodium chloride as injection were added to obtain 0.01 mg/ml reserpine solution B. In addition to special labeling, other chemicals were purchased from UZhiYi Biotechnology Co. Ltd. (Guangzhou, China). The mice were forced to swim, and the time from the beginning of swimming to the ﬁrst stop swimming was recorded, which is helpful to judge the effect of the SD model in mice. In this case, a water tank with a smooth sidewall and a suitable size was prepared with water temperature kept at 25°C. First, the mice were put into a water tank for 30 s, and then the mice were acclimated to the water tank environment. The mice that could not swim or could not swim very well were excluded, and the order of the mice that were put into the water tank was recorded. After the mice returned to their normal state, they were placed in the tank again in the same order as before, and the total duration of swimming–struggling–ﬂoating immobility was recorded and a statistical analysis of the results performed. BALB/C-Nu Mice Tumorigenesis Experiment When C57BL/6 mice were induced with SD for 1 week, precultured hepa1-6 cell suspension (1 × 107/ml) was subcutaneously injected into the armpit of BALB/C nude mice at 0.1 ml. Each mouse was inoculated with 1 × 106 cells. The mental status and tumorigenesis of the nude mice were observed every day. When the diameter of the subcutaneous tumor was Animals and Groups Animals and Groups p Forty male SPF C57BL/6 mice (4 weeks old, 18–20 g) and three male SPF BALB/C-Nu (4 weeks old, 16–18 g) were purchased October 2021 \| Volume 12 \| Article 757194 Frontiers in Pharmacology \| www.frontiersin.org 3 Exosome Regulates Spleen Deﬁciency HCC Wang et al. TABLE 1 \| Spleen deﬁciency rating scale. Item One point Two-point Three-point Four-point Odor No unusual Faint odor Serious odor Extremely odor Mental state Normal Tiredness Drowsy Irritability Body cold and heat Normal Crouch Serious cold Extremely cold Respiratory status Normal Pant Shortness of breath Faint breath Hair color Normal No bright Shaggy hair Rarer and yellow Stool Normal Slight wettability Serious wettability Extreme wettability Food intake Normal Reduce food intake by 50% Reduce food intake by 75% Barely food intake The calculated sum of the scores of each item (total score ≤6, no SD; 7 ≤total scores ≤12, mild SD; 13 ≤total scores ≤18, typical SD; 19 ≤total scores ≤24, severe SD). TABLE 1 \| Spleen deﬁciency rating scale. Item One point Two-point Three-point Four-point Odor No unusual Faint odor Serious odor Extremely odor Mental state Normal Tiredness Drowsy Irritability Body cold and heat Normal Crouch Serious cold Extremely cold Respiratory status Normal Pant Shortness of breath Faint breath Hair color Normal No bright Shaggy hair Rarer and yellow Stool Normal Slight wettability Serious wettability Extreme wettability Food intake Normal Reduce food intake by 50% Reduce food intake by 75% Barely food intake The calculated sum of the scores of each item (total score ≤6, no SD; 7 ≤total scores ≤12, mild SD; 13 ≤total scores ≤18, typical SD; 19 ≤total scores ≤24, severe SD). medium, and these were then beaten into a single-cell suspension, stained using trypan blue stain, and counted. The cells were inoculated into a 96-well plate, and another blank control well was set up, which was only adding a complete culture medium, and cultured in the incubator (37°C, 5% CO2, saturation humidity) for 24 h. The cells were cultured to form a monolayer covering the bottom of the well. The culture solution was then removed and added with 10 μL PBS (control group), serum exosomes of liver cancer, serum exosome of liver cancer with SD, and serum exosome of liver cancer with SD that combined with 50 ng/ml CTLA-4, 100 ng/ml CTLA-4, or 200 ng/ml CTLA- 4 inhibitor. Apoptosis Assay Apoptosis of HepG2 cells was detected by Annexin V–FITC/PI double-labeled ﬂow detector. The cells were cultured in a DMEM medium containing 10% fetal bovine serum (FBS) at 37°C, 5% CO2, and saturated humidity for the logarithmic phase. The cell concentration was adjusted to 1 × 104/ml. The cells were inoculated into a six-well cell plate with each well about 2 ml. Then, added with 10 µL PBS (control group), serum exosomes of liver cancer, serum exosome of liver cancer with SD, and serum exosome of liver cancer with SD that combined with 200 ng/ml CTLA-4 inhibitor. According to the instructions of the Annexin V–FITC/PI double staining kit, the cells were collected 24 h later, digested by trypsin, then centrifuged to collect the cells. After resuspending the cells with 1 ml precooled PBS, 100 μL of cell suspension solution was transferred to a 5-ml culture tube and added with 5 μL Annexin V–FITC and 5 μL PI. After incubation at room temperature for 15 min in the dark, the cells were added with 400 μL of combined buffer and analyzed by ﬂow cytometry. Cell Migration Assay HepG2 cells (HepG2, HepG2 + HCC exosome, HepG2 + SD- HCC exosome, HepG2 + SD-HCC exosome + CTLA-4 inhibitor) were selected in the logarithmic phase. The cells were digested with trypsin, centrifuged, and washed twice with PBS. The concentration of cells was adjusted to 2 × 105/ml in a serum- Establishment of Liver Cancer Model One week after the SD model was established, the orthotopic liver cancer models were established in the liver cancer group and the spleen liver cancer group. Mice were anesthetized with 1% pentobarbital sodium solution (50 mg/kg). At the same time, the BALB/C nude mice were killed by cervical dislocation, the tumor was quickly removed under aseptic conditions, the necrotic tissue was removed, and the ﬂesh-like tissue was cut into 1-mm3- sized pieces in PBS solution. The skin was incised at the xiphoid process and the left lobe of the liver pulled out of the abdominal cavity. The 1-mm3-sized tumor tissue was placed in the cannula (5 mm from the tip of the cannula), and the cannula was inserted into the liver surface at an angle of 10 degrees, then the tumor tissue was implanted under the liver capsule. Absorbable gelatin sponge was applied to the bleeding area, and the liver lobe was sent back to the abdominal cavity. Penicillin solution was used to ﬂush the abdominal cavity, and absorbable suture was used to close the abdominal cavity. The mice were closely monitored for survival and weighed daily. After the liver cancer model was established for 28 days, blood and tissue samples were collected. Extraction of Exosomes The serum exosomes of mice were extracted by ultracentrifugation. The blood (500 µL) was collected in a centrifuge tube, then centrifuged at 4°C for 5 min at 3,000 r/ min after the blood had coagulated. The supernatant serum was transferred to a new centrifuge tube and centrifuged at 4°C for 10 min at 300 × g. The supernatant was centrifuged at 4°C for 10 min at 2000 × g, then the precipitates were discarded, and the supernatant was further centrifuged for 70 min at 10000 × g. After this step, the supernatant was transferred to a new high speed centrifuge tube, centrifuged at 4°C for 70 min at 100,000 × g. The supernatant was discarded and added with the appropriate amount of PBS buffer to make the exosomes suspended in the PBS buffer. The suspension was centrifuged at 4°C for 70 min at 100,000 × g, and the supernatant was discarded. The bottom precipitated exosomes were suspended in 200 μL of 4°C precooled PBS buffer and kept at −80°C for future usage. Animals and Groups After culturing at 37°C for 72 h, 50 μL (1 mg/ml) of MTT was added and cocultured for 4 h, then the supernatant was discarded and replaced with 150 μL DMSO solution. The plate was placed in the microwell plate oscillator to oscillate for 15 min, and the blue-purple crystal dissolved completely. The OD values of each well were measured at 570 nm wavelength by enzyme- linked immunosorbent assay. Each group was tested with 12 repeating wells. about 1 cm at 2 weeks, the tumor could be used to establish the liver cancer model. MTT Assay HepG2 cells were digested with 0.25% trypsin at the logarithmic growth stage, digestion was stopped with serum-containing October 2021 \| Volume 12 \| Article 757194 Frontiers in Pharmacology \| www.frontiersin.org 4 Wang et al. Exosome Regulates Spleen Deﬁciency HCC free medium. The transwell chamber was placed in a 24-well plate, 100 μL cell suspension was added in the transwell chamber, then along with 5 µL PBS (control group), serum exosomes of liver cancer, serum exosome of liver cancer with SD, and serum exosome of liver cancer with SD that combined with 200 ng/ml CTLA-4 inhibitor were added. 600 μL medium containing 10% FBS was added in the lower chamber and incubated for 24 h at 37°C in 5% CO2. After removing the transwell chamber and discarding the culture ﬂuid from the well, it was rinsed with PBS and 4% paraformaldehyde for 30 min. The chamber was dried properly and dyed with crystal violet for 30 min, using a cotton swab to gently remove the non-penetrating cells from the surface of the membrane, and washed twice with PBS. Under the microscope, ﬁve high-power (×400) views were randomly selected to observe the cells in each group. The number of transmembrane cells was used to express the ability of locomotion. Cells of each group were set into three double wells. labeled, and set aside. The histomorphological structure was observed under the microscope. Western Blotting Assay g Firstly, liver tissue of the normal group and SD group, and liver cancer tissue of the liver cancer group and SD liver cancer group were extracted. The tissues were split with RIPA for 30 min, and then the split solution was transferred to a centrifuge tube and centrifuged for 10 min at 4°C, 12,000 rpm. The supernatant (protein sample) was packed in a 0.5-ml centrifuge tube and stored at −20°C. The concentration of protein was determined by the BCA (bicinchoninic acid) method. Immunohistochemical Assay The tissue was ﬁxed using 10% formaldehyde, dehydrated from low concentration alcohol to high concentration alcohol, made transparent using xylene, embedded in parafﬁn, and sectioned. The tissue slices were placed in the oven at 60°C for 30 min, and then were taken out, cooled, dewaxed, and hydrated with high concentration alcohol to low concentration alcohol, which were then was incubated with 3% hydrogen peroxide at room temperature for 5–10 min. The slices were repaired by microwave in 0.01 mol/L citric acid buffer (pH 6.0) for 30 min and sealed with 5% normal sheep serum for 30 min at room temperature. The sheep serum was removed from the section and added to the diluted primary antibody (1:250). Then, this was incubated overnight at 4°C. The diluted secondary antibody was added and incubated at 37°C for 30 min. This was then rinsed with PBS for 2 min (three times). A moderate amount of Streptomyces antibiotic protein peroxidase was added and was then incubated at 37°C for 30 min and rinsed with PBS for 3 min (two times). These were then stained with DAB color for 5 min and rinsed thoroughly with distilled water. Restaining, dehydration, transparency, and sealing were performed. Five high- power microscopic views were randomly selected from each section to observe the staining of the cells. Wound-Healing Assays Lines were drawn across the back of the six-well plate, and 5 × 105 cells were inoculated into each well. The cell processing methods are the same as the Apoptosis Assay. On the next day, the back of the six-well plate was scratched using the tip of a pipette. The wells were then washed three times with sterile PBS to remove the cells below the underline to ensure that the remaining space was clearly visible. Then, the serum-free medium was replaced, and the cells were cultured in a 5% CO2 incubator at 37°C. At 0, 24 h, and 48 h, the wounds were observed and photographed under a microscope. The width of the wounds at each time was recorded. After the addition of 20 g of the sample, the protein was separated by 10% SDS-PAGE and transferred to the PVDF membrane by the wet transfer membrane. When the membrane transfer is completed, the membrane is cut into different bands according to the target protein molecule of the target protein. Sealing ﬂuid containing 5% skimmed milk powder was used to seal this for 2 h at room temperature. The diluted (1:1000) primary antibody (CTLA-4, CD80, CD86, PD-1, PD-L1, PTEN, CD44, and β-Actin) was added and rested at 4°C overnight. It was then rinsed with TBST three times (10°min/time). An HRP-labeled anti-rabbit or anti-rat IgG was used as the secondary antibody (1:10000) and incubated at room temperature for 1 hour. This was rinsed again with TBST for three times (10 min/time). Finally, chemiluminescence detection (light-proof operation) was performed, and β-actin was used as the internal control. The ratio of various proteins to β-actin indicates the relative expression level of each protein. Cell Invasion Assay A serum-free medium containing ﬁbronectin was added to the lower chamber of the transwell cell membrane. At 4°C, Matrigel was melted overnight, diluted in the serum-free medium, and applied to the upper surface of the transwell chamber at an amount of 100 μL Matrigel (1 mg/ml) per well. This was set in a 37°C incubator for 30 min. The other experimental steps and cell processing methods were the same as the Cell Migration Assay. The invasive ability was expressed by the number of transmembrane cells. The experiment was repeated three times. H&E Staining of Tissue Sections g The liver tissues of the normal group, SD group, liver cancer group, and SD liver cancer group were selected. After the tissue was ﬁxed with 10% formaldehyde, it was put into alcohol ranging from low-concentration alcohol to high-concentration alcohol for dehydration treatment. The tissue block was immersed in xylene for transparency and then embedded in parafﬁn. The parafﬁn wax was removed with xylene before staining. Then the slides were washed through the high concentration to low concentration of alcohol, and ﬁnally immersed into distilled water. The slices were dyed in hematoxylin solution for 5 min, ﬂushed with water for 1 hour, then dehydrated with 70 and 90% alcohol for 10 min and stained by eosin for 2 minutes. After staining, the slides were dehydrated with alcohol from low concentration to high concentration and then made transparent by using xylene. Finally, the slices are sealed, Frontiers in Pharmacology \| www.frontiersin.org Real-Time Quantitative PCR Analysis Real-Time Quantitative PCR Analysis For liver tissue of the normal group and SD group, liver cancer tissue of liver cancer group and SD liver cancer group, the total RNA was extracted according to the instructions of the TRIzol RNA extraction kit. RNA concentrations were measured using an October 2021 \| Volume 12 \| Article 757194 Frontiers in Pharmacology \| www.frontiersin.org 5 Exosome Regulates Spleen Deﬁciency HCC Wang et al. FIGURE 1 \| Preparation of orthotopic transplantation liver cancer model with SD. (A) Flow chart; (B) transplantation process: 1) shave and disinfect the surgical area, 2) squeeze out the liver lobe, 3) inoculate the tumor mass, 4) suture the wound; (C) comparison of tumor size; (D) bodyweight change curve; (E) SD score statistics; (F) changes in the diet of mice during the preparation of SD model; (G) stop swimming time statistics. n 3, p < 0.05, p < 0.01, *p < 0.001. IGURE 1 \| Preparation of orthotopic transplantation liver cancer model with SD. (A) Flow chart; (B) transplantation process: 1) shave and disinfect the surgical rea, 2) squeeze out the liver lobe, 3) inoculate the tumor mass, 4) suture the wound; (C) comparison of tumor size; (D) bodyweight change curve; (E) SD score statistics; F) changes in the diet of mice during the preparation of SD model; (G) stop swimming time statistics. n 3, p < 0.05, p < 0.01, p < 0.001. FIGURE 1 \| Preparation of orthotopic transplantation liver cancer model with SD. (A) Flow chart; (B) transplantation process: 1) shave and disinfect the surgical area, 2) squeeze out the liver lobe, 3) inoculate the tumor mass, 4) suture the wound; (C) comparison of tumor size; (D) bodyweight change curve; (E) SD score statistics; (F) changes in the diet of mice during the preparation of SD model; (G) stop swimming time statistics. n 3, p < 0.05, p < 0.01, p < 0.001. ultraviolet photometer, and mRNA was reverse-transcribed into cDNA by reverse transcription kit then stored at −20°C after the reaction. GAPDH was used as the internal control for the relative quantitative analysis. Then, PCR ampliﬁcation was performed using cDNA as a template. The total reaction system of real-time PCR was 20 L. The ampliﬁcation procedures were pre-denaturation in 95°C, 2 min; denaturation in 95°C, 20 s; annealing in 60°C, 20 s; and extension in 72°C, 30 s, altogether 40 cycles. Real-Time Quantitative PCR Analysis At 72°C for 10 min after the last cycle, the relative expression levels of CTLA-4, PD-1, PD-L1, PTEN, and CD44 mRNA were analyzed by the 2−△△Ct analytical method. The experiment was repeated three times independently. Genespeciﬁc primers are listed at supplementary material. measurement. The data are expressed as mean ± standard deviation. The differences between the two groups were tested by Student’s t-test, and the differences among many groups were compared by variance analysis. Groups tested with p < 0.05 showed that the difference was statistically signiﬁcant. All the tests were repeated three times independently. Establishment of the SD-HCC Model With Reserpine After passing the quarantine period, the mice were treated with reserpine (0.1 mg/kg) daily for 14 days. Orthotopic liver cancer transplantation was performed 14 days later, and the specimens were collected after 49 days. The ﬂow chart is shown in Statistical Analysis SPSS 26.0 software was used for data analysis and Prism GraphPad 5.0 software was used for mapping and October 2021 \| Volume 12 \| Article 757194 Frontiers in Pharmacology \| www.frontiersin.org 6 Wang et al. Exosome Regulates Spleen Deﬁciency HCC FIGURE 2 \| Exosome veriﬁcation. (A) TEM images of 1) HCC exosomes, 2) SD-HCC exosomes; (B) exosomal protein level detection band chart; (C) size distribution of exosomes; (D) histogram of exosomal protein levels. n 3, p < 0.05, **p < 0.001. FIGURE 2 \| Exosome veriﬁcation. (A) TEM images of 1) HCC exosomes, 2) SD-HCC exosomes; (B) exosomal protein level detection band chart; (C) size distribution of exosomes; (D) histogram of exosomal protein levels. n 3, p < 0.05, *p < 0.001. FIGURE 3 \| In vitro evaluation of cell proliferation of exosomes. (A) Relative cell viability of HepG2 after 72 h incubation of HCC exosomes and SD-HCC exosomes; (B) the Annexin V/PI apoptosis assay was measured by FCM analysis of HepG2 cells after being treated with each group for 24 h. n 3, p < 0.001. 1) HepG2, 2) HepG2 + HCC exosome, 3) HepG2 + SD-HCC exosome, 4) HepG2 + SD-HCC exosome + CTLA-4 inhibitor. FIGURE 3 \| In vitro evaluation of cell proliferation of exosomes. (A) Relative cell viability of HepG2 after 72 h incubation of HCC exosomes and SD-HCC exosomes; (B) the Annexin V/PI apoptosis assay was measured by FCM analysis of HepG2 cells after being treated with each group for 24 h. n 3, p < 0.001. 1) HepG2, 2) HepG2 + HCC exosome, 3) HepG2 + SD-HCC exosome, 4) HepG2 + SD-HCC exosome + CTLA-4 inhibitor. FIGURE 3 \| In vitro evaluation of cell proliferation of exosomes. (A) Relative cell viability of HepG2 after 72 h incubation of HCC exosomes and SD-HCC exosomes; (B) the Annexin V/PI apoptosis assay was measured by FCM analysis of HepG2 cells after being treated with each group for 24 h. n 3, p < 0.001. 1) HepG2, 2) HepG2 + HCC exosome, 3) HepG2 + SD-HCC exosome, 4) HepG2 + SD-HCC exosome + CTLA-4 inhibitor. October 2021 \| Volume 12 \| Article 757194 Frontiers in Pharmacology \| www.frontiersin.org 7 Exosome Regulates Spleen Deﬁciency HCC Wang et al. FIGURE 4 \| Effect of exosomes of SD on growth and invasion of HCC cells. Statistical Analysis (A) Cell migration experiment, (B) histogram of migration experiment statistics, (C) cell invasion test, (D) invasion experiment statistics histogram, (E) cell scratch test, (F) scratch experiment statistics histogram. n 3, p < 0.05, p < 0.01,p < 0.001, 1) HepG2, 2) HepG2 + HCC exosome, 3) HepG2 + SD-HCC exosome, 4) HepG2 + SD-HCC exosome + CTLA-4 inhibitor. FIGURE 4 \| Effect of exosomes of SD on growth and invasion of HCC cells. (A) Cell migration experiment, (B) histogram of migration experiment statistics, (C) cell invasion test, (D) invasion experiment statistics histogram, (E) cell scratch test, (F) scratch experiment statistics histogram. n 3, p < 0.05, p < 0.01,p < 0.001, 1) HepG2, 2) HepG2 + HCC exosome, 3) HepG2 + SD-HCC exosome, 4) HepG2 + SD-HCC exosome + CTLA-4 inhibitor. Figure 1A. The SD score (Luo et al., 2017) is an important method for assessing SD by evaluating scores for factors including body odor, mental state, chill and fever, respiration, fur, feces, and appetite. The SD scores assessment showed that the SD group had lower scores than the control group (p < 0.001) (Figure 1E). Compared with the control group and HCC group, the weight of the mice in the SD-HCC and SD groups was lower and signiﬁcantly different (p < 0.01) during 5–49 days (Figure 1D). The SD group consumed less food per day than the control group (Figure 1F). The forced swim test revealed that the stop swimming time of the SD group was shorter for the ﬁrst time (Figure 1G). The preparation process of orthotopic transplantation liver cancer model is as follows: ﬁrst, mouse HCC hepa1-6 cells were inoculated subcutaneously into nude mice. When a 1 cm3 tumor was formed, the tumor was taken out and cut into 1-mm3-sized pieces for use. The operation area in the C57BL/6 black mouse was shaved and disinfected. A 0.5–0.8 mm incision was made near the breastbone, and the liver was extruded. The spare tumor tissue was inoculated into the liver lobe with a trocar. After pressing the hemostatic, the liver lobe was returned to the body, and the incision was sutured. The animals were dissected 28 days after the operation. The liver tumor grew well, and the tumor size of the mice in the HCC group was signiﬁcantly smaller than that in the SD-HCC group, which demonstrated that the orthotopic transplantation model was successfully prepared. Exosomes Extraction and Veriﬁcation We chose mice from the HCC group and the SD-HCC group. After the preparation of the model was completed, blood was taken, and exosomes were extracted. We examined the morphology of HCC exosomes and SD-HCC exosomes by using transmission electron microscopy (TEM). The results displayed exosomes as spherical, membrane-bound vesicles (Figure 2A). The hydrodynamic diameter of the HCC and SD-HCC exosomes was measured to be 43 and 68 nm by dynamic light scattering (Figure 2C). Detecting the components of exosomes, we found that the levels of CTLA- 4 and AKT in serum exosomes in SD-HCC were higher than those in the HCC group (p < 0.001). The contents of PD-1 and PTEN were higher than those in the HCC group, and the difference was signiﬁcant (p < 0.05), suggesting that the exosomes were successfully extracted, and SD could affect the expression of CTLA-4 and other proteins in the exosomes (Figures 2B,D). Statistical Analysis The transplantation process and tumor size comparison pictures are shown in Figures 1B,C. Frontiers in Pharmacology \| www.frontiersin.org Proliferation Inhibition To explore the relationship between SD and cell proliferation in vitro, we generated cell growth curves evaluated by MTT. October 2021 \| Volume 12 \| Article 757194 Frontiers in Pharmacology \| www.frontiersin.org 8 Exosome Regulates Spleen Deﬁciency HCC Wang et al. FIGURE 5 \| Liver tissue slice morphology and molecular mechanism of liver tumor occurrence and development. (A) H&E staining observation of morphological changes of liver tissue sections of mice in different treatment groups, (B) immunohistochemical detection of CTLA-4 and other immune checkpoint protein distribution, (C)Western blot detection of CTLA-4 and other immune checkpoint-related protein band diagram, (D) histogram, (E) RT-PCR detects the expression of mRNA related to immune checkpoints. n 3, p < 0.01,p < 0.001, 1) control group, 2) SD group, 3) HCC group, 4) SD-HCC group. FIGURE 5 \| Liver tissue slice morphology and molecular mechanism of liver tumor occurrence and development. (A) H&E staining observation of morphological changes of liver tissue sections of mice in different treatment groups, (B) immunohistochemical detection of CTLA-4 and other immune checkpoint protein distribution, (C)Western blot detection of CTLA-4 and other immune checkpoint-related protein band diagram, (D) histogram, (E) RT-PCR detects the expression of mRNA related to immune checkpoints. n 3, p < 0.01,***p < 0.001, 1) control group, 2) SD group, 3) HCC group, 4) SD-HCC group. DISCUSSION HCC is one of the most malignant cancers and is the third leading cause of cancer-related death due to its high recurrence and poor prognosis (Chen et al., 2016). HBV infection is a major risk factor for HCC, and Chinese HCC patients account for 50% of HCC patients in the world (Bray et al., 2018; Feng et al., 2019). Currently, treatment strategies for HCC patients include surgery, liver transplantation, chemotherapy, radiotherapy, biological therapy, and targeted molecular therapy, but the overall effect is not satisfactory due to the highly metastatic nature of HCC (Couri and Pillai, 2019). Therefore, it is necessary to search for reliable biomarkers and therapeutic targets to monitor the progress of HCC. The imbalance of the body’s internal environment is closely related to the occurrence and development of cancer, and genetic inheritance can only explain 5% of the occurrence of cancer (Bhattacharjee et al., 2013; Craig et al., 2020; Llovet et al., 2021). Eventually, changes in the body’s internal environment are unfavorable to the survival of normal cells but appropriate to the survival of tumor cells. “Bian Zheng Lun Zhi,” the core of TCM therapy systems, is based on syndrome differentiation, which helps to guide the design of individualized treatments (Ji et al., 2016; Zhang et al., 2020). Syndromes such as loose stools, abdominal distension after meals, loss of appetite, sallow complexion, weight loss, general weakness, and/or low disease resistance could be summarized as SD in TCM In recent years, tumor immunotherapy has been the focus of research which has brought great beneﬁts to cancer patients. However, due to a variety of complex factors, it is easy for tumors to escape from the host immune system during the treatment process, leading to treatment failure (Whiteside et al., 2011). Moreover, tumor immunotherapy also induces some serious toxic events and autoimmune diseases, such as pituitary hypophysitis, autoimmune hepatitis, pneumonia, and so on (Cogdill et al., 2017; Couzin-Frankel, 2017). Therefore, it is very necessary to ﬁnd out the cancer-related immune mechanism. Exosomes (30–150 nm) are considered as messengers between cells, carrying a large number of macromolecules, including proteins, mRNAs, lipids, and miRNAs, which constitutes an important part of the tumor immune microenvironment (Wen et al., 2016; Bach et al., 2017; Ruivo et al., 2017; Su et al., 2021). H&E Staining of Liver Tumor Hematoxylin and eosin (H&E) staining of the liver tissues from the HCC and SD-HCC groups showed all sizes of carcinoma cells with some multinucleated giant tumor cells, and the funicular slices were distributed and accumulated irregularly without normal hepatocyte construction (Figure 5A). To some extent, the results indicated that the degree of malignancy of the tumor tissue was higher in the SD-HCC group than in the HCC group. Cell Migration and Invasion the scratches were made. The differences are statistically signiﬁcant (p < 0.001) (Figures 4E,F). Cell Migration and Invasion Brieﬂy, 10 µl of PBS, liver cancer mouse serum, SD liver cancer mouse serum, SD liver cancer mouse serum plus CTLA-4 inhibitors were added separately into a 96-well plate with HepG2 cells. The results revealed that HCC cell proliferation was promoted by SD liver cancer mouse serum compared with liver cancer mouse serum in 48 and 72 h. The addition of CTLA-4 inhibitors could partially reverse this change (Figure 3A). Apoptosis study of HepG2 cells tested by ﬂow cytometry showed that SD liver cancer mouse serum could induce least miniature cells apoptosis compared with liver cancer mouse serum. The addition of CTLA-4 inhibitors could partially reverse cell apoptosis (Figure 3B). g To study the effect of SD on the migration and invasion of liver cancer cells, the HepG2 cells were treated with SD liver cancer mouse serum or liver cancer mouse serum for 48 h. The cell scratch method and transwell test were used to detect the migration and invasion ability of HepG2 cells after drug treatment. We found that compared with the HCC group and the SD-HCC add CTLA-4 inhibitor groups, the SD-HCC group had a stronger migration ability (Figures 4A,B) and stronger invasion ability (Figures 4C,D). In the scratch experiment, we observed that the diameter of the scratches in the SD-HCC group was narrower than in the other three groups at 24 and 48 h after October 2021 \| Volume 12 \| Article 757194 Frontiers in Pharmacology \| www.frontiersin.org 9 Exosome Regulates Spleen Deﬁciency HCC Wang et al. theoretical system. In the clinical investigation of 767 cases of gastric cancer patients, 32.86% of patients showed the syndrome of SD and stomach cold syndrome (Sun et al., 2010). SD syndrome is also considered to be the key pathogenesis of colorectal cancer (Sun et al., 2016a). It has been reported that traditional Chinese medicine for invigorating the spleen helps in delaying the pathological process of HCC cachexia induced by ascites by reducing the levels of IL-Lα, IL-6, and TNFα and inhibiting the activation of the ubiquitin–proteasome pathway (Sun et al., 2016b). Meanwhile, traditional Chinese medicine in invigorating the spleen could prolong the survival time and decrease tumor metastasis in the liver in mice, which may be associated with enhancing the expression of PTEN in the liver (Yin et al., 2008). Therefore, the internal environment of SD is especially vital for the occurrence and development of immune recognition disorders of HCC. Molecular Mechanism of Liver Tumor Occurrence and Development In order to verify the inﬂuence of the internal environment of SD on liver tumor occurrence and development, we established liver cancer models and SD liver cancer models in the early stage. The results of immunohistochemistry and WB (Western Blot) showed that both the SD group and liver cancer group mice had signiﬁcantly higher (p < 0.01) levels of CTLA-4 and PD-1 compared with the normal group. In addition, the liver cancer mice with SD had signiﬁcantly higher levels of CTLA-4 and PD-1 proteins than those in the liver cancer group only (p < 0.01) (Figures 5B–D). The mRNA expression in tissues as analyzed by the RT-PCR test showed a similar trend (Figure 5E). All these results suggest that the internal environment of SD may affect liver tumor occurrence and development by disturbing CTLA-4 and other series of proteins and genes. In our study, reserpine was used to establish a model of SD syndrome referring to the previous researches (Zhao et al., 2011; Luo et al., 2017). Chronic rather than acute doses of reserpine were used to induce the syndrome of SD in the mice in this study. In our analysis, we referred to each factor in the SD scores assessment (Luo et al., 2017) and found that the score of the SD group was higher than that of the control group. During the establishment of the model of SD syndrome, there were also signiﬁcant differences between the SD group and the control group in the amount of daily food intake and the time to stop swimming. In addition, compared with the control group, the weight of the mice in the SD group decreased signiﬁcantly, and this effect continued throughout the modeling process. The results of H&E staining showed that the malignant degree of tumor in the SD-HCC group was higher than that in the HCC group. These results indicated that the SD model was successfully established, and the factor of SD indeed affected the growth of the HCC mice. Frontiers in Pharmacology \| www.frontiersin.org DISCUSSION These characteristics of exosomes are consistent with the results of other studies, which demonstrates that we successfully extracted exosomes of HCC. Studies have found that tumor-derived exosomes carry immunosuppressive proteins, such as PD-1, CTLA-4, FasL, TRAIL, etc. (Whiteside, 2013; Ukrainskaya et al., 2019; Benecke et al., 2021). Detecting the components of exosomes, we found the presence of PD-1, CTLA-4, PTEN, and AKT. Interestingly, the level of CTLA-4 of the SD-HCC group was the highest, which may indicate that CTLA-4 plays an important role in exosome HCC with SD syndrome. CTLA-4 is highly expressed in lymphocytes CD4 + and CD8 + T (Phung et al., 2020), competing with the T-cell costimulator CD28 for binding to CD80 and CD86 with a higher afﬁnity, which conveys inhibitory signals within the T cells (Van Coillie et al., 2020; Goenka et al., 2021). We examined liver tumor tissues to verify this relationship between CTLA-4 and T cells in the internal environment of SD. The results of immunohistochemistry and WB showed that CTLA-4, CD80, and CD86 were higher in the HCC and SD-HCC groups than in the other groups, and it was signiﬁcantly higher in the SD-HCC group. The mRNA expression showed the same results. These data may indicate that T-cell activation and function were restricted. It has been reported that mutations in the CTLA-4 gene are associated with an increased risk of human autoimmune disorders (Ueda et al., 2003; Schubert et al., 2014). In immunotherapy of cancer, the use of blocking anti–CTLA-4 and anti–PD-1 antibodies have yielded promising results, and the 2018 Nobel Prize was granted to Jim P. Allison and Tasuku Honjo, who are the pioneers in this ﬁeld (Wolchok, 2018). In the cell proliferation experiment, compared with the serum of HCC mice at 48 and 72 h, the serum of SD-HCC mice promoted the proliferation of HCC cells and the addition of CTLA-4 inhibitor could partially reverse this change. Detecting apoptosis on HepG2 cells showed that SD-HCC mice serum can induce the least cell apoptosis compared with liver cancer mouse serum and when added into CTLA-4 inhibitors could partially reverse cells apoptosis. In order to study the effect of SD on the migration and invasion of HCC cells, cell scratch assay and transwell test were used after drug treatment. Compared with the HCC group and the CTLA-4 inhibitor group, the SD-HCC group had stronger migration ability and stronger invasion ability. DISCUSSION These results suggest that SD may boost the occurrence and development of liver cancer through exosome CTLA-4. g g y In conclusion, in the HCC mouse model, we found that SD plays a key role in the occurrence and development of immune recognition disorders of HCC. In addition, at the molecular level, the exosome CTLA-4 expression in the SD- HCC group was upregulated, which played a powerful role in regulating the growth, self-renewal, and metastasis of HCC. SD also promoted the PTEN/CD44 pathway process. These important ﬁndings suggest that the internal environment of SD exosome CTLA-4 promotes the metastasis of liver cancer by regulating the PTEN/CD44 pathway. However, the molecular mechanism of how CTLA-4 regulates the PTEN/CD44 pathway remains unclear and needs further study. DATA AVAILABILITY STATEMENT The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors. PTEN is an important tumor suppressor protein, and a loss in its function is associated with malignant transformation, progression, chemotherapy response, and survival of a variety of cancers (Su et al., 2016; Wang et al., 2018a; Luongo et al., 2019; Raffone et al., 2019). PTEN has an important effect on regulating the PI3K/Akt/mTOR signaling pathway, which plays an important role in the proliferation and maintenance of CSC self-renewal, according to reports (Su et al., 2016; Yang et al., 2016; Luongo et al., 2019). The relationship between PTEN and PI3K/Akt/mTOR can be partly illustrated in our study according DISCUSSION In addition, at the molecular level, the exosome CTLA-4 expression in the SD- HCC group was upregulated, which played a powerful role in regulating the growth, self-renewal, and metastasis of HCC. SD also promoted the PTEN/CD44 pathway process. These important ﬁndings suggest that the internal environment of SD exosome CTLA-4 promotes the metastasis of liver cancer by regulating the PTEN/CD44 pathway. However, the molecular mechanism of how CTLA-4 regulates the PTEN/CD44 pathway remains unclear and needs further study. to the result of detecting exosome components, which displayed the presence of PTEN and Akt in the exosomes and was higher in the SD-HCC group than in the HCC group. Growing evidence has indicated that PTEN has speciﬁc functions to interfere with stem/progenitor cells, including regulating the differentiation of neural SCs, promoting the differentiation of mesenchymal SCs, and maintaining the balance between proliferation and differentiation of hematopoietic SCs (Su et al., 2016). It is well known that CSCs have the characteristics of self-renewal and differentiation (Luongo et al., 2019; Nero et al., 2019; Cetintas and Batada, 2020). There are multiple biomarkers of CSCs, including CD133, SOX2, BMI1, CD44, Nanog, and ABCG2 (Hu et al., 2020). The PTEN/CD44 signaling pathway plays an important role in maintaining the characteristics of CSCs, involved in tumorigenesis, invasion and metastasis, and immune escape (Luongo et al., 2019; Nero et al., 2019; Cetintas and Batada, 2020). In this study, the expression of PTEN and CD44 is the highest in the SD-HCC group than in the other groups according to the results of immunohistochemistry, WB, and mRNA. We inferred that the SD factor could promote the expression of PTEN and CD44 and thus has a positive effect on the differentiation of CSCs. Besides liver cancer, the current treatments of various types of other cancers are also waiting for better outcomes. Although there are multiple forms of technologies those were explored, e.g., photoacoustic technology, reactive oxygen species, and cytotoxic peroxynitrite (ONOO−), the efﬁciency of cancer treatments is still far from satisfactory (Wang et al., 2021; Zhang et al., 2021). Here, our ﬁndings from exosomes may provide some hints from a new angle for high efﬁciency of treatment of various cancers. for separation of the exosomes. Exosomes were observed as spherical, membrane-bound vesicles by using TEM. The hydrodynamic diameter of the exosomes was measured as 43 and 68 nm by dynamic light scattering. DISCUSSION Exosomes are nanoscale membranous vesicles secreted from intracellular multivesicular bodies (MVBs) or late endosomes into the extracellular space through extracellular action (Su et al., 2021). CD81, CD63, and TSG101 have become the most commonly used exosomal labeling proteins (Pegtel and Gould, 2019). In our study, after the establishment of the model, we selected the serum of the HCC group and SD-HCC group mice October 2021 \| Volume 12 \| Article 757194 Frontiers in Pharmacology \| www.frontiersin.org 10 Wang et al. Exosome Regulates Spleen Deﬁciency HCC to the result of detecting exosome components, which displayed the presence of PTEN and Akt in the exosomes and was higher in the SD-HCC group than in the HCC group. Growing evidence has indicated that PTEN has speciﬁc functions to interfere with stem/progenitor cells, including regulating the differentiation of neural SCs, promoting the differentiation of mesenchymal SCs, and maintaining the balance between proliferation and differentiation of hematopoietic SCs (Su et al., 2016). It is well known that CSCs have the characteristics of self-renewal and differentiation (Luongo et al., 2019; Nero et al., 2019; Cetintas and Batada, 2020). There are multiple biomarkers of CSCs, including CD133, SOX2, BMI1, CD44, Nanog, and ABCG2 (Hu et al., 2020). The PTEN/CD44 signaling pathway plays an important role in maintaining the characteristics of CSCs, involved in tumorigenesis, invasion and metastasis, and immune escape (Luongo et al., 2019; Nero et al., 2019; Cetintas and Batada, 2020). In this study, the expression of PTEN and CD44 is the highest in the SD-HCC group than in the other groups according to the results of immunohistochemistry, WB, and mRNA. We inferred that the SD factor could promote the expression of PTEN and CD44 and thus has a positive effect on the differentiation of CSCs. Besides liver cancer, the current treatments of various types of other cancers are also waiting for better outcomes. Although there are multiple forms of technologies those were explored, e.g., photoacoustic technology, reactive oxygen species, and cytotoxic peroxynitrite (ONOO−), the efﬁciency of cancer treatments is still far from satisfactory (Wang et al., 2021; Zhang et al., 2021). Here, our ﬁndings from exosomes may provide some hints from a new angle for high efﬁciency of treatment of various cancers. In conclusion, in the HCC mouse model, we found that SD plays a key role in the occurrence and development of immune recognition disorders of HCC. REFERENCES Hu, H., Luo, S. J., Cao, Z. R., Wu, Y., Mo, Z., Wang, Y., et al. (2020). Depressive Disorder Promotes Hepatocellular Carcinoma Metastasis via Upregulation of ABCG2 Gene Expression and Maintenance of Self-Renewal. J. Cancer 11 (18), 5309–5317. doi:10.7150/jca.45712 Bach, D. H., Hong, J. Y., Park, H. J., and Lee, S. K. (2017). The Role of Exosomes and miRNAs in Drug-Resistance of Cancer Cells. Int. J. Cancer 141 (2), 220–230. doi:10.1002/ijc.30669 Ji, Q., Luo, Y. Q., Wang, W. H., Liu, X., Li, Q., and Su, S. B. (2016). Research Advances in Traditional Chinese Medicine Syndromes in Cancer Patients. J. Integr. Med. 14 (1), 12–21. doi:10.1016/S2095-4964(16)60237-6 Integr. Med. 14 (1), 12–21. doi:10.1016/S2095-4964(16)60237-6 Benecke, L., Coray, M., Umbricht, S., Chiang, D., Figueiró, F., and Muller, L. (2021). Exosomes: Small EVs with Large Immunomodulatory Effect in Glioblastoma. Int. J. Mol. Sci. 22 (7). doi:10.3390/ijms22073600 Lee, Y. R., Chen, M., and Pandolﬁ, P. P. (2018). The Functions and Regulation of Lee, Y. R., Chen, M., and Pandolﬁ, P. P. (2018). The Functions and Regulation of the PTEN Tumour Suppressor: New Modes and Prospects. Nat. Rev. Mol. Cel Biol 19 (9), 547–562. doi:10.1038/s41580-018-0015-0 the PTEN Tumour Suppressor: New Modes and Prospects. Nat. Rev. Mol. Cel Biol 19 (9), 547–562. doi:10.1038/s41580-018-0015-0 Bhattacharjee, P., Banerjee, M., and Giri, A. K. (2013). Role of Genomic Instability in Arsenic-Induced Carcinogenicity. A Review. Environ. Int. 53, 29–40. doi:10.1016/j.envint.2012.12.004 Llovet, J. M., De Baere, T., Kulik, L., Haber, P. K., Greten, T. F., Meyer, T., et al. (2021). Locoregional Therapies in the Era of Molecular and Immune Treatments for Hepatocellular Carcinoma. Nat. Rev. Gastroenterol. Hepatol. 18 (5), 293–313. doi:10.1038/s41575-020-00395-0 Bray, F., Ferlay, J., Soerjomataram, I., Siegel, R. L., Torre, L. A., and Jemal, A. (2018). Global Cancer Statistics 2018: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J. Clin. 68 (6), 394–424. doi:10.3322/caac.21492 Luo, H., Chen, Y., Sun, B., Xiang, T., and Zhang, S. (2017). Establishment and Evaluation of Orthotopic Hepatocellular Carcinoma and Drug-Induced Hepatocellular Carcinoma in Mice with Spleen-Deﬁciency Syndrome in Traditional Chinese Medicine. Afr. J. Tradit Complement. Altern. Med. 14 (1), 165–173. doi:10.21010/ajtcam.v14i1.18 Cetintas, V. B., and Batada, N. N. (2020). Is There a Causal Link between PTEN Deﬁcient Tumors and Immunosuppressive Tumor Microenvironment? J. Transl Med. 18 (1), 45. doi:10.1186/s12967-020-02219-w Chen, W., Zheng, R., Baade, P. D., Zhang, S., Zeng, H., Bray, F., et al. (2016). Cancer Statistics in China, 2015. REFERENCES CA Cancer J. Clin. 66 (2), 115–132. doi:10.3322/ caac.21338 Luongo, F., Colonna, F., Calapà, F., Vitale, S., Fiori, M. E., and De Maria, R. (2019). PTEN Tumor-Suppressor: The Dam of Stemness in Cancer. Cancers (Basel) 11 (8). doi:10.3390/cancers11081076 Mahoney, K. M., Rennert, P. D., and Freeman, G. J. (2015). Combination Cancer Immunotherapy and New Immunomodulatory Targets. Nat. Rev. Drug Discov. 14 (8), 561–584. doi:10.1038/nrd4591 Chen, Y., Sun, B. G., Zhang, S. J., Chen, Z. X., Hardi, C. F., and Xiang, T. (2014). Observations of TCRVβ Gene Expression in Rats with Dampness Syndrome. Evid. Based Complement. Alternat Med. 2014, 373608. doi:10.1155/2014/ 373608 Maker, A. V., Attia, P., and Rosenberg, S. A. (2005). Analysis of the Cellular Mechanism of Antitumor Responses and Autoimmunity in Patients Treated with CTLA-4 Blockade. J. Immunol. 175 (11), 7746–7754. doi:10.4049/ jimmunol.175.11.7746 Cogdill, A. P., Andrews, M. C., and Wargo, J. A. (2017). Hallmarks of Response to Immune Checkpoint Blockade. Br. J. Cancer 117 (1), 1–7. doi:10.1038/ bjc.2017.136 Couri, T., and Pillai, A. (2019). Goals and Targets for Personalized Therapy for HCC. Hepatol. Int. 13 (2), 125–137. doi:10.1007/s12072-018-9919-1 Mo, Z., Cao, Z., Yu, L., Wang, Y., Li, P., Lin, Y., et al. (2020). An Integrative Analysis Reveals the Potential Mechanism between Herbal Medicine Yinchen and Immunoregulation in Hepatocellular Carcinoma. Biomed. Res. Int. 2020, 8886914. doi:10.1155/2020/8886914 Couzin-Frankel, J. (2017). Autoimmune Diseases Surface after Cancer Treatment. Science 358 (6365), 852. doi:10.1126/science.358.6365.852 Science 358 (6365), 852. doi:10.1126/science.358.6365.852 Nero, C., Ciccarone, F., Pietragalla, A., and Scambia, G. (2019). PTEN and Gynecological Cancers. Cancers (Basel) 11 (10). doi:10.3390/ cancers11101458 Craig, A. J., von Felden, J., Garcia-Lezana, T., Sarcognato, S., and Villanueva, A. (2020). Tumour Evolution in Hepatocellular Carcinoma. Nat. Rev. Gastroenterol. Hepatol. 17 (3), 139–152. doi:10.1038/s41575-019-0229-4 Curran, M. A., Montalvo, W., Yagita, H., and Allison, J. P. (2010). PD-1 and CTLA- 4 Combination Blockade Expands Inﬁltrating T Cells and Reduces Regulatory T and Myeloid Cells within B16 Melanoma Tumors. Proc. Natl. Acad. Sci. U S A. 107 (9), 4275–4280. doi:10.1073/pnas.0915174107 Ou, W., Thapa, R. K., Jiang, L., Soe, Z. C., Gautam, M., Chang, J. H., et al. (2018). Regulatory T Cell-Targeted Hybrid Nanoparticles Combined with Immuno- Checkpoint Blockage for Cancer Immunotherapy. J. Control. Release 281, 84–96. doi:10.1016/j.jconrel.2018.05.018 Pegtel, D. M., and Gould, S. J. (2019). Exosomes. Annu. Rev. Biochem. 88, 487–514. doi:10.1146/annurev-biochem-013118-111902 Feng, R. M., Zong, Y. N., Cao, S. M., and Xu, R. H. (2019). ETHICS STATEMENT The animal study was reviewed and approved by the clinical research and animals ethics committee of the ﬁrst afﬁliated hospital, Sun Yat-Sen University. October 2021 \| Volume 12 \| Article 757194 Frontiers in Pharmacology \| www.frontiersin.org 11 Exosome Regulates Spleen Deﬁciency HCC Wang et al. AUTHOR CONTRIBUTIONS 81903967, 81873248), Project of Inheriting Famous TCM Masters of Guangdong Provincial Administration of Traditional Chinese Medicine (No. (2020)1), the Scientiﬁc Research Project of Guangdong Provincial Administration of Traditional Chinese Medicine (No. 20211070). YW, PL, XL, SZ, and LY: conceptualization, methodology, data collection, data analysis, manuscript writing and revision; ZM, ZC, JL, MZ and SM: methodology, data collection. All authors approved the ﬁnal version of the manuscript. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar.2021.757194/ full#supplementary-material This study was supported by the China Postdoctoral Science Foundation (Nos 2020M673026, 2021T140778), the National Natural Science Foundation of China (Nos 8210153649, This study was supported by the China Postdoctoral Science Foundation (Nos 2020M673026, 2021T140778), the National Natural Science Foundation of China (Nos 8210153649, FUNDING The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar.2021.757194/ full#supplementary-material REFERENCES EB- virus Latent Membrane Protein 1 Potentiates the Stemness of Nasopharyngeal Carcinoma via Preferential Activation of PI3K/AKT Pathway by a Positive Feedback Loop. Oncogene 35 (26), 3419–3431. doi:10.1038/onc.2015.402 Su, R., Nan, H., Guo, H., Ruan, Z., Jiang, L., Song, Y., et al. (2016). Associations of Components of PTEN/AKT/mTOR Pathway with Cancer Stem Cell Markers and Prognostic Value of These Biomarkers in Hepatocellular Carcinoma. Hepatol. Res. 46 (13), 1380–1391. doi:10.1111/hepr.12687 Yin, L. R., Chen, Z. X., Zhang, S. J., Sun, B. G., Liu, Y. D., and Huang, H. Z. (2008). Expression of Phosphatase and Tensin Homolog Deleted on Chromosome Ten in Liver of Athymic Mice with Hepatocellular Carcinoma and the Effect of Fuzheng Jiedu Decoction. World J. Gastroenterol. 14 (1), 108–113. doi:10.3748/ wjg.14.108 Su, T., Zhang, P., Zhao, F., and Zhang, S. (2021). Exosomal MicroRNAs Mediating Crosstalk between Cancer Cells with Cancer-Associated Fibroblasts and Tumor-Associated Macrophages in the Tumor Microenvironment. Front. Oncol. 11, 631703. doi:10.3389/fonc.2021.631703 Yu, L., Wang, Z., Mo, Z., Zou, B., Yang, Y., Sun, R., et al. (2021). Synergetic Delivery of Triptolide and Ce6 with Light-Activatable Liposomes for Efﬁcient Hepatocellular Carcinoma Therapy. Acta Pharmaceutica Sinica B. doi:10.1016/j.apsb.2021.02.001 Sun, B., Luo, H., Deng, L., Zhang, S., and Chen, Z. (2016). The Study on Mechanism of the Modiﬁed Chinese Herbal Compound, Jianpijiedu, on a Mouse Model of Hepatic Carcinoma Cachexia. Mol. Med. Rep. 14 (4), 3113–3121. doi:10.3892/ mmr.2016.5602 Yu, Y., Nangia-Makker, P., Farhana, L., G Rajendra, S., Levi, E., and Majumdar, A. P. (2015). miR-21 and miR-145 Cooperation in Regulation of colon Cancer Stem Cells. Mol. Cancer 14, 98. doi:10.1186/s12943-015-0372-7 Sun, D. Z., Liu, L., Jiao, J. P., Wei, P. K., Jiang, L. D., and Xu, L. (2010). Syndrome Characteristics of Traditional Chinese Medicine: Summary of a Clinical Survey in 767 Patients with Gastric Cancer. Zhong Xi Yi Jie He Xue Bao 8 (4), 332–340. doi:10.3736/jcim20100406 Zhang, W. L., Li, N., Shen, Q., Fan, M., Guo, X. D., Zhang, X. W., et al. (2020). Establishment of a Mouse Model of Cancer Cachexia with Spleen Deﬁciency Syndrome and the Effects of Atractylenolide I. Acta Pharmacol. Sin 41 (2), 237–248. doi:10.1038/s41401-019-0275-z Sun, X. G., Lin, X. C., Diao, J. X., Yu, Z. L., and Li, K. (2016). Pi (Spleen)-Deﬁciency Syndrome in Tumor Microenvironment Is the Pivotal Pathogenesis of Colorectal Cancer Immune Escape. Chin. J. Integr. Med. 22 (10), 789–794. REFERENCES Current Cancer Situation in China: Good or Bad News from the 2018 Global Cancer Statistics? Cancer Commun. (Lond) 39 (1), 22. doi:10.1186/s40880-019-0368-6 Phung, C. D., Pham, T. T., Nguyen, H. T., Nguyen, T. T., Ou, W., Jeong, J. H., et al. (2020). Anti-CTLA-4 Antibody-Functionalized Dendritic Cell-Derived Exosomes Targeting Tumor-Draining Lymph Nodes for Effective Induction of Antitumor T-Cell Responses. Acta Biomater. 115, 371–382. doi:10.1016/ j.actbio.2020.08.008 Gish, R. G., Porta, C., Lazar, L., Ruff, P., Feld, R., Croitoru, A., et al. (2007). Phase III Randomized Controlled Trial Comparing the Survival of Patients with Unresectable Hepatocellular Carcinoma Treated with Nolatrexed or Doxorubicin. J. Clin. Oncol. 25 (21), 3069–3075. doi:10.1200/JCO.2006.08.4046 Quezada, S. A., Peggs, K. S., Curran, M. A., and Allison, J. P. (2006). CTLA4 Blockade and GM-CSF Combination Immunotherapy Alters the Intratumor Balance of Effector and Regulatory T Cells. J. Clin. Invest. 116 (7), 1935–1945. doi:10.1172/JCI27745 Goenka, R., Xu, Z., Samayoa, J., Banach, D., Beam, C., Bose, S., et al. (2021). CTLA4-Ig-Based Bifunctional Costimulation Inhibitor Blocks CD28 and ICOS Signaling to Prevent T Cell Priming and Effector Function. J. Immunol. 206 (5), 1102–1113. doi:10.4049/jimmunol.2001100 October 2021 \| Volume 12 \| Article 757194 Frontiers in Pharmacology \| www.frontiersin.org 12 Wang et al. Exosome Regulates Spleen Deﬁciency HCC Raffone, A., Travaglino, A., Saccone, G., Campanino, M. R., Mollo, A., De Placido, G., et al. (2019). Loss of PTEN Expression as Diagnostic Marker of Endometrial Precancer: A Systematic Review and Meta-Analysis. Acta Obstet. Gynecol. Scand. 98 (3), 275–286. doi:10.1111/aogs.13513 Whiteside, T. L. (2013). Immune Modulation of T-Cell and NK (Natural Killer) Cell Activities by TEXs (Tumour-derived Exosomes). Biochem. Soc. Trans. 41 (1), 245–251. doi:10.1042/BST20120265 Whiteside, T. L., Mandapathil, M., Szczepanski, M., and Szajnik, M. (2011). Mechanisms of Tumor Escape from the Immune System: Adenosine- Producing Treg, Exosomes and Tumor-Associated TLRs. Bull. Cancer 98 (2), E25–E31. doi:10.1684/bdc.2010.1294 Ruivo, C. F., Adem, B., Silva, M., and Melo, S. A. (2017). The Biology of Cancer Exosomes: Insights and New Perspectives. Cancer Res. 77 (23), 6480–6488. doi:10.1158/0008-5472.CAN-17-0994 Schubert, D., Bode, C., Kenefeck, R., Hou, T. Z., Wing, J. B., Kennedy, A., et al. (2014). Autosomal Dominant Immune Dysregulation Syndrome in Humans with CTLA4 Mutations. Nat. Med. 20 (12), 1410–1416. doi:10.1038/nm.3746 Wolchok, J. (2018). Putting the Immunologic Brakes on Cancer. Cell 175 (6), 1452–1454. doi:10.1016/j.cell.2018.11.006 Yang, C. F., Yang, G. D., Huang, T. J., Li, R., Chu, Q. Q., Xu, L., et al. (2016). REFERENCES doi:10.1007/s11655-015-2086-5 Zhang, Y., He, X., Zhang, Y., Zhao, Y., Lu, S., Peng, Y., et al. (2021). Native Mitochondria-Targeting Polymeric Nanoparticles for Mild Photothermal Therapy Rationally Potentiated with Immune Checkpoints Blockade to Inhibit Tumor Recurrence and Metastasis. Chem. Eng. J. 424, 130171. doi:10.1016/j.cej.2021.130171 Ueda, H., Howson, J. M., Esposito, L., Heward, J., Snook, H., Chamberlain, G., et al. (2003). Association of the T-Cell Regulatory Gene CTLA4 with Susceptibility to Autoimmune Disease. Nature 423 (6939), 506–511. doi:10.1038/nature01621 Zhao, N., Zhang, W., Guo, Y., Jia, H., Zha, Q., Liu, Z., et al. (2011). Effects on Neuroendocrinoimmune Network of Lizhong Pill in the Reserpine Induced Rats with Spleen Deﬁciency in Traditional Chinese Medicine. J. Ethnopharmacol 133 (2), 454–459. doi:10.1016/j.jep.2010.10.016 Ukrainskaya, V. M., Rubtsov, Y. P., Knorre, V. D., Maschan, M. A., Gabibov, A. G., and Stepanov, A. V. (2019). The Role of Tumor-Derived Vesicles in the Regulation of Antitumor Immunity. Acta Naturae 11 (4), 33–41. doi:10.32607/20758251-2019-11-4-33-41 Van Coillie, S., Wiernicki, B., and Xu, J. (2020). “Molecular and Cellular Functions of CTLA-4,” in Regulation of Cancer Immune Checkpoints: Molecular and Cellular Mechanisms and Therapy. Editor J. Xu (Singapore: Springer Singapore), 7–32. doi:10.1007/978-981-15-3266-5_2 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. Wang, L., Zuo, X., Xie, K., and Wei, D. (2018). The Role of CD44 and Cancer Stem Cells. Methods Mol. Biol. 1692, 31–42. doi:10.1007/978-1-4939-7401-6_3 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. Wang, X., Cao, X., Sun, R., Tang, C., Tzankov, A., Zhang, J., et al. (2018). Clinical Signiﬁcance of PTEN Deletion, Mutation, and Loss of PTEN Expression in De Novo Diffuse Large B-Cell Lymphoma. Neoplasia 20 (6), 574–593. doi:10.1016/ j.neo.2018.03.002 Wang, Z., Zhan, M., Li, W., Chu, C., Xing, D., Lu, S., et al. (2021). Photoacoustic Cavitation-Ignited Reactive Oxygen Species to Amplify Peroxynitrite Burst by Photosensitization-free Polymeric Nanocapsules. Angew. Chem. Int. Ed. Engl. 60 (9), 4720–4731. doi:10.1002/anie.202013301 Copyright © 2021 Wang, Li, Mao, Mo, Cao, Luo, Zhou, Liu, Zhang and Yu. REFERENCES 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. Wen, S. W., Sceneay, J., Lima, L. G., Wong, C. S., Becker, M., Krumeich, S., et al. (2016). The Biodistribution and Immune Suppressive Effects of Breast Cancer- Derived Exosomes. Cancer Res. 76 (23), 6816–6827. doi:10.1158/0008- 5472.CAN-16-0868 October 2021 \| Volume 12 \| Article 757194 Frontiers in Pharmacology \| www.frontiersin.org 13 13
https://openalex.org/W3162049491	https://figshare.com/articles/journal_contribution/How_working_memory_capacity_modulates_the_time_course_of_indirect_replies_comprehension_an_event-related_potential_study/14605852/1/files/28039294.pdf	English	null	How working memory capacity modulates the time course of indirect replies comprehension: an event-related potential study	Language, cognition and neuroscience	2,021	cc-by	2,945	1. The rating tests added for experiment materials In order to explore whether participants could predict the reply type and whether they could understand what Speaker B’s meaning according to Speaker A’s question and the first two parts of the utterance, we added three rating tests for the materials used in the present study. In the first rating, we removed Speaker B’s whole utterance (ZERO). In the second rating, the first part of Speaker B’s utterance (e.g, “A: What does Liming usually do ?/Is Liming an interesting person? B: He ” as in Table 1) was presented (ONE). In the third rating, the first two parts of Speaker B’s utterance (e.g, “A: What does Liming usually do ?/Is Liming an interesting person? B: He every day” as in Table 1 ) was presented (TWO). Twenty participants were asked to rate the reply type of Speaker B’s utterance (1 representing direct reply, 2 representing not sure and 3 representing indirect reply). If the participants rated it as indirect reply, they were asked to type the meanings conveyed by Speaker B. 1 The rating results showed that for the direct reply condition, the rate of replies judged as the direct reply (Directness Rate) was 61%, 85% and 87% for the ZERO, ONE and TWO tests, respectively, and the rate of replies judged as the indirect reply (Indirectness Rate) was 7%, 4% and 5%, respectively. For the indirect reply condition, the rate of replies judged as the direct reply was 79%, 59% and 48% for the ZERO, ONE and TWO tests, respectively, and the rate of replies judged as the indirect reply was 7%, 27% and 43%, respectively. The rate of correctly understanding the meaning of the indirect replies was 1.5%, 13.7% and 25.7% for the ZERO, ONE and TWO tests, respectively. Table 1 presented the Mean (SD) of rating results. of the indirect replies was 1.5%, 13.7% and 25.7% for the ZERO, ONE and TWO p , , tests, respectively. Table 1 presented the Mean (SD) of rating results. tests, respectively. Table 1 presented the Mean (SD) of rating results. Table S1 The results of rating tests for experiment materials Directness Rate Indirectness Rate Condition ZERO ONE TWO ZERO ONE TWO Direct 61%(23%) 85%(17%) 87%(13%) 7%(8%) 4%(8%) 5%(8%) Indirect 79%(19%) 59%(27%) 48%(22%) 7%(10%) 27%(21%) 43%(20%) Table S1 The results of rating tests for experiment materials 2. The ERP analysis at the first two parts of the target utterance We analyze ERP results of the first and the second part of the reply utterances. The preprocessing and statistics analysis procedure was the same as that for the third part. As the duration time of the first part and the second part including the blank was 1400 ms, the electrophysiological data were segmented from 200 ms (-200 ms) prior to the onset of the first part to 1400 ms after the onset of the first part. The mean amplitude of -200 ms to 0 ms served as a baseline. The time windows of P200 (180-300 ms), N400 (300-500 ms), and late positivity (500-700 ms) were selected for statistical analysis. Figure S1 showed the ERP waveforms and topography of the first two parts of the target sentence. 2 At the first part of the target utterance, in the time window of P200 (180-300 ms), the interaction between Reply and Anteriority was significant [F (2, 76) = 5.67, MSE = 2 1.91, p = 0.018]. Simple-effect tests showed that no significant difference between direct and indirect reply was found at the anterior, central and posterior regions (ps > 0.1). In the time window of N400 (300-500 ms), the main effect of Group was significant [F (1, 38) = 9.40, MSE = 42.07, p = 0.004]. High span readers elicited a larger N400 than low span readers in both direct reply and indirect reply conditions. No other effect was significant at this time widow (ps > 0.1). In the time window of 500-700 ms, the main effect of Group was significant [F (1, 38) = 5.40, MSE = 33.531, p = 0.026]. High span readers elicited a larger late negativity than low span readers in both direct reply and indirect reply conditions. The interaction between Reply and Anteriority was significant [F (2, 76) = 4.91, MSE = 2.19, p = 0.024]. Simple-effect tests showed that no significant difference between direct and indirect reply was found at the anterior, central and posterior regions (ps > 0.1). 1.91, p = 0.018]. Simple-effect tests showed that no significant difference between direct and indirect reply was found at the anterior, central and posterior regions (ps > 0.1). In the time window of N400 (300-500 ms), the main effect of Group was significant [F (1, 38) = 9.40, MSE = 42.07, p = 0.004]. 2. The ERP analysis at the first two parts of the target utterance High span readers elicited a larger N400 than low span readers in both direct reply and indirect reply conditions. No other effect was significant at this time widow (ps > 0.1). In the time window of 500-700 ms, the main effect of Group was significant [F (1, 38) = 5.40, MSE = 33.531, p = 0.026]. High span readers elicited a larger late negativity than low span readers in both direct reply and indirect reply conditions. The interaction between Reply and Anteriority was significant [F (2, 76) = 4.91, MSE = 2.19, p = 0.024]. Simple-effect tests showed that no significant difference between direct and indirect reply was found at the anterior, central and posterior regions (ps > 0.1). At the second part of the target utterance, in the time window of P200 (880-1000ms), the interaction between Reply and Anteriority was significant [F (2, 76) = 4.95, MSE = 2.01, p = 0.016]. Simple-effect tests showed that no significant difference between direct and indirect reply was found at the anterior, central and posterior regions (ps > 0.1). In the time window of N400 (1000-1200 ms), the interaction between Reply and Anteriority was significant [F (2, 76) = 4.83, MSE = 2.64, p = 0.017]. Simple-effect tests showed that no significant difference between direct and indirect reply was found at the anterior, central and posterior regions (ps > 0.1). In the time window of 1200-1400 ms, the interaction between Reply and Anteriority was significant [F (2, 76) = 3.59, MSE = 2.41, p = 0.043]. Simple-effect tests showed that no 3 significant difference between direct and indirect reply was found at the anterior, central and posterior regions (ps > 0.1). 4 significant difference between direct and indirect reply was found at the anterior, central and posterior regions (ps > 0.1). The N400 effect has been associated with the integration difficulty of current information to prior contexts (Coulson & Van Petten, 2002; Pynte et al., 1996; Weiland et al., 2014) and semantic prediction benefits/costs (e.g., Brothers, Wlotko, Warnke, &Kuperberg, 2020; Kuperberg, Brothers &Wlotko, 2020; Liao & Lau 2020).The prolonged N400 effect at the first part of the target utterance for high span readers may indicate that the high span readers were actively integrating the upcoming reply sentences with their context questions. 2. The ERP analysis at the first two parts of the target utterance Another possibility might be that high span readers were more inclined to anticipate Speaker B’s reply when they read Speaker A’s question both for direct reply and indirect reply since they could use 4 The N400 effect has been associated with the integration difficulty of current information to prior contexts (Coulson & Van Petten, 2002; Pynte et al., 1996; Weiland et al., 2014) and semantic prediction benefits/costs (e.g., Brothers, Wlotko, Warnke, &Kuperberg, 2020; Kuperberg, Brothers &Wlotko, 2020; Liao & Lau 2020).The prolonged N400 effect at the first part of the target utterance for high span readers may indicate that the high span readers were actively integrating the upcoming reply sentences with their context questions. Another possibility might be that high span readers were more inclined to anticipate Speaker B’s reply when they read Speaker A’s question both for direct reply and indirect reply since they could use The N400 effect has been associated with the integration difficulty of current information to prior contexts (Coulson & Van Petten, 2002; Pynte et al., 1996; general world knowledge and contextual information to generate more bridging inferences for building more elaborate representations (e.g., George, Mannes, & Hoffman, 1997; Singer, Andruslak, Reisdorf, & Black, 1992). As we add fillers to prevent participants from forming particular predictions, participants could not make accurate predictions for the upcoming reply. Thus, when they encounter the first part of the reply utterance, they might maintain multiple predictions about the upcoming information, reflected by the prolonged N400. These results might indicate that the high span readers prepare better for the upcoming information than the low span readers. However, as no significant main effect of Reply or interactions involving Reply was found, our results did not support an earlier difference on the expectations of the two kinds of replies. 3. Principal Component Analysis (PCA) In order to clarify whether the high span readers' Reply effects were a long-lasting positivity or three distinct components, a Principal Component Analysis (PCA) was conducted for the high span readers and the low span readers, respectively. 5 Temporospatial PCA is a technique which extracts linear combinations of data points that meet certain criteria that tend to distinguish between consistent patterns of electrocortical activity (Dien & Frishkoff, 2005). This method is a useful tool for statistical decomposition of ERPs and help to disentangle temporally overlapping components (Dien & Frishkoff, 2005; Foti, Weinberg, Dien, & Hajcak, 2011; Macnamara, Ochsner, & Hajcak, 2011). The two-step temporospatial PCA was 5 conducted by the ERP PCA toolkit 2.83 and following published guidelines (Dien, 2010; Dien et al., 2005; Dien, Khoe, & Mangun, 2007), and a temporal PCA was firstly performed on the data to capture variance across time points. Sixty electrodes (VEOG/HEOG/CB1/CB2/M1/M2) were deleted from further analysis) were included in the analysis of the electrophysiological waveforms across the scalp. PCA was performed in -200–1300 ms time interval after stimulus onset. The input to the temporal PCA consisted of 751 variables (time points) by 2400 observations (60 electrodes by 20 participants by Reply [Direct Reply vs. Indirect Reply]). Promax rotation was used, and 22/21 temporal factors (TFs, for the High span and the low span Group, respectively) were extracted based on the resulting Scree plot (Cattell, 1966). For each TF, this analysis yielded factor scores for each combination of electrode, participant, and condition, representing the amount of activity in the original data captured by that factor. Thereafter, separate spatial PCAs were performed on each TF retained in the previous step to reduce spatial dimensions of datasets. This step used all electrodes sites as variables and considered all participants, conditions, and temporal factor scores as observations. Infomax rotation was used and based on the averaged Scree plot for all 22/21 temporal factors, five spatial factors (SFs) were extracted, yielding 110/105 unique factors combinations (TFSF, for the High span and the low span Group, respectively). The covariance matrix and Kaiser normalization were used for each PCA. Of these, 34/31 factor combinations (for the high span and low span group, respectively) that accounted for more than 1% of the variance were retained for 6 further examination. We looked for any correspondence between the temporospatial factors and the deflections in the grand-average waveforms. 3. Principal Component Analysis (PCA) Figure S2 showed the factor loadings of the high and low span readers that could be considered consistent with the ERP components found in the two group, respectively. For the high span readers, five combinations were the most consistent with the morphology of the P200, P300 and delayed LPC (Table S1). Specifically, TF1SF2 (accounting for 4.31% of the variance) was maximal at electrode PO3 with a peak latency at 1300 ms, resembling the delayed LPC. TF2SF1 (accounting for 6.07% of the variance), TF2SF2 (accounting for 4.71% of the variance) and TF3SF2 (accounting for 3.97% of the variance) were all considered together as part of the P300, for their positive polarity, maximal peak channel (FCZ , CPZ and POZ, respectively, which were similar to the distribution of the P300 effect), and peak latency (648 ms , 648 ms and 398 ms, respectively). Additionally, TF8SF1 (accounting for 1.07% of the variance) was maximal at electrode CPZ with a peak latency at 224 ms, resembling the P200. For the low span readers, TF1SF3 (accounting for 8.90% of the variance) was maximal at electrode POZ with a peak latency at 1202 ms, resembling the delayed LPC. Thus, these factor combinations were determined as the PCA-derived P200, P300, delayed LPC and their voltages were obtained for statistical analysis. 7 Table S2. PCA factor combinations selected for statistical analysis Group Factor Number PCA factor Associated ERP component Peak channel Peak latency (ms) Variance explained (%) High span 2 6 7 12 36 TF1SF2 TF2SF1 TF2SF2 TF3SF2 TF8SF1 Delayed LPC P300 P300 P300 P200 PO3 FCZ CPZ POZ CPZ 1300 648 648 398 224 4.31 6.07 4.71 3.97 1.07 Low span 3 TF1SF3 Delayed LPC POZ 1202 8.90 For each temporospatial factor, paired t-tests were performed with Reply (Direct Table S2. PCA factor combinations selected for statistical analysis Table S2. PCA factor combinations selected for statistical analysis Group Factor Number PCA factor Associated ERP component Peak channel Peak latency (ms) Variance explained (%) High span 2 6 7 12 36 TF1SF2 TF2SF1 TF2SF2 TF3SF2 TF8SF1 Delayed LPC P300 P300 P300 P200 PO3 FCZ CPZ POZ CPZ 1300 648 648 398 224 4.31 6.07 4.71 3.97 1.07 Low span 3 TF1SF3 Delayed LPC POZ 1202 8.90 For each temporospatial factor, paired t-tests were performed with Reply (Direct 8 Reply, Indirect Reply) as a within-subject factor and voltages as dependent variables. Reply, Indirect Reply) as a within-subject factor and voltages as dependent variables. The results showed that for the high span readers, compared to direct replies, indirect replies elicited a larger delayed LPC for TF1SF2 (t19 = -4.53, p < .001), a larger P300 for TF2SF1 (t19 = -2.88, p = .010) , TF2SF2 (t19 = -2.69, p = .015) and TF3SF2 (t19 = -3.66, p = .002), and a larger P200 for TF8SF1 (t19 = -2.42, p = .026). For the low span readers, indirect replies elicited a larger delayed LPC than direct replies for TF1SF3 (t19 = -2.61, p = .017). Therefore, based on the results of PCA, the P200, P300 and the delayed LPC seem to be three different components, and represent different cognitive processes. We linked the P200 to automatic attention allocation for initial pragmatic processing of indirect replies (Chen, Wang, & Yang, 2014; Chen & Yang, 2015; Ding, Wang, & Yang, 2015, 2016；Regel & Gunter, 2017; Regel et al., 2014) and rapid establishment of expectation (León et al., 2010). The P300 was related to immediate integration and updating of Speakers’ meaning when the indirect replies were encountered (e.g., Bartholow, et al., 2001; Bartholow, et al., 2003; Chen, 2018; Cowles, Kluender, Kutas, & Polinsky, 2007; Donchin & Coles, 1988; Polich, 2003, 2007; Van Duynslaeger, et al., 2008; Van Duynslaeger, et al., 2007). The delayed LPC was associated with an attempt to infer other’s intention/goal and late pragmatic inference for deriving speakers’ intended meanings (Jiang & Pell, 2016a, 2016b; Jiang & Zhou, 2015). Cattell, R. B. (1966). The scree test for the number of factors. Multivariate behavioral research, References: Cattell, R. B. (1966). The scree test for the number of factors. Multivariate behavioral research, 9 1(2), 245-276. 1(2), 245-276. Dien, J. (2010). Evaluating two‐step PCA of ERP data with geomin, infomax, oblimin, promax, and varimax rotations. Psychophysiology, 47(1), 170-183. Dien, J., Beal, D. J., & Berg, P. (2005). Optimizing principal components analysis of event-related potentials: matrix type, factor loading weighting, extraction, and rotations. Clinical N h i l 116(8) 1808 1825 potentials: matrix type, factor loading weighting, extraction, and rotations. Clinical Dien, J., Khoe, W., &Mangun, G. R. (2007). Evaluation of PCA and ICA of simulated ERPs: Promax vs. Infomax rotations. Human Brain Mapping, 28(8), 742-763. Foti, D., Weinberg, A., Dien, J., &Hajcak, G. (2011). Event‐related potential activity in the basal ganglia differentiates rewards from nonrewards: Temporospatial principal components analysis and source localization of the feedback negativity. Human Brain Mapping, 32(12), 2207-2216. ganglia differentiates rewards from nonrewards: Temporospatial principal components analysis and source localization of the feedback negativity. Human Brain Mapping, 32(12), 2207-2216. MacNamara, A., Ochsner, K. N., &Hajcak, G. (2010). Previously reappraised: the lasting effect of description type on picture-elicited electrocortical activity. Social Cognitive and Affective Neuroscience, 6(3), 348-358. Papo, D., Baudonniere, P. M., Hugueville, L., &Caverni, J. P. (2003). Feedback in hypothesis testing: an ERP study. Journal of Cognitive Neuroscience, 15(4), 508-522. Papo, D., Baudonniere, P. M., Hugueville, L., &Caverni, J. P. (2003). Feedback in hypothesis testing: an ERP study. Journal of Cognitive Neuroscience, 15(4), 508-522. Papo, D., Caverni, J. P., Douiri, A., Podlipsky, I., &Baudonnière, P. M. (2007). Time-varying spectral entropy differentiates between positive and negative feed back-related EEG activity in a hypothesis testing paradigm. International Journal of Psychophysiology, 66(3), 183-195. Spencer, K. M., Dien, J., &Donchin, E. (2001). Spatiotemporal analysis of the late ERP responses to deviant stimuli. Psychophysiology, 38(2), 343-358. 10 Wambacq, I. J., &Jerger, J. F. (2004). Processing of affective prosody and lexical-semantics in spoken utterances as differentiated by event-related potentials. Cognitive Brain Research, 20(3), 427-437. Research, 20(3), 427-437. Yuan, J., Meng, X., Yang, J., Yao, G., Hu, L., & Yuan, H. (2012). The valence strength of unpleasant emotion modulates brain processing of behavioral inhibitory control: Neural Yuan, J., Meng, X., Yang, J., Yao, G., Hu, L., & Yuan, H. (2012). The valence strength of correlates. Biological Psychology, 89(1), 240-251. correlates. Biological Psychology, 89(1), 240-251. 11 11
W4391751222.txt	https://www.nature.com/articles/s41598-024-54260-0.pdf	en		Novel nomograms based on microvascular invasion grade for early-stage hepatocellular carcinoma after curative hepatectomy	Scientific reports	2,024	cc-by	6,558	www.nature.com/scientificreports OPEN Novel nomograms based on microvascular invasion grade for early‑stage hepatocellular carcinoma after curative hepatectomy Hengkai Chen 1,2,6, Honghao Ye 3,4,6, Linfang Ye 5,6, Fangzhou Lin 3,4, Yingjun Shi 3,4, Aoxue Zhong 3,4, Guoxian Guan 1,2* & Jinfu Zhuang 1,2* Microvascular invasion (MVI) is a critical risk factor for postoperative recurrence of hepatocellular carcinoma (HCC). This study aimed to firstly develop and validate nomograms based on MVI grade for predicting recurrence, especially early recurrence, and overall survival in patients with early-stage HCC after curative resection. We retrospectively reviewed the data of patients with early-stage HCC who underwent curative hepatectomy in the First Affiliated Hospital of Fujian Medical University (FHFU) and Mengchao Hepatobiliary Hospital of Fujian Medical University (MHH). Kaplan–Meier curves and Cox proportional hazards regression models were used to analyse disease-free survival (DFS) and overall survival (OS). Nomogram models were constructed on the datasets from the 70% samples of and FHFU, which were validated using bootstrap resampling with 30% samples as internal validation and data of patients from MHH as external validation. A total of 703 patients with early-stage HCC were included to create a nomogram for predicting recurrence or metastasis (DFS nomogram) and a nomogram for predicting survival (OS nomogram). The concordance indexes and calibration curves in the training and validation cohorts showed optimal agreement between the predicted and observed DFS and OS rates. The predictive accuracy was significantly better than that of the classic HCC staging systems. Keywords Nomogram, Microvascular invasion grade, Early-stage HCC, Early recurrence Abbreviations HCC Hepatocellular carcinoma BCLC Barcelona Clinic Liver Cancer OS Overall survival MVI Microvascular invasion FHFU The First Affiliated Hospital of Fujian Medical University MMH Mengchao Hepatobiliary Hospital of Fujian Medical University CT Computed tomography MRI Magnetic resonance imaging AFP α-Fetoprotein DFS Disease-free survival C-index Concordance index ALP Alkaline phosphatase LDL Low-density lipoprotein 1 Department of Colorectal Surgery, The First Affiliated Hospital of Fujian Medical University, 20th, Chazhong Road, Fuzhou 350005, China. 2Department of Colorectal Surgery, National Regional Medical Center, Binhai Campus of the First Affiliated Hospital, Fujian Medical University, Fuzhou 350212, China. 3Fuzhou University, Fuzhou 350108, China. 4Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou 350025, China. 5Zhongshan Hospital Xiamen University, Xiamen 361004, China. 6These authors contributed equally: Hengkai Chen, Honghao Ye and Linfang Ye. *email: Gxguan1108@163.com; zjf2611@fjmu.edu.cn Scientific Reports \| (2024) 14:3470 \| https://doi.org/10.1038/s41598-024-54260-0 1 Vol.:(0123456789) www.nature.com/scientificreports/ Apo-A1 Apolipoprotein A1 TT Thrombin time MCH Mean corpuscular haemoglobin PAB Prealbumin AFU α-Fucosidase CI Confidence interval AJCC American Joint Committee on Cancer staging system JIS Japan Integrated Staging Score HKLC Hong Kong Liver Cancer prognostic classification scheme HBV Hepatitis B virus Hepatocellular carcinoma (HCC) is among the most frequent causes of cancer-related deaths worldwide1. Despite remarkable improvements in comprehensive HCC treatment, radical surgical resection and liver transplantation are considered the only curative treatments for patients with HCC classified as early-stage (stages 0 and A) according to the Barcelona Clinic Liver Cancer (BCLC) staging system. However, postoperative recurrence and metastasis rates of patients with early HCC vary from 50 to 70%2, resulting in poor overall survival (OS). Early recurrence after liver resection for HCC is the leading cause of death during the first 2 years3. Therefore, developing a model for predicting postoperative recurrence, especially early recurrence, for patients with early-stage HCC to guide risk stratification and treatment is urgently needed. Microvascular invasion (MVI), a mass of cancer cells in the vascular cavity with adhesion to endothelial cells, and only visible under a m icroscope4, has been reported by previous studies to be an indicator of early invasive manifestation of HCC. It is a crucial independent predictive factor for early recurrence and poor OS among patients with HCC who underwent hepatectomy or received liver transplantation. Most patients with BCLC early-stage HCC with early recurrence are pathologically verified as MVI positive 5–7. Moreover, a previous study found that more invading tumor cells and multiple-invaded microvessels might be related to poor survival and recurrence rates4. These findings suggest that the BCLC staging system should reappraise HCC based on the presence or grade of MVI to distinguish the biological behavior of early-stage HCC. MVI is graded according to the number of cancer cells and the distance of MVI to the tumor according to the Standard for Diagnosis and Treatment of Primary Liver Cancer8. Although predictive models for postoperative early recurrence in patients with HCC have been established, a predictive model for patients with early BCLC stage HCC patients according to the MVI grade has not been reported. Therefore, we retrospectively investigated the clinical and histopathological characteristics of patients with early HCC after curative hepatectomy from two centers to establish a prognostic nomogram based on MVI grade to predict early recurrence and OS. Methods Patients and study design The database was retrospectively derived from patients with HCC who underwent hepatectomy at the First Affiliated Hospital of Fujian Medical University (FHFU) and the Mengchao Hepatobiliary Hospital of Fujian Medical University (MMH) from March 2015 to March 2020. The inclusion criteria for patients with HCC patients in this study were: (1) early-stage HCC (BCLC stage 0 or A) diagnosis that was confirmed by postoperative pathology; (2) Child–Pugh A or B liver function before surgery; (3) R0 surgical resection of the tumor with curative intent; (4) all patients who survived for at least 30 days after surgery; (5) no preoperative anticancer treatments that could introduce any bias; and (6) clinicopathological data and follow-up information were available. Patients with the following criteria were excluded: (1) recurrent HCC, (2) combined hepatocellular cholangiocarcinoma, (3) previous history of malignancy, and (4) age < 18 years. R0 surgical resection was defined as complete tumor resection with histopathologically tumorfree resection margins. Nomogram models were constructed on the datasets from the FHFU, which were also validated using bootstrap resampling as internal validation, and the dataset from the MMH was used for external validation. This study was approved by the Ethics Review Committee of the First Affiliated Hospital of Fujian Medical University and the Ethics Review Committee of Mengchao Hepatobiliary Hospital of Fujian Medical University. Written informed consent was obtained from all subjects before the operation. All procedures were performed in accordance with the Declaration of Helsinki. Clinical variables Demographic, laboratory, and HCC pathological data were collected. The laboratory tests included various tests for routine blood parameters, full sets of tests for blood clotting, full sets of tests for blood biochemistry, and hepatitis virus markers. Imaging data included, but were not limited to, the number of tumors, presence of satellite nodules, the diameter of the largest nodule, tumor capsule, and cirrhosis based on preoperative contrastenhanced computed tomography (CT) or magnetic resonance imaging (MRI). The diagnosis and grade of MVI were confirmed according to the Standard for Diagnosis and Treatment of Primary Liver Cancer4 by 2 independent pathologists. In case of any doubt, the final decision was determined after MDT discussion. Briefly, the grade of MVI is defined as follows: M0: no MVI; M1: the number of MVI is < 5 and at a distance of ≤ 1 cm from the tumor; M2: the number of MVI is > 5 or at a distance of > 1 cm from the tumor (Fig. 1)4. Scientific Reports \| Vol:.(1234567890) (2024) 14:3470 \| https://doi.org/10.1038/s41598-024-54260-0 2 www.nature.com/scientificreports/ Figure 1. The grade of MVI Standard for Diagnosis and Treatment of Primary Liver Cancer. (a) M0: no MVI; (b) M1: the number of MVI is < 5 and at a distance of ≤ 1 cm from the tumor; (c) M2: the number of MVI is > 5 or at a distance of > 1 cm from the tumor. Follow‑up During the follow-up, serum alpha-fetoprotein (AFP) levels were measured, and ultrasonography, CT, or MRI of the chest and abdomen was done once every 2 months for the first 2 years after surgery. For patients who were free of cancer recurrence 2 years after surgery, a 6-month interval surveillance was performed. Disease-free survival (DFS) was defined as the duration from the first surgery to the first recurrence, metastasis, or death. OS was defined as the duration from the first surgery to death or the last follow-up. Statistical analysis Continuous variables are expressed as mean ± standard deviation. Chi-squared or Fisher’s exact tests were used to assess differences in categorical variables. The Wilcoxon rank-sum test was used to compare continuous variables between groups. In this study, the cut-off values of continuous variables were established using the X-tile software version 3.6.1 (Yale University School of Medicine, New Haven, Connecticut, United States) for widely clinical application. For DFS and OS curves during follow-up, Kaplan–Meier curves, log-rank Mantel-Cox test, and Cox proportional hazards regression analyses were used. Nomograms were generated using the rms package in R software version 3.5.2 (R Foundation for Statistical Computing, Vienna, Austria)9. The predictive accuracy and discriminative ability of the nomogram were assessed using the concordance index (C-index) and calibration curves. The larger the C-index, the more accurate the prognostic prediction is. A value of P < 0.05 was considered significant. All covariates met the Cox proportional hazard assumption, as determined by the Schoenfeld residuals. Variance inflator factor was used to assess multicollinearity in all estimated models; there was no indication of multicollinearity. Scientific Reports \| (2024) 14:3470 \| https://doi.org/10.1038/s41598-024-54260-0 3 Vol.:(0123456789) www.nature.com/scientificreports/ Ethics approval and consent to participate This study was approved by the Ethics Review Committee of the First Affiliated Hospital of Fujian Medical University and the Ethics Review Committee of Mengchao Hepatobiliary Hospital of Fujian Medical University. Written informed consent was obtained from all subjects before the operation. All procedures were performed in accordance with the Declaration of Helsinki. Consent for publication Written informed consent was obtained from every patient with HCC to perform tumor resection for analysis and publication. Results Characteristics of patients in study and validation cohorts Overall, 703 patients (490 from FHFU used as training cohort, and 213 from MMH used as external validation cohort) with BCLC early-stage HCC were included. The baseline characteristics of the two cohorts are shown in Supplementary Table 1. The average age of the entire cohort was 53.7 ± 10.9 years, with a male to female ratio of 4.72:1 (580/130). The average follow-up time for all patients was 18.8 ± 10.2 months. The 8-month, 1-, 2-, and 3-year recurrence or metastasis rates were 5.8%, 8.1%, 11.8%, and 12.7%, respectively. The 8-month, 1-, 2-, and 3-year survival rates were 1.7%, 2.4%, 3.9%, and 4.2%, respectively. Among them, M1 was observed in 173 cases (24.6%), while M2 was observed in 102 cases (14.5%). Between-group differences in sex, age, operative time, follow-up time, ASA scores, laboratory, and HCC pathological data were not significant (Supplementary Table 1). The association of MVI grade relative to OS or DFS is shown in Fig. 2. The Kaplan–Meier curves of OS and DFS showed that the M2 group had significantly poorer outcomes than the M1 and M0 groups (both P < 0.001). The univariate analysis results for DFS and OS in the study cohort are shown in Table 1. Multivariate analysis revealed that eight factors including neutrophils, alkaline phosphatase (ALP), urea, low-density lipoprotein (LDL), apolipoprotein A1 (Apo-A1), thrombin time (TT), tumor size, and MVI grade were independent prognostic factors for DFS, while six factors including TT, MVI grade, mean corpuscular haemoglobin (MCH), monocyte, prealbumin (PAB) and α-fucosidase (AFU) were prognostic factors for OS (Table 1). Therefore, these variables were included in the subsequent analysis to establish predictive models. Establishment of nomogram model for postoperative early‑relapse/OS and evaluation of its discriminability and calibration Based on the independent prognostic factors, nomograms for DFS and OS in the study cohort were established (Fig. 3). The results are shown in Supplementary Table 2. The C-index of the nomogram for DFS was 0.775 (95% confidence interval [CI] 0.720–0.830). The C-index for OS was 0.812 (95% CI 0.732–0.892). The validation showed excellent consistency between the observed and predicted 8-month, 1-, 2-and 3-year DFS, and 8-month, 1-, 2- and 3-year OS (Fig. 3); with a C-index of 0.865 (95% CI 0.806–0.924) for DFS and a C-index of 0.839 for OS (95% CI 0.675–1.00) in the internal validation cohort, and with a C-index of 0.857 (95% CI 0.763–0.951) for DFS and a C-index of 0.842 (95% CI 0.708–0.970) for OS in the external validation cohort (Supplementary Table 2). Calibration curves of internal verification and external verification with slopes closed to 1 and all p-value greater than 0.05 in the Hosmer and Lemeshow test, showed good consistency between the observed and predicted events (Fig. 4). Taken together, the nomogram models were able to accurately predict postoperative relapse and OS in patients with BCLC early-stage HCC. Figure 2. Kaplan–Meier estimates of the prognosis of patients with early-stage HCC according to MVI grade. (a) The MVI grade satisfactorily determined the disease-free survival (DFS) in the whole cohort; (b) The MVI grade satisfactorily determined the overall survival (OS) in the whole cohort. Scientific Reports \| Vol:.(1234567890) (2024) 14:3470 \| https://doi.org/10.1038/s41598-024-54260-0 4 www.nature.com/scientificreports/ DFS Clinical parameter HR (95% CI) OS p value HR (95% CI) p value Univariate analysis Age, year 0.25 (0.08–0.77) 0.015 0.98 (0.95–1) 0.091 Sex, male/female 0.81 (0.45–1.46) 0.998 0.88 (0.41–1.9) 0.741 BCLC staging system, (0/A) 3.04 (0.75–12.33) 0.122 2.74 (0.68–11.29) 0.995 WBC, ≤ 3.7/ > 3.7 × 109/L 2.41 (0.98–6.02) 0.055 1.21 (1.06–1.5.2) 0.013 PLT, ≤ 160/ > 160 × 109/L 1.76 (1.12–2.77) 0.018 1.00 (1.00–1.00) 0.027 Hb, ≤ 125/ > 125 × 109/L 2.05 (0.81–5.06) 0.133 0.99 (0.97–1.12) 0.312 Hematocrit, ≤ 42.2/ > 42.2% 1.56 (0.67–2.32) 0.073 0.98 (0.91–1.12) 0.692 MCV, ≤ 87/ > 87fL 0.66 (0.42–1.03) 0.061 0.95 (0.90–1.01) 0.055 MCH, ≤ 31.5/ > 31.5 pg 0.45 (0.28–0.72) < 0.001 0.85 (0.76–0.96) 0.007 Lymphocyte, ≤ 1.5/ > 1.5 × 109/L 0.93 (0.60–1.45) 0.732 0.86 (0.52–1.55) 0.585 Neutrophil, ≤ 3.3/ > 3.3 × 109/L 2.02 (1.01–3.80) 0.045 1.43 (1.14–1.72) 0.002 Monocyte, ≤ 0.5/ > 0.5 × 109/L 1.54 (0.89–2.60) 0.126 7.51 (1.63–15.86) 0.012 LR, ≤ 41.6/ > 41.6% 0.56 (0.33–0.96) 0.036 0.95 (0.92–0.99) 0.011 NR, ≤ 47.8/ > 47.8% 1.60 (0.97–2.52) 0.068 1.05 (1.00–1.10) 0.033 RDW, ≤ 13.6/ > 13.6% 1.81 (1.12–3.01) 0.016 1.12 (0.89–1.56) 0.292 0.533 RBC, ≤ 4.5/ > 4.5 × 109/L 1.35 (0.86–2.63) 0.152 1.22 (0.66–2.24) AFP, < 400/ ≥ 400 μg/L 2.01 (1.20–3.26) 0.005 1.00 (1.00–1.00) < 0.001 ALT, < 61/ ≥ 61 U/L 13.12 (1.85–5.40) < 0.001 1.00 (0.99–1.00) 1.000 HBV DNA < 50/ ≥ 50 × 109 IU/mL 1.74 (1.12–2.96) 0.016 1.00 (1.00–1.00) 0.971 Albumin, < 50/ ≥ 50 g/L 0.47 (0.23–0.93) 0.031 0.89 (0.82–0.97) 0.008 Scr, < 76/ ≥ 76 μmol/L 0.62 (0.34–1.12) 0.123 0.98 (0.96–1.02) 0.240 γ-GGT, < 34/ ≥ 34U/L 2.10 (1.12–3.95) 0.021 1.00 (1.00–1.00) 0.541 ALP, < 76/ ≥ 76& < 117 ≥ 117U/L 4.75 (2.61–8.56) < 0.001 1.00 (1.00–1.00) 0.182 TBil, < 16.4/ ≥ 16.4 μmol/L 1.44 (0.88–2.12) 0.169 0.97 (0.93–1.24) 0.313 DBil, < 5.6/ ≥ 5.6 μmol/L 1.52 (0.97–2.30) 0.065 0.95 (0.83–1.10) 0.415 IBil, < 6.6/ ≥ 6.6 μmol/L 1.55 (0.83–2.70) 0.178 0.94 (0.86–1.00) 0.182 TBA, < 7.2/ ≥ 7.2 μmol/L 2.33 (1.20–4.32) 0.011 0.95 (0.89–1.01) 0.061 TP, < 77 / ≥ 77 g/L 0.26 (0.07–1.01) 0.049 1.00 (0.95–1.10) 0.706 ALB, < 37/ ≥ 37 g/L 1.40 (0.23–0.93) 0.031 0.89 (0.82–0.97) 0.008 GLB, < 28.2/ ≥ 28.2 g/L 1.42 (0.88–2.10) 0.168 1.00 (0.96–1.06) 0.727 ALB/GLB < 2.2/ ≥ 2.2 0.39 (0.19–0.82) 0.013 1.73 (0.26–1.30) 0.186 PAB, ≤ 200/ > 200 mg/L 0.32 (0.14–0.73) 0.007 0.99 (0.99–1.00) 0.0014 AFU, < 38/ ≥ 38 g/L 4.12 (1.62–10.04) 0.003 1.02 (1.00–1.03) 0.032 ADA, < 7/ ≥ 7U/L 1.92 (1.25–3.04) 0.008 1.12 (0.99–1.32) 0.063 LDH, < 212/ ≥ 212U/L 2.02 (1.12–3.76) 0.033 1.00 (1.00–1.00) < 0.001 Urea, < 4/ ≥ 4& < 6.9/ ≥ 6.9 mmol/L 0.22 (0.08–0.59) 0.003 0.95 (0.76–1.22) 0.623 Uric acid, < 379/ ≥ 379 μmol/L 1.64 (0.99–2.62) 0.053 1.01 (0.99–1.03) 0.192 GLU, < 4.9/ ≥ 4.9 mmol/L 0.60 (0.38–0.94) 0.025 0.97 (0.78–1.22) 0.821 TCHO, < 3.4/ ≥ 3.4 mmol/L 1.95 (0.74–4.78) 0.184 1.02 (0.75–1.40) 0.990 TG, < 1.1/ ≥ 1.1 mmol/L 0.60 (0.37–0.98) 0.043 0.51 (0.24–1.18) 0.081 HDL, < 1/ ≥ 1 mmol/L 0.70 (0.43–1.12) 0.156 1.25 (0.48–3.05) 0.702 LDL, < 3/ ≥ 3 mmol/L 2.08 (1.22–3.16) 0.005 0.98 (0.65–1.52) 0.905 Apo-A1, < 83/ ≥ 83 g/L 0.45 (0.22–0.91) 0.028 1.00 (0.99–1.01) 0.760 Apo-B, < 113/ ≥ 113 g/L 2.01 (1.14–3.66) 0.027 1.00 (0.99–1.01) 0.708 Calcium, < 2.5/ ≥ 2.5 mmol/L 1.75 (0.24–12.4) 0.590 2.01 (0.09–4.01) 0.660 Phosphorus, < 1.1/ ≥ 1.1 mmol/L 1.72 (1.03–3.01) 0.043 10.01 (1.82–20.14) 0.008 Magnesium, < 0.8/ ≥ 0.8 mmol/L 0.70 (0.38–1.25) 0.212 2.10 (0.02–4.24) 0.751 Kalium, < 4.5/ ≥ 4.5 mmol/L 1.84 (1.01–3.23) 0.044 3.72 (1.65–8.62) 0.003 Natrium, < 141/ ≥ 141 mmol/L 0.57 (0.36–0.89) 0.014 0.99 (0.87–1.15) 0.860 Chlorine, < 102/ ≥ 102 mmol/L 0.46 (0.29–0.73) < 0.001 0.93 (0.84–1.02) 0.151 0.025 TT, < 20/ ≥ 20 s 0.46 (0.22–0.95) 0.035 0.75 (0.59–0.96) FIB, < 2.8/ ≥ 2.8 g/L 2.10 (1.34–3.37) 0.002 1.81 (1.40–2.34) < 0.001 APTT, < 25.7/ ≥ 25.7 s 0.59 (0.37–0.92) 0.020 1.01 (0.93–1.15) 0.960 Continued Scientific Reports \| (2024) 14:3470 \| https://doi.org/10.1038/s41598-024-54260-0 5 Vol.:(0123456789) www.nature.com/scientificreports/ DFS OS Clinical parameter HR (95% CI) p value HR (95% CI) p value PT, < 11.3/ ≥ 11.3 s 1.52 (0.94–2.57) 0.092 1.00 (0.76–1.41) 0.933 Tumor size, < 5/ ≥ 5& < 10/ ≥ 10centimiter 3.82 (2.12–6.84) < 0.001 1.22 (1.14–1.35) < 0.001 Tumor number, single/multiple 0.62 (0.20–12.0) 0.415 1.55 (0.55–4.36) 0.408 0.525 Satellite nodules, yes/no 1.72 (1.13–2.61) 0.022 1.20 (0.69–2.1) MVI, M0/M1/M2 2.60 (1.51–4.45) < 0.001 2.20 (1.61–3.05) < 0.001 Tumor capsule, yes/no 0.87 (0.67–1.15) 0.308 0.62 (0.20–2.01) 0.415 Cirrhosis, yes/no 0.77 (0.46–1.32) 0.313 0.54 (0.25–1.15) 0.107 Multivariate analysis Neutrophil 0.34 (0.19–0.60) < 0.001 ALP 4.41 (2.05–9.62) < 0.001 – – Urea 0.46 (0.26–0.80) 0.007 – – LDL 2.15 (1.34–3.67) 0.003 – – Apo-A1 0.32 (0.16–0.66) 0.002 – – TT 0.34 (0.16–0.70) 0.003 0.92 (0.87–0.97) 0.003 Tumor size 2.20 (1.29–3.82) 0.008 - - MVI grade 2.31 (1.25–4.16) 0.009 3.71 (1.75–7.85) < 0.001 MCH – – 0.67 (0.54–0.83) < 0.001 Monocyte – – 4.67 (2.37–9.68) < 0.001 PAB – – 0.56 (0.38–0.84) 0.005 AFU – – 0.71 (0.61–0.82) < 0.001 Table 1. Univariate and multivariate of clinical parameters associated with DFS and OS in early-stage HCC patients after R0 resection. BCLC staging system Barcelona Clinic Liver Cancer staging system, WBC white blood cell, PLT platelet, Hb hemoglobin, MCV mean corpuscular volume, MCH mean corpuscular hemoglobin, LR lymphocyte ratio, NR neutrophil ratio, MR monocyte ratio, RDW red blood cell distribution width, MPC mean platelet volume, RBC red blood cell, AFP α-fetoprotein, ALT alanine aminotransferase, HBV DNA level hepatitis B virus deoxyribonucleic acid level, Scr Serum creatinine, γ-GTTγ-glutamyl transpeptidase, ALP alkaline phosphatase, TBil total bilirubin, DBil direct bilirubin, IBil indirect bilirubin, TBA total bile acid, TP total protein, ALB albumin, GLB globumin, PAB prealbumin, AFU α-fucosidase, ADA adenosine deaminase, LDH lactate dehydrogenase, GLU glucose, TCHO total cholesterol, TG triglyceride, HDL high-density lipoprotein, LDL low-density lipoprotein, Apo-A1 apolipoprotein A1, Apo-B apolipoprotein B, TT thrombin time, FIB fibrinogen, APTT activated partial thromboplastin time, PT prothrombin time, MVI microvascular invasion. Comparison of predictive accuracy between the nomogram models and the classical staging systems The predictive value of the constructed model, in terms of clinical practicability, was compared with that of the 8th edition American Joint Committee on Cancer (AJCC) staging system, the BCLC staging system, the Japan Integrated Staging Score (JIS) and the Hong Kong Liver Cancer prognostic classification scheme (HKLC). The results are shown in Supplementary Table 2. In the training cohort, the C-index of the nomogram for DFS and OS was 0.775 and 0.812, respectively, which was significantly higher than the AJCC (DFS: 0.591; OS: 0.588), BCLC (DFS: 0.601; OS: 0.599), JIS (DFS: 0.589; OS: 0.592), and HKLC (DFS: 0.595; OS: 0.612) staging systems. Similarly, in the validation cohort, the C-index of the nomogram for DFS (internal cohort: 0.865; external cohort: 0.857) and OS (internal cohort: 0.839; external cohort: 0.842), was also significantly higher than the AJCC (internal cohort: 0.622, external cohort: 0.586 for DFS; and internal cohort: 0.615, external cohort: 0.578 for OS), BCLC (internal cohort: 0.602, external cohort: 0.574 for DFS; and internal cohort: 0.608, external cohort: 0.571 for OS), JIS (internal cohort: 0.606, external cohort: 0.581 for DFS; and internal cohort: 0.599, external cohort: 0.574 for OS), HKLC (internal cohort: 0.625, external cohort: 0.558 for DFS; and internal cohort: 0.619, external cohort: 0.541 for OS) staging systems. Overall, the nomogram models exhibited superior predictive accuracy to that of these authoritative staging systems for DFS and OS. Discussion Although patients with BCLC early-stage HCC typically have a more favorable prognosis compared to those with late-stage HCC characterized by macrovascular invasion and multiple intrahepatic metastases, a significant proportion of patients still experience recurrence and metastasis. The presence of macrovascular invasion is universally recognized as a highly influential factor in predicting poor prognosis among patients with early-stage HCC4,8,9. Moreover, recent research has demonstrated a strong correlation between the grade of macrovascular invasion and postoperative recurrence, particularly early r ecurrence8,10–13. Neither of the two most commonly used pathological staging systems for hepatocellular carcinoma (HCC) incorporates the presence of MVI as a criterion. Currently, there is a lack of reported predictive models utilizing the MVI grading system to identify Scientific Reports \| Vol:.(1234567890) (2024) 14:3470 \| https://doi.org/10.1038/s41598-024-54260-0 6 www.nature.com/scientificreports/ Figure 3. Nomograms for predicting disease-free survival (DFS) and overall survival (OS) in patients with early-stage HCC after curative hepatectomy. (a) DFS; (b) OS. MCH, mean corpuscular hemoglobin; ALP, alkaline phophatase; PAB, prealbumin; AFU, α-fucosidase; Apo-A1, apolipoprotein A1; TT, thrombin time; MVI, microvascular invasion. patients with early-stage HCC who are at a high risk of recurrence or have a poor prognosis. The development of such a model would be advantageous in order to establish a system for early and continuous monitoring or prompt postoperative adjuvant therapy for HCC patients. Consequently, nomograms were developed utilizing the MVI grading system to predict recurrence and overall survival in early-stage HCC patients who underwent curative surgery. Subsequent validation demonstrated a favorable concordance between the nomogram predictions and observed outcomes in terms of predictive probability. Furthermore, our nomograms exhibited superior predictive efficacy compared to the conventional BCLC and AJCC staging systems. The prognosis of patients with HCC is mainly affected by: (1) patient factors, such as immune function, nutritional state, liver function, and status of hepatitis virus infection; (2) tumor factors, such as tumor diameter, MVI classification, and satellite nodules; and (3) factors of treatment, in particularly adjuvant treatment after surgery. In our study, nine of the twelve risk factors associated with recurrence or OS were patient factors, including neutrophil, monocyte, ALP, PAB, MCH, Urea, LDL, Apo-A1, and TT levels, while three factors were tumor-related factors including tumor size, MVI classification, and AFU. These results indicate that the prognosis of HCC is a multifactorial and complex process. Scientific Reports \| (2024) 14:3470 \| https://doi.org/10.1038/s41598-024-54260-0 7 Vol.:(0123456789) www.nature.com/scientificreports/ Figure 4. Calibration curves for predicting disease-free survival (DFS) and overall survival (OS) using the nomograms. (a) 8-month, 1, 2, and 3-year DFS in the training cohort; (b) 8-month, 1, 2, and 3-year DFS in the internal validation cohort; (c) 8-month, 1, 2, and 3-year DFS in the external validation cohort; (d) 8-month, 1, 2, and 3-year OS in the training cohort; (e) 8-month, 1, 2, and 3-year OS in the internal validation cohort; (f) 8-month, 1, 2, and 3-year OS in the external validation cohort. The histopathological types and grades of MVI serve as indicators of the histopathological transformations that transpire when a cancer embolus within a vessel develops into a satellite lesion or a metastatic site. Scientific Reports \| Vol:.(1234567890) (2024) 14:3470 \| https://doi.org/10.1038/s41598-024-54260-0 8 www.nature.com/scientificreports/ Consequently, the histopathological type of MVI can be employed as a morphological marker for assessing the biology and advancement of HCC4,14,15. The detectability rate of MVI in patients with early-stage HCC ranges from 12.4% to 33.1%, and the prognostic significance of MVI in this patient population following curative surgery is still a matter of d ebate16–18. In our study, we found that MVI was an independent risk factor associated with DFS and OS (Fig. 2, P < 0.001), with a detection rate of 39.1% (275/703). Recently, studies indicated that the tumor microenvironment in MVI-positive HCC patients was more immunosuppressive than that in MVInegative HCC patients. This environment could promote tumor progression by activating signaling pathways that enhance tumor cell proliferation, migration, and angiogenesis including HIF-1 pathway, Wnt pathway, MAPK pathway, and Ras pathway19–21. Additionally, it recruits inhibitory immune cells and upregulates immune checkpoints to mediate immune escape in t umors22–24. Ultimately, these mechanisms jointly contribute to recurrence and metastasis in MVI-positive HCC patients after curative hepatectomy. The relationship between tumor size and patient prognosis is widely acknowledged, particularly in cases of HCC. Tumor enlargement has been consistently associated with a poor prognosis in HCC patients, leading to the establishment of cut-off values in various guidelines to predict prognosis. This is due to the non-linear nature of the relationship between tumor size and poor prognosis. For the purpose of this study, the cut-off values of 5 and 10 cm were utilized. Notably, our study revealed that tumors with a diameter exceeding 10 cm were identified as a significant risk factor for recurrence. Interestingly, despite AFP being widely recognized as a conventional clinical marker for diagnosing and prognosticating patients with HCC, our study found that it did not independently correlate with prognosis in early-stage HCC following curative hepatectomy. This observation may be attributed to the limited sensitivity of AFP in predicting the prognosis of early-stage HCC. Previous reports have indicated that AFP remains undetectable in approximately 30–35% of individuals with primary HCC, while elevated AFP levels can also be observed in individuals with normal h ealth25. It is noteworthy that AFU emerged as a significantly independent factor associated with OS in early-stage HCC. Existing literature reports AFU as a specific marker for HCC, demonstrating superior sensitivity and specificity compared to AFP in the diagnosis of HCC. Particularly, AFU exhibits high accuracy in distinguishing AFP-negative cases and early-stage HCC. Consequently, the dynamic monitoring of AFU holds immense importance in the diagnosis and prognosis of early-stage HCC26. Besides that, ALP is also a valuable predictor of early-stage HCC patients’ DFS after curative hepatectomy in our nomogram. ALP is an important indicator of liver function and highly associated with some hepatic diseases including hepatitis, cirrhosis and HCC27. It has recently been reported that ALP levels could be used to monitor and predict recurrence and metastasis in HCC patients28. Previous studies have found that high level of ALP was related to tumor cell proliferation and epithelial-mesenchymal transition (EMT)29,30. Besides, the liver is highly susceptible to oxidative stress-induced damage, with ALP serving as a dependable and sensitive marker for assessing oxidative stress. The detrimental effects of oxidative stress on hepatocytes encompass lipid, protein, and DNA impairment, ultimately leading to liver injury31. Consequently, these processes can eventually promote the metastasis and recurrence of HCC. Previous research has indicated a correlation between immune function and nutritional status and the prognosis of patients diagnosed with H CC32–35. Within our nomogram models, we have identified several influential immune and nutritional indices, namely neutrophil, monocyte, MCH, PAB, and urea, which can effectively predict prognosis. Notably, patients with low levels of neutrophils and urea, indicative of inadequate protein intake, exhibit a poorer prognosis. The tumor microenvironment is a crucial factor in the development of tumors. The immune and nutritional status, as components of the tumor microcirculation, undoubtedly impact the prognosis of patients with HCC. A growing body of evidence indicates a significant association between fundamental nutritional status, systemic inflammation, and the long-term prognosis of individuals diagnosed with cancer36–39. The presence of malnutrition and compromised immune function not only impacts the efficacy of treatment in individuals diagnosed with malignant tumors, but also increases the susceptibility of patients with HCC to relapse and metastasis36. In recent times, metabolic disorders, specifically lipid metabolism disorders, have gained prominence as a crucial microenvironment contributing to the development of HCC40,41. In this study, LDL and Apo-A1, serving as indicators of hepatic lipid metabolism, emerged as significant prognostic factors for early-stage HCC. It is well-established that alterations in liver lipid metabolism are intricately linked to the onset of liver cancer, and it is plausible that non-alcoholic fatty liver disease may be recognized as a principal etiological factor for primary liver cancer in the future42. Furthermore, prior research has demonstrated that lipid metabolism disorders can facilitate the proliferation of tumor cells by impeding the apoptosis of hepatocellular carcinoma cells, consequently leading to an unfavorable p rognosis43. However, there remains scope for additional enhancements. Initially, our model predominantly relies on datasets obtained retrospectively from two Chinese institutions. Despite the satisfactory performance of the models, certain indices were not consistently gathered in certain countries, such as South Africa. The inclusion of additional cohorts from other institutions across regions may improve the predictive accuracy and universality of models. Furthermore, our models would benefit from external validation using a completely unseen and more diversity dataset, for a more accurate description of models performance. Second, though the sample size in this study is adequate, a larger sample size in conjunction with meaningful information including postoperative adjuvant treatment collected in the future may improve the accuracy of our results. Third, although, HBV infection could strongly influence on prognosis in HCC patients, the HBV-related HCC accounted for most of HCC patients in our country, which leaded it limited influence in our models. Cohort with different eitologies, multi-population and across regions will be included in the future to determine the influence of HBV infection on the prognosis in early-stage HCC patients after curative resection. In summary, we developed and validated nomograms for predicting recurrence, especially early recurrence, and OS in patients with early-stage HCC after curative surgery. The predictive performances were superior to the common typical HCC staging systems, and they can establish patients with a high risk of recurrence or poor Scientific Reports \| (2024) 14:3470 \| https://doi.org/10.1038/s41598-024-54260-0 9 Vol.:(0123456789) www.nature.com/scientificreports/ prognosis to provide comprehensive and accurate guidance of postoperative monitoring and adjuvant therapy after surgery, such as developing the optimal frequency of follow-up examination and individualized postoperative treatment strategies, thereby resulting in improved clinical outcomes in this group of patients. Data availability All data generated or analysed during this study are included in this published article. Received: 15 October 2023; Accepted: 10 February 2024 References 1. Siegel, R. L. et al. Cancer statistics, 2023. CA Cancer J. Clin. 73, 17–48. https://doi.org/10.3322/caac.21763 (2023). 2. Dhir, M. et al. A review and update of treatment options and controversies in the management of hepatocellular carcinoma. Ann. Surg. 263, 1112–1125. https://doi.org/10.1097/sla.0000000000001556 (2016). 3. Poon, R. T. et al. Different risk factors and prognosis for early and late intrahepatic recurrence after resection of hepatocellular carcinoma. Cancer 89, 500–507 (2000). 4. Janssen, K. J. et al. Missing covariate data in medical research: To impute is better than to ignore. J. Clin. Epidemiol. 63, 721–727. https://doi.org/10.1016/j.jclinepi.2009.12.008 (2010). 5. Adam, R. et al. Resection or transplantation for early hepatocellular carcinoma in a cirrhotic liver: Does size define the best oncological strategy?. Ann. Surg. 256, 883–891. https://doi.org/10.1097/SLA.0b013e318273bad0 (2012). 6. Harrell, F. E., Lee, K. L. & Mark, D. B. Multivariable prognostic models: Issues in developing models, evaluating assumptions and adequacy, and measuring and reducing errors. Stat. Med. 15, 361–387. https://doi.org/10.1002/(sici)1097-0258(19960229)15:4% 3c361::Aid-sim168%3e3.0.Co;2-4 (1996). 7. Zhou, J. et al. Guidelines for the Diagnosis and Treatment of Primary Liver Cancer (2022 Edition). Liver Cancer 12, 405–444. https://doi.org/10.1159/000530495 (2023). 8. Iguchi, T. et al. New pathologic stratification of microvascular invasion in hepatocellular carcinoma: Predicting prognosis after living-donor liver transplantation. Transplantation 99, 1236–1242. https://doi.org/10.1097/tp.0000000000000489 (2015). 9. Tsilimigras, D. I. et al. Utilizing machine learning for pre- and postoperative assessment of patients undergoing resection for BCLC-0, A and B hepatocellular carcinoma: Implications for resection beyond the BCLC guidelines. Ann. Surg. Oncol. 27, 866–874. https://doi.org/10.1245/s10434-019-08025-z (2020). 10. Banerjee, S. et al. A computed tomography radiogenomic biomarker predicts microvascular invasion and clinical outcomes in hepatocellular carcinoma. Hepatology 62, 792–800. https://doi.org/10.1002/hep.27877 (2015). 11. Chan, A. W. H. et al. Development of pre and post-operative models to predict early recurrence of hepatocellular carcinoma after surgical resection. J. Hepatol. 69, 1284–1293. https://doi.org/10.1016/j.jhep.2018.08.027 (2018). 12. Erstad, D. J. & Tanabe, K. K. Prognostic and therapeutic implications of microvascular invasion in hepatocellular carcinoma. Ann. Surg. Oncol. 26, 1474–1493. https://doi.org/10.1245/s10434-019-07227-9 (2019). 13. Roayaie, S. et al. A system of classifying microvascular invasion to predict outcome after resection in patients with hepatocellular carcinoma. Gastroenterology 137, 850–855. https://doi.org/10.1053/j.gastro.2009.06.003 (2009). 14. Feng, L. H. et al. Novel microvascular invasion-based prognostic nomograms to predict survival outcomes in patients after R0 resection for hepatocellular carcinoma. J. Cancer Res. Clin. Oncol. 143, 293–303. https://d oi.o rg/1 0.1 007/s 00432-0 16-2 286-1 (2017). 15. Zhao, H. et al. Prognostic value of a novel risk classification of microvascular invasion in patients with hepatocellular carcinoma after resection. Oncotarget 8, 5474–5486. https://doi.org/10.18632/oncotarget.12547 (2017). 16. Huang, C. et al. Microvascular invasion has limited clinical values in hepatocellular carcinoma patients at Barcelona Clinic Liver Cancer (BCLC) stages 0 or B. BMC Cancer 17, 58. https://doi.org/10.1186/s12885-017-3050-x (2017). 17. Shindoh, J. et al. Microvascular invasion does not predict long-term survival in hepatocellular carcinoma up to 2 cm: Reappraisal of the staging system for solitary tumors. Ann. Surg. Oncol. 20, 1223–1229. https://doi.org/10.1245/s10434-012-2739-y (2013). 18. Wang, H., Wu, M. C. & Cong, W. M. Microvascular invasion predicts a poor prognosis of solitary hepatocellular carcinoma up to 2 cm based on propensity score matching analysis. Hepatol. Res. 49, 344–354. https://doi.org/10.1111/hepr.13241 (2019). 19. Mavragani, C. P. et al. Type I and II interferon signatures can predict the response to anti-TNF agents in inflammatory bowel disease patients: Involvement of the microbiota. Inflamm. Bowel Dis. 26, 1543–1553. https://doi.org/10.1093/ibd/izaa216 (2020). 20. Cai, M.A.-O. et al. T-cell exhaustion interrelates with immune cytolytic activity to shape the inflamed tumor microenvironment. J. Pathol. 251, 147–159. https://doi.org/10.1002/path.5435 (2020). 21. Dang, H. et al. LINC01419 promotes cell proliferation and metastasis in hepatocellular carcinoma by enhancing NDRG1 promoter activity. Cell. Oncol. (Dordr.) 43, 931–947. https://doi.org/10.1007/s13402-020-00540-6 (2020). 22. Lim, C. J. et al. Multidimensional analyses reveal distinct immune microenvironment in hepatitis B virus-related hepatocellular carcinoma. Gut 68, 916–927. https://doi.org/10.1136/gutjnl-2018-316510 (2019). 23. Kurebayashi, Y. et al. Landscape of immune microenvironment in hepatocellular carcinoma and its additional impact on histological and molecular classification. Hepatology https://doi.org/10.1002/hep.29904 (2018). 24. Yang, J. et al. The effects of several postoperative adjuvant therapies for hepatocellular carcinoma patients with microvascular invasion after curative resection: A systematic review and meta-analysis. Cancer Cell. Int. 21, 92. https://doi.org/10.1186/s12935- 021-01790-6 (2021). 25. Li, J. et al. A novel prognostic index-neutrophil times γ-glutamyl transpeptidase to lymphocyte ratio (NγLR) predicts outcome for patients with hepatocellular carcinoma. Sci. Rep. 7, 9229. https://doi.org/10.1038/s41598-017-09696-y (2017). 26. Wang, K. et al. Alpha-1-fucosidase as a prognostic indicator for hepatocellular carcinoma following hepatectomy: A large-scale, long-term study. Br J Cancer 110, 1811–1819. https://doi.org/10.1038/bjc.2014.102 (2014). 27. Wu, S. J. et al. Prognostic value of alkaline phosphatase, gamma-glutamyl transpeptidase and lactate dehydrogenase in hepatocellular carcinoma patients treated with liver resection. Int. J. Surg. 36, 143–151. https://doi.org/10.1016/j.ijsu.2016.10.033 (2016). 28. Yoon, J. H. et al. Early extrahepatic recurrence as a pivotal factor for survival after hepatocellular carcinoma resection: A 15-year observational study. World J. Gastroenterol. 28, 5351–5363. https://doi.org/10.3748/wjg.v28.i36.5351 (2022). 29. Yamamoto, K., Awogi, T., Okuyama, K. F. & Takahashi, N. 17Nuclear localization of alkaline phosphatase in cultured human cancer cells. Med. Electron Microsc. 36, 47–51. https://doi.org/10.1007/s007950300006 (2003). 30. Iwadate, Y., Matsutani, T., Hirono, S., Shinozaki, N. & Saeki, N. 18Transforming growth factor-β and stem cell markers are highly expressed around necrotic areas in glioblastoma. J. Neurooncol. 129, 101–107. https://doi.org/10.1007/s11060-016-2145-6 (2016). 31. Ping, L.A.-O. et al. 19Systematic oxidative stress indexes associated with the prognosis in patients with T lymphoblastic lymphoma/ leukemia. Oxid. Med. Cell. Longev. 2022, 2679154. https://doi.org/10.1155/2022/2679154 (2022). 32. Fan, W. et al. Neutrophil-to-lymphocyte and platelet-to-lymphocyte ratios as predictors of survival and metastasis for recurrent hepatocellular carcinoma after transarterial chemoembolization. PLoS One 10, e0119312. https://doi.org/10.1371/journal.pone. 0119312 (2015). Scientific Reports \| Vol:.(1234567890) (2024) 14:3470 \| https://doi.org/10.1038/s41598-024-54260-0 10 www.nature.com/scientificreports/ 33. Goh, B. K. et al. Significance of neutrophil-to-lymphocyte ratio, platelet-to-lymphocyte ratio and prognostic nutrition index as preoperative predictors of early mortality after liver resection for huge (≥10 cm) hepatocellular carcinoma. J. Surg. Oncol. 113, 621–627. https://doi.org/10.1002/jso.24197 (2016). 34. Liao, W. et al. Preoperative neutrophil-to-lymphocyte ratio as a new prognostic marker in hepatocellular carcinoma after curative resection. Transl. Oncol. 7, 248–255. https://doi.org/10.1016/j.tranon.2014.02.011 (2014). 35. Lin, Z. X. et al. Lymphocyte-to-monocyte ratio predicts survival of patients with hepatocellular carcinoma after curative resection. World J. Gastroenterol. 21, 10898–10906. https://doi.org/10.3748/wjg.v21.i38.10898 (2015). 36. Huang, P. Y. et al. Predictive effects of inflammatory scores in patients with BCLC 0-A hepatocellular carcinoma after hepatectomy. J. Clin. Med. https://doi.org/10.3390/jcm8101676 (2019). 37. Galizia, G. et al. Inflammatory and nutritional status is a predictor of long-term outcome in patients undergoing surgery for gastric cancer. Validation of the Naples prognostic score. Ann. Ital. Chir. 90, 404–416 (2019). 38. Kosuga, T. et al. Value of prognostic nutritional index as a predictor of lymph node metastasis in gastric cancer. Anticancer Res. 39, 6843–6849. https://doi.org/10.21873/anticanres.13901 (2019). 39. Bai, X. & Feng, L. Correlation between prognostic nutritional index, glasgow prognostic score, systemic inflammatory response, and TNM staging in colorectal cancer patients. Nutr. Cancer 72, 1170–1177. https://doi.org/10.1080/01635581.2019.1675725 (2020). 40. Chen, K. et al. Advancing the understanding of NAFLD to hepatocellular carcinoma development: From experimental models to humans. Biochim. Biophys. Acta Rev. Cancer 1871, 117–125. https://doi.org/10.1016/j.bbcan.2018.11.005 (2019). 41. Shi, C. et al. Acetaminophen aggravates fat accumulation in NAFLD by inhibiting autophagy via the AMPK/mTOR pathway. Eur. J. Pharmacol. 850, 15–22. https://doi.org/10.1016/j.ejphar.2019.02.005 (2019). 42. Dou, X. et al. Glutathione disulfide sensitizes hepatocytes to TNFα-mediated cytotoxicity via IKK-β S-glutathionylation: A potential mechanism underlying non-alcoholic fatty liver disease. Exp. Mol. Med. 50, 1–16. https://doi.org/10.1038/s12276-017-0013-x (2018). 43. Bai, Y. et al. ApoM is an important potential protective factor in the pathogenesis of primary liver cancer. J. Cancer 12, 4661–4671. https://doi.org/10.7150/jca.53115 (2021). Acknowledgements This work was supported by the Talent programs granted from The First Affiliated Hospital of Fujian Medical University (Grant number YJRC3600), Joint Funds for the innovation of Science and Technology, Fujian Province (Grant number 2020Y9127) and the National Natural Science Youth Foundation of China (Grant number 82303728) for data collection and analysis. Author contributions Z.J.F., G.G.X. and C.H.K. made the conception and design. Y.H.H., and Y.L.F. developed the methodology. Y.H.H., L.F.Z., S.Y.J. and Z.A.X. collected the data. C.H.K., Y.H.H. and Y.L.F. analysed and interpreted the data. Z.J.F., G.G.X. and C.H.K. wrote, reviewed, and/or revised the manuscript. Z.J.F. and G.G.X. supervised the study. Competing interests The authors declare no competing interests. Additional information Supplementary Information The online version contains supplementary material available at https://doi.org/ 10.1038/s41598-024-54260-0. 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https://openalex.org/W4389307531	https://www.scielo.br/j/rlae/a/FMsqpfJWPwW5F4Zxyqfz37c/?lang=es&format=pdf	es		Clasificación de riesgo y tiempo puerta-antibiótico en pacientes con sospecha de sepsis	Revista latino-americana de enfermagem	2,023	cc-by	7,060	Rev. Latino-Am. Enfermagem 2023;31:e4064 DOI: 10.1590/1518-8345.6635.4064 www.eerp.usp.br/rlae Artículo Original Clasificación de riesgo y tiempo puerta-antibiótico en pacientes con sospecha de sepsis Ana Paula Souza Lima1 https://orcid.org/0000-0003-2587-8496 Destacados: (1) La realización de la clasificación de riesgo Gláucio de Oliveira Nangino2 repercute favorablemente en el manejo de la sepsis. (2) https://orcid.org/0000-0002-0409-066X Fabiana Fernandes Rego Soares Cuanto mayor la prioridad clínica, más corto el tiempo 3 puerta-antibiótico. (3) El tiempo puerta-antibiótico no https://orcid.org/0000-0002-7322-326X Joyce de Carvalho Xavier difirió entre los grupos clasificado y no clasificado. (4) La 3 clasificación de riesgo asertiva es más importante que su https://orcid.org/0000-0003-4653-8431 propia implementación. Maria Cláudia Martins4 https://orcid.org/0000-0003-0136-9777 Arnaldo Santos Leite5 Objetivo: evaluar la asociación entre la clasificación de riesgo y https://orcid.org/0000-0003-4856-4166 el tiempo puerta-antibiótico en pacientes con sospecha de sepsis. Método: estudio de cohorte retrospectivo, con una muestra de 232 pacientes con sospecha de sepsis atendidos en el departamento de emergencias. Se dividieron en 2 grupos: con y sin clasificación de riesgo. Una vez identificado el tiempo puerta-antibiótico, se realizó un análisis de varianza de un factor con la prueba post hoc de Bonferroni o la prueba t de Student independiente para variables cuantitativas continuas; pruebas de correlación de Pearson, correlación biserial puntual o correlación biserial para análisis de asociación; y procedimiento de bootstrap cuando no había distribución normal de variables. Para el análisis de los datos se utilizó el software 1 2 Hospital da Polícia Militar de Minas Gerais, Centro de Terapia puerta-antibiótico no difirió entre el grupo que recibió clasificación Hospital da Polícia Militar de Minas Gerais, Diretoria Técnica, de riesgo en comparación con el que no fue clasificado. El tiempo Belo Horizonte, MG, Brasil. 3 Hospital da Polícia Militar de Minas Gerais, Unidade de Internação, Belo Horizonte, MG, Brasil. 4 5 Statistical Package for the Social Sciences. Resultados: el tiempo Intensiva, Belo Horizonte, MG, Brasil. Hospital da Polícia Militar de Minas Gerais, Centro de Terapia puerta-antibiótico fue significativamente más corto en el grupo que recibió una clasificación de riesgo de alta prioridad. Conclusión: no hubo asociación entre el tiempo puerta-antibiótico y si se realizó o Intensiva, Belo Horizonte, MG, Brasil. no la clasificación de riesgo, ni con la hospitalización en enfermería Universidade Federal de Minas Gerais, Faculdade de y en unidad de cuidados intensivos, ni con la duración de la estancia Medicina, Belo Horizonte, MG, Brasil. hospitalaria. Se observó que cuanto mayor era la prioridad, más corto era el tiempo puerta-antibiótico. Descriptores: Sepsis; Triaje; Servicio de Urgencia en Hospital; Antibacterianos; Enfermería de Urgencia; Cuidados Críticos. Cómo citar este artículo Lima APS, Nangino GO, Soares FFR, Xavier JC, Martins MC, Leite AS. Risk classification and door-toantibiotic time in patients with suspected sepsis. Rev. Latino-Am. Enfermagem. 2023;31:e4065 [cited Available from: . https://doi.org/10.1590/1518-8345.6635.4065 URL ]. mes día año Rev. Latino-Am. Enfermagem 2023;31:e4064. 2 Introducción de sepsis, ya que esto podría comprometer el desempeño general del proceso de triaje y retrasar el proceso de La sobrepoblación de los servicios de emergencia atención de otros pacientes en situaciones tan graves ha mostrado un crecimiento exponencial desde la década como esta enfermedad. Sin embargo, ante la sospecha de 1990 en varios países, y sigue siendo un fenómeno de sepsis, el profesional puede proceder con el protocolo mundial llamando al médico, quien dará seguimiento o no(10). . En Brasil, para reorganizar la prestación de (1-2) estos servicios y minimizar los riesgos y daños causados En 2018, el Departamento de Emergencias (DE) de la por el hacinamiento, se propuso, por medio de la Política institución participante implementó el Sistema Manchester Nacional de Humanización, la acogida con clasificación de Clasificación de Riesgo con el fin de garantizar que de riesgo la atención médica se produzca de acuerdo al tiempo . (3-4) Entre los protocolos de clasificación de riesgo de respuesta determinado por la gravedad clínica del existentes, se destaca el Protocolo de Manchester, paciente. En septiembre de 2019 se implementó el desarrollado por enfermeros y médicos en el Reino protocolo de manejo de la sepsis en este DE, conformado Unido. Su estrategia es establecer, entre los pacientes por un consultorio médico y una Sala de Observación y que acuden al departamento de emergencias, cuáles Clasificación de Riesgo, esta última abierta de lunes a deben tener prioridad en la atención, en base a criterios sábado de 9 h a 21 h. En su momento, se estableció que clínicos. La metodología de este protocolo se basa en la se debe dar la sospecha de sepsis a todo paciente que queja principal del paciente, la cual dirige al enfermero a presente al menos dos signos de Síndrome de Respuesta un flujograma del estado clínico. Cada flujograma contiene Inflamatoria Sistémica (SRIS), tales como: hipotermia (< discriminadores que guían la investigación y, dependiendo 35ºC) o hipertermia (> 37,8ºC), leucocitosis (> 1200 o de las respuestas del paciente, se clasifica la gravedad desviación a la izquierda > 10%) o leucopenia (< 4000), o riesgo clínico . taquicardia (> 90 lpm) y taquipnea (> 20 rpm); y un (5) Actualmente, una de las principales causas de criterio de disfunción orgánica: oliguria, hipotensión morbilidad y mortalidad a nivel mundial es la sepsis. (presión arterial sistólica < 90 mmHg), disminución La sepsis es la presencia de una disfunción orgánica del nivel de conciencia, disnea o desaturación (<92%). potencialmente mortal causada por una respuesta También se determinó que por cada paciente con sospecha desregulada del hospedero a la infección (6). Una de sepsis se llenaría un Formulario de Seguimiento de extrapolación de datos de países de altos ingresos sugiere Sepsis, en el que se registraría toda la información de la estimaciones globales de 31,5 millones de casos de sepsis atención inicial del paciente. y 19,4 millones de casos de sepsis grave, con un potencial de 5,3 millones de muertes al año(7). Considerando que el tiempo es un factor determinante para mejores resultados en el contexto de la sepsis y que Cuanto más temprano y más asertivo sea el enfoque la clasificación de riesgo puede priorizar la atención al terapéutico, mayores serán las posibilidades de obtener paciente, surge la siguiente pregunta: ¿la presencia de resultados positivos, con una mejor supervivencia de los una clasificación de riesgo en un DE reduce el TPA en pacientes con sepsis. En pacientes con alta probabilidad de pacientes con sospecha de sepsis? sepsis o posible shock séptico, se recomienda administrar El objetivo general del estudio es evaluar la asociación un antimicrobiano inmediatamente o dentro de una hora entre la clasificación de riesgo y el TPA en pacientes con después de que se reconoce o sospecha la sepsis(8). Se sospecha de sepsis. Los objetivos específicos incluyen considera tiempo puerta-antibiótico (TPA) al tiempo en verificar si hubo diferencia en el tiempo de administración horas desde la llegada del paciente al departamento de de antibióticos desde que el paciente ingresó al DE, emergencias hasta el inicio de la administración del primer dependiendo de si se realizó o no la clasificación de riesgo antibiótico . y según la clasificación por colores (prioridad), comparar (9) La sospecha inicial de sepsis basada en los el TPA con el resultado del paciente (ingreso a la Unidad flujogramas/discriminadores del Protocolo de Manchester de Cuidados Intensivos - UCI, ingreso a la enfermería, alta es bastante sensible, y generalmente determinará el inicio o muerte), y verificar la asociación entre TPA y duración del protocolo de sepsis. Por otro lado, se debe prestar de la estancia hospitalaria. atención al paciente sin disfunción orgánica clínicamente aparente, y que puede clasificarse en la prioridad clínica Método menos urgente, lo que permitiría atención médica en 120 minutos, y podría conllevar a la no adherencia al Tipo de estudio protocolo. Sin embargo, no es función del profesional responsable de la clasificación de riesgo abrir el protocolo Este es un estudio de cohorte retrospectivo. www.eerp.usp.br/rlae Lima APS, Nangino GO, Soares FFR, Xavier JC, Martins MC, Leite AS. Lugar variables: estancia en UCI, estancia en enfermería, alta, muerte y duración de la estancia. También se analizaron El estudio se realizó en un hospital público de mediana las siguientes variables: sexo, edad, Clasificación de complejidad en Belo Horizonte, Minas Gerais, Brasil. Riesgo, color de clasificación de riesgo, foco de infección Actualmente cuenta con 82 camas, 10 de las cuales están en y antibiótico utilizado. UCI, y brinda apoyo a pacientes de una amplia gama de clínicas. Recolección de datos Período La recolección de datos se realizó de diferentes Se consideró el período comprendido entre el 1 er fuentes. En primer lugar, se hizo un rastreo de los de septiembre de 2019 y el 30 de septiembre de 2021. formularios de seguimiento de sepsis, que incluyen la siguiente información: edad y sexo del paciente, si existía Población clasificación de riesgo, el color de la Clasificación de Riesgo (refleja la prioridad de atención), el foco de sospecha de Personas atendidas en el DE del hospital entre el 1er infección, el momento de inicio de la administración del de septiembre de 2019 y el 30 de septiembre de 2021, antibiótico y el antibiótico administrado. Este formulario para lograr una muestra representativa. se completa para todos los pacientes que reciben atención en el DE tan pronto como surge la sospecha de sepsis. Criterios de selección El período de dos años para la búsqueda de individuos (septiembre de 2019 a septiembre de 2021) se debe a Se incluyeron individuos adultos atendidos en el DE que este formulario fue insertado de manera concomitante del hospital, para quienes se planteó la sospecha de sepsis con la implementación del Protocolo de Manejo de la y se completó el formulario específico de la Institución, Sepsis en septiembre de 2019. A todos los pacientes se les denominado Formulario de Seguimiento de Sepsis. calculó el TPA para comparar entre los grupos de estudio Se excluyeron los pacientes que tuvieron iniciado el y control. El TPA es el comprendido entre el ingreso del protocolo de sepsis, pero posteriormente se descartó el paciente al DE (momento en que se abre el formulario de diagnóstico de infección, así como aquellos individuos remitidos atención en el mostrador) y el inicio de la administración a cuidados paliativos exclusivos, independientemente de del antibiótico. La hora de ingreso del paciente al DE haber recibido terapia antibiótica inicial. fue identificada en el Sistema Integrado de Gestão de Se consideraron pérdidas aquellos individuos cuyo Assistência à Saúde (SIGAS) del Instituto de Previdência horario de sospecha de sepsis o de administración de dos Servidores Militares de Minas Gerais (IPSM). La hora antibiótico no fue registrado. de administración del antibiótico fue identificada en el formulario de seguimiento de sepsis. Definición de la muestra La información del paciente sometido a la Clasificación de Riesgo, así como el color de la Clasificación, fueron Para definir la muestra se adoptó el valor crítico confirmados directamente en el registro de asistencia para el nivel de confianza del 95% (Za/2 = 1,96), con un archivado en el Serviço de Arquivo Médico e Estatística margen de error del 2,1% y con base en una población (SAME). finita de 260 individuos atendidos en el mismo período en el DE del hospital. La información relacionada con el resultado clínico del paciente (ingreso a la UCI o a enfermería, alta hospitalaria El grupo de estudio estuvo formado por pacientes o muerte) se recopiló de la historia clínica del paciente sometidos a la Clasificación de Riesgo, teniendo como grupo mediante el Sistema Integrado de Gestão à Saúde (SIGS). control a los pacientes no sometidos a ella. El grupo con Toda la información recopilada se ingresó a una base clasificación de riesgo estuvo conformado por los pacientes de datos organizada en una hoja de cálculo y se exportó ingresados en el DE de lunes a sábado entre las 9 h y las 21 al paquete estadístico para su análisis. h, y el grupo control lo conformaron los ingresados en el DE los domingos y otros días hábiles entre las 21 h y las 9 h. Variables del estudio Tratamiento y análisis de los datos Las variables cuantitativas continuas del resultado del estudio se compararon mediante un análisis de varianza El TPA se consideró como el resultado primario. de un factor (ANOVA one-way) con post hoc de Bonferroni Como resultados secundarios se incluyeron las siguientes para comparaciones múltiples o la prueba t de Student www.eerp.usp.br/rlae 3 Rev. Latino-Am. Enfermagem 2023;31:e4064. 4 independiente cuando las comparaciones se realizaron estudio y de la Comissão Nacional de Ética em Pesquisa solo entre dos grupos. Los análisis de asociación se (CONEP) – Certificado de Apresentação de Apreciação realizaron mediante pruebas de correlación de Pearson, Ética (CAAE) nº 57746822.8.0000.5155. correlación biserial puntual o correlación biserial, según Por tratarse de datos recopilados de forma el tipo de variables: continuas o discretas/categóricas, retrospectiva, se renunció al Formulario de Consentimiento respectivamente. Como algunas variables no presentaron Libre e Informado. Sin embargo, se realizó el Término una distribución normal, evaluadas mediante la prueba de Compromiso de Uso de Datos (TCUD), ya que la de Shapiro-Wilk, se utilizó el procedimiento de bootstrap investigación involucró el uso y recolección de información con 500 repeticiones de muestreo simple. Este es un de la base de datos de la Institución y registros de método robusto y confiable para proporcionar intervalos los pacientes. de confianza válidos cuando no se observa una distribución normal de los residuos y/o la muestra es pequeña . (11) El riesgo para los sujetos de la investigación implicó la posible exposición de información relacionada con sus Las características sociodemográficas y clínicas de la datos contenidos en registros médicos y fichas de consulta. muestra se expresaron como media (mínimo – máximo) El equipo de investigadores se compromete a minimizar o frecuencia absoluta y relativa. Los resultados de los este riesgo, divulgando los resultados únicamente en análisis de comparación se expresaron como media eventos científicos y publicándolos en revistas indexadas. ± desviación estándar, intervalo de confianza de las Además, para preservar la privacidad de los individuos, diferencias entre medias y valor p. El nivel de significancia se mantuvo el anonimato de los datos recopilados. adoptado fue alfa inferior al 5%. Los datos fueron analizados utilizando el software Resultados Statistical Package for the Social Sciences – SPSS (versión 20.0). Durante el período estudiado se realizaron 140.879 consultas en el DE del hospital, de las cuales 28.026 Aspectos éticos (19,9%) estuvieron sujetas a Clasificación de Riesgo. Se identificaron un total de 260 pacientes elegibles para Este estudio fue desarrollado de acuerdo con lo el estudio, con sospecha de sepsis. Se excluyeron 28 dispuesto en las Resoluciones nº 466/2012 y 510/2016 del pacientes (16 pacientes en cuidados paliativos, 10 sin Consejo Nacional de Salud, que establecen lineamientos información sobre la hora del inicio de la administración de y estándares para las investigaciones con seres antibióticos y 2 cuya infección fue descartada). Por tanto, humanos se analizaron 232 pacientes (Figura 1). Después de la . Fue sometido al análisis y aprobación del (12-13) Comité de Ética en Investigación de la Institución del hospitalización, se siguió a 182 pacientes. 140.879 asistencias 140.619 no elegibles 16 palliative cares 28 eliminados 10 sin información sobre la hora de inicio del antibiótico 260 incluidos 232 analizados 2 infección descartada 182 hospitalizados y acompañados 28 ingresados en outra institución 22 fueron dados alta del departamento de emergencias Figura 1 - Flujograma de la composición de la muestra de este estudio. Belo Horizonte, MG, Brasil, 2022 La Tabla 1 contiene las características sociodemográficas y clínicas de la muestra. www.eerp.usp.br/rlae Lima APS, Nangino GO, Soares FFR, Xavier JC, Martins MC, Leite AS. 5 Tabla 1 - Características sociodemográficas y clínicas de Los sitios de infección identificados como probables en los pacientes con sospecha de sepsis (n= 232). Belo pacientes con sospecha de sepsis fueron: pulmonar (33,2%), Horizonte, MG, Brasil, 2022 urinario (32,3%), abdominal (9,1%), indefinido (3,9%), otros Variables Edad Estadística descriptiva (17,6%), y no informado (3,9%). Los antibióticos utilizados 68,2 (15,0 – 101,0) en la terapia fueron: ceftriaxona (29,7%), ceftriaxona con Sexo (n / %)† azitromicina (13,8%), piperacilina con tazobactam (7,8%), Femenino 115 / 49,6 Masculino 117 / 50,4 meropenem (7,3%), ceftriaxona con metronidazol (6%), amoxicilina con ácido clavulánico (5,6%), cefepima (4,3%), Clasif.‡ de Riesgo (n / %)† Sí otros (19,5%), no informado (6%). 97 / 41,8 No El TPA no difirió entre el grupo que recibió Clasificación 135 / 58,2 de Riesgo y el grupo no clasificado: 133,2 ± 94,8 vs 152,8 ± Color de la Clasif. Riesgo (n / %) ‡ † Azul 5 / 2,2 108,0 (IC:95%: -9,4 a 39,1; p= 0,175) y 67,6 ± 48,3 vs 87,0 Verde 16 / 6,9 ± 118,8 (IC:95%: -2,8 a 21,4; p= 0,118), respectivamente. Amarillo 37 / 15,9 El promedio de TPA fue significativamente menor Naranja 38 / 16,4 Rojo 1 / 0,4 (p<0,01) en el grupo que recibió una Clasificación de No clasificado Riesgo de alta prioridad (con color naranja o rojo), en 135 / 58,2 comparación con los grupos de prioridad intermedia Hospitalización (n / %)† (clasificado como amarillo), baja prioridad (clasificados UCI§ 105 / 45,2 Enfermería 77 / 33,2 como verde o azul), y aquellos que no recibieron una No hospitalizado 50 / 21,6 Clasificación de Riesgo, como se muestra en la Tabla 2. La Tabla 3 muestra solo los pacientes que ingresaron Alta hospitalaria con vida (n / %)† Sí 147 / 63,3 en el hospital de referencia y fueron seguidos (n=182). No 35 / 15,1 Pacientes no acompañados 50 / 21,6 El TPA no fue diferente entre los grupos de hospitalización n = Muestra; †n / % = Frecuencia Absoluta / Frecuencia Relativa; ‡Clasif. = Clasificación; §UCI = Unidad de Cuidados Intensivos. Datos expresados como: promedio (mínimo – máximo) o frecuencia absoluta/relativa en enfermería y en la UCI. Lo mismo se observó con relación a los grupos que fueron dados de alta vivos o no (p>0,05 para todas las comparaciones). Tabla 2 – Comparación del TPA* entre las clasificaciones de prioridad para atención / colores entre sospechosos de sepsis (n†= 232). Belo Horizonte, MG, Brasil, 2022 Clasificación de prioridad para atención/colores TPA(min)§ Promedio ±DP\|\| Promedio de las diferencias (IC95%‡) Sin Clasificación de Riesgo Baja prioridad (Azul/Verde) Prioridad intermedia (Amarillo) Alta prioridad (Naranja/Rojo) Sin Clasificación de Riesgo 157,3 ± 8,6¶ ------- -35,5 (-99,9 a 18,1) 15,8 (-19,0 a 47,3) 60,1 (36,3 a 83,0) Baja prioridad (Azul/Verde) 192,8 ± 21,7¶ 35,5 (-18,1 a 99,9) ------- 51,3 (-6,4 a 114,1) 95,6 (42,3 a 155,8) Prioridad intermedia (Amarillo) 141,4 ± 16,4¶ -15,8 (-47,3 a 19,0) -51,3 (-114,1 a 6,4) ------- 44,3 (13,6 a 75,2) Alta prioridad (Naranja/Rojo) 97,2 ± 15,9 -60,1 (-83,0 a -36,3) -95,6 (-155,8 a -42,3) -44,3 (-75,2 a -13,6) TPA = Tiempo Puerta-Antibiótico; †n = Muestra; ‡IC95% = Intervalo de Confianza del 95%; §min = minutos; \|\|DP = Desviación Estándar; ¶p (nivel de significancia) <0,01 comparado al grupo de alta prioridad de atención (Naranja/Rojo). Datos expresados como: promedio ± desviación estándar; promedio de las diferencias (intervalo de confianza del 95% de las comparaciones) Tabla 3 – Valores promedios e IC95%* de las asociaciones entre el TPA† y la hospitalización (enfermería y UCI‡) y alta hospitalaria (n§= 182). Belo Horizonte, MG, Brasil, 2022 Variables Hospitaliz ** Enfermería UCI‡ Alta hospitalaria con vida Sí No TPA† (min)\|\| promedio ± desviación estándar [IC95%* comparaciones] (p-valor¶) 146,05 ± 109,65 152,66 ± 101,15 [-37,72 a 26,22] (0,686) 148,39 ± 109,86 154,23 ± 84,92 [-25,97 a 39,09] (0,732) IC95% = Intervalo de Confianza del 95%; †TPA = Tiempo Puerta-Antibiótico; ‡UCI = Unidad de Cuidados Intensivos; §n = Muestra; \|\|min = Minutos; ¶ p-valor = Valor de p (nivel de significancia); *Hospitaliz.= Hospitalización. Datos expresados como: promedio ± desviación estándar; [intervalo de confianza del 95% de las comparaciones entre los grupos (valor de p)] www.eerp.usp.br/rlae Rev. Latino-Am. Enfermagem 2023;31:e4064. 6 No hubo asociación entre el TPA y la realización o no de la Clasificación de Riesgo (rbp= -0,09; IC95%: -0,22 a prioridad (niveles 4 y 5 = verde y azul en el sistema de Manchester)(17). 0,04; p=0,177), tampoco con el tiempo de hospitalización Los principales sitios sospechosos de infección fueron (r= -0,06; IC95%: -0,13 a 0,11; p=0,430). Sin embargo, pulmonar (33,2%) y urinario (32,3%), similar a resultados al analizar la Clasificación de Riesgo por severidad/color encontrados en otros estudios. Uno de ellos demostró que con el TPA, se observó una asociación de baja magnitud, casi la mitad de los casos de sepsis eran secundarios a inversa y estadísticamente significativa (rb= -0,14; infecciones pulmonares (49%)(18). El predominio del foco IC95%: -0,26 a -0,01; p=0,045), demostrando que, a pulmonar en pacientes con sepsis puede ser un reflejo de mayor gravedad, menor es el TPA. que la mayoría de la población analizada estaba compuesta por personas de edad avanzada, y este grupo tiene una Discusión mayor tasa de enfermedades crónicas y, normalmente, está más predispuesto a infecciones respiratorias. Los La mayor parte de la población del estudio estuvo formada por personas mayores, con una edad media de 68,2 años, y una ligera mayoría masculina (50,4%). Estos hallazgos corroboran los de otros estudios focos urinarios y abdominales suelen alternarse como segundo y tercer sitio de infección más frecuentes(19). Se destacó el uso del antibiótico ceftriaxona en el . El tratamiento inicial de cuadros sépticos. Se aplicó solo hecho de que las personas mayores de 60 años sean más en el 29,7% de los casos, y asociado a azitromicina afectadas se explica por la presencia de enfermedades y metronidazol en el 13,8% y 6,0% de los casos, crónicas, comorbilidades, fragilidad y deterioro funcional respectivamente. (14-15) (inmunosenescencia característica de las personas El uso temprano de antimicrobianos puede mejorar mayores). A los cambios actuales en las características el pronóstico de los pacientes con sepsis, por lo que guías demográficas se suman el envejecimiento de la población internacionales recomiendan el uso empírico de antibióticos y el aumento de la esperanza de vida(16). de amplio espectro, aunque advierten contra su utilización La mayoría de los pacientes de este estudio (58,2%) excesiva, que comúnmente se asocia con el desarrollo no fueron sometidos a clasificación de riesgo. Este dato de resistencia bacteriana(20). Por tanto, la recomendación puede justificarse por el hecho de que la mayor parte latinoamericana destaca que la terapia combinada del período de recolección de datos ocurrió durante la debe reservarse para situaciones en las que datos de la pandemia de COVID-19 (Coronavirus – 2019). Durante microbiota local o de la situación clínica sugieran una mayor este período, fue necesario dividir el acceso al DE en dos: probabilidad de infección por gérmenes resistentes(21). uno para atención de pacientes con sospecha de COVID-19 Además, recomienda suspender la terapia antibiótica y otro para otras afecciones. El servicio de Clasificación empírica tan pronto como se identifique el patógeno y de Riesgo quedó solo para pacientes que no presentaron su perfil de sensibilidad antimicrobiana, así como una signos gripales, sin sospecha de COVID-19. Las personas mejoría clínica significativa, reforzando la importancia de con sospecha de COVID-19 representaron un porcentaje la reevaluación continua y el control del foco. significativo de las visitas generadas en el DE del hospital durante el período estudiado. Entre los pacientes incluidos en el estudio, el 78,4% ingresó en el hospital participante. Estos individuos fueron Entre los pacientes sometidos a Clasificación de analizados después de la hospitalización, identificándose Riesgo, la mayoría fueron clasificados como color naranja el 57,7% y el 42,3% de ellos ingresados en UCI y en (16,4%) o amarillo (15,9%). Una revisión sistemática enfermería, respectivamente. En cuanto al resultado de encontró que la sensibilidad de la detección de alta las hospitalizaciones, el 80,7% de los pacientes fueron prioridad para los resultados de enfermedades críticas dados de alta vivos y el 19,3% fallecieron. Otros pacientes variaba y era, en promedio, menor para la sepsis grave no pudieron ser monitoreados durante la hospitalización, (36 a 74%), lo que revela un desafío para los enfermeros desconociéndose su evolución hospitalaria, ya que fueron de Clasificación de Riesgo. Un total de 20 estudios capturó ingresados en otras instituciones, lo que representó el datos de sensibilidad y especificidad para las asignaciones 21,6% de la muestra inicialmente analizada en el DE. Cabe de niveles de clasificación para el resultado de la admisión destacar que la muestra estuvo conformada por pacientes hospitalaria. La sensibilidad de asignar a los pacientes con sospecha de sepsis. Un estudio multicéntrico realizado hospitalizados una prioridad más alta (niveles 1 a 3 = en Brasil demostró que un tercio de las camas de cuidados rojo, naranja y amarillo en el sistema de Manchester) fue intensivos están ocupadas por pacientes sépticos, con una relativamente alta, y sólo 3 de 20 estudios informaron tasa de mortalidad del 55,7%(22). menos del 70%. Sin embargo, todos los estudios Un estudio sobre el análisis de tendencia de la informaron ingresos hospitalarios de pacientes de baja mortalidad por sepsis en Brasil y por regiones de 2010 www.eerp.usp.br/rlae Lima APS, Nangino GO, Soares FFR, Xavier JC, Martins MC, Leite AS. a 2019 señaló que, en comparación con otros países, posterior a la utilizada en el hospital en el momento de la tanto desarrollados como subdesarrollados, las muertes investigación, presenta el discriminador “posible sepsis” en por sepsis en Brasil están en una tendencia global de alta 40 flujogramas. Este discriminador indica la evaluación del prevalencia, con una tasa de mortalidad de 22,8 muertes pulso, la temperatura, el cambio en el nivel de conciencia, por 100 mil habitantes(16). la frecuencia respiratoria y la presión arterial sistólica. Poco más de una quinta parte de los pacientes Los parámetros se establecen en el protocolo y, ante del estudio (21,6%) no fueron admitidos en el hospital alguna alteración, orienta al clasificador a seleccionar el participante. Por tanto, en este estudio no se analizaron referido discriminador. Los criterios establecidos asignan sus circunstancias, situaciones clínicas y condiciones de una alta sensibilidad para la detección precoz de la sepsis alta. Cabe señalar que la población estuvo compuesta y determinan una prioridad clínica muy urgente (naranja). por pacientes con sospecha de sepsis. Se cree que, para Cabe mencionar que, en este estudio, los pacientes la porción que fue dada de alta del DE sin indicación de clasificados como naranja presentaban alguna condición hospitalización (19,2% de la muestra inicial), se descartó crítica que señalaba al clasificador la prioridad clínica de sepsis y/o fueron pacientes estables, con indicación de muy urgente. Sin embargo, el protocolo de clasificación tratamiento en domicilio y seguimiento ambulatorio. Hay utilizado en ese momento no incluía el discriminador un estudio que señala que altas del DE de pacientes con “posible sepsis”. Se cree que esta actualización permite definición clínica de sepsis reflejan diagnósticos erróneos, al clasificador dirigir más rápidamente dichas condiciones juicios de tratamiento inadecuados, preferencias del para que el equipo de atención pueda desencadenar paciente o triaje adecuado de pacientes de bajo riesgo la secuencia de atención que implica la investigación, para tratamiento en ambulatorio el diagnóstico y el inicio temprano del tratamiento. . (23) El TPA promedio fue menor entre los pacientes Otros estudios también demostraron que el tiempo sometidos a Clasificación de Riesgo en comparación con de la administración de antibiótico fue más corto en los no clasificados, como se especifica en la Tabla 2, pero los pacientes clasificados como de mayor prioridad, sin significación estadística. Las pautas internacionales en comparación con los demás(26-27). Asimismo, se ha para el manejo de la sepsis recomiendan, para adultos demostrado que la clasificación de los pacientes en con posible shock séptico o alta probabilidad de sepsis, prioridades de atención más bajas es un factor de riesgo la administración de antimicrobianos inmediatamente, independiente de retraso en la administración de la idealmente dentro de 1 hora después del reconocimiento, primera dosis de antibiótico(28). y dentro de las 3 horas para pacientes adultos con posible sepsis sin shock . El TPA no fue diferente entre los pacientes ingresados en la UCI o en enfermería, ni se asoció con la supervivencia (8) En un estudio de 10.811 pacientes elegibles, el TPA hospitalaria. Sin embargo, para este análisis no se tuvieron mediano fue de 166 minutos (rango intercuartil, 115-230 en cuenta varias particularidades epidemiológicas que minutos) y la letalidad a 1 año fue del 19%. Después del pueden influir en el desenlace del paciente, como, por ajuste, cada hora adicional desde la llegada al DE hasta ejemplo, comorbilidades preexistentes. Tampoco se evaluó el inicio de los antibióticos se asoció con un aumento del la asertividad en el diagnóstico de sepsis y shock séptico, 10% (intervalo de confianza del 95% [IC del 95%] 5-14; así como el uso de antimicrobianos, que pueden influir P <0,001) en la letalidad a 1 año(24). Por el contrario, una directamente en la evaluación de la mortalidad. Además, encuesta realizada en un Departamento de Emergencias se sabe que el período de hospitalización de los pacientes con pacientes con shock séptico no encontró asociación con sepsis es un factor de gran influencia en su pronóstico, entre la letalidad hospitalaria y el tiempo desde el triaje de ya que la estancia del paciente, especialmente en la urgencias hasta la administración de antibióticos durante UCI, requiere de un mayor número de procedimientos las primeras 6 horas de reanimación y cuidados intensivos, lo que puede derivar en otros . (25) En este estudio, el TPA fue significativamente menor focos infecciosos(29). en el grupo que recibió una Clasificación de Riesgo de alta Otro factor relevante en los resultados del estudio prioridad en comparación con los demás, estableciéndose es la implementación del protocolo gestionado como la relación inversa, es decir, a mayor prioridad, menor estrategia que alinea las mejores evidencias en el el TPA. Este resultado apunta a la importancia de una tratamiento de la sepsis con acciones organizadas para Clasificación de Riesgo asertiva. El procedimiento tiene el reconocimiento y tratamiento, capacitación de equipos, como objetivo la identificación temprana del paciente que composición de equipos de profesionales para apoyar presenta posibilidad de empeoramiento clínico y establece las acciones, así como medición de los indicadores para prioridad para la atención de las condiciones más graves. evaluar resultados(30). Un estudio demostró que esta La versión actualizada del Sistema de Triaje de Manchester, estrategia aumentaba ocho veces las posibilidades del www.eerp.usp.br/rlae 7 Rev. Latino-Am. Enfermagem 2023;31:e4064. 8 paciente de recibir tratamiento en una hora, además de significativamente con un TPA más bajo, en comparación reducir la mortalidad en un 10,33% con los pacientes no clasificados. No hubo asociación entre . (31) Aparte del TPA, no se han estudiado resultados a el TPA y la hospitalización en UCI o en enfermería, ni con corto plazo. El efecto de confusión puede aumentar con la supervivencia hospitalaria. Tampoco hubo asociación la duración de la estancia hospitalaria. La Clasificación entre el TPA y la duración de la estancia hospitalaria. de Riesgo, según la rutina del lugar de estudio, se Los resultados de este estudio permiten evaluar la interrumpió de 21 h a 9 h de lunes a sábado, y no relevancia de realizar clasificación de riesgo en el DE y su ocurrió los domingos, lo que pudo haber influido en que impacto favorable en el manejo de pacientes con sepsis. no se encontró diferencia estadística en el TPA entre los También resaltan la importancia de ampliar las discusiones pacientes sometidos o no a la Clasificación de Riesgo. Este y estudios sobre los procesos de Clasificación de Riesgo, hecho representa una de las limitaciones de este estudio, ya que la clasificación asertiva con determinación de la ya que los momentos en los que no estaba disponible la prioridad clínica adecuada contribuye en gran medida a la Clasificación de Riesgo, es decir, los fines de semana y conducción oportuna de los manejos clínicos. Sin embargo, lo periodo nocturno, son tradicionalmente periodos de es evidente que realizar una clasificación asertiva es menor oferta de recursos, pero también de menor flujo de mucho más importante que su propia implementación. pacientes. Como no fue posible determinar el tiempo de espera en la Clasificación de Riesgo, tampoco es posible Referencias inferir su influencia en los resultados, como por ejemplo retrasos en la conducta. 1. Jobé J, Ghuysen A, D’Orio V. Advanced nurse triage Otra limitación del estudio es su diseño retrospectivo for emergency department. Rev Med Liege [Internet]. y observacional, por lo que los individuos no fueron 2018 [cited 2021 Oct 10];73(5-6):229-36. Available asignados a los grupos de estudio o de control según from: https://www.rmlg.ulg.ac.be/aboel.php?num_ criterios específicos, sino únicamente debido a las id=3006&langue=EN condiciones impuestas por la disponibilidad de recursos 2. Morley C, Unwin M, Peterson GM, Stankovich J, humanos en momentos específicos y por el fuerte impacto Kinsman L. Emergency department crowding: A systematic resultante de la pandemia de COVID-19. review of causes, consequences and solutions. PLoS One El estudio se llevó a cabo en un único centro y con [Internet]. 2018 [cited 2021 Oct 15];13(8):e0203316. un tamaño de participantes relativamente pequeño. https://doi.org/10.1371/journal.pone.0203316 El carácter retrospectivo del estudio y la posible mala 3. Ministério da Saúde (BR), Secretaria de Atenção à documentación provocaron la falta de cierta información, Saúde. Política Nacional de Humanização da Atenção e lo que dificultó la selección de los pacientes. Otra Gestão do SUS. Acolhimento e classificação de risco nos limitación fue el hecho de que el período de inclusión serviços de urgência [Internet]. Brasília: Ministério da coincidió con la pandemia de COVID-19. En ese momento, Saúde; 2009 [cited 2021 Oct 16]. Available from: https:// la Clasificación de Riesgo estaba dirigida únicamente a bvsms.saude.gov.br/bvs/publicacoes/acolhimento_ pacientes sin síntomas similares a los de la gripe. De esta classificaao_risco_servico_urgencia.pdf manera, hubo una reducción significativa en el número de 4. Sacoman TM, Beltrammi DGM, Andrezza R, Cecílio LCO, pacientes clasificados, lo que se reflejó en la estructura Reis AAC. Implementation of the Manchester Risk del estudio, en el que la mayoría de la población no fue Classification System in emergency municipal network. sometida a Clasificación de Riesgo, pero también permitió Saude Debate [Internet]. 2019 [cited 2021 Oct la composición de un grupo de control expresivo. 25];43(121):354-67. https://doi.org/10.1590/01031104201912105 Conclusión 5. Grupo Brasileiro de Classificação de Risco. Diretrizes para implementação do Sistema Manchester de Este estudio mostró que el TPA no difirió entre el Classificação de Risco nos pontos de atenção às urgências grupo que recibió clasificación de riesgo en comparación e emergências: como implementar o Sistema Manchester con el grupo que no fue clasificado. Por otro lado, se de Classificação de Risco em sua instituição de saúde observó que existe asociación entre la Clasificación [Internet]. 2.ed. Belo Horizonte: Grupo Brasileiro de Riesgo por prioridad/colores y el TPA. En otras de Classificação de Risco; 2015 [cited 2021 Oct 20]. palabras, cuanto mayor sea la prioridad clínica recibida Available from: https://www.gbcr.org.br/wp-content/ en la Clasificación de Riesgo, menor será el TPA. uploads/2021/03/DIRETRIZES.pdf La alta prioridad de atención según el Protocolo de 6. Singer M, Deutschman CS, Seymour CW, Shankar-Hari M, Manchester de Clasificación de Riesgo también se asoció Annane D, Bauer M, et al. The Third International Consensus www.eerp.usp.br/rlae Lima APS, Nangino GO, Soares FFR, Xavier JC, Martins MC, Leite AS. Definitions for Sepsis and Septic Shock (Sepsis-3). JAMA 2020 [cited 2022 Sep 25];15(1):e022765215. https:// [Internet]. 2016 [cited 2021 Nov 4];315(8):801-10. doi.org/10.1371/journal.pone.0227652 https://doi.org/10.1001/jama.2016.0287 15. Crilly J, Robinson J, Sharman V, Cross J, Romero B, 7. Fleischmann C, Scherag A, Adhikari NK, Hartog CS, Teasdale T, et al. Recognition, response and outcomes Tsaganos T, Schlattmann P, et al. Assessment of Global of sepsis: a dual site retrospective observational study. Incidence and Mortality of Hospital-treated Sepsis: Current Int Emerg Nurs [Internet]. 2019 [cited 2022 Oct 18];46(9): Estimates and Limitations. Am J Respir Crit Care Med 100782. https://doi.org/10.1016/j.ienj.2019.06.005 [Internet]. 2016 Feb [cited 2021 Nov 7];193(3):259-72. 16. Almeida NRC, Pontes GF, Jacob FL, Deprá JVS, Porto https://doi.org/10.1164/rccm.201504-0781OC JPP, Lima FR, et al. Analysis of trends in sepsis mortality 8. Evans L, Rhodes A, Alhazzani W, Antonelli M, in Brazil and by regions from 2010 to 2019. Rev Saude Coopersmith CM, French C, et al. Surviving sepsis Publica [Internet]. 2022 [cited 2022 Oct 18];56:25. campaign: international guidelines for management https://doi.org/10.11606/s1518-8787.2022056003789 of sepsis and septic shock 2021. Intensive Care Med 17. Hinson JS, Martinez DA, Cabral S, George K, Whalen M, [Internet]. 2021 [cited 2021 Nov 25];47(11):1181-247. Hansoti B, et al. Triage performance in emergency https://doi.org/10.1007/s00134-021-06506-y medicine: a systematic review. Ann Emerg Med [Internet]. 9. Levy MM, Evans LE, Rhodes A. The Surviving Sepsis 2019 [cited 2022 Sep 4];74(1):140-52. https://doi. Campaign Bundle: 2018 update. Intensive Care Med org/10.1016/j.annemergmed.2018.09.022 [Internet]. 2018 [cited 2021 Nov 25];44(6):925-8. 18. Morello LG, Dalla-Costa LM, Fontana RM, Oliveira ACS https://doi.org/10.1007/s00134-018-5085-0 Netto, Petterle RR, Conte D, et al. Assessment of clinical 10. Grupo Brasileiro de Classificação de Risco; Instituto and epidemiological characteristics of patients with and Latino Americano de Sepse. Associação entre Sistema without sepsis in intensive care units of a tertiary hospital. Manchester de Classificação de Risco e Protocolo de Sepse Einstein (São Paulo) [Internet]. 2019 [cited 2022 Sep [Internet]. Belo Horizonte: REDEC; 2017 [cited 2021 Dec 6];17(2):eAO4476. https://doi.org/10.31744/einstein_ 5]. Available from: http://redec.com.br/blog/wp-content/ journal/2019AO4476 uploads/2019/04/Nota_Tecnica_Sepse_e_Ilas_oficial.pdf 19. Abe T, Ogura H, Kushimoto S, Shiraishi A, Sugiyama T, 11. LaFleur BJ, Greevy RA. Introduction to permutation Deshpande GA, et al. Variations in infection sites and and resampling-based hypothesis tests. J Clin Child Adolesc mortality rates among patients in intensive care units Psychol. [Internet]. 2009 [cited 2021 Dec 9];38(2):286-94. with severe sepsis and septic shock in Japan. J Intensive https://doi.org/10.1080/15374410902740411 Care [Internet]. 2019 [cited 2022 Sep 6];7:28. https:// 12. Ministério da Saúde (BR), Conselho Nacional de doi.org/10.1186/s40560-019-0383-3 Saúde. Resolução nº 466, de 12 de dezembro de 2012. 20. Peng Z, Niu Z, Zhang R, Pan L, Feng H, Zhou Y, Trata sobre as diretrizes e normas regulamentadoras et al. Antimicrobial Step-Down Therapy versus Conventional de pesquisa envolvendo seres humanos. Diário Oficial Antimicrobial Therapy in the Treatment of Patients with da União [Internet]. 2013 Jun 13 [cited 2021 Dec Sepsis. Dis Markers [Internet]. 2022 [cited 2023 Jul 11];12(1):59-62. Available from: https://conselho.saude. 27];2022:3117805. https://doi.org/10.1155/2022/3117805 gov.br/ultimas_noticias/2013/06_jun_14_publicada_ 21. Instituto Latino Americano de Sepse. Guia Prático resolucao.html de Terapia Antimicrobiana na Sepse [Internet]. 2. ed. 13. Ministério da Saúde (BR), Conselho Nacional de São Paulo: ILAS; 2022 [cited 2022 Oct 18]. Available Saúde. Resolução nº 510, de 7 de abril de 2016. Dispõe from: https://ilas.org.br/wp-content/uploads/2022/02/ sobre as normas aplicáveis a pesquisas em Ciências Guia_ATM_final.pdf Humanas e Sociais cujos procedimentos metodológicos 22. Machado FR, Cavalcanti AB, Bozza FA, Ferreira EM, envolvam a utilização de dados diretamente obtidos com Carrara FSA, Sousa JL, et al. The epidemiology of sepsis os participantes ou de informações identificáveis ou que in Brazilian intensive care units (the Sepsis PRE-valence possam acarretar riscos maiores do que os existentes Assessment Database, SPREAD): an observational study. na vida cotidiana. Diário Oficial da União [Internet] Lancet Infect Dis [Internet]. 2017 [cited 2022 Sep 8];17:1180-9. 2016 May 24 [cited 2021 Dec 11];98(1):44-6. Available https://doi.org/10.1016/S1473-3099(17)30322-5 from: https://cep.ensp.fiocruz.br/sites/default/files/ 23. Peltan ID, McLean SR, Murnin E, Butler AM, reso510_2016_chs.pdf Wilson EL, Samore MH, et al. Prevalence, characteristics, 14. Husabo G, Nilsen RM, Flaatten H, Solligard E, Frich JC, and outcomes of emergency department discharge Bondevik GT, et al. Early diagnosis of sepsis in emergency among patients with sepsis. JAMA Netw Open [Internet]. departments, time to treatment, and association with 2022 [cited 2022 Sep 10];5(2):e2147882. https://doi. mortality: an observational study. PLoS One [Internet]. org/10.1001/jamanetworkopen.2021.47882 www.eerp.usp.br/rlae 9 10 Rev. Latino-Am. Enfermagem 2023;31:e4064. 24. Peltan ID, Brown SM, Bledsoe JR, Sorensen J, 31. Borguezam CB, Sanches CT, Albaneser SPR, Samore MH, Allen TL, et al. ED Door-to-Antibiotic Time Moraes URO, Grion CMC, Kerbauy G. Managed clinical and Long-term Mortality in Sepsis. Chest [Internet]. protocol: impact of implementation on sepsis treatment 2019 [cited 2022 Oct 12];155(5):938-46. https://doi. indicators. Rev Bras Enferm [Internet]. 2021 [cited 2023 org/10.1016/j.chest.2019.02.008 Jul 27];74(2):e20200282. Available from: https://doi. 25. Puskarich MA, Trzeciak S, Shapiro NI, Arnold RC, org/10.1590/0034-7167-2020-0282 Horton JM, Studnek JR, et al. Association between timing of antibiotic administration and mortality from septic shock in patients treated with a quantitative resuscitation protocol. Crit Care Med [Internet]. 2011[cited 2022 Sep 10];39(9):2066-71. https://doi.org/10.1097/ ccm.0b013e31821e87ab 26. Nevill A, Kuhn L, Thompson J, Morphet J. The influence of nurse allocated triage category on the care of patients with sepsis in the emergency department: A retrospective review. Australas Emerg Care [Internet]. 2021[cited 2022 Sep 14];24(2):121-6. Available from: https://www.sciencedirect.com/science/article/pii/ S2588994X20300956 27. Ryan K, Greenslade J, Williams J. Examining the association between triage streamed treatment location and time to appropriate antibiotics in emergency department patients with septic shock. Emerg Med Australas [Internet]. 2020 [cited 2022 Oct 15];32(6):1008-14. https://doi. org/10.1111/1742-6723.13552 28. Joseph JV, Madhiyazhagan M, Roshan R, Dhanapal SG, Arul S, Abhilash KPP. Factors affecting the time to first dose antibiotic in sepsis in acute emergency. Indian J Crit Care [Internet]. 2021 [cited 2022 Sep 20];25(10):1155-60. https://doi.org/10.5005/jp-journals-10071-23994 29. Westphal GA, Pereira AB, Fachin SM, Barreto ACC, Bornschein ACGJ, Caldeira M Filho, et al. Characteristics and outcomes of patients with community-acquired and Contribución de los autores Concepción y dibujo de la pesquisa: Ana Paula Souza Lima, Gláucio de Oliveira Nangino, Fabiana Fernandes Rego Soares, Joyce de Carvalho Xavier, Maria Cláudia Martins, Arnaldo Santos Leite. Obtención de datos: Ana Paula Souza Lima, Gláucio de Oliveira Nangino, Fabiana Fernandes Rego Soares, Joyce de Carvalho Xavier, Maria Cláudia Martins, Arnaldo Santos Leite. Análisis e interpretación de los datos: Ana Paula Souza Lima, Gláucio de Oliveira Nangino, Fabiana Fernandes Rego Soares, Joyce de Carvalho Xavier, Maria Cláudia Martins, Arnaldo Santos Leite. Análisis estadístico: Ana Paula Souza Lima, Gláucio de Oliveira Nangino, Fabiana Fernandes Rego Soares, Joyce de Carvalho Xavier, Maria Cláudia Martins, Arnaldo Santos Leite. Redacción del manuscrito: Ana Paula Souza Lima, Gláucio de Oliveira Nangino, Fabiana Fernandes Rego Soares, Joyce de Carvalho Xavier, Maria Cláudia Martins, Arnaldo Santos Leite. Revisión crítica del manuscrito en cuanto al contenido intelectual importante: Ana Paula Souza Lima, Gláucio de Oliveira Nangino, Fabiana Fernandes Rego Soares, Joyce de Carvalho Xavier, Maria Cláudia Martins, Arnaldo Santos Leite. Todos los autores aprobaron la versión final del texto. hospital-acquired sepsis. Rev Bras Ter Intensiva [Internet]. Conflicto de intereses: los autores han declarado 2019 [cited 2022 Sep 22];31(1):71-8. Available from: que no existe ningún conflicto de intereses. https://www.scielo.br/j/rbti/a/DMwbCZckQ5J3jft88cLg p7c/?lang=en 30. Instituto Latino Americano de Sepse. Roteiro de implementação de protocolo assistencial gerenciado de sepse: programa de melhoria de qualidade [Internet]. São Paulo: ILAS; 2019 [cited 2023 Jul 27]. Available from: https://ilas.org.br/wp-content/uploads/2022/05/ roteiro-de-implementacao-isbn-1.pdf Recibido: 22.12.2022 Aceptado: 03.09.2023 Editora Asociada: Evelin Capellari Cárnio Autor de correspondencia: Ana Paula Souza Lima E-mail: anapsouzal@yahoo.com.br https://orcid.org/0000-0003-2587-8496 Copyright © 2023 Revista Latino-Americana de Enfermagem Este es un artículo de acceso abierto distribuido bajo los términos de la Licencia Creative Commons CC BY. Esta licencia permite a otros distribuir, mezclar, ajustar y construir a partir de su obra, incluso con fines comerciales, siempre que le sea reconocida la autoría de la creación original. Esta es la licencia más servicial de las ofrecidas. Recomendada para una máxima difusión y utilización de los materiales sujetos a la licencia. www.eerp.usp.br/rlae
https://openalex.org/W2624632270	https://europepmc.org/articles/pmc5461223?pdf=render	English	null	Manifestations of HIV stigma and their impact on retention in care for people transitioning from prisons to communities	Health & justice	2,017	cc-by	9,555	RESEARCH ARTICLE Open Access © 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. Abstract Background: While most people living with HIV who are incarcerated in United States receive appropriate HIV care while they are in prison, interruptions in antiretroviral therapy and virologic failure are extremely common after they are released. The purpose of this study was to describe whether and how HIV stigma influences continuity of care for people living with HIV while they transition from prison to community settings. Methods: We conducted semi-structured, telephone-based interviews with 32 adults who received HIV care while residing in a Wisconsin state prison, followed by a second interview 6 months after they returned to their home community. Interview transcripts were analyzed by an interdisciplinary research team using conventional content analysis. We identified themes based on commonly-reported experiences that were characterized as internalized stigma, perceived stigma, vicarious stigma, or enacted stigma. Results: All four forms of HIV stigma appeared to negatively influence participants’ engagement in community- based HIV care. Mechanisms described by participants included care avoidance due to concerns about HIV status disclosure and symptoms of depression and anxiety caused by internalized stigma. Supportive social relationships with clinic staff, professional case managers and supportive peers appeared to mitigate the impact of HIV stigma by increasing motivation for treatment adherence. Conclusions: HIV stigma is manifest in several different forms by people living with HIV who were recently incarcerated, and are perceived by patients to negatively influence their desire and ability to engage in HIV care. By being cognizant of the pervasive influence of HIV stigma on the lives of criminal justice involved adults, HIV care providers and clinical support staff can ameliorate important barriers to optimal HIV care for a vulnerable group of patients. Keywords: Human immunodeficiency virus, Antiretroviral therapy, Incarceration, Stigma, Social support Springer, Friedland, Doros, Pesanti, & Altice, 2007). A major challenge, however, is ensuring that HIV treatment continues without interruption after release. The process of community re-entry poses significant challenges to managing HIV, including risk of substance use relapse, poor access to mental health care, and lack of employment, housing, trans- portation, and education (Brinkley-Rubinstein, 2013; Small, Wood, Betteridge, Montaner, & Kerr, 2009). Engagement in HIV care, including attendance at scheduled clinic appoint- ments and adherence to antiretroviral therapy tends to be inconsistent during the re-entry period, leading to frequent lapses in treatment and virologic failure (Baillargeon et al., 2009; Meyer et al., 2014a; Springer et al., 2004). Health and Justice Health and Justice Kemnitz et al. Health and Justice (2017) 5:7 DOI 10.1186/s40352-017-0054-1 Manifestations of HIV stigma and their impact on retention in care for people transitioning from prisons to communities Rebecca Kemnitz1, Theresa C. Kuehl1, Karli R. Hochstatter1, Emily Barker1, Anna Corey1, Elizabeth A. Jacobs1, Michael D. Repplinger1, William J. Ehlenbach1, David W. Seal2, James M. Sosman1 and Ryan P. Westergaard1* * Correspondence: rpw@medicine.wisc.edu 1University of Wisconsin Madison School of Medicine and Public Health, Madison, WI, USA Full list of author information is available at the end of the article Background The U.S. prison population carries an excess burden of HIV/AIDS compared to the general population (Maruschak, 2012). An estimated 1.5% of incarcerated U.S. adults are living with HIV, which is approximately three times greater than the prevalence of the general population. Antiretroviral therapy is effectively administered in most prison settings, and those receiving HIV care in prison tend to have good treatment outcomes (Meyer et al., 2014a; * Correspondence: rpw@medicine.wisc.edu 1University of Wisconsin Madison School of Medicine and Public Health, Madison, WI, USA Full list of author information is available at the end of the article Page 2 of 11 Page 2 of 11 Page 2 of 11 Kemnitz et al. Health and Justice (2017) 5:7 Stigma associated with one’s positive HIV status is one of the numerous individual-level, social, and structural factors that negatively influences the health of formerly incarcerated people. As defined by Goffman (1963), stigma is “an attribute that links a person to an undesir- able stereotype, leading other people to reduce the bearer from a whole and usual person to a tainted, dis- counted one” (p. 3). HIV-related stigma was concep- tualized by Parker and Aggleton (2003) as both a manifestation and a driver of social inequality, represent- ing a structural issue that leads to health disparities. Evi- dence that HIV stigma shapes the ways people living with HIV engage in care is common in the literature. In a meta-analysis of 75 studies from 32 countries, Katz et al. (2013) found that HIV stigma was consistently associ- ated with poorer antiretroviral adherence. Research de- lineating mechanisms by which stigma leads to poor HIV treatment outcomes is less common, but several formative studies suggest its effects are multi-faceted and complex. A qualitative study by Haley et al. (2014) suggested that HIV stigma negatively influenced many aspects of respondents’ lives, including acting as a trig- ger for substance use and impairing one’s ability to prioritize HIV care. Brinkley-Rubinstein (2015) described intersecting influences of racial stigma, HIV stigma and incarceration stigma, which drive people to disengage from usual sources of health care. Wisconsin Department of Corrections (DOC). Inclusion criteria specify that participants must be 18 years of age or older, English-speaking, HIV-positive and intend to re- ceive HIV care in the community upon release. Informed consent, participant demographic information, and con- tact information necessary for ensuring study follow-up after release are obtained at an in-person clinic visit. Data collection d Semi-structured interview guides were developed with open-ended questions that were informed by the situated information-motivation-behavioral skills (sIMB) framework of behavioral determinants of retention in HIV care (Amico, 2011). The main outcome of interest was continuity of HIV care in the community after re- lease from prison. Participants were invited to complete two interviews: the first interview occurred within one month prior to release and the follow-up interview approximately six months after the release date. The pre-release interview provided a baseline understanding of the participants’ experiences with HIV care prior to and during the current incarceration period and antici- pated barriers to HIV care continuity upon release. The post-release interviews were designed to provide insight into participants’ experiences transitioning to commu- nity care after release. Full text of the interview guides used for this study is presented in the Appendix. Background Subsequent study assessments are conducted via tele- phone over an unmonitored line in the Health Services Unit of the participant’s prison facility. The first 2 years of this 5-year study involved intensive data collection using numerous modalities, both before and after release from prison. All eligible DOC patients receiving HIV care were recruited for the study, and those expecting to be released during the first phase were invited to complete monthly questionnaires and a longer post-release interview during the first six months after their release date. To encourage follow-up in the study after release, participants were given the option to receive a cellular phone with unlimited voice and text for the duration of the study or monetary compensation of $50 per month, with the value of either option total- ing approximately $400 per participant. The analysis presented in this paper uses data derived from the pre- release and post-release interviews conducted among the participants enrolled during the first phase of the study. A sample size of 20–30 participants was expected to be necessary for the qualitative component of the study in order to achieve theme saturation. Quantitative data ob- tained through questionnaires, electronic health records, and HIV surveillance systems are still being collected, and will be analyzed and disseminated in future reports. To contribute to our growing understanding of how the experience of HIV stigma influences the HIV-related health of criminal justice-involved adults, we conducted a qualitative analysis of interviews with 32 individuals who received HIV care while in prison and were subsequently released. Through paired interviews scheduled several weeks before and six months after they were released, we aimed to study in-depth the experiences of people who engaged in HIV care both while incarcerated and in the community. This qualita- tive analysis is a component of a larger, mixed-methods study that longitudinally examines the HIV treatment outcomes of a cohort of patients involved with the criminal justice system. The overall aim of the parent study is to identify modifiable risk factors for suboptimal engagement in HIV care and lapses in antiretroviral adher- ence during the transition from prisons to communities. Participants Beginning in October 2013, a research assistant screened all patients referred for HIV care by the Wisconsin DOC for eligibility in the study. To date, 131 of 185 eligible indi- viduals (71%) have been approached during a clinic visit, and of these, 128 (98%) provided informed consent to par- ticipate. Of the 3 refusals, 1 individual stated he did not have time to participate because of a work-release pro- gram, 1 did not provide a reason for refusal, and 1 stated “I do not want to talk about HIV any more than I have to.” The first 32 participants who were released from prison during the intensive data collection phase were interviewed. The sample size was slightly larger than expected because a number of participants were unavail- able to complete the planned post-release interview. Twenty-three participants (72%) completed both a pre- release and post-release interview, 8 completed a pre- release interview only, and 1 completed a post-release interview only. Of the 8 participants who failed to complete the post-release interview, 6 were determined to have been re-incarcerated, 1 was discovered to have sold the study phone, and 1 was simply lost to follow-up. Data analysis The first 5 de-identified interview transcripts were read and discussed by an interdisciplinary research team to develop familiarity with the early data and agree upon an analytic approach. The team was comprised of a so- cial psychologist, three physicians, two medical students and two research assistants with training in social work and public health. The qualitative technique used was conventional content analysis (Hsieh & Shannon, 2005). A standard codebook was developed and used by the research team to identify common themes that appeared in the text of the interview transcripts. p The role of HIV stigma in continuity of HIV care was not selected as a focus of the interviews a priori by the research team, but rather emerged as an important and pervasive theme from the experiences reported by early participants. To explore stigma-related content emerging from our early analysis, we identified passages of text appearing to connote experiences of stigma and devel- oped supplemental stigma-related codes to be used data analysis. The supplemental codebook divided stigma into the four types of stigma established by Churcher (2013), and included the main headings of internalized, per- ceived, enacted, and vicarious stigma. Each main heading was further subdivided into 5–10 smaller categories to more specifically characterize the context of the stigma reported. For example, perceived stigma could reflect specific interpersonal encounters or general perceptions about public opinions, and therefore multiple codes were developed to capture these distinctions. The mean participant age was 41 years (range 19 to 68). Twenty-seven participants were male, three were female, and two were transgender women. Twenty-two parti- cipants were African American, eight were White/non- Hispanic, and two self-identified as White and Hispanic/ Latino. Nine of the male participants self-identified as men who have sex with men (MSM). Nine participants reported a history of injection drug use. Two declined to answer about previous injection drug use. Study setting and participants The Wisconsin Transitions Study is an ongoing obser- vational, prospective cohort study initiated in 2013 to comprehensively evaluate barriers to engagement in HIV care after release from prison. While incarcerated, all participants receive HIV care at the University of Wisconsin HIV/AIDS Comprehensive Care Clinic, the sole provider of antiretroviral treatment for the Each participant was asked the same initial questions, with follow-up questions and probes informed by their Kemnitz et al. Health and Justice (2017) 5:7 Page 3 of 11 initial responses. A research assistant with a background in social work and additional training in qualitative research methods conducted all of the pre-release and post-release interviews. Initial questions asked during the pre-release interview included “Tell me about your prior- ities for your first few months after going home” and “How will these affect your HIV medical care?” A typical question at the post-release interview was “Tell me about your transition from HIV care in prison to HIV care in the community.” Using these questions and subsequent probes for further explanations or clarification of partici- pants’ responses, we sought to understand participants’ at- titudes and motivation to seek HIV care and how these factors shaped their care-seeking behavior. Interviews lasted between 30 and 60 min, were digitally audio- recorded directly from the telephone and later profession- ally transcribed. The research assistant reviewed audio recordings and collaborated with the transcriptionist to ensure accuracy of the transcripts, responding to queries related to recorded speech that was difficult to under- stand. Once verified, transcripts were imported into a data analysis software package, NVivo Version 10 (QRS Inter- national, Doncaster, Australia). All interviews were coded individually by at least two members of the research team, who engaged in an itera- tive process of coding to achieve theme saturation (Morse, 1995). Any discrepancies in codes were resolved via con- sensus, referring back to the transcripts and discussing codes until there was 100% agreement on designated codes. When possible, interviews were analyzed as pairs, representing the pre-release and post-release interview from each individual participant. The research team finally met to reach consensus about the most important themes occurring throughout the dataset and selected illustrative quotes to aid in the presentation of the findings. Experiences of HIV stigma Experiences of HIV stigma All four types of stigma were experienced by numerous participants. Internalized stigma was reported most frequently; this code appeared 222 times, in 41 of the 55 transcripts analyzed. Vicarious stigma was mentioned least frequently (3 codes in 3 transcripts). In many cases, multiple forms of stigma were reported to co-occur: Participants who experienced externalized stigma often Kemnitz et al. Health and Justice (2017) 5:7 Page 4 of 11 Page 4 of 11 antiretroviral drugs with them when they were around friends, leading them to miss doses if they were away from home for an extended period. One participant de- scribed this type of situation while living with the sister of a former partner. also reported high levels of internalized stigma. In nearly all cases, reported stigma was directly linked by the par- ticipants as reasons for poor adherence to care, includ- ing missed appointments or missed doses of medication. Descriptions of perceived and experienced HIV stigma were not substantially different when taken from pre- release versus post-release interviews. Most participants had been incarcerated multiple times in the past, and recounted their prior transitions in HIV care similarly in the pre-release and post-release interviews. So they were always curious about me, so when [HIV care organization] dropped off those meds, she Googled it on her phone and saw that the pharmacy was part of the [HIV care organization] network, and basically she just tried to talk to me about it, but I found it offensive because I just, I didn’t want her to know. That wasn’t her business. So, I stopped taking the meds. (44 year-old man, pre-release interview) Internalized stigma Nearly all participants (n = 30) reported some form of internalized stigma, represented as status disclosure con- cerns, expressions of shame related to HIV risk behav- iors, or negatively connoted internal representations of HIV. Several participants cited adverse emotional re- sponses to diagnosis as a reason for missed appoint- ments. One participant reported: Placement in a Transitional Living Program (“halfway house”) was particularly problematic for several partici- pants because the dormitory-style residence offered lim- ited privacy. It’s easier for me to just carry the pills in my bag versus I got the bottles that they came in with the names on the bottles, they run into the wrong person so there’s somebody being nosy going through your stuff and they don’t know what this is and then they Google the name and then there go everything that the pill is. (37 year-old man, post-release interview) I would just get depressed, so sometimes I would just make up excuses, just like, oh, I’m just tired, and, because I really didn’t want to face what I had to do because it was just, it was just another reminder in my face that, you know, what I carry. (24 year-old trans- gender woman, pre-release interview) The most commonly reported instances of internalized stigma stemmed from concerns about disclosure of HIV status. In some cases, prior negative experiences of dis- closing their HIV status to others contributed to feelings of shame and anxiety. These emotional responses influ- enced future behaviors and social interactions, at times leading to avoidance of situations in which the confiden- tiality of their HIV status might be threatened. Fear of disclosure was a frequently cited barrier to keeping ap- pointments with HIV care providers. Discomfort related to the public nature of clinic waiting rooms was a com- mon example of this: Vicarious stigma Vicarious stigma, referring to stories or events of HIV stigma witnessed by the participant, was mentioned by only three participants. A participant’s godfather was HIV positive and discussed the stigma he faced, shaping the way that the participant anticipated stigma he might face. He states: My godfather, he used to tell me a lot of things because he got his HIV through a blood transfusion, and he used to tell me that people would treat him different. People never wanted to sit by him. People never wanted to, you know, be around him and they didn’t want to talk to him for the simple fact he was HIV positive. (28 year-old man, pre-release interview) You know, they were just ignorant to the disease as much as I was or anybody else was back then, so now I understand that. But it hurt me a lot, kind of turned me away from my family as far as, you know, resentment and, when I did think about all of that together, I just wanted to just drown it out, and I do drugs to drown it out. (51 year-old man, pre-release interview) The other 2 participants’ reporting the experience of vicarious stigma discussed the general tendency for people around them to speak negatively about people living with HIV. Participants exposed to vicarious stigma frequently described experiences of internalized stigma as well. They reported that exposure to others’ negative HIV disclosure experiences caused them to anticipate negative or discriminatory responses to disclosure of their own HIV status. Perceived stigma P d Basically I put my life on hold as far as medications and stuff because I was thinking about what other people say about me. (43 year-old man, post-release interview) Basically I put my life on hold as far as medications and stuff because I was thinking about what other people say about me. (43 year-old man, post-release interview) Common examples of enacted stigma were instances of refusal to touch, hug, or shake hands with an HIV- positive individual, isolation behavior, or throwing away dishes or silverware used by the participant. Participants discussed losing friends, being rejected by family, and being rejected from social situations or gatherings as a result of their HIV status. Participants described others’ responses to disclosure of their status as expressions of pity or fear. Participants endorsed perceptions of public disapproval of HIV risk behaviors and judgment of people living with HIV as be- ing personally responsible for their illness. Within like a year, I knew that something had to be done about it in my area, my community, because everybody was judging. There’s no reason to judge. It’s like, why would you judge someone like that? If you don’t judge someone with diabetes or leukemia or anything else, why would you judge somebody with HIV? (61 year-old man, pre-release interview). I had told [my daughter’s mother] that I was sick and that her daughter was playing by me and she told me not to touch her daughter, and I . . . I stood there in shock. I’m like, what are you talking about? She’s like, you’re sick so I don’t want you to touch my daughter and, you know, that kind of hurt me, you know. And I felt like, dang, you know, I’m sick, so everybody’s looking at me different, you know. (28 year-old man, pre-release interview) Participants who reported higher levels of perceived stigma commonly also reported high levels of internalized stigma and expressed negative attitudes about their HIV status. Negative attitudes, in turn, often corresponded with lower apparent motivation to engage in care seeking behaviors and avoidance of people and locations associ- ated with HIV services. Unpleasant thoughts and experi- ences related to HIV were also described as triggers for unhealthy behaviors such as drug and alcohol misuse. Perceived stigma P d Perceived stigma refers to the anticipated responses of others to the participants’ HIV status and was the sec- ond most common reported type of stigma by study participants (n = 21). Reports of perceived stigma were classified as social reactions to HIV status, interpersonal experiences surrounding HIV, and perceived public attitudes about HIV. In the prison setting, this type of stigma could manifest as hypervigilance and anxiety due to participants’ fears of social sanctions or even violence resulting from disclosure of their HIV status. I don’t want to have to go somewhere where I don’t feel comfortable . . . I’m going to walk into this room and I know why everybody there, and everybody know why I’m there. I guess I can say I do privy myself of what people think of me, you know, how did he get it, what they’re thinking in their head when I walk through the door. Those are a lot of questions in the back of my head that I’m thinking, you know, I guess I’m a self-preserved type of person. (44 year-old man, pre-release interview) What I mean by keeping it hidden is, you know, holding it in and not letting others know about it or what they would think about me or what they would say about me or, you know, the kind of treatment that I would get if [other incarcerated men] would, you know, try to kill me or beat me up or, you know, just the derogatory name-calling, everything. (44 year-old man, pre-release interview) After release, participants continued to fear disclosure of their HIV status to peers, criminal justice staff (e.g. probation or parole officers), and even close contacts. Engagement in HIV care and antiretroviral adherence Fearing disclosure of one’s HIV status to family or friends was linked to poor medication adherence. Partic- ipants avoided disclosure by not bringing pill cases or Page 5 of 11 Page 5 of 11 Kemnitz et al. Health and Justice (2017) 5:7 way she really treat me, you know, as far as [not] shaking my hand and some, it don’t really bother me, but I know she, either she’s not educated about HIV or she has got an attitude toward people with HIV, I don’t know. (64 year old man, post-release interview) could be influenced by stigma, even if participants were not actually treated in a negative or harmful way. Enacted stigma Enacted stigma, or specific lived experiences perceived to reflect stigmatization of HIV, were described by several participants (n = 9) as a cause of psychological distress and served to reinforce the internalization of HIV stigma. Drawing distinctions between perceived stigma and enacted stigma is difficult in this study – all the data de- rive from participants’ self-report and cannot be verified by other sources. Despite this inherent limitation, we iden- tified numerous examples of participants observing others’ behavior that appeared to be driven by HIV stigma. Discussion In this analysis of semi-structured interviews among a cohort of people living with HIV who are released from prison, we found evidence supporting a pervasive impact of HIV stigma, which influences the way people seek and receive care during the re-entry period. Internalized stigma, in particular, was cited as an important barrier to engagement in HIV care upon release from prison by the majority of participants. The real and/or perceived social consequences of identifying as a person living with HIV seemed to affect nearly all of participants’ experi- ences navigating the health care system and the social interactions of their daily lives. Additionally, participants reported positive relation- ships with HIV care providers that mitigated the intern- alization of HIV stigma. Many participants discussed their care providers as the first people to help them accept their diagnosis, often through normalization tech- niques that demonstrated HIV-positive individuals living long and generally healthy lives after receiving their HIV diagnosis. When care was established and the relation- ship built, participants reported fewer concerns regard- ing status disclosure or discomfort with the clinic waiting room. Participants responded positively when asked about their clinic experience, citing good relation- ships with their provider and the clinic staff. For ex- ample, one participant reported arriving early to chat with a front desk staff member with whom he had developed a positive relationship. In spite of disclosure concerns expressed by many participants, this type of relationship with clinic staff encouraged participants to continue to receive care and attend appointments. Our findings are consistent with previous conceptuali- zations of the role of HIV stigma in HIV care. Earnshaw and Chaudoir (2009) developed the HIV Stigma Frame- work to understand the mechanisms through which stigma works and its relevant consequences. The mani- festations of stigma documented through our analysis mirror the mechanisms of HIV stigma in their proposed model, in which stigma can be “anticipated,” “enacted” (experienced) and “internalized” by people living with HIV, to the detriment of their physical health, mental health, and social relationships. During the study period, several participants received additional services through a federally funded demon- stration project that was implemented by the Wisconsin Division of Public Health AIDS/HIV Program. The pro- ject provided funding to HIV care sites for hiring add- itional support staff, called “linkage-to-care specialists,” to provide high-intensity case management and social support. Social support mitigates the effects of HIV stigma Social support mitigates the effects of HIV stigma Participants described receiving beneficial social support from numerous sources, including family or friends, HIV care providers, and case managers. Family and [My parole officer] knows about my HIV status. And she never said anything, but I can tell her actions, the Kemnitz et al. Health and Justice (2017) 5:7 Page 6 of 11 Page 6 of 11 friends provided mixed levels of support. Although in some cases, family and friends reinforced feelings of stigmatization, they frequently played a positive role in participants’ care. Supportive family and peers often reminded participants to take medication, provided transportation to appointments, or provided a stable home environment where medication could be stored and integrated into a daily routine. If family members accepted an individual’s status, that individual was more likely to accept their status, manage their HIV, and ad- here to treatment. One participant described the role his friend’s acceptance plays in his keeping medical appoint- ments after he was released: who reported no missed clinic appointments cited the assistance from their linkage-to-care specialist as the main reason they were able to stay in care, often due to the transportation assistance they received. One 45 year- old man estimated that 90% of the financial help he re- ceived post-incarceration came from his linkage-to-care specialist’s assistance. Similarly, a 26 year-old man, dis- cussed how his relationship with his linkage-to-care spe- cialist prior to the date of his release from prison helped build his skills and confidence to manage his HIV: I’m really glad that they brought [linkage-to-care specialist] into us, so now I have someone I can call and say, hey, I need to set up health insurance. Um, hey, I need to set up an appointment with dental… Once I know where to go with all this, once I’m educated on this, I can handle it myself… But I just need her to educate me. (26 year-old man, pre-release interview) It means, staying on track means to, you know, go to my appointments, you know, then like I was telling you that, you know, the friend that I’m talking about she is understanding, so do a lot of talking about, you know, her situation. So she probably still is the only one that really accept me. (42 year-old man, post-release interview) Discussion Linkage-to-care specialists provided assistance beyond what is typically provided by medical case managers. For example, they helped participants sign up for government benefits, find housing, utilize the food pantry and other services offered at the clinic, and pro- vided transportation to clinic appointments. Participants Our study adds new empirical data in support of prior efforts to document and measure the role of stigma in the lives of people living with HIV. The Internalized AIDS-Related Stigma Scale, developed by Kalichman et al. (2009), is a validated measure of participants’ identifi- cation with common internal representations of HIV, such as “being a bad person” or “unclean” (Kipp et al., 2015). The shame and fear of status disclosure has been linked to adverse mental health outcomes, psychological distress, and substance use, which are well-described Kemnitz et al. Health and Justice (2017) 5:7 Page 7 of 11 Page 7 of 11 barriers to HIV care (Alonzo & Reynolds, 1995; Earnshaw, Smith, Chaudoir, Amico, & Copenhaver, 2013; Pulerwitz, Michaelis, Weiss, Brown, & Mahendra, 2010; Small et al., 2009; Taylor, 2001). among our participants and directly compare our find- ings to prior published work. An additional limitation of the study was inherent in the convenience sampling method for participant recruitment. All participants interviewed resided in a single U.S. state, and the majority lived in a single large city after they were released. Their experiences may therefore be imperfectly representative of individuals who receive HIV care in other regions of the U.S. or in other countries. Racial dis- parities in incarceration and HIV are particularly severe in Wisconsin: One analysis indicated that 12.8% of the state’s African American adult male population was incarcerated at the time of the 2010 U.S. Census, compared to a na- tional average of 6.7% (Pasawarat & Quinn, 2013). African Americans make up 44% of new HIV diagnoses in Wisconsin in 2014, but comprise less than 7% of the over- all population (Wisconsin Department of Health Services, 2015). Our study sample, which was comprised predomin- antly of African American men, reflects the experience of a highly disadvantaged community of extraordinary public health importance. y Social support and increased social network size have been demonstrated in prior research to be resiliency fac- tors against internalized stigma (Beals, Peplau, & Gable, 2009; Earnshaw, Lang, Lippitt, Jin, & Chaudoir, 2015; Logie & Gadalla, 2009; Sowell & Phillips, 2010). Discussion Partici- pants in this study who received high levels of social sup- port in the reintegration process reported greater success in medication adherence and fewer missed appointments with HIV care providers. Social support, especially from objective professional sources such as linkage-to-care spe- cialists, served as an important resiliency factor that miti- gated stigma and helped participants navigate both anticipated and unanticipated barriers to care upon re- lease. A previous study evaluating the Wisconsin linkage- to-care specialist intervention found that social support provided by professional case managers helped to moti- vated and enable clients to adhere to HIV care, leading to improved outcomes such as undetectable viral load (Broaddus, Hanna, Schumann, & Meier, 2015). Contrary to expectations, we did not find that partici- pants’ descriptions of stigma were unique in the context of the pre-release versus the post-release interviews. The longitudinal study data did not illuminate, for example, in- stances of anticipated HIV stigma during the pre-release period, and enacted stigma during the post-release period. The reason for this was that nearly all participants had been incarcerated and released multiple times in the past, and had experienced relevant transitions in HIV care prior to enrollment in the study. Finally, the “linkage to care specialist” intervention occurring in Wisconsin during the study period provided a level of social and material sup- port that is not typically available to most patients who require HIV care after release from prison. While this may limit the generalizability of our findings, our results suggest that similar interventions could play a beneficial role in overcoming HIV stigma and other barriers to care in diverse settings. Our findings have several implications for the delivery of HIV care to patients who were recently incarcerated. Sensitivity to the experiences of HIV stigma should inform the design of waiting room environments to provide greater privacy. When alteration of the clinic environment is not feasible, our study indicates that supportive relation- ships with staff and a welcoming social environment can ameliorate disclosure concerns among patients who at- tend clinic appointments. This is also supported by a re- cent study by Rozanova, Brown, Bhushan, Marcus, and Altice (2015), who demonstrated important benefits of es- tablishing a trusting relationship between criminal justice- involved patients and their providers. Discussion In such trusting relationships, patients are more likely to be honest about their adherence and any challenges they face and pro- viders will have a more realistic understanding of barriers that are modifiable and can be addressed (Joachim & Acorn, 2000; Vanable, Carey, Blair, & Littlewood, 2006). Clinical providers should also support patients by encour- aging them to involve supportive family members or friends in their care, and to be aware of the level of social support an individual receives from their personal rela- tionships (Aberg et al., 2014). 4. [If known positive before incarceration] b. Where will you stay? b. Where will you stay? Conclusions HIV-related stigma is an important example of the numer- ous, complex psychosocial factors that can contribute to suboptimal engagement in HIV care for adults transition- ing from prison to the general community. Stigma influ- ences care-seeking behavior through internalization of negative attitudes about HIV and contributes to significant anxiety related to disclosure of HIV status. Individual-level interventions providing social support may mitigate the psychological distress associated with the manifestations of HIV stigma. Acknowledgment of HIV stigma in crim- inal justice settings should also inform clinic-level and systems-level change to enhance supportive care environ- ments and reduce unnecessary barriers to care. Our study has several important limitations. In-depth examination of HIV stigma was not specified as a re- search question in the original study protocol, but rather emerged as a common and influential barrier to care through the semi-structured interviews. As such, we did not incorporate existing validated measures of HIV stigma into the scheduled study assessments, which would have allowed us to more rigorously assess stigma Page 8 of 11 Kemnitz et al. Health and Justice (2017) 5:7 5. Did you ever see a doctor or other provider for HIV care before you were incarcerated? 6. What makes a good HIV doctor? What is the most important characteristic about a HIV doctor, to you? 7. In general, what encourages you to keep coming in for regular care? a. Once finding out your status, how long was it before you started HIV medication? b. How many different HIV medications have you tried? c. Have you experienced negative side effects? d. What was the longest period you went without taking meds? What was going on during that time? How was your health during that time? e. What motivates you to take your meds? f. How will it be to take your HIV meds once you rejoin the community? 5. Did you ever see a doctor or other provider for HIV care before you were incarcerated? 6. What makes a good HIV doctor? What is the most important characteristic about a HIV doctor, to you? 7. In general, what encourages you to keep coming in for regular care? 5. Did you ever see a doctor or other provider for HIV care before you were incarcerated? Interview Guide Pre-release interview guide Interview Guide Pre-release interview guide Interview Guide Pre-release interview guide 6. What makes a good HIV doctor? What is the most important characteristic about a HIV doctor, to you? 7. In general, what encourages you to keep coming in for regular care? 1. Tell me a little bit about yourself. 1. Tell me a little bit about yourself. a. How long have you been incarcerated? How much have you been able to keep in contact with friends and family while you’ve been incarcerated? a. Once finding out your status, how long was it before you started HIV medication? b. How many different HIV medications have you tried? 2. If you feel comfortable, will you share a little bit about how you found out that you were positive? c. Have you experienced negative side effects? d. What was the longest period you went without taking meds? What was going on during that time? How was your health during that time? a. What did you do after hearing the news? e. What motivates you to take your meds? b. How were you feeling? Do you still feel that way now? f. How will it be to take your HIV meds once you rejoin the community? c. Did you tell anyone? If so, who did you share with? 3. Tell me about what it has been like for you to come to this clinic for HIV treatment. Are there skills or strategies you have developed to re- member to take your meds? Will those work in the com- munity? How so or why not? a. What have you liked about receiving treatment at this clinic? b. What haven’t you liked? c. What has your doctor helped you with the most? a. What have you liked about receiving treatment at this clinic? 8. Tell me about your experience taking medication for HIV. b. What haven’t you liked? c. What has your doctor helped you with the most? a. Where are you heading to? Is that a place you’ve lived before? 4. [If known positive before incarceration] 9. Tell me about your plans for release. 9. Tell me about your plans for release. a. What are your top priorities? p b. What was the clinic/hospital like? How did it compare to your care at UW? b. What resources do you have to help you achieve those priorities? c. How frequently would you see your HIV doctor when you were living in the community? c. Thinking about these other priorities in your life, how will these affect your HIV medical care? Why? d. Since the first time you saw a doctor about your HIV, what is the longest period of time you have gone without seeing a doctor for your HIV or getting your HIV labs done? d. Do you have a place in mind where you plan to receive HIV care? Is that a place you have been before? How are you feeling about going there? e. Do you have an appointment with an HIV provider in the community? [If YES] c. What do you anticipate doing the first few days? a. How would you describe your relationship with that provider? 9. Tell me about your plans for release. i. How long should those things be available? call and a longer call at 6 months, where we’ll ask a lot of questions like we did today. Where do you expect to be in 6 months? Medication adherence post-release a. Were there important people in your life that encouraged you to stay engaged in your HIV care? Post-Release Interview Guide Tell me about the clinic where you went for HIV care. Was there anything about the clinic that made it easier for you to keep your appointment? 1. It has been about 6 months since you were released from prison. What have those 6 months been like for you? a. What were your first few days like? i. Are you where you thought you’d be? b. What things were harder than you expected them to be? What things were easier? c. What has been the biggest success for you in the last 6 months? i. What has made you the happiest? ii. What has been the best thing that happened to you since you rejoined? 1. It has been about 6 months since you were released from prison. What have those 6 months been like for you? a. What were your first few days like? a. Were there things that made it hard to keep your appointments with your HIV provider? What helped you overcome these challenges? i. Are you where you thought you’d be? y g b. How many scheduled appointments did you miss with your provider? Share with me why you were unable to make the appointment(s). b. What things were harder than you expected them to be? What things were easier? c. Describe your relationship with your HIV provider. c. What has been the biggest success for you in the last 6 months? [If participant did not attend appointment with HIV provider]. [If participant did not attend appointment with HIV provider]. i. What has made you the happiest? What were the main reasons you did not see an HIV provider after you were released from prison? What things might encourage you to get back into care? ii. What has been the best thing that happened to you since you rejoined? 2. What did you find helpful during your transition? Transition to HIV care in community 4. Tell me about your transition from getting treatment for HIV in prison, to getting HIV care in the community. How does this compare to what you expected it to be like? b. Is there anything that you fear about the next 6 months? 5. [If participant attended an appointment with an HIV provider] Post-Release Interview Guide Icebreaker/General reflection on first 6 months 6. [If participant remained in care & continued ART] i. Did you meet those people after your release or did you know them before? What did they offer you? Did you encounter any challenges that made it hard to stay on your medications? b. What have you needed that you have received? a. What helped you overcome these challenges? b. How many days did you miss your meds? i. What do you wish you knew before leaving? [If participant discontinued ART for 3 days or more]. Tell me about what was going on in your life at the time you stopped taking medications for HIV. 3. Some people we’ve talked to said there were a lot more challenges that they anticipated initially. What challenges do you suppose they experienced? a. What challenges did you experience? a. What were the main reasons you did not take your meds? i. Thinking about your life before you were incarcerated – your life while incarcerated – and today, how do they compare? b. Is there anything you can think of that would have made it easier for you to keep taking your medications? [If participant indicates a gap in care] [If participant indicates a gap in care] f. How will you get to that appointment? a. Thinking about this period of time, tell me more about what else was happening in your life. 10. Tell me about your priorities for your first few months. b. Probe for more information about work, housing, incarceration, substance use, relationships, etc. 11. If a clinic or community program was going to develop a program to help people stay in regular HIV care after they are released from prison, what would be the most helpful things for the program to offer? Why are these things the most important? p c. What was your biggest priority in your life at that time? c. What was your biggest priority in your life at that time? d. Did you feel healthy/well during those times? d. Did you feel healthy/well during those times? e. What was it that got you back into regular care? Are there any skills or strategies you have developed that help you keep coming in for regular HIV care visits? 12. Our hope is that you will participate in the study for the next 6 months. That will involve a monthly Kemnitz et al. Health and Justice (2017) 5:7 Page 9 of 11 call and a longer call at 6 months, where we’ll ask a lot of questions like we did today. Where do you expect to be in 6 months? a. How will your health be? b. Is there anything that you fear about the next 6 months? i. How long should those things be available? Role of study 1University of Wisconsin Madison School of Medicine and Public Health, Madison, WI, USA. 2Tulane University School of Public Health, New Orleans, LA, USA. 1University of Wisconsin Madison School of Medicine and Public Health, Madison, WI, USA. 2Tulane University School of Public Health, New Orleans, University of Wisconsin Madison School of Medicine and Public Health, Madison, WI, USA. 2Tulane University School of Public Health, New Orleans, LA, USA. 10.What has it been like for you to be part of this study? Received: 20 October 2016 Accepted: 25 May 2017 Received: 20 October 2016 Accepted: 25 May 2017 a. How would your first 6 months have been if you were not doing the study? Availability of data and materials The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. b. [If used study cell] Alonzo, A. A., & Reynolds, N. R. (1995). Stigma, HIV and AIDS: An exploration and elaboration of a stigma trajectory. Social Science & Medicine, 41(3), 303–315. c. Did you use your study cell phone for reasons other than the study? Amico, K. R. (2011). A situated-information motivation behavioral skills model of care initiation and maintenance (sIMB-CIM): An IMB model based approach to understanding and intervening in engagement in care for chronic medical conditions. Journal of Health Psychology, 16(7), 1071–1081. doi:10.1177/1359105311398727. d. How are you feeling about completing the study today? Baillargeon, J., Giordano, T. P., Rich, J. D., Wu, Z. H., Wells, K., Pollock, B. H., & Paar, D. P. (2009). Accessing antiretroviral therapy following release from prison. Jama, 301(8), 848–857 doi:301/8/848 [pii]. Competing interests All authors have no competing interests to declare. All authors have no competing interests to declare. All authors have no competing interests to declare. 8. [If applicable] Upon your release, you were connected to a linkage to care specialist [insert specialists name here]. Describe your relationship with them. In what ways were they helpful? Did you find them to be unhelpful? 9 [If li bl ] We attest that this work has not been presented or published in any other form previously. Authors’ contributions b. Do you recall your most recent CD4 count and viral load? RW, JS and DS conceived of and designed the study and obtained funding support. RK composed the first draft of the manuscript and was responsible for integrating the contributions of all the co-authors. TK and KH coordinated day- to-day aspects of the study and conducted interviews. RK, EB, AC, EJ and RW conducted qualitative analysis. MR, WE and EJ provided substantive revisions to the manuscript. All authors reviewed and approved of the final manuscript. c. If problems with your health in the future, what will you do? i. How does your health impact your life? Ethics approval and consent to participate pp p p The study protocol was approved by the Health Sciences Institutional Review Board at the University of Wisconsin-Madison. A Certificate of Confidentiality was obtained through the National Institute on Drug Abuse. You spoke a few times about your parole officer. Please describe your relationship with them. In what ways were they helpful to you? Did you find them to be unhelpful? Does your parole officer know your HIV positive? Abbreviations d D. P. (2009). Accessing antiretroviral therapy following release from prison. Jama, 301(8), 848–857 doi:301/8/848 [pii]. Beals, K. P., Peplau, L. A., & Gable, S. L. (2009). Stigma management and well- being: The role of perceived social support, emotional processing, and suppression. Personality and Social Psychology Bulletin, 35(7), 867–879. doi:10.1177/0146167209334783. Brinkley-Rubinstein, L. (2013). Incarceration as a catalyst for worsening health. AIDS: acquired immunodeficiency syndrome; ART: antiretroviral therapy; DOC: Department of Corrections; HIV: human immunodeficiency virus; Beals, K. P., Peplau, L. A., & Gable, S. L. (2009). Stigma management and well- being: The role of perceived social support, emotional processing, and suppression. Personality and Social Psychology Bulletin, 35(7), 867–879. doi:10.1177/0146167209334783. Beals, K. P., Peplau, L. A., & Gable, S. L. (2009). Stigma management and well- being: The role of perceived social support, emotional processing, and suppression. Personality and Social Psychology Bulletin, 35(7), 867–879. doi:10.1177/0146167209334783. MSM: men who have sex with men; sIMB: situated information, motivation, and behavioral skills model Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Acknowledgments Brinkley-Rubinstein, L. (2013). Incarceration as a catalyst for worsening health. Health & Justice, 1(1), 3. doi:10.1186/2194-7899-1-3. The authors are most grateful to the study participants who shared their time and experiences for this research. We also thank the staff and administration of the Bureau of Health Services at the Wisconsin Department of Corrections, in particular Lori Alsum, Beth Dittman, Ryan Holzmacher and James Greer. Without their support, this research would not have been possible. Brinkley-Rubinstein, L. (2015). Understanding the effects of multiple stigmas among formerly incarcerated HIV-positive African American men. AIDS Education and Prevention, 27(2), 167–179. doi:10.1521/aeap.2015.27.2.167. Broaddus, M. R., Hanna, C. R., Schumann, C., & Meier, A. (2015). "she makes me feel that I'm not alone": Linkage to care specialists provide social support to people living with HIV. AIDS Care, 27(9), 1104–1107. doi:10.1080/09540121. 2015.1028882. General health/HIV knowledge b. Looking back and recalling what helped you or what brought challenge, if someone were to create a system to help people transition back to the community, what should that program offer? General health/HIV knowledge 7. How often do you think about your health, specifically HIV, these days as compared to 6 months ago? Page 10 of 11 Kemnitz et al. Health and Justice (2017) 5:7 Page 10 of 11 Page 10 of 11 a. How confident are you that you can manage your HIV care for the next year? On a scale of 1 to 10, 1 being “not confident at all” and 10 being “totally confident”. Where would you put yourself? Consent for publication Written informed consent for publication of direct quotations was obtained from individual study participants at the time of enrollment. y p y p 9. [If applicable] References Aberg, J. A., Gallant, J. E., Ghanem, K. G., Emmanuel, P., Zingman, B. S., Horberg, M. A., & Infectious Diseases Society of, A. (2014). Primary care guidelines for the management of persons infected with HIV: 2013 update by the HIV medicine association of the Infectious Diseases Society of America. Clinical Infectious Diseases, 58(1), e1–34. doi:10.1093/cid/cit665. Aberg, J. A., Gallant, J. E., Ghanem, K. G., Emmanuel, P., Zingman, B. S., Horberg, M. A., & Infectious Diseases Society of, A. (2014). Primary care guidelines for the management of persons infected with HIV: 2013 update by the HIV medicine association of the Infectious Diseases Society of America. Clinical Infectious Diseases, 58(1), e1–34. doi:10.1093/cid/cit665. i. How would the first 6 months be different for people who did not participate in the study? b. [If used study cell] Funding Th d This study was funded by the National Institutes of Health/National Institute on Drug Abuse grants K23DA032306 and R01DA030770. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of this manuscript. Churcher, S. (2013). Stigma related to HIV and AIDS as a barrier to accessing health care in Thailand: A review of recent literature. WHO South-East Asia Journal of Public Health, 2(1), 12. doi:10.4103/2224-3151.115829. Page 11 of 11 Page 11 of 11 Page 11 of 11 Kemnitz et al. Health and Justice (2017) 5:7 Earnshaw, V. A., & Chaudoir, S. R. (2009). From conceptualizing to measuring HIV stigma: A review of HIV stigma mechanism measures. AIDS and Behavior, 13(6), 1160–1177. doi:10.1007/s10461-009-9593-3. Earnshaw, V. A., Lang, S. M., Lippitt, M., Jin, H., & Chaudoir, S. R. (2015). HIV stigma and physical health symptoms: Do social support, adaptive coping, and/or identity centrality act as resilience resources? AIDS and Behavior, 19(1), 41–49. doi:10.1007/s10461-014-0758-3. Earnshaw, V. A., Smith, L. R., Chaudoir, S. R., Amico, K. R., & Copenhaver, M. M. (2013). HIV stigma mechanisms and well-being among PLWH: A test of the HIV stigma framework. AIDS and Behavior, 17(5), 1785–1795. doi:10.1007/ s10461-013-0437-9. Goffman, E. (1963). Stigma: Notes on the management of spoiled identity. Englewood Cliffs, New Jersey: Prentice-Hall. Haley, D. F., Golin, C. E., Farel, C. E., Wohl, D. A., Scheyett, A. M., Garrett, J. J., et al. (2014). Multilevel challenges to engagement in HIV care after prison release: A theory-informed qualitative study comparing prisoners' perspectives before and after community reentry. BMC Public Health, 14, 1253. doi:10.1186/1471- 2458-14-1253. Hsieh, H. F., & Shannon, S. E. (2005). Three approaches to qualitative content analysis. Qualitative Health Research, 15(9), 1277–1288. doi:10.1177/1049732305276687. Joachim, G., & Acorn, S. (2000). Living with chronic illness: The interface of stigma and normalization. The Canadian Journal of Nursing Research, 32(3), 37–48. Kalichman, S. C., Simbayi, L. C., Cloete, A., Mthembu, P. P., Mkhonta, R. N., & Ginindza, T. (2009). Measuring AIDS stigmas in people living with HIV/AIDS: The internalized AIDS-related stigma scale. AIDS Care, 21(1), 87–93. doi:10. 1080/09540120802032627. Katz, I. T., Ryu, A. E., Onuegbu, A. G., Psaros, C., Weiser, S. D., Bangsberg, D. R., & Tsai, A. C. (2013). Impact of HIV-related stigma on treatment adherence: Systematic review and meta-synthesis. Journal of the International AIDS Society, 16(3 Suppl 2), 18640. doi:10.7448/IAS.16.3.18640. Kipp, A. M., Audet, C. M., Earnshaw, V. A., Owens, J., McGowan, C. Vanable, P. A., Carey, M. P., Blair, D. C., & Littlewood, R. A. (2006). Impact of HIV-related stigma on health behaviors and psychological adjustment among HIV-positive men and women. AIDS and Behavior, 10(5), 473–482. doi:10.1007/s10461-006-9099-1. Wisconsin Department of Health Services. (2015). Wisconsin HIV/AIDS surveillance annual report. Retrieved from https://www.dhs.wisconsin.gov/publications/ p0/p00484.pdf. Taylor, B. (2001). HIV, stigma and health: Integration of theoretical concepts and the lived experiences of individuals. Journal of Advanced Nursing, 35(5), 792–798. Taylor, B. (2001). HIV, stigma and health: Integration of theoretical concepts and the lived experiences of individuals. Journal of Advanced Nursing, 35(5), 792–798. Vanable, P. A., Carey, M. P., Blair, D. C., & Littlewood, R. A. (2006). Impact of HIV-related stigma on health behaviors and psychological adjustment among HIV-positive men and women. AIDS and Behavior, 10(5), 473–482. doi:10.1007/s10461-006-9099-1. Wisconsin Department of Health Services. (2015). Wisconsin HIV/AIDS surveillance annual report. Retrieved from https://www.dhs.wisconsin.gov/publications/ p0/p00484.pdf. Funding Th d C., & Wallston, K. A. (2015). Re-validation of the van Rie HIV/AIDS-related stigma scale for use with people living with HIV in the United States. PloS One, 10(3), e0118836. doi:10.1371/journal.pone.0118836. Logie, C., & Gadalla, T. M. (2009). Meta-analysis of health and demographic correlates of stigma towards people living with HIV. AIDS Care, 21(6), 742–753. doi:10.1080/09540120802511877. Maruschak, L. (2012). HIV in prisons, 2001–2010. Washington, D.C.: Bureau of Justice Statistics Retrieved from https://www.bjs.gov/content/pub/pdf/hivp10.pdf. Meyer, J. P., Cepeda, J., Springer, S. A., Wu, J., Trestman, R. L., & Altice, F. L. (2014a). HIV in people reincarcerated in Connecticut prisons and jails: An observational cohort study. Lancet HIV, 1(2), e77–e84. doi:10.1016/S2352-3018(14)70022-0. Morse, J. (1995). The significance of saturation. Qualitative Health Research, 5(2), 147–149. Parker, R., & Aggleton, P. (2003). HIV and AIDS-related stigma and discrimination: A conceptual framework and implications for action. Social Science & Medicine, 57(1), 13–24. Pasawarat, J., & Quinn, L. M. (2013). Wisconsin’s mass incarceration of African American males: Workforce challenges for 2013. Retrieved from http://www4. uwm.edu/eti/2013/BlackImprisonment.pdf. Pulerwitz, J., Michaelis, A., Weiss, E., Brown, L., & Mahendra, V. (2010). Reducing HIV-related stigma: Lessons learned from horizons research and programs. Public Health Reports, 125(2), 272–281. doi:10.1177/003335491012500218. Rozanova, J., Brown, S. E., Bhushan, A., Marcus, R., & Altice, F. L. (2015). Effect of social relationships on antiretroviral medication adherence for people living with HIV and substance use disorders and transitioning from prison. Health Justice, 3, 18. doi:10.1186/s40352-015-0030-6. Small, W., Wood, E., Betteridge, G., Montaner, J., & Kerr, T. (2009). The impact of incarceration upon adherence to HIV treatment among HIV-positive injection drug users: A qualitative study. AIDS Care, 21(6), 708–714 doi:912522130 [pii]. Sowell, R. L., & Phillips, K. D. (2010). Understanding and responding to HIV/AIDS stigma and disclosure: An international challenge for mental health nurses. Issues in Mental Health Nursing, 31(6), 394–402. doi:10.3109/ 01612840903497602. Springer, S. A., Friedland, G. H., Doros, G., Pesanti, E., & Altice, F. L. (2007). Antiretroviral treatment regimen outcomes among HIV-infected prisoners. HIV Clinical Trials, 8(4), 205–212 doi:4T88607W588531KX [pii]. Springer, S. A., Pesanti, E., Hodges, J., Macura, T., Doros, G., & Altice, F. L. (2004). Effectiveness of antiretroviral therapy among HIV-infected prisoners: Reincarceration and the lack of sustained benefit after release to the community. Clinical Infectious Diseases, 38(12), 1754–1760. doi:10.1086/421392.
https://openalex.org/W4309399156	https://ebooks.uni-lj.si/ZalozbaUL/catalog/download/374/701/8153?inline=1	Slovene	null	Digitalna podpora slovenskemu jeziku v stiku	Založba Univerze v Ljubljani (University of Ljubljana Press) eBooks	2,022	cc-by-sa	3,438	Simpozij OBDOBJA 41 Simpozij OBDOBJA 41 Simpozij OBDOBJA 41 j j Nataša Pirih Svetina, Ina Ferbežar (ur.): Na stičišču svetov: slovenščina kot drugi in tuji jezik. Obdobja 41. Ljubljana: Založba Univerze v Ljubljani, 2022. https://centerslo.si/simpozij-obdobja/zborniki/obdobja-41/ 2 Zaradi konciznosti večkrat uporabljamo krajši, četudi površinski termin zamejci in zamejska raba, ki jo kontrastiramo s t. i. osrednjo rabo, tj. jezikovno normo slovenščine oz. osrednjim kontinuumom slovenščine. Damjan Popič Digitalna podpora slovenskemu jeziku v stiku objavljeno v: Nataša Pirih Svetina, Ina Ferbežar (ur.): Na stičišču svetov: slovenščina kot drugi in tuji jezik. Obdobja 41. Ljubljana: Založba Univerze v Ljubljani, 2022. https://centerslo.si/simpozij-obdobja/zborniki/obdobja-41/ j j Nataša Pirih Svetina, Ina Ferbežar (ur.): Na stičišču svetov: slovenščina kot drugi in tuji jezik. Obdobja 41. Ljubljana: Založba Univerze v Ljubljani, 2022. https://centerslo.si/simpozij-obdobja/zborniki/obdobja-41/ © Univerza v Ljubljani, Filozofska fakulteta, 2022. © Univerza v Ljubljani, Filozofska fakulteta, 2022. Obdobja (e-ISSN 2784-7152) Simpozij OBDOBJA 41 1 V času priprave prispevka je uradna predstavitev orodja načrtovana za jesen 2022. DIGITALNA PODPORA SLOVENSKEMU JEZIKU V STIKU Damjan Popič Filozofska fakulteta, Univerza v Ljubljani, Ljubljana damjan.popic@ff.uni-lj.si DOI:10.4312/Obdobja.41.281-290 V prispevku predstavljamo spletno aplikacijo Loris za pomoč govorcem slovenščine na območju jezikovnega stika med slovenščino in italijanščino. Loris je nastal z namenom, da uporabnikom prek spletnega vmesnika hipno in samodejno poda povratne informacije o njihovem besedilu, pri tem pa jim pomaga predvsem pri jezikovni rabi, ki je tipična za območje jezikovnega stika in je v osrednjem standardu slovenščine zaznamovana ali nepoznana. Loris, jezikovne tehnologije, spletna orodja za slovenščino This article describes Loris, the online language assistant for Slovenian speakers living in the language-contact region between Slovenian and Italian. The tool was constructed to provide instant feedback to users about their language use via an online interface, and to thereby draw users’ attention to any regional linguistic elements that may be marked or unknown in the central Slovenian standard. Loris, language technology, online tools for Slovenian 3 Številni vplivi italijanskega jezika so seveda močno vidni tudi pri govorcih slovenščine na slovenski strani meje, vendar niso pod vplivom slovenskega šolskega sistema, v katerem se perpetuirajo še restriktivnejši jezikovni vzorci in elementi (npr. tintenblic ‛edigs’, gumica ‛radirka’, kažistran ‛zaznamek’ ipd.). 1 Uvod V prispevku predstavljamo zasnovo1 novega jezikovnega orodja za slovenščino, ki nastaja v sodelovanju med Slovenskim raziskovalnim inštitutom (SLORI) in Centralnim uradom za slovenski jezik Avtonomne dežele Furlanije - Julijske krajine (CU), namenjeno pa je predvsem govorcem slovenščine na področju jezikovnega stika med slovenščino in italijanščino.2 Kot osnova orodja Loris služijo obsežne podatkovne baze o rabi slovenščine na območju jezikovnega stika, ki se nenehno posodabljajo, zajemajo pa predvsem paronime, kalke in druge zaznamovane jezikovne pojave, tem pa se pridružujejo tudi drugi dostopni normativni podatki. Pri uporabi zaznamovanih jezikovnih elementov uporabniki dobijo povratne informacije o tem, da gre za (morebiten) odstop od osrednjega standarda, obenem pa prejmejo tudi informacije o tem, kateri jezikovni elementi so morda v posameznem primeru ustreznejši, nezaznamovani in standardni. Orodje je tako namenjeno predvsem uporabnikom slovenščine na območju jezikovnega stika med italijanščino in slovenščino, pri katerih 281 Simpozij OBDOBJA 41 je slovenščina sicer nominalno prvi oz. materni jezik, vendar njihova sporazumevalna zmožnost v slovenščini v različnih govornih položajih ni na enaki ravni. V prispevku predstavljamo gradivno osnovo in vsebinsko ogrodje Lorisa, obenem pa tudi tehnično ozadje delovanja aplikacije in možnosti za njen nadaljnji razvoj. 2 Komu je Loris namenjen (in zakaj) Kot je bilo že izpostavljeno, je Loris primarno namenjen skupnosti govorcev slovenščine, ki živi na območju jezikovnega stika med slovenščino in italijanščino, zlasti na italijanski strani meje,3 in zlasti v Italiji rojenim uporabnikom slovenščine, ki se opredeljujejo za (zamejske) Slovence in Slovenke. Podatki kažejo (gl. Grgič 2017), da prihaja na območju slovenske poselitve v Italiji do gradualnega razhajanja jezikovnega koda z osrednjim standardom slovenščine, tako da se »lokalne rabe včasih že močno distancirajo od slovenskega jezikovnega kontinuuma« (Grgič 2017: 95). Pretekle raziskave (Mezgec 2016; Grgič 2017; Jagodic idr. 2017; Melinc Mlekuž 2019) izpostavljajo predvsem dve značilnosti razhajanja zamejske slovenščine z osrednjim standardom oz. vzpostavitve paralelne rabe, in sicer 1) odmik same rabe in nastanek nove, regionalne rabe ter 2) omejenost govornih položajev, v katerih se slovenščina v tem okolju uporablja, pri čemer je verjetno slednji vidik tudi (vsaj delni) povzročitelj prvega: [N]a naselitvenem območju slovenske manjšine v Italiji [so se] že uveljavili procesi, ki imajo nekatere značilnosti pidžinizacije, folklorizacije, okamnitve, oslabitve in opuščanja (zamenjave) slovenskega jezika. Ti procesi niso posledica nezadostne pravno-formalne zaščite ali nižjega statusa oz. prestiža manjšinskega jezika, ampak predvsem pomanjkanja pasivne (input) in aktivne (output) izpostavljenosti različnim rabam slovenskega splošnosporazumevalnega jezika in drugih idiomov slovenskega jezikovnega kontinuuma (Grgič 2017: 97). Slovenska skupnost v Italiji je tako razvila specifične jezikovne poteze, ki so bile predvsem v zadnjih dveh desetletjih predmet številnih raziskav (Mezgec 2016; Grgič 2017; Jagodic idr. 2017; Melinc Mlekuž 2019). Te analize so potrdile prisotnost tovrstnih pojavov, niso je mogle kvantitativno meriti, razen na sorazmerno majhnem (in posledično manj reprezentativnem) vzorcu. S sodobnimi metodami korpusnega jezikoslovja lahko na širšem segmentu besedil preverimo, katere izbire govork in govorcev slovenščine v Italiji se razlikujejo od izbir govork in govorcev slovenščine v Sloveniji ter posledično od besedil, ki nastajajo v primerljivih okoliščinah in s primerljivim namenom. Na podlagi tega lahko spremljamo stopnjo oddaljenosti dveh ali več kodov, variant oz. idiomov znotraj danega jezikovnega kontinuuma. To najbolje opazimo pri besedilih, v katerih je pričakovana variantnost minimalna – to so strukturirana besedila z ustaljenimi sporazumevalnimi vzorci in visoko koncentracijo terminov za poimenovanje iste 282 Simpozij OBDOBJA 41 Simpozij OBDOBJA 41 predmetnosti (eno- ali istoreferenčni termini oz., drugače rečeno, termini z eno ali isto referenco na obeh straneh meje). 4 www.jeziknaklik.it 5 Avtist je na območju jezikovnega stika med slovenščino in italijanščino paronim, saj se zaradi vpliva italijanščine (autista) uporablja v pomenu ‛voznik’ oz. ‛šofer’. Pri tem je nujno opozoriti, da so vsa poimenovanja v menijih v aplikaciji na dan priprave prispevka začasna in služijo zgolj prikazu delovanja. 3 Zasnova in namen orodja Loris Namen orodja je dvojni, in sicer želimo 1) pripraviti orodje, ki bo hipno in uporabniku prijazno pomagalo pri pisanju v slovenščini, obenem pa želimo 2) tudi napraviti čim bolj enovit in povezujoč jezikovni portal, ki bi združeval večje število jezikovnih virov, seveda glede na njihovo tehnično in avtorskopravno razpoložljivost. To pomeni, da poskušamo na enem mestu zbrati čim več jezikovnih virov in tehnologij, ki so sicer razpršeni po različnih infrastrukturnih centrih. Glede na zasnovo so ciljni uporabniki torej predvsem govorci slovenščine na območju jezikovnega stika med slovenščino in italijanščino, vendar želimo, da je sistem odprt in univerzalen, tako da Namen orodja je dvojni, in sicer želimo 1) pripraviti orodje, ki bo hipno in uporabniku prijazno pomagalo pri pisanju v slovenščini, obenem pa želimo 2) tudi napraviti čim bolj enovit in povezujoč jezikovni portal, ki bi združeval večje število jezikovnih virov, seveda glede na njihovo tehnično in avtorskopravno razpoložljivost. To pomeni, da poskušamo na enem mestu zbrati čim več jezikovnih virov in tehnologij, ki so sicer razpršeni po različnih infrastrukturnih centrih. Glede na zasnovo so ciljni uporabniki torej predvsem govorci slovenščine na območju jezikovnega stika med slovenščino in italijanščino, vendar želimo, da je sistem odprt in univerzalen, tako da lahko služi vsem govorcem slovenščine. Na ta način se Loris vpenja v ogrodje portala Jezik na klik,4 ki je zbirališče prosto dostopnih jezikovnih virov, orodij in gradiva, ki nastaja v okviru CU in SLORI. 2 Komu je Loris namenjen (in zakaj) S korpusnojezikoslovno raziskavo terminologije, vezane na epidemijo covida-19 (Grgič, Popič 2022), smo preverili, do kolikšne stopnje variantnosti prihaja v najsodobnejših zamejskih javnih besedilih, in sicer smo zgradili korpus iz dveh letnikov Primorskega dnevnika (2020 in 2021; okoli 20 milijonov besed). Korpusnojezikoslovna analiza je pokazala, da prihaja na področju terminologije in tudi v širši javni rabi do znatnega razhajanja med zamejskim in osrednjim jezikovnim standardom. Jezikovno (in specifično leksikalno) variantnost običajno pričakujemo pri manj formalnih sporazumevalnih kodih, pri referiranju na različno predmetnost/ pojavnost in seveda pri jezikovnih različicah (idiomih), ki jih sami govorci in govorke prepoznavajo kot lokalne/regionalne (Fought 2006: 37). Korpusna analiza pa je pokazala, da je leksikalna variantnost v besedilih, ki nastajajo na območju poselitve slovenske skupnosti v Italiji, znatna tudi takrat, ko gre za strokovno terminologijo (in bi torej pričakovali večjo enotnost), za ubesedovanje predmetnosti/pojavnosti, ki se na obeh straneh meje ne razlikuje, ter za besedila, ki jih govorci in govorke dojemajo kot standardna. 4 www.jeziknaklik.it 3.1 Zasnova Loris je zasnovan kot spletna platforma, ki se nenehno širi ter dodaja funkcionalnosti in vsebino. Uporabnik v glavno okence vpiše ali prilepi besedilo, s klikom na gumb pa Loris besedilo pregleda in uporabniku poda povratne informacije o njem. Slika 1 prikazuje delovno različico uporabniškega vmesnika orodja Loris in povratne informacije za vneseno besedo avtist.5 283 Simpozij OBDOBJA 41 Slika 1: Uporabniški vmesnik orodja Loris. Slika 1: Uporabniški vmesnik orodja Loris. Slika 1: Uporabniški vmesnik orodja Loris. Kot prikazuje Slika 1, orodje uporabnika opozori s podčrtovanjem jezikovnih elementov, ob prehodu kurzorja se prikaže pojavno okence z osnovnimi informacijami, v stranskem, informativnem delu pa so podane morebitne dodatne informacije. Pri zaznamovani leksiki, kot v primeru avtist na sliki, uporabnik prejme informacijo o tem, da je leksem zaznamovan, kaj pomeni v osrednjem standardu slovenščine, obenem pa dobi še nekaj tipičnih primerov rabe. Povratne informacije so seveda odvisne od tipa jezikovnega pojava, ki ga orodje prepozna. V naslednjem odseku so predstavljeni tipi jezikovnih pojavov, ki jih orodje prepozna oz. zajema. 6 Stanje na dan 20. 8. 2022. 3.2 Gradivna osnova Lorisa Orodje Loris temelji na spiskih besedišča, zajema pa naslednje kategorije:6 1) paronime in kalke, Orodje Loris temelji na spiskih besedišča, zajema pa naslednje kategorije:6 1) paronime in kalke, 2) toponime, 2) toponime, ) p , 3) normirano terminologijo na področju Furlanije - Julijske krajine (terminološke komisije pri CU),i 3) normirano terminologijo na področju Furlanije - Julijske krajine (terminološke komisije pri CU),i 4) leksiko, klasificirano kot prepovedano (SP 2001),i 5) leksiko, klasificirano kot nepravilno (SP 2001), 6) leksiko z normativno oznako (Sloleks), 7) sopomenke (CJVT), 8) črkovalnik (hunspell). 3.2.1 Paronimi7 Paronimi so z vidika uporabniške izkušnje za orodje Loris bistvenega pomena, zato je v okviru priprave baze za orodje pripisanih tudi več informacij, ki lahko uporabniku povedo, zakaj je določen jezikovni element (lahko) zunaj okvirov zamejske skupnosti razumljen drugače. Informacije, vključene v podatkovno bazo Lorisa, na primeru paronima abonma, podaja Slika 2. Slika 2: Prikaz informacij o leksemu abonma. Slika 2: Prikaz informacij o leksemu abonma. 8) črkovalnik (hunspell). Kot lahko vidimo, Loris zajema raznorodne kategorije, število kategorij pa se bo s časom še povečevalo, zlasti z vključitvijo strojno naučenih modelov. Podatki v orodju, ki niso statični oz. prevzeti iz drugih jezikovnih virov (pravopisa, Sloleksa ali Sopomenk 1.0), so bili pridobljeni (in se tudi posodabljajo) na naslednje načine: 284 Simpozij OBDOBJA 41 –– s pregledom obstoječih raziskav, ki se dotikajo zamejske jezikovne rabe; –– s pregledom prevajalske in lektorske prakse v okviru CU in SLORI; –– s korpusnojezikoslovno primerjavo besedil Primorskega dnevnika in drugih korpusov ter jezikovnih virov za slovenščino; –– z odzivnim nabiranjem primerov besedja med pripadniki skupnosti, s čimer se poskušamo pri izdelavi jezikovnega orodja čim bolj vpeti v skupnost, hkrati pa omogočiti tudi takojšnjo odzivnost. –– z odzivnim nabiranjem primerov besedja med pripadniki skupnosti, s čimer se poskušamo pri izdelavi jezikovnega orodja čim bolj vpeti v skupnost, hkrati pa omogočiti tudi takojšnjo odzivnost. 7 Zaradi želje po čim manjšem številu kategorij in posledično tudi datotek uporabljamo paronim kot krovni termin za vse pojave jezikovnega stikanja. 8 To so Duino/Devin, Trieste/Trst, Barcola/Barkovlje, Roiano/Rojan, San Giovanni/Sveti Ivan, San Giacomo/Sveti Jakob, Coloncovez/Kolonkovec, San Vito/Sveti Vid, Guardiella/Vrdela, Miramare/ Miramar, Conconello/Ferlugi, Cattinara/Katinara/Četnara, Longera/Lonjer, Banne/Bani, Basovizza/ Bazovica, Gropada/Gropada, Santa Croce/Križ, Villa Opicina/Opčine, Padriciano/Padriče, Prosecco/ Prosek, Trebiciano/Trebče, Malchina/Mavhinje, Aurisina/Nabrežina, Aurisina Stazione/Nabrežina Postaja, Aurisina Cave/Nabrežina Kamnolomi, Villaggio del Pescatore/Ribiško naselje, San Pelagio/ Šempolaj, Visogliano/Vižovlje, Bagnoli della Rosandra/Boljunec, Grozzana/Gročana, Caresana/ Mačkovlje, Pesek/Pesek, San Giuseppe della Chiusa/Ricmanje, Monrupino/Repentabor, Sales/Salež, Gorizia/Gorica, Lucinico/Ločnik, Oslavia/Oslavje, Doberdò del Lago/Doberdob, Vallone/Dol, Jamiano/ Jamlje, Marcottini/Poljane, Gabria/Gabrje, Savogna d’Isonzo/Sovodnje ob Soči, San Floriano del Collio/Števerjan, Cormons/Krmin, Ronchi dei Legionari/Ronke, Staranzano/Štarancan, Monfalcone/ Tržič, Vencò/Jenkovo, Malborghetto/Naborjet, Collio/Brda, Cividale del Friuli/Čedad, Firenze/Firence, Val Canale/Kanalska dolina, Napoli/Neapelj, Aquileia/Oglej, Sottolongera/Podlonjer, Roma/Rim, Val Rosandra/Dolina Glinščice, Sella Nevea/Na Žlebeh, Duino Aurisina/Devin - Nabrežina, Malborghetto Valbruna/Naborjet - Ovčja vas. Slika 2: Prikaz informacij o leksemu abonma. Kot lahko vidimo, so v podatkovno bazo vključene potrebne informacije, da lahko uporabnik prepozna potencialno napačno oz. zaznamovano rabo leksema abonma. 7 Zaradi želje po čim manjšem številu kategorij in posledično tudi datotek uporabljamo paronim kot krovni termin za vse pojave jezikovnega stikanja. 285 Simpozij OBDOBJA 41 Valbruna/Naborjet - Ovčja vas. 3.2.2 Toponimi Druga kategorija, ki je prav tako močno vezana na okolje jezikovnega stika med slovenščino in italijanščino, zajema toponime, in sicer slovenska imena krajev s slovensko poselitvijo v Italiji ter nekaterih krajev s slovenskimi oblikami imen (npr. Rim). Orodje uporabnika pri rabi italijanskega imena opozori, da obstaja tudi slovensko ime. Pri tem je nujno opozoriti, da so v orodje vključena zgolj zemljepisna imena, ki so v rabi ustaljena in pri katerih ni potencialnih dvojnic, zlasti pa tista, ki jih v celoti sprejema skupnost. S seznama 476 zemljepisnih imen jih je bilo v orodje Loris za zdaj vključenih 64.8 Informacije o toponimih in njihov prikaz ponazarja Slika 3. Slika 3: Prikaz informacij o toponimu Monfalcone v orodju Loris. 8 To so Duino/Devin, Trieste/Trst, Barcola/Barkovlje, Roiano/Rojan, San Giovanni/Sveti Ivan, San Giacomo/Sveti Jakob, Coloncovez/Kolonkovec, San Vito/Sveti Vid, Guardiella/Vrdela, Miramare/ Miramar, Conconello/Ferlugi, Cattinara/Katinara/Četnara, Longera/Lonjer, Banne/Bani, Basovizza/ Bazovica, Gropada/Gropada, Santa Croce/Križ, Villa Opicina/Opčine, Padriciano/Padriče, Prosecco/ Prosek, Trebiciano/Trebče, Malchina/Mavhinje, Aurisina/Nabrežina, Aurisina Stazione/Nabrežina Postaja, Aurisina Cave/Nabrežina Kamnolomi, Villaggio del Pescatore/Ribiško naselje, San Pelagio/ Šempolaj, Visogliano/Vižovlje, Bagnoli della Rosandra/Boljunec, Grozzana/Gročana, Caresana/ Mačkovlje, Pesek/Pesek, San Giuseppe della Chiusa/Ricmanje, Monrupino/Repentabor, Sales/Salež, Gorizia/Gorica, Lucinico/Ločnik, Oslavia/Oslavje, Doberdò del Lago/Doberdob, Vallone/Dol, Jamiano/ Jamlje, Marcottini/Poljane, Gabria/Gabrje, Savogna d’Isonzo/Sovodnje ob Soči, San Floriano del Collio/Števerjan, Cormons/Krmin, Ronchi dei Legionari/Ronke, Staranzano/Štarancan, Monfalcone/ Tržič, Vencò/Jenkovo, Malborghetto/Naborjet, Collio/Brda, Cividale del Friuli/Čedad, Firenze/Firence, Val Canale/Kanalska dolina, Napoli/Neapelj, Aquileia/Oglej, Sottolongera/Podlonjer, Roma/Rim, Val Rosandra/Dolina Glinščice, Sella Nevea/Na Žlebeh, Duino Aurisina/Devin - Nabrežina, Malborghetto Valbruna/Naborjet - Ovčja vas. Slika 3: Prikaz informacij o toponimu Monfalcone v orodju Loris. Slika 3: Prikaz informacij o toponimu Monfalcone v orodju Loris. 286 Simpozij OBDOBJA 41 Simpozij OBDOBJA 41 Simpozij OBDOBJA 41 Kot lahko vidimo, orodje ob prepoznanem italijanskem poimenovanju poda infor macije o slovenski ustreznici ter poimenovanjih za prebivalca, prebivalko, pridevnik na -ski/ški, občino in pokrajino. 3.2.3 Normirana terminologija Pri normirani terminologiji gre za termine, ki so potrjeni v okviru terminoloških komisij, ki delujejo v zamejskem okolju pod okriljem Centralnega urada za slovenski jezik Avtonomne pokrajine Furlanije - Julijske krajine. Ker govorci slovenščine v Italiji v številnih vidikih živijo in delujejo v dvojnem sistemu ter so razpeti med Slovenijo in Italijo, mora slovenščina poimenovati številne koncepte, ki jih v slovenskem sistemu ni. Terminološke komisije (računovodska, šolska ipd.) s številnimi pridruženimi strokovnjaki skrbijo za čim boljšo poenotenost terminologije na teh področjih, hkrati pa tudi za posledično poenotenost prevodov italijanskih uradovalnih besedil v slovenščino. Zapisniki terminoloških komisij so dostopni na portalu Jezik na klik, medtem ko neposredni rezultat njihovih naporov odseva tudi v Zvezku normiranih terminov,9 ki ga pripravlja CU. Ta je vključen tudi v orodje Loris, in sicer kot neposredna povezava, krajša leksika, ki jo orodje lahko prepozna, pa je vključena tudi neposredno kot podatkovna baza. 9 https://www.jeziknaklik.it/zvezek/ 3.2.4 Prepovedana in nepravilna leksika (SP 2001) ter normativno označena leksika (Sloleks) in črkovalnik Ker se pri izdelavi orodja Loris oziramo po vseh dostopnih virih, ki so lahko pišočim v pomoč, smo vanj vključili tudi leksiko, ki ni neposredno povezana s pojavi jezikovnega stika. Tako je dodana tudi leksika, ki je v Slovenskem pravopisu 2001 označena z normativno oznako in jo je mogoče posledično tudi izluščiti s portala Fran. Normativno vrednotenemu besedju iz pravopisnega priročnika smo dodali tudi vse leme iz oblikoslovnega leksikona Sloleks, ki imajo normativno oznako. Tri oblike v treh naštetih kategorijah in njihov prikaz ponazarja Slika 4, obenem pa lahko vidimo tudi predloge črkovalnika. 287 Simpozij OBDOBJA 41 Slika 4: Normativne oznake in popravek črkovalnika v Lorisu. Slika 4: Normativne oznake in popravek črkovalnika v Lorisu. Kot prikazuje Slika 4, so pravopisno normirane oblike označene enako, ne glede na vrsto normativne oznake v pravopisnem slovarju, normirano besedje iz Sloleksa pa se vidno razlikuje. Razlog za to je, da gre pri oblikah iz SP 2001 za končni seznam, medtem ko bomo poskušali morebitno dodatno normativno označeno besedje v poznejših različicah Sloleksa sproti tudi dodajati. Zaradi splošne uporabnosti je orodju dodan tudi prosto dostopen črkovalnik hunspell, ki ga želimo v prihodnosti ravno tako posodabljati na podlagi frekvenčnih seznamov korpusov slovenščine. 10 https://viri.cjvt.si/sopomenke/slv/about 11 Portal je zasnovan tako, da se leksikalni predlogi beležijo v posebno bazo, ki je prikazana tudi na uporabniških straneh orodja. Na ta način lahko uporabniki vidijo, kateri primeri so v obdelavi in kateri so bili sprejeti v podatkovno bazo orodja. Predlogi za tehnične izboljšave bodo – če so seveda tehnično izvedljivi – upoštevani pri naslednjih večjih posodobitvah orodja. 3.2.5 Sopomenke Kot osnovo za nabor smo izluščili sopomenke iz jezikovnega vira Sopomenke 1.0,10 pri čemer smo primerjali podatke iz korpusa Primorskega dnevnika z referenčnim korpusom Gigafida 2.0. Primerjali smo pogostnost posameznih lem in tistim, ki v zamejski rabi izkazujejo bistveno višjo frekvenco, pripisali sopomenke, izluščene neposredno iz vira. Slika 5 prikazuje (nedokončni) nabor sopomenk za glagol dobiti, ki se v zamejskem okolju pogosto uporablja tudi v pomenu ‛najti’, zaradi česar bo verjetno deležen obravnave v kateri od drugih kategorij, za zdaj pa ostaja kot iztočnica za sopomenko. 10 https://viri.cjvt.si/sopomenke/slv/about 10 https://viri.cjvt.si/sopomenke/slv/about 288 Simpozij OBDOBJA 41 Slika 5: Primer prikaza sopomenk za glagol dobiti. Slika 5: Primer prikaza sopomenk za glagol dobiti. Kot prikazuje slika, ob prehodu kurzorja pridobimo sopomenke za iskano iztočnico, pri tem pa so podane vse potencialne sopomenke, zaradi samodejne priprave vira Sopomenke 1.0 pa lahko s tem pride tudi do določene mere šuma, kar pa lahko zaradi pomanjkanja konteksta seveda povzroči težave. Zaradi tega bodo sopomenke v Lorisu ročno izbrane in prilagojene. JAGODIC, Devan, KAUČIČ BAŠA, Majda, DAPIT, Roberto, 2017: Jezikovni položaj Slovencev v Italiji. Zaira Vidau, Norina Bogatec (ur.): Skupnost v središču Evrope: Slovenci v Italiji od padca Berlinskega zidu do izzivov tretjega tisočletja. Trst: ZTT, Slori. 66–88. 4 Sklep V prispevku smo predstavili novo orodje, namenjeno takojšnji pomoči piscem in zasnovano kot zbirališče jezikovnih virov in podatkov, zlasti za področje jezikovnega stika med slovenščino in italijanščino. Orodje smo zasnovali tako, da bi uporabnikom pomagali preseči razpršenost jezikovnih virov za slovenščino tako, da lahko o jezikovnih elementih (v trenutni obliki na ravni leksike) na enem mestu in takoj dobijo povratne informacije ter jim za to ni treba obiskati več portalov oz. uporabiti več orodij. Ta zasnova je morda največja prednost Lorisa, hkrati pa najbrž tudi največja hiba: Loris je po eni strani dober ravno toliko, kot je dobra njegova podatkovna baza, po drugi pa tudi toliko, kot odgovarja na potrebe uporabnikov – te se bodo sčasoma še natančneje definirale, saj je Loris zasnovan tako, da lahko uporabniki hipno poročajo o pomanjkljivostih ter vsebinskih in uporabniških vidikih, ki jih pri orodju pogrešajo.11 Kot smo prikazali v prispevku, orodje za zdaj temelji na obsežnih spiskih, ki po eni strani terjajo procesno moč, hkrati pa tudi dolgotrajno prilagajanje vsakovrstnih 289 Simpozij OBDOBJA 41 jezikovnih virov, ki jih želimo vpeljati v orodje, obenem pa je ta postopek neizbežen ob vsaki posodobitvi izvornega jezikovnega vira. Naš namen v prihodnosti je orodje zasnovati oz. preoblikovati tako, da bo vanj mogoče vpeljati jezikovne vire prek API-jev (vmesnikov za namensko programiranje), tako da bi lahko nanj z nekaj prilagajanja »priklopili« tudi druge primerne obstoječe jezikovne vire (npr. Vejico 1.0, Sopomenke 1.0 ipd.), vendar pa bodo morali to obstoječi viri (oz. njihovi avtorji, lastniki in financerji) tudi omogočati. MELINC MLEKUŽ, Maja, 2019: Sporazumevalna zmožnost v šolah s slovenskim učnim jezikom v Italiji. Razprave in gradivo 83. 67–82. MEZGEC, Maja, 2016: Linguistic landscape as a mirror: the case of the Slovene minority in Italy. Razprave in gradivo 77. 67–85. GRGIČ, Matejka, POPIČ, Damjan, 2022 (v tisku): Procesi jezikovnega separatizma pri čezmejnih jezikovnih manjšinah: prevzemanje, prilagajanje in prevajanje terminologije med Slovenci in Slovenkami v Italiji. Annales. GRGIČ, Matejka, 2017: Italijansko-slovenski jezikovni stik med ideologijo in pragmatiko. Jezik in slovstvo LXII/1. 89–98. FOUGHT, Carmen, 2006: Language and ethnicity. Cambridge: Cambridge University Press. Literatura FOUGHT, Carmen, 2006: Language and ethnicity. Cambridge: Cambridge University Press. GRGIČ, Matejka, 2017: Italijansko-slovenski jezikovni stik med ideologijo in pragmatiko. Jezik in slovstvo LXII/1. 89–98. GRGIČ, Matejka, POPIČ, Damjan, 2022 (v tisku): Procesi jezikovnega separatizma pri čezmejnih jezikovnih manjšinah: prevzemanje, prilagajanje in prevajanje terminologije med Slovenci in Slovenkami v Italiji. Annales. JAGODIC, Devan, KAUČIČ BAŠA, Majda, DAPIT, Roberto, 2017: Jezikovni položaj Slovencev v Italiji. Zaira Vidau, Norina Bogatec (ur.): Skupnost v središču Evrope: Slovenci v Italiji od padca Berlinskega zidu do izzivov tretjega tisočletja. Trst: ZTT, Slori. 66–88. MELINC MLEKUŽ, Maja, 2019: Sporazumevalna zmožnost v šolah s slovenskim učnim jezikom v Italiji. Razprave in gradivo 83. 67–82. MEZGEC, Maja, 2016: Linguistic landscape as a mirror: the case of the Slovene minority in Italy. Razprave in gradivo 77. 67–85. 290
https://openalex.org/W2886592161	https://www.mdpi.com/2073-4441/10/8/1065/pdf	English	null	Dynamics and Functional Potential of Stormwater Microorganisms Colonizing Sand Filters	Water	2,018	cc-by	11,740	Received: 29 June 2018; Accepted: 9 August 2018; Published: 10 August 2018 Received: 29 June 2018; Accepted: 9 August 2018; Published: 10 August 2018 Abstract: Stormwater management is increasingly relying on engineered inﬁltration systems (EIS) to reduce the volume and improve the quality of managed stormwater. Yet, EIS in the ﬁeld will be colonized by a diverse array of environmental microorganisms that change the physiochemical properties of the EIS and provide a habitat for microorganisms with harmful or beneﬁcial qualities. Understanding factors inﬂuencing the composition and stability of microbial communities could open up strategies for more efﬁcient management of stormwater. Here, we analyzed the potential pathogenic and metabolic capabilities of stormwater microorganisms colonizing idealized EIS (i.e., sand columns) under laboratory conditions over time. The diversity of microbial communities was analyzed using 16S rRNA gene sequencing, and potential pathogens and denitrifying microbes were identiﬁed from taxonomic match to known species. Denitriﬁcation potential as determined by nosZ abundance was also assessed with quantitative polymerase chain reaction PCR. Our ﬁndings demonstrate that replicate microbial communities colonizing sand columns change in a similar way over time, distinct from control columns and the source community. Potential pathogens were initially more abundant on the columns than in the stormwater but returned to background levels by 24 days after inoculation. The conditions within sand columns select for potential denitrifying microorganisms, some of which were also potential pathogens. These results demonstrate that a diverse suite of stormwater microorganisms colonize sand ﬁlters, including a transient population of potential pathogens and denitriﬁers. Manipulating the inoculating microbial community of EIS could prove an effective mechanism for changing both potential pathogens and denitrifying bacteria. Keywords: 16S rRNA gene; microbial community; diversity; potential pathogen; denitriﬁcation water water Water 2018, 10, 1065 Water 2018, 10, 1065 2 of 17 Nitrogen and nitrate removal in EIS is variable, and often below ideal efﬁciencies [2,7,8]. Along with physiochemical properties of the media promoting nutrient removal, the microbially mediated process of denitriﬁcation transforms bioavailable nitrogen (nitrate) into various gaseous forms (N2, N2O) that are removed from the stormwater. Denitrifying bacteria must either be present within the EIS during installation or colonize EIS from the environment. Denitriﬁcation potential within EIS will be impacted by the colonization, growth, and dynamics of these microorganisms. Colonization of a robust denitrifying community within the ﬁlter will depend on the presence of microorganisms in the bioﬁlm with these capabilities from the inoculating water. Conditions promoting denitriﬁcation provide a selective advantage to microorganisms with this potential function, increasing the potential for denitriﬁcation over time. For EIS to be maximally effective at nutrient removal, it is important to understand the factors that inﬂuence the presence of denitrifying bacteria. y g Pathogens are an important contaminant of stormwater that can be managed effectively with EIS, although their fate within EIS is not well understood. The fate of pathogens within EIS can be inﬂuenced by colonizing microorganisms in many ways. Physical factors, such as mechanical ﬁltering and water velocity have a strong impact on the initial retention of pathogens within the EIS [9]. Bioﬁlms created by colonizing microorganisms within EIS can alter the physical environment to further inﬂuence pathogen retention [10]. Pathogen retention efﬁciency within EIS may change over time, as the properties of the EIS are altered by the dissolved material, particles, and microorganisms that pass through [3]. Once trapped on the EIS, pathogen survival is also impacted by predators and other organisms competing for nutrients [5,11]. Conditions, such as the presence of a protective bioﬁlm or suitable energy and nutrients for growth, can also promote their survival. These pathogens could eventually be transported out of the EIS during the next storm if survival is high, negating the short-term beneﬁcial effects of retention with the EIS. Given the importance of the colonizing community in the biotransformation of many pollutants in stormwater, it is important to understand the factors that inﬂuence EIS microbial community assembly and succession. Within any ecosystem, both selective (“niche”) and neutral factors can impact microbial community assembly [12,13]. 1. Introduction Urbanization has signiﬁcantly increased the area of impervious surfaces that prevent natural groundwater recharge, resulting in large volumes of stormwater that need to be managed [1]. Stormwater can transport pathogens, nutrients, such as nitrogen, and other contaminants from these surfaces to surrounding water bodies if not properly managed [2]. Engineered inﬁltration systems (EIS) can promote groundwater recharge and reduce the concentration of contaminants through physical ﬁltration, chemical reactions and biological transformations [3,4]. Biotransformation of nutrients and removal of pathogens is inﬂuenced by microbial communities colonizing engineered inﬁltration systems (EIS), and these processes are not well understood [5]. Microbial bioﬁlms, or microorganisms attaching to the surface of the media which typically secrete a protective extra polysaccharide layer, are an important aspect of biotransformation and contaminant removal [6]. Understanding the factors that promote efﬁcient and effective contaminant removal in EIS will aid stormwater management efforts and improve surface water quality in surrounding areas. www.mdpi.com/journal/water Water 2018, 10, 1065; doi:10.3390/w10081065 www.mdpi.com/journal/water Water 2018, 10, 1065 Selective factors will be highly variable in the ﬁeld, as EIS conﬁgurations and environmental conditions experienced will vary over time and from site to site [3]. Neutral processes inﬂuencing microbial community composition in EIS include random ﬂuctuations in populations abundances (i.e., drift) and movement of organisms into the EIS from other areas (i.e., dispersal; [13]). Both drift and dispersal could have a large impact on community composition but are often overlooked as compared to selective factors. Some studies indicate that neutral factors could have a large role in shaping the microbial community. For example, historical contingency, or the order in which microorganisms arrive, has been shown to play a large role in the resulting community [14,15] and has also been shown to impact interactions [16]. Both niche and neutral factors have been shown to impact microbial community assembly on sand ﬁlters [17]. Understanding the inﬂuence of selection versus neutral factors will be important for engineering the microbial community of EIS to enhance biotransformation since efforts could be thwarted if drift or dispersal drive the community away from a desired state. Thus, while studies have focused on how environmental conditions impact the resulting community [18], few have investigated the impact of drift and dispersal alone on the resulting community composition. Given the importance of the colonizing microbial community in determining the fate of nutrients and pathogens within EIS, we examined the potential for denitriﬁcation and pathogen survival in experimental EIS initiated with stormwater inoculum. Additionally, we investigated the successional dynamics of the microbial community within idealized EIS under experimental conditions, from inoculation through 24 days post-colonization, to determine whether historical contingency has a sustained impact on microbial community composition. Sand is the most common EIS media, so sand ﬁlters were used as the model experimental EIS. Here, we use microbial community analysis of the 16S ribosomal RNA gene (16S rRNA) as a proxy for microbial community composition and infer potential functions from taxonomic predictions, including potential pathogenicity and 3 of 17 Water 2018, 10, 1065 denitriﬁcation, and validated denitriﬁcation potential by quantifying gene abundance for a key gene in the denitriﬁcation pathway (nosZ). denitriﬁcation, and validated denitriﬁcation potential by quantifying gene abundance for a key gene in the denitriﬁcation pathway (nosZ). 2.1. Sampling Site and Protocol Water was collected along Stony Run (1983 Remington Ave, Baltimore, MD 21211, USA; latitude N 39◦19′36.172′′, longitude W 76◦37′32.355′′) during a storm event on 29 September 2016. The air temperature at time of collection was 65 ◦F (18.3 ◦C), and the total rainfall in the previous 48 h was 3.1 inches (7.87 cm) (Baltimore-Washington International Airport weather station). A storm drain outfall empties directly into Stony Run at this location. Two types of samples were collected; water for inoculation of experimental columns and water for analysis of the microbial community in the stream and outfall discharge. For inoculation of experimental columns, 1 L of water was collected directly from the outfall. Stormwater from the outfall was not ﬁltered, collected in a 1 L carboy, transported back to the lab on ice, stored at 4 ◦C until use two days later. Water samples for microbial community analysis were taken from the outfall and approximately 50 ft upstream and downstream from the outfall and from the inoculum immediately before being added to the column. After collection, 50 mL of water was ﬁltered through a 0.22 µm polyethersulfone ﬁlter (MilliporeSigma, Inc., Burlington, MA, USA ) using a peristaltic pump. Filters were stored at −80 ◦C until DNA extraction. The microbial community composition of water used for inoculation and in the stream nearby provides a comparison to determine how much the column communities deviate from the original community structure. 2.2. Column Description, Set-Up, and Operation A 24-day study was designed to investigate the dynamics of microbial communities colonizing sand columns inoculated with stormwater. Disposable polypropylene chromatograph columns (14 cm depth, 20 mL bed volume, 1.5 cm end ﬁtting) including a 30 µm polyethylene ﬁlter at the bottom (BioRad, Inc., Hurcules, CA, USA) were rinsed with deionized water and autoclaved. Fifty to seventy mesh sand (SiO2, 212–300 µm) was rinsed with sterile, deionized water three times and dried for about 24 h (105 ◦C) and autoclaved as previously described [19]. 9.4 g (approximately 6 cm depth) of sand was packed into each column. All of the columns were autoclaved again before inoculation to ensure a sterile environment inside the columns. Twenty columns were initiated on day one, grouped into four sampling time-points with ﬁve columns per time-point (Figure S1). Each time-point consisted of three stormwater columns (A, B, and C), non-inoculated control column, and one Pseudomonas aeruginosa positive control column. Columns were inoculated with an approach velocity of 15 cm/h for 3 h using a 24-channel peristaltic pump resulting in approximately 78 mL total volume added to each column. This simulates a common storm of 0.75 cm/h intensity and 3 h duration (return period < 1 year [20]) concentrated by a factor of 20, which resembles a typical bioretention area sized at 5% of the drainage area [11,21]. The top of each column served as the inlet and was uncovered. During the inoculation, P. aeruginosa overnight culture, sterile synthetic stormwater (SS), and approximately 78 mL stormwater were added by recycling liquid from 1 L bottles. After inoculation, the ﬁrst group of columns (Day 1) was collected. Sterile synthetic stormwater was added to the columns under the same simulated storm velocity and duration on days 3, 6, 10, 13, 17, and 20. During simulated storm events, a total of 78 mL of sterile synthetic stormwater was pipetted directly into each column intermittently to evenly wet the surface and avoid contamination. Columns were sacriﬁced on days 10, 17, and 24 before each simulated storm event. Water 2018, 10, 1065 4 of 17 2.4. DNA Extraction and 16S rRNA Gene Library Protocol All water samples collected in the ﬁeld were ﬁltered through 0.22 µm polyethersulfone ﬁlters (MilliporeSigma, Inc.) and stored for DNA extraction. The inoculation sample was ﬁltered and stored for DNA extraction as described previously. The DNA was extracted using the PowerWater kit (Qiagen, Hilden, Germany). The sand from each column was poured into a sterile 50 mL falcon tube and votexed. Three replicate three-gram sub-samples were added to 15 mL sterile falcon tubes. The columns were homogenized to remove the effect of depth when sampling replicates, as organisms can deposit differently throughout the length of the column [23]. Tubes were immediately frozen at −80 ◦C until DNA extraction. DNA was extracted using the PowerSoil kit (Qiagen), following the manufacture’s protocol plus 20 µL proteinase K and a 65 ◦C incubation step before bead-beating to promote additional cell lysis. The 16S rRNA gene was ampliﬁed using primers U515F and E786R [24] modiﬁed as previously described [25]. Modiﬁcation provided overhanging adapters used as the primer-binding site for a second step PCR reaction, adding sample- speciﬁc barcodes and adapters appropriate for Illumina MiSeq sequencing. Sample indices and binding sites are added in the second step. A mock community positive control [26] and PCR negative controls were also ampliﬁed and sequenced. Replicates from group A stormwater columns were sequenced twice to control for sequencing batch variability. DNA sequencing was performed at the Genetic Research Core Facility at Johns Hopkins University. Illumina data has been submitted to the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) under study accession number PRJNA482666. 2.3. Media and Culture Conditions Synthetic stormwater (SS) was used to simulate storm events. SS was formulated from a previous recipe [22]. The media consisted of 5 mM NaCl, 0.75 mM CaCl2, 0.075 mM MgCl2, 0.30 mM Na2SO4, 1 mM NaHCO3, 0.15 mM NaNO3, 0.07 NH4Cl, and 0.02 mM Na2HPO4 (pH ca. 7).) Carbon was added in the form of yeast extract (3 g/L) as well as 0.0015% (by weight) peptone, 0.0011% meat extract, and 0.0003% urea. All media was ﬁlter sterilized through a 0.2 µm polyethersulfone ﬁlter (MilliporeSigma, Inc., Burlington, MA, USA) and stored at 4 ◦C until use. Bioﬁlm-forming Pseudomonas aeruginosa served as a positive control. P. aeruginosa was stored in 10% glycerol (v/v) at −80 ◦C until use. Glycerol stocks were regrown on Luria Broth (LB) agar plates and incubated at 37 ◦C overnight. A single colony was picked to inoculate 1 L LB. The culture was allowed to grow overnight at 37 ◦C before inoculation onto the columns. 2.5. Sequence Analysis and Quality Control Samples were processed with the bioinformatics platform QIIME2 [27] using the program DADA2 [28] to remove sequencing artifacts and chimeras. We analyzed the composition of a positive control (mock community) to ensure the resulting processed sequence data represented the input community as accurately as possible. The mock community was comprised of puriﬁed DNA templates of known sequence and concentration, as previously described [26]. We compared the resulting sequence read count for each mock community template to the expected read count for samples without mismatches in the primer binding site (Figure S1). We expected the input concentration of template to explain a large proportion of the variation in the resulting read count for the mock community templates without primer binding site mismatches (R2 = 0.74). However, with the default DADA2 parameters in QIIME2, one mock community template was ﬂagged as chimeric and removed. Additionally, a number of DNA sequences found in this library were not mock community sequences, including some non-16S rRNA gene sequences. We changed the DADA2 parameters to require chimeras to be 10-fold less abundant than parent sequences. We also used mothur [29] to align operational taxonomic unit (OTU) representative sequences to the Silva alignment [30] subset to the sequenced region. To remove non-16S rRNA sequences, any sequence shorter than 250 bp or missing data within the ﬁrst 5 bp of the alignment was removed from the ﬁnal analysis. With these changes, 5 of 17 Water 2018, 10, 1065 the relationship between observed and expected mock community templates improved to R2 = 0.88. We also used the OTU calling program dbOTU plug-in in QIIME2 to create operational taxonomic units (OTUs) from closely related, similarly distributed sequences [31]. The greengenes classiﬁer distributed with QIIME2 was used for taxonomic classiﬁcation. Multiple sequence alignment and phylogenetic trees were generated with the programs MAFFT [32] and FastTree2 [33], respectively within QIIME2. Bray-Curtis, Jaccard, Unweighted and Weighted Unifrac distances were calculated in QIIME2 subsampled to 49,950 counts (lowest non-negative sample library read count). Principle coordinate analysis plots were visualized using EMPeror [34]. Bray-Curtis distances were used in the analysis, but the results were similar with other distance metrics. OTU tables collapsed by taxonomy created with QIIME2 were used as the input for the program FAPROTAX [35] to predict functional information and potential pathogens. OTU tables were normalized to the total read count for each library before running FAPROTAX. 2.5. Sequence Analysis and Quality Control Negative controls were included at every step of processing, from DNA extraction through the library preparation. A subset of samples was sequenced in both sequencing runs to verify that methodological errors did not impact our results. Negative and positive controls samples were distinct from the majority of environmental samples (Figure S2a). Clustering was not driven by batch effects, as replicates from the same samples processed in different batches clustered together (Figure S2c,d). 3. Results 3.1. Stormwater-Inoculated Column Communities Are Distinct from Positive and Non-Inoculated Control Column Communities 2.7. Quantitative Polymerase Chain Reaction (qPCR) A quantitative Polymerase Chain Reaction (qPCR) protocol was developed to quantify the number of 16S rRNA and nosZ gene copies within the columns. 16S rRNA templates were created as previously described [26] from 16S rRNA gene amplicon from a freshwater lake sample cloned into Escherichia coli with TOPO Blunt End cloning kit (Invitrogen, Carlsbad, CA, USA). NosZ templates were created by amplifying with nosZ primers (nosZ Forward: 5′-CGYTGTTCMTCGACAGCCAG-3′; nosZ Reverse: 5′- CATGTGCAGNGCRTGGCAGA-3′) using DNA extracted from a Pseudomonas aerugniosa culture, puriﬁed with Zymo PCR clean-up kit. Templates were quantiﬁed using the High Sensitivity DNA assay on a Bioanalyzer (Agilent; Santa Clara, CA, USA). A standard curve was made to determine the relationship between concentration and the threshold value (Cq). PCR was carried with SsoAdvanced Universal SYBR® Green Supermix (Biorad; Hercules, CA, USA) according to the manufacture’s protocol on the RealTime PCR thermocycler (BioRad). 2.6. Statistical Analysis Biological columns replicates (A, B, C) and their technical replicates (1–7) were analyzed. Statistical signiﬁcance of distances between column samples from day 17 and 24 plus the initially sampled and inoculated outfall samples was carried out with permanova and analysis of similarity program ANOSIM [36] analysis in QIIME2. Data from the last two time-points were aggregated because positive and non-inoculated columns did not have multiple biological replicates per time-point. Exported Bray-Curtis distance matrices were used to test the average distances between technical replicates, biological replicates, and source community using Welch two sample t-test and Wilcoxon rank sum test in R [37]. 3.1.1. Bacterial Growth on Columns To determine whether bacteria could successfully colonize the sand columns, the change in 16S rRNA copy number, which corresponds to bacterial concentration, over the 24-day experimental period was determined by qPCR (16S rRNA gene copies/µL). Non-inoculated control columns, positive 6 of 17 Water 2018, 10, 1065 control P. aeruginosa columns, and replicate stormwater-inoculated columns all showed an increase in 16S rRNA copy number over the experimental period (Figure 1). Although microorganisms were not intentionally added to the non-inoculated columns and media and tubing were sterilized before use, some level of contamination was expected. The non-inoculated columns represent the microbial community coming from the equipment, reagents or laboratory environment. Pseudomonas columns also became contaminated with a different set of microorganisms that could have been the same as the non-inoculated control or come from within the Pseudomonas culture itself if it had low levels of contamination. The non-inoculated control had the lowest measurable cell concentration on day 1 (1.47 × 107 copies/g), but increased to levels slightly exceeding the stormwater columns by day 24. In contrast, Pseudomonas columns on Day 24 had the highest measurable cell concentration for the entire experimental period (5.9 × 1011 copies/g). The high cell concentration on the Pseudomonas columns likely results from the high initial loading of Pseudomonas cells from the culture. Overall, this demonstrates that microbial growth, rather than just deposition of dead or dormant cells, inﬂuences the community composition on all columns. Water 2018, 10, x FOR PEER REVIEW 6 of 18 microorganisms were not intentionally added to the non-inoculated columns and media and tubing were sterilized before use, some level of contamination was expected. The non-inoculated columns represent the microbial community coming from the equipment, reagents or laboratory environment. Pseudomonas columns also became contaminated with a different set of microorganisms that could have been the same as the non-inoculated control or come from within the Pseudomonas culture itself if it had low levels of contamination. The non-inoculated control had the lowest measurable cell concentration on day 1 (1.47 × 107 copies/g), but increased to levels slightly exceeding the stormwater columns by day 24. In contrast, Pseudomonas columns on Day 24 had the highest measurable cell concentration for the entire experimental period (5.9 × 1011 copies/g). The high cell concentration on the Pseudomonas columns likely results from the high initial loading of Pseudomonas cells from the culture. 3.1.1. Bacterial Growth on Columns Overall, this demonstrates that microbial growth, rather than just deposition of dead or dormant cells, influences the community composition on all columns. Figure 1. Change in bacterial cell concentration within environmental and control columns over time, as estimated by 16S rRNA gene copy number/µL. Abbreviations: S (blue), stormwater-inoculated columns; P (orange), Pseudomonas aeruginosa -inoculated columns; N (gray), non-inoculated columns. Significantly more cells were added in the Pseudomonas columns on Day 1 than in the non-inoculated or stormwater columns. X-axis; Days-days of the experiment from 1 (first day) to 24 (last day). Y-axis; cell concentration as measured by quantitative PCR of 16S rRNA gene copies per gram of sediment in the columns. Figure 1. Change in bacterial cell concentration within environmental and control columns over time, as estimated by 16S rRNA gene copy number/µL. Abbreviations: S (blue), stormwater-inoculated columns; P (orange), Pseudomonas aeruginosa -inoculated columns; N (gray), non-inoculated columns. Signiﬁcantly more cells were added in the Pseudomonas columns on Day 1 than in the non-inoculated or stormwater columns. X-axis; Days-days of the experiment from 1 (ﬁrst day) to 24 (last day). Y-axis; cell concentration as measured by quantitative PCR of 16S rRNA gene copies per gram of sediment in the columns. Figure 1. Change in bacterial cell concentration within environmental and control columns over time, as estimated by 16S rRNA gene copy number/µL. Abbreviations: S (blue), stormwater-inoculated columns; P (orange), Pseudomonas aeruginosa -inoculated columns; N (gray), non-inoculated columns. Significantly more cells were added in the Pseudomonas columns on Day 1 than in the non-inoculated or stormwater columns. X-axis; Days-days of the experiment from 1 (first day) to 24 (last day). Y-axis; cell concentration as measured by quantitative PCR of 16S rRNA gene copies per gram of sediment in the columns. Figure 1. Change in bacterial cell concentration within environmental and control columns over time, as estimated by 16S rRNA gene copy number/µL. Abbreviations: S (blue), stormwater-inoculated columns; P (orange), Pseudomonas aeruginosa -inoculated columns; N (gray), non-inoculated columns. Signiﬁcantly more cells were added in the Pseudomonas columns on Day 1 than in the non-inoculated or stormwater columns. X-axis; Days-days of the experiment from 1 (ﬁrst day) to 24 (last day). Y-axis; cell concentration as measured by quantitative PCR of 16S rRNA gene copies per gram of sediment in the columns. 3.1.2. Bacterial Community Composition on Columns 3.1.2. Bacterial Community Composition on Columns Columns were colonized by a diverse range of microbial taxa (Figure 2). Water sampled up- stream from the stormwater outfall contained a majority of Proteobacteria (45%) and Saccharibacteria, formerly Candidate division TM7, (26%). Outfall samples used as inoculum for stormwater columns had a higher percentage of Proteobacteria (76%) and less Saccharibacteria (4%). Samples downstream f h f ll i bl i h l i il d h i il Columns were colonized by a diverse range of microbial taxa (Figure 2). Water sampled up-stream from the stormwater outfall contained a majority of Proteobacteria (45%) and Saccharibacteria, formerly Candidate division TM7, (26%). Outfall samples used as inoculum for stormwater columns had a higher percentage of Proteobacteria (76%) and less Saccharibacteria (4%). Samples downstream from the 7 of 17 Water 2018, 10, 1065 outfall were variable, with some samples more similar to stream and others more similar to outfall samples. Initially, stormwater columns were more similar to the outfall community but diverged by the end of the 24-day experimental period. Stormwater-inoculated columns still had a large percentage of Proteobacteria (51%), but more Bacteroidetes (18%) and Firmicutes (9%), and less Saccharibacteria than the outfall community. Non-inoculated column samples were dominated by Firmicutes (42%), Proteobacteria (34%), and Bacteroidetes (11%). In contrast, Pseudomonas columns were dominated by Proteobacteria (71%) and Bacteroidetes (25%). Negative PCR samples had more Actinobacteria and Planctomycetes than other samples, along with common contaminant genera Halomonas and Shewanella. Water 2018, 10, x FOR PEER REVIEW 7 of 18 Saccharibacteria than the outfall community. Non-inoculated column samples were dominated by Firmicutes (42%), Proteobacteria (34%), and Bacteroidetes (11%). In contrast, Pseudomonas columns were dominated by Proteobacteria (71%) and Bacteroidetes (25%). Negative PCR samples had more Actinobacteria and Planctomycetes than other samples, along with common contaminant genera Halomonas and Shewanella. Figure 2. Taxa plots (phylum level) of microbial communities across replicate stormwater columns, Pseudomonas columns, non-inoculated columns, field samples, and controls. Legend provides phylum level classification although phyla comprising less than 1% of the samples overall are not listed. Sample type is listed about each group of samples (SC, Stormwater inoculated columns by day; IN, Stormwater column inoculum; Field, Field samples; Non-inoculated, Non-inoculated columns; Pseudomonas, Pseudomonas columns; SS, Synthetic Stormwater media; Controls, PCR control samples). 3.1.2. Bacterial Community Composition on Columns 3.1.2. Bacterial Community Composition on Columns Sample names (X-axis) include column replicate (A, B, C) and time-point (1–4) for stormwater columns, inoculum type (N, non-inoculated; P, Pseudomonas) and time-point (1–4) for non-inoculated and Pseudomonas columns, and short descriptors for other samples (DN, Downstream of outfall; Out, Outfall; Up, Upstream of outfall; M, Mix9 positive control; CONT, negative controls; IN, inoculum). Technical replicates for the same column are displayed individually. Figure 2. Taxa plots (phylum level) of microbial communities across replicate stormwater columns, Pseudomonas columns, non-inoculated columns, ﬁeld samples, and controls. Legend provides phylum level classiﬁcation although phyla comprising less than 1% of the samples overall are not listed. Sample type is listed about each group of samples (SC, Stormwater inoculated columns by day; IN, Stormwater column inoculum; Field, Field samples; Non-inoculated, Non-inoculated columns; Pseudomonas, Pseudomonas columns; SS, Synthetic Stormwater media; Controls, PCR control samples). Sample names (X-axis) include column replicate (A, B, C) and time-point (1–4) for stormwater columns, inoculum type (N, non-inoculated; P, Pseudomonas) and time-point (1–4) for non-inoculated and Pseudomonas columns, and short descriptors for other samples (DN, Downstream of outfall; Out, Outfall; Up, Upstream of outfall; M, Mix9 positive control; CONT, negative controls; IN, inoculum). Technical replicates for the same column are displayed individually. Figure 2. Taxa plots (phylum level) of microbial communities across replicate stormwater columns, Pseudomonas columns, non-inoculated columns, field samples, and controls. Legend provides phylum level classification although phyla comprising less than 1% of the samples overall are not listed. Sample type is listed about each group of samples (SC, Stormwater inoculated columns by day; IN, Stormwater column inoculum; Field, Field samples; Non-inoculated, Non-inoculated columns; Pseudomonas, Pseudomonas columns; SS, Synthetic Stormwater media; Controls, PCR control samples). Sample names (X-axis) include column replicate (A, B, C) and time-point (1–4) for stormwater columns, inoculum type (N, non-inoculated; P, Pseudomonas) and time-point (1–4) for non-inoculated and Pseudomonas columns, and short descriptors for other samples (DN, Downstream of outfall; Out, Outfall; Up, Upstream of outfall; M, Mix9 positive control; CONT, negative controls; IN, inoculum). Technical replicates for the same column are displayed individually. Figure 2. Taxa plots (phylum level) of microbial communities across replicate stormwater columns, Pseudomonas columns, non-inoculated columns, ﬁeld samples, and controls. Legend provides phylum level classiﬁcation although phyla comprising less than 1% of the samples overall are not listed. 3.1.2. Bacterial Community Composition on Columns 3.1.2. Bacterial Community Composition on Columns Sample type is listed about each group of samples (SC, Stormwater inoculated columns by day; IN, Stormwater column inoculum; Field, Field samples; Non-inoculated, Non-inoculated columns; Pseudomonas, Pseudomonas columns; SS, Synthetic Stormwater media; Controls, PCR control samples). Sample names (X-axis) include column replicate (A, B, C) and time-point (1–4) for stormwater columns, inoculum type (N, non-inoculated; P, Pseudomonas) and time-point (1–4) for non-inoculated and Pseudomonas columns, and short descriptors for other samples (DN, Downstream of outfall; Out, Outfall; Up, Upstream of outfall; M, Mix9 positive control; CONT, negative controls; IN, inoculum). Technical replicates for the same column are displayed individually. A statistical analysis was used to determine whether the microbial communities in the stormwater-inoculated columns, non-inoculated columns, and Pseudomonas columns were significantly different after 17 days of incubation. The Bray-Curtis distance between microbial communities that developed on the non-inoculated columns was significantly different (permanova and analysis of similarity with ANOSIM p-value ≤ 0.005) from the community that developed on the stormwater- and Pseudomonas- inoculated columns (Figure 3). Additionally, the stormwater columns were significantly different (permanova and ANOSIM p-value ≤ 0.005) from the field and initial inoculum samples. Only the Pseudomonas columns and field samples were not significantly different, likely because they lack statistical power from the small sample set (sample size = 9). The complex community that developed on the stormwater-inoculated columns did not resemble either the initial inoculum or the non-inoculated columns in either phylum-level composition (Figure 2) or specific taxa A statistical analysis was used to determine whether the microbial communities in the stormwater-inoculated columns, non-inoculated columns, and Pseudomonas columns were signiﬁcantly different after 17 days of incubation. The Bray-Curtis distance between microbial communities that developed on the non-inoculated columns was signiﬁcantly different (permanova and analysis of similarity with ANOSIM p-value ≤0.005) from the community that developed on the stormwater- and Pseudomonas- inoculated columns (Figure 3). Additionally, the stormwater columns were signiﬁcantly different (permanova and ANOSIM p-value ≤0.005) from the ﬁeld and initial inoculum samples. Only the Pseudomonas columns and ﬁeld samples were not signiﬁcantly different, likely because they lack statistical power from the small sample set (sample size = 9). The complex community that developed on the stormwater-inoculated columns did not resemble either the initial inoculum or the non-inoculated columns in either phylum-level composition (Figure 2) or speciﬁc taxa. 3.1.2. Bacterial Community Composition on Columns 3.1.2. Bacterial Community Composition on Columns 8 of 17 8 of 18 Water 2018, 10, 1065 Water 2018, 10, x FOR -Curtis distance between different inoculum types from the last t dian, interquartile range and outliers distances between all column (Neg) column distance (b) Pseudomonas aeruginosa (Pseu), and mns (Env). For each group, the left most comparison represents th other comparisons are between-group comparisons. All pairwi s were statistically significantly different with both permanova and A -Curtis distance between different inoculum types from the last t dian, interquartile range and outliers distances between all colum ted (Neg) column distance (b) Pseudomonas aeruginosa (Pseu), and mns (Env). For each group, the left most comparison represents th ther comparisons are between-group comparisons. All pairwise compa tistically signiﬁcantly different with both permanova and ANOSIM (p Figure 3. Bray-Curtis distance between different inoculum types from the last two time-points combined. Median, interquartile range and outliers distances between all column samples and (a) non-inoculated (Neg) column distance (b) Pseudomonas aeruginosa (Pseu), and (c) Stormwater inoculated columns (Env). For each group, the left most comparison represents the within-group distances, and other comparisons are between-group comparisons. All pairwise comparisons between groups were statistically significantly different with both permanova and ANOSIM (p-value ≤ 0.005). Figure 3. Bray-Curtis distance between different inoculum types from the last two time-points combined. Median, interquartile range and outliers distances between all column samples and (a) non-inoculated (Neg) column distance (b) Pseudomonas aeruginosa (Pseu), and (c) Stormwater inoculated columns (Env). For each group, the left most comparison represents the within-group distances, and other comparisons are between-group comparisons. All pairwise comparisons between groups were statistically signiﬁcantly different with both permanova and ANOSIM (p-value ≤0.005). Figure 3. Bray-Curtis distance between different inoculum types from the last two time-points combined. Median, interquartile range and outliers distances between all column samples and (a) non-inoculated (Neg) column distance (b) Pseudomonas aeruginosa (Pseu), and (c) Stormwater inoculated columns (Env). For each group, the left most comparison represents the within-group distances, and other comparisons are between-group comparisons. All pairwise comparisons between groups were statistically significantly different with both permanova and ANOSIM (p-value Figure 3. Bray-Curtis distance between different inoculum types from the last two time-points combined. Median, interquartile range and outliers distances between all column samples and (a) non-inoculated (Neg) column distance (b) Pseudomonas aeruginosa (Pseu), and (c) Stormwater inoculated columns (Env). 3.2.1. Technical Variability Is Less Than Biological Variability between Replicate Columns .2.1. Technical Variability Is Less Than Biological Variability between Replicate Columns 3.2.1. Technical Variability Is Less Than Biological Variability between Replicate Columns 3.2.1. Technical Variability Is Less Than Biological Variability between Replicate Columns To understand the inﬂuence of drift on microbial community structure variability, we compared the variability across biological replicates of stormwater-inoculated columns to the variability across technical replicates. The median Bray-Curtis distance between biological replicates (i.e., samples from different columns incubated for the same amount of time with the same inoculum) was greater than the median distance between technical replicates (i.e., different DNA extractions or libraries from the same column; Figure 4). The average Bray-Curtis distance between biological replicates was greater than the average distance between technical (p-value < 0.001) at all weeks, demonstrating that a portion of variability between biological replicates cannot be explained by technical reproducibility. Additionally, the communities shifted away from the inoculum community by day 10 and changed slowly after that point. The average distance between biological replicates was signiﬁcantly lower (p < 0.001) than the distance to the inoculum community between days 10 to 24 (Figure 4), although the difference was not signiﬁcant at day 1. Thus drift, as measured by the distance between biological replicates, is signiﬁcant, but small compared to the difference between the source community and the communities later time-points. To understand the influence of drift on microbial community structure variability, we compared the variability across biological replicates of stormwater-inoculated columns to the variability across technical replicates. The median Bray-Curtis distance between biological replicates (i.e., samples from different columns incubated for the same amount of time with the same inoculum) was greater than the median distance between technical replicates (i.e., different DNA extractions or libraries from the same column; Figure 4). The average Bray-Curtis distance between biological replicates was greater than the average distance between technical (p-value < 0.001) at all weeks, demonstrating that a portion of variability between biological replicates cannot be explained by technical reproducibility. Additionally, the communities shifted away from the inoculum community by day 10 and changed slowly after that point. The average distance between biological replicates was significantly lower (p < 0.001) than the distance to the inoculum community between days 10 to 24 (Figure 4), although the difference was not significant at day 1. Thus drift, as measured by the distance between biological replicates, is significant, but small compared to the difference between the source community and the communities later time-points. Figure 4. 3.2.1. Technical Variability Is Less Than Biological Variability between Replicate Columns .2.1. Technical Variability Is Less Than Biological Variability between Replicate Columns The average Bray-Curtis distance between technical replicates (T), biological replicates (B) and the distance it diverged from the starting inoculum community (S) each day. Numbers following single letter comparison group designations indicate the day of the experiment (e.g., S24 is the distance between 24-day columns and inoculum community). More similar communities have a lower Bray-Curtis distance. The average distance between technical replicates is significantly different than the average distance between biological replicates. Average distances between biological and technical replicates are statistically significantly different (t-test and Wilcoxon rank sum p-value < 0.001) for all days. Average distances between biological replicates and the inoculum community are statistically significantly different from average distances between technical and biological replicates (p < 0.001) for all days, except for the day 1 samples. Figure 4. The average Bray-Curtis distance between technical replicates (T), biological replicates (B) and the distance it diverged from the starting inoculum community (S) each day. Numbers following single letter comparison group designations indicate the day of the experiment (e.g., S24 is the distance between 24-day columns and inoculum community). More similar communities have a lower Bray-Curtis distance. The average distance between technical replicates is signiﬁcantly different than the average distance between biological replicates. Average distances between biological and technical replicates are statistically signiﬁcantly different (t-test and Wilcoxon rank sum p-value < 0.001) for all days. Average distances between biological replicates and the inoculum community are statistically signiﬁcantly different from average distances between technical and biological replicates (p < 0.001) for all days, except for the day 1 samples. Figure 4. The average Bray-Curtis distance between technical replicates (T), biological replicates (B) and the distance it diverged from the starting inoculum community (S) each day. Numbers following single letter comparison group designations indicate the day of the experiment (e.g., S24 is the distance between 24-day columns and inoculum community). More similar communities have a lower Bray-Curtis distance. The average distance between technical replicates is significantly different than the average distance between biological replicates. Average distances between biological and technical replicates are statistically significantly different (t-test and Wilcoxon rank sum p-value < 0.001) for all days. Average distances between biological replicates and the inoculum community are statistically significantly different from average distances between technical and biological replicates (p < 0.001) for all days, except for the day 1 samples. Figure 4. 3.1.2. Bacterial Community Composition on Columns 3.1.2. Bacterial Community Composition on Columns For each group, the left most comparison represents the within-group distances, and other comparisons are between-group comparisons. All pairwise comparisons between groups were statistically signiﬁcantly different with both permanova and ANOSIM (p-value ≤0.005). Water 2018, 10, 1065 Water 2018, 10, x FOR 9 of 17 9 of 18 3.2. Microbial Community Succession on Stormwater Columns 3.2. Microbial Community Succession on Stormwater Columns 3.2. Microbial Community Succession on Stormwater Columns 3.2. Microbial Community Succession on Stormwater Columns 3.2.2. Stability of Stormwater-Inoculated Columns over Time The community structure in stormwater-inoculated columns became more stable over time. The mean Bray-Curtis distance between samples from day 1 and samples from other time-points was large (0.88–0.93), suggesting a rapid change in community structure by day 10. Between day 17 and 24, the average distance between samples from different time-points (0.44) became similar to the average distances between biological replicates from the same time-point (0.37–0.49) (Figure 5). If all columns types (stormwater inoculated, Pseudomonas-inoculated and non-inoculated) were becoming more similar to each other over time, this would suggest that contamination from reagents or equipment resulted in the similarity observed between replicate columns (e.g., high dispersal resulting in homogenization). However, the statistically signiﬁcantly different community structure between columns with different inoculum-types (Figure 3) demonstrates dispersal of the lab environment to the columns is not high enough to cause the observed the similarity between replicate columns. Water 2018, 10, x FOR PEER REVIEW 10 of 18 3.2.2. Stability of Stormwater-Inoculated Columns over Time The community structure in stormwater-inoculated columns became more stable over time. The mean Bray-Curtis distance between samples from day 1 and samples from other time-points was large (0.88–0.93), suggesting a rapid change in community structure by day 10. Between day 17 and 24, the average distance between samples from different time-points (0.44) became similar to the average distances between biological replicates from the same time-point (0.37–0.49) (Figure 5). If all columns types (stormwater inoculated, Pseudomonas-inoculated and non-inoculated) were becoming more similar to each other over time, this would suggest that contamination from reagents or equipment resulted in the similarity observed between replicate columns (e.g., high dispersal resulting in homogenization). However, the statistically significantly different community structure between columns with different inoculum-types (Figure 3) demonstrates dispersal of the lab environment to the columns is not high enough to cause the observed the similarity between replicate columns. Figure 5. Heatmap of mean Bray-Curtis distance between time-points from stormwater inoculated columns. Colors indicate mean distances between biological replicates (diagonal) or all sample comparisons between different time-points, with red indicating more similar and yellow indicating more different. The last two time-points are as similar between time-points as within time-points, suggesting that the community is stabilizing. Labels indicate Day (D) in experiment 1, 10, 17, and 24. 3.2.3. Dynamics of Potential Pathogens and Denitrifying Bacteria D1 D10 D17 D24 D1 D10 D17 D24 0.4 0.6 0.8 Value Color Key Figure 5. 3.2.1. Technical Variability Is Less Than Biological Variability between Replicate Columns .2.1. Technical Variability Is Less Than Biological Variability between Replicate Columns The average Bray-Curtis distance between technical replicates (T), biological replicates (B) and the distance it diverged from the starting inoculum community (S) each day. Numbers following single letter comparison group designations indicate the day of the experiment (e.g., S24 is the distance between 24-day columns and inoculum community). More similar communities have a lower Bray-Curtis distance. The average distance between technical replicates is signiﬁcantly different than the average distance between biological replicates. Average distances between biological and technical replicates are statistically signiﬁcantly different (t-test and Wilcoxon rank sum p-value < 0.001) for all days. Average distances between biological replicates and the inoculum community are statistically signiﬁcantly different from average distances between technical and biological replicates (p < 0.001) for all days, except for the day 1 samples. Water 2018, 10, 1065 10 of 17 10 of 17 3.2.2. Stability of Stormwater-Inoculated Columns over Time 3.2.2. Stability of Stormwater-Inoculated Columns over Time Heatmap of mean Bray-Curtis distance between time-points from stormwater inoculated columns. Colors indicate mean distances between biological replicates (diagonal) or all sample comparisons between different time-points, with red indicating more similar and yellow indicating more different. The last two time-points are as similar between time-points as within time-points, suggesting that the community is stabilizing. Labels indicate Day (D) in experiment 1, 10, 17, and 24. D1 D10 D17 D24 D1 D10 D17 D24 0.4 0.6 0.8 Value Color Key 0.4 0.6 0.8 Value Color Key D1 D10 Figure 5. Heatmap of mean Bray-Curtis distance between time-points from stormwater inoculated columns. Colors indicate mean distances between biological replicates (diagonal) or all sample comparisons between different time-points, with red indicating more similar and yellow indicating more different. The last two time-points are as similar between time-points as within time-points, suggesting that the community is stabilizing. Labels indicate Day (D) in experiment 1, 10, 17, and 24. 3.2.3. Dynamics of Potential Pathogens and Denitrifying Bacteria Figure 5. Heatmap of mean Bray-Curtis distance between time-points from stormwater inoculated columns. Colors indicate mean distances between biological replicates (diagonal) or all sample comparisons between different time-points, with red indicating more similar and yellow indicating more different. The last two time-points are as similar between time-points as within time-points, suggesting that the community is stabilizing. Labels indicate Day (D) in experiment 1, 10, 17, and 24. We also investigated changes in the functional potential of microb after inoculation. Using the taxonomic classification from the 16S r 3.2.3. Dynamics of Potential Pathogens and Denitrifying Bacteria We also investigated changes in the functional potential of microbial communities on columns after inoculation. Using the taxonomic classiﬁcation from the 16S rRNA gene sequences and a database linking taxonomy to function (software program FAPROTAX [24]), we found 10 potential denitrifying taxa. All potentially denitrifying taxa were from Alpha-, Beta-, and Gamma-Proteobacteria. Using the same method, we identiﬁed 24 potentially pathogenic taxa within the dataset. Stenotrophomonas acidaminiphila [38] was the most abundant potential pathogen. Interestingly, this species also has denitriﬁcation capabilities, although it was not ﬂagged as a potential denitriﬁer, but rather only nitrate-respiration. Although the pathogenicity of this species has not been evaluated, some of its closest relatives are opportunistic pathogens [39,40]. Acinetobacter johnsonii was the 11 of 17 Water 2018, 10, 1065 second most abundant potential pathogen identiﬁed in the stormwater columns, which was on average 1.85-fold more abundant on columns than in the inoculum samples. A. johnsonii can be found in environmental samples [41], on the human skin [42], and associated with disease [43,44]. Other relatively abundant potentially pathogenic taxa were also classiﬁed as Stenotrophomonas or Acinetobacter. The potential pathogen OTU composition was slightly different between stormwater inoculated, and non-inoculated samples but the same S. acidaminiphila was the most abundant potential pathogen OTU in both (Figure S3). This suggests that this potential pathogen could have come from the lab. Other OTUs are also found in both samples but at different relative abundances. Both potentially pathogenic and denitrifying microorganisms initially increased in relative abundance on the columns but declined from the peak by day 24 (Figure 6). The relative abundance of potential pathogens on stormwater columns was high on day 1 and was maintained through day 10 (Figure 6c). Potential pathogens increased on the non-inoculated columns at day 10 as well. In contrast, the relative abundance of potential denitrifying taxa was high on day 1 in stormwater columns but decreased immediately (Figure 6a). Non-inoculated columns showed a peak at day 10 in denitrifying taxa. Both potentially pathogenic and denitrifying microorganisms decrease in relative abundance from their peak by day 17 and 24 (Figure 6a). This suggests that these microorganisms are initially selected for under the conditions within the column, but that this selection pressure is decreased as the community stabilizes by day 24. Water 2018, 10, x FOR PEER REVIEW 12 of 18 Figure 6. We also investigated changes in the functional potential of microb after inoculation. Using the taxonomic classification from the 16S r 3.2.3. Dynamics of Potential Pathogens and Denitrifying Bacteria Relative (a,c) and total (b,d) abundance of potential denitrifying (a,b) and pathogenic (c,d) microbial taxa over time within non-inoculated, (red) and stormwater inoculated (blue) columns. (a) The relative abundance of potential denitrifying taxa within the community over time. (b) The abundance of denitrifying taxa within the sand columns over time. The total number of 16S rRNA gene copies per gram was multiplied by the fraction of the total community to provide a quantitative measure of changes of potential through time. (c) The relative abundance of potentially pathogenic taxa within the community over time. (d) The abundance of potentially pathogenic taxa within the sand columns over time. The total number of 16S rRNA gene copies per gram was multiplied by the fraction of the total community to provide a quantitative measure of changes of potential through time. 3.2.4. Changes in the Abundance of Denitrification Potential over Time The o e t atio of the o Z e e a key e y e i the de it ifi atio ath ay a a e ed Figure 6. Relative (a,c) and total (b,d) abundance of potential denitrifying (a,b) and pathogenic (c,d) microbial taxa over time within non-inoculated, (red) and stormwater inoculated (blue) columns. (a) The relative abundance of potential denitrifying taxa within the community over time. (b) The abundance of denitrifying taxa within the sand columns over time. The total number of 16S rRNA gene copies per gram was multiplied by the fraction of the total community to provide a quantitative measure of changes of potential through time. (c) The relative abundance of potentially pathogenic taxa within the community over time. (d) The abundance of potentially pathogenic taxa within the sand columns over time. The total number of 16S rRNA gene copies per gram was multiplied by the fraction of the total community to provide a quantitative measure of changes of potential through time. Figure 6. Relative (a,c) and total (b,d) abundance of potential denitrifying (a,b) and pathogenic (c,d) microbial taxa over time within non-inoculated, (red) and stormwater inoculated (blue) columns. (a) The relative abundance of potential denitrifying taxa within the community over time. (b) The abundance of denitrifying taxa within the sand columns over time. The total number of 16S rRNA gene copies per gram was multiplied by the fraction of the total community to provide a quantitative measure of changes of potential through time. 3.2.4. Changes in the Abundance of Denitriﬁcation Potential over Time 3.2.4. Changes in the Abundance of Denitriﬁcation Potential over Time The concentration of the nosZ gene, a key enzyme in the denitriﬁcation pathway, was assessed with quantitative PCR (Figure 7). nosZ is the gene encoding nitrous oxide reductase, capable of mediating the conversion of nitrous oxide (N2O) to N2 as the ﬁnal step in denitriﬁcation. The number of copies of nosZ increased throughout the experiment in both non-inoculated columns and stormwater-inoculated columns. The non-inoculated control samples start out with few copies of nosZ but become colonized with organisms containing nosZ genes. By day 24 after inoculation, the concentration of nosZ gene copies in the non-inoculated columns is greater than the stormwater-inoculated samples. While the potential denitrifying microorganisms predicted from the taxonomic classiﬁcation show a decrease in the abundance of denitrifying taxa by the 24-day time-point, the trend in the nosZ signal shows a continuous increase in the potential for denitriﬁcation over time. Water 2018, 10, x FOR PEER REVIEW 13 of 18 Figure 7. Total abundance of nosZ over time within non-inoculated (Neg) and environmental columns. Env-stormwater inoculated columns; Neg-Non-inoculated columns. Figure 7. Total abundance of nosZ over time within non-inoculated (Neg) and environmental columns. Env-stormwater inoculated columns; Neg-Non-inoculated columns. Figure 7. Total abundance of nosZ over time within non-inoculated (Neg) and environmental columns. Env-stormwater inoculated columns; Neg-Non-inoculated columns. Figure 7. Total abundance of nosZ over time within non-inoculated (Neg) and environmental columns. Env-stormwater inoculated columns; Neg-Non-inoculated columns. Water 2018, 10, 1065 Water 2018, 10, 1065 12 of 17 The relative abundance of potential pathogens and denitrifying microorganisms was transformed by the concentration of 16S rRNA copies to provide a quantitative estimate of total abundance. While potential pathogens made up a relatively large proportion of the total input community on stormwater columns on day 1 (Figure 6c), the overall bacterial cell concentration was lower than at later time-points (Figure 6d). However, both potential pathogens and denitrifying taxa expanded within the column on day 10, reaching a maximum between day 10 and 17 (Figure 6b,d). This initial increase was not maintained, and both types decreased from their peak by day 24. We also investigated changes in the functional potential of microb after inoculation. Using the taxonomic classification from the 16S r 3.2.3. Dynamics of Potential Pathogens and Denitrifying Bacteria (c) The relative abundance of potentially pathogenic taxa within the community over time. (d) The abundance of potentially pathogenic taxa within the sand columns over time. The total number of 16S rRNA gene copies per gram was multiplied by the fraction of the total community to provide a quantitative measure of changes of potential through time. 3.2.4. Changes in the Abundance of Denitrification Potential over Time Figure 6. Relative (a,c) and total (b,d) abundance of potential denitrifying (a,b) and pathogenic (c,d) microbial taxa over time within non-inoculated, (red) and stormwater inoculated (blue) columns. (a) The relative abundance of potential denitrifying taxa within the community over time. (b) The abundance of denitrifying taxa within the sand columns over time. The total number of 16S rRNA gene copies per gram was multiplied by the fraction of the total community to provide a quantitative measure of changes of potential through time. (c) The relative abundance of potentially pathogenic taxa within the community over time. (d) The abundance of potentially pathogenic taxa within the sand columns over time. The total number of 16S rRNA gene copies per gram was multiplied by the fraction of the total community to provide a quantitative measure of changes of potential through time. 4. Discussio 4. Discussion Here, we focused on the impact of neutral processes on the resulting community under identical environmental conditions. Since the distance in the microbial community between biological replicates was greater than the distance between technical replicates, drift between identical columns inﬂuenced the resulting community structure. Drift between biological replicates could be due to random ﬂuctuations in population abundances between replicate columns or introduced by chance during inoculation. But these distances were small compared to the differences in community structure between columns with different inoculum types. Previous work has found that environmental conditions inﬂuence the community structure of sand ﬁlters in drinking water treatment [18], although they did not separate the impact of historical contingency and migration from inﬂuent water on ﬁlter community composition, or have the opportunity to investigate replicates. Drift and historical contingency could undermine efforts to engineering speciﬁc communities to improve desired biotransformations, such as denitriﬁcation or pathogen retention. While we demonstrate that drift is not an important factor over the short term, it could become more important over the lifespan of the EIS. Future work should focus on the relative contribution of variable environmental conditions on shaping the microbial community structure compared to neutral processes over a typical life-span of EIS. The microbial community on the EIS over time was determined by the initial inoculum, suggesting that initial seeding could be an effective mechanism for altering the resulting microbial community on EIS. The seeded microbial community can change both the chemistry and the hydrology within the system, which could have a feedback mechanism on the resulting community. Seeding the microbial community with speciﬁc microorganisms has been successful in altering the resulting community with nitrifying communities in drinking water sand ﬁltration [46]. However, our results suggest it will be difﬁcult to control the direction the community takes within the environment without more understanding about the selective conditions of the EIS since all experimental communities in our experiment diverged substantially from their initial state. To successfully manipulate the community, seeding the sand with a culture or consortium with the desired function, such increased denitriﬁcation or pathogen retention and removal, would likely result in loss of the majority of the seeded microorganisms from the community. This could limit the potential impact speciﬁc strains could have within EIS. 4. Discussio 4. Discussion This work demonstrates how neutral factors, such as drift and initial inoculum, shape the microbial community composition within idealized EIS systems in the absence of other factors influencing the microbial community. A portion of the variation in community composition across replicate columns cannot be explained by technical variability, suggesting that drift has a significant impact on community structure. However, this difference is small in comparison to the differences observed between communities on columns with different inocula. Biological replicates became more similar to each other over time, and were distinct from the source community, demonstrating that selection by the unique conditions of the experiment allowed for the expansion of the same subset of This work demonstrates how neutral factors, such as drift and initial inoculum, shape the microbial community composition within idealized EIS systems in the absence of other factors inﬂuencing the microbial community. A portion of the variation in community composition across replicate columns cannot be explained by technical variability, suggesting that drift has a signiﬁcant impact on community structure. However, this difference is small in comparison to the differences observed between communities on columns with different inocula. Biological replicates became more 13 of 17 Water 2018, 10, 1065 similar to each other over time, and were distinct from the source community, demonstrating that selection by the unique conditions of the experiment allowed for the expansion of the same subset of stormwater taxa in each column. The conditions in the column transiently selected potentially pathogenic taxa but resulted in a decrease in abundance by the 24-day time-point. Column conditions continuously selected for taxa that were capable of denitriﬁcation over the 24-day experiment. y p y p While the experimental conditions do not mimic the environmental conditions experienced by microbial communities in EIS in the ﬁeld, the controlled conditions provide insight into the factors that impact community assembly. We found that neutral processes, including historical contingency and drift, can signiﬁcantly inﬂuence the resulting community structure. While there has been a great deal of discussion about whether niche (e.g., environmental conditions) or neutral factors (e.g., drift, migration) dominate community assembly processes [12], both are likely to have some inﬂuence on the resulting community. A previous study of the microbial community on slow sand ﬁlters found evidence for both niche and neutral processes impacting the microbial community structure [45]. Funding: This research was funded by the National Science Foundation, Division of Chemical, Bioengineering, Environmental, and Transport Systems (CBET), grant award number 1511915. A.N.F. was supported by an Integrative Graduate Education and Research Traineeship (IGERT) fellowship from the National Science Foundation, Division of Graduate Education, grant award number 1069213. Author Contributions: Conceptualization, A.N.F. and S.P.P.; Formal analysis, A.N.F., Y.Z. and S.P.P.; Funding acquisition, S.P.P.; Methodology, A.N.F. and Y.Z.; Supervision, A.N.F., E.G.S. and S.P.P.; Validation, S.P.P.; Visualization, A.N.F. and S.P.P.; Writing—original draft, S.P.P.; Writing—review and editing, A.N.F., Y.Z., E.G.S. and S.P.P. 4. Discussio 4. Discussion Future work is needed to determine whether the communities eventually become similar regardless of inoculum over the normal operation period of a typical EIS in the ﬁeld or with the migration of other microorganisms on to the ﬁlters, as would be expected under normal operating conditions. Microbial seed cultures, like the Pseudomonas and stormwater communities seeded in this experiment, were an important determinant of the ﬁnal microbial community structure in this experimental system. More work is needed to determine whether seed cultures could be an effective mechanism for manipulating the resulting microbial community within EIS as compared to selective pressures or high dispersal rates into the system. Pathogenic taxa were transiently selected for within our experimental system. In this case, the most abundant potential human pathogen, Stenotrophomonas acidaminiphila, could also denitrify, Water 2018, 10, 1065 14 of 17 demonstrating that conditions promoting beneﬁcial functions for one type of pollutant (nitrogen) might negatively impact other pollutants (pathogens). The initial expansion then contraction of this population on sterile sand media suggests its role as an early colonizer in primary succession of sand surfaces. The conditions of the column selected for the expansion of potentially denitrifying taxa, as assessed by both the presence of the nosZ gene and potentially denitrifying taxa. Gene abundance has been shown to correspond to denitriﬁcation rates within certain environments but not others [7,47,48]. We did not measure the removal of nitrate within our columns with this experiment to connect denitriﬁcation potential and nutrient remediation. Denitriﬁcation is not as phylogenetically conserved as other metabolic processes [49], which might have caused the prediction tool we used based on phylogeny to miss the dynamics of nosZ on the non-inoculated columns. The potential for denitriﬁcation, as assessed through the concentration of nosZ genes in the columns, increased in both the environmental and non-inoculated samples. Interestingly, the non-inoculated columns had a higher ﬁnal concentration of nosZ than the stormwater columns, demonstrating the importance of inoculum in shaping the structure and potential function of the microbial community. The high organic carbon content of our synthetic stormwater media could have selected for of potential denitriﬁers, as denitriﬁcation potential and denitrifying populations increased with organic carbon concentrations [7]. More work is needed to determine whether this copy number difference results in a measurable change in nitrogen removal from the columns and how to inﬂuence the community toward greater denitriﬁcation under EIS conditions. plementary Materials: The following are available online at http://www.mdpi.com/2073-4441/10/8/1065 References 1. Council, N.R. Urban Stormwater Management in the United States; The National Academies Press: Washington, DC, USA, 2009; p. 610. [CrossRef] 2. Fraley-McNeal, L.; Schueler, T.; Winer, R. National Pollutant Removal Performance Database, Version 3; Center for Watershed Protection: Ellicott City, MD, USA, 2007; pp. 1–10. 3. Grebel, J.E.; Mohanty, S.K.; Torkelson, A.A.; Boehm, A.B.; Higgins, C.P.; Maxwell, R.M.; Nelson, K.L.; Sedlak, D.L. Engineered Inﬁltration Systems for Urban Stormwater Reclamation. Environ. Eng. Sci. 2013, 30, 437–454. [CrossRef] 4. Bekele, E.; Page, D.; Vanderzalm, J.; Kaksonen, A.; Gonzalez, D. Water Recycling via Aquifers for Sustainable Urban Water Quality Management: Current Status, Challenges and Opportunities. Water 2018, 10, 457. [CrossRef] 5. Haig, S.J.; Schirmer, M.; D’Amore, R.; Gibbs, J.; Davies, R.L.; Collins, G.; Quince, C. Stable-isotope probing and metagenomics reveal predation by protozoa drives E-coli removal in slow sand ﬁlters. ISME J. 2015, 9, 797–808. [CrossRef] [PubMed] 6. Afrooz, A.R.M.N.; Boehm, A.B. Escherichia coli Removal in Biochar-Modiﬁed Bioﬁlters: Effects of Bioﬁlm. PLoS ONE 2016, 11, e0167489. [CrossRef] [PubMed] 7. Waller, L.J.; Evanylo, G.K.; Krometis, L.A.H.; Strickland, M.S.; Wynn-Thompson, T.; Badgley, B.D. Engineered and Environmental Controls of Microbial Denitriﬁcation in Established Bioretention Cells. Environ. Sci. Technol. 2018, 52, 5358–5366. [CrossRef] [PubMed] 8. Morse, N.R.; McPhillips, L.E.; Shapleigh, J.P.; Walter, M.T. The Role of Denitriﬁcation in Stormwater Detention Basin Treatment of Nitrogen. Environ. Sci. Technol. 2017, 51, 7928–7935. [CrossRef] [PubMed] 9. Sasidharan, S.; Bradford, S.A.; Torkzaban, S.; Ye, X.Y.; Vanderzalm, J.; Du, X.Q.; Page, D. Unraveling the complexities of the velocity dependency of E-coli retention and release parameters in saturated porous media. Sci. Total Environ. 2017, 603, 406–415. [CrossRef] [PubMed] 10. Bozorg, A.; Gates, I.D.; Sen, A. Impact of bioﬁlm on bacterial transport and deposition in porous media. J. Contam. Hydrol. 2015, 183, 109–120. [CrossRef] [PubMed] 11. Zhang, L.; Seagren, E.A.; Davis, A.P.; Karns, J.S. Long-Term Sustainability of Escherichia Coli Removal in Conventional Bioretention Media. J. Environ. Eng.-ASCE 2011, 137, 669–677. [CrossRef] 12. Wennekes, P.L.; Rosindell, J.; Etienne, R.S. The Neutral-Niche Debate: A Philosophical Perspective. Acta Biotheor. 2012, 60, 257–271. [CrossRef] [PubMed] 13. Nemergut, D.R.; Schmidt, S.K.; Fukami, T.; O’Neill, S.P.; Bilinski, T.M.; Stanish, L.F.; Knelman, J.E.; Darcy, J.L.; Lynch, R.C.; Wickey, P.; et al. Patterns and Processes of Microbial Community Assembly. Microbiol. Mol. Biol. Rev. 2013, 77, 342–356. [CrossRef] [PubMed] 14. Teles, F.R.; Teles, R.P.; Uzel, N.G.; Song, X.Q.; Torresyap, G.; Socransky, S.S.; Haffajee, A.D. Water 2018, 10, 1065 Water 2018, 10, 1065 Acknowledgments: Part of this research project was conducted using computational resources at the Maryland Advanced Research Computing Center (MARCC). The authors would like to thank Kai Loon Chen (former PI) and Yusong Li (co-PI) for their efforts in obtaining funding for this work and Kaiyi Zhang for his help with the column experiments. Conﬂicts of Interest: The authors declare no conﬂict of interest. 5. Conclusions This work shows that replicate sand ﬁlters inoculated with the same community and incubated under controlled laboratory conditions change in a similar manner over a 24-day period. The largest changes to the community composition occur within the ﬁrst 10 days, then the community changes slowly, even as growth remains constant. Columns inoculated with nothing or with a single isolate maintained a distinct community from the stormwater inoculated column communities. Potential pathogens and denitrifying microorganisms become more abundant on the columns as compared to both the inoculum and the day 1 communities, suggesting speciﬁc growth within the columns. Potential pathogens decrease by the end of the 24-day experiment as other microorganisms become more abundant. Denitriﬁers continued to increase in abundance over the entire 24-day period. This work demonstrates that neutral processes of drift, historical contingency and migration have a signiﬁcant impact on the resulting microbial community structure, although the impact of drift is small compared to historical contingency over 24 days in the absence of additional migration. Future work needs to be done to determine the relative importance of these processes as compared to selective pressures imposed by different chemical and physical environments in shaping the community colonizing EIS. Our results suggest that management strategies manipulating inoculum could promote lasting change to microbial community structure and function, although it may be difﬁcult to maintain a speciﬁc community composition within these systems unless the community is well adapted to the conditions within the EIS. Author Contributions: Conceptualization, A.N.F. and S.P.P.; Formal analysis, A.N.F., Y.Z. and S.P.P.; Funding acquisition, S.P.P.; Methodology, A.N.F. and Y.Z.; Supervision, A.N.F., E.G.S. and S.P.P.; Validation, S.P.P.; Visualization, A.N.F. and S.P.P.; Writing—original draft, S.P.P.; Writing—review and editing, A.N.F., Y.Z., E.G.S. and S.P.P. Funding: This research was funded by the National Science Foundation, Division of Chemical, Bioengineering, Environmental, and Transport Systems (CBET), grant award number 1511915. A.N.F. was supported by an Integrative Graduate Education and Research Traineeship (IGERT) fellowship from the National Science Foundation, Division of Graduate Education, grant award number 1069213. 15 of 17 References Early microbial succession in redeveloping dental bioﬁlms in periodontal health and disease. J. Periodontal Res. 2012, 47, 95–104. [CrossRef] [PubMed] 15. David, L.; Maurice, C.; Carmody, R.; Gootenberg, D.; Button, J.; Wolfe, B.; Ling, A.; Devlin, A.; Varma, Y.; Fischbach, M.; et al. Diet rapidly and reproducibly alters the human gut microbiome. Nature 2014, 505, 559–563. [CrossRef] [PubMed] 16. Vannette, R.L.; Fukami, T. Historical contingency in species interactions: Towards niche-based predictions. Ecol. Lett. 2014, 17, 115–124. [CrossRef] [PubMed] 17. Vignola, M.; Werner, D.; Wade, M.J.; Meynet, P.; Davenport, R.J. Medium shapes the microbial community of water ﬁlters with implications for efﬂuent quality. Water Res. 2018, 129, 499–508. [CrossRef] [PubMed] 18. Albers, C.N.; Ellegaard-Jensen, L.; Harder, C.B.; Rosendahl, S.; Knudsen, B.E.; Ekelund, F.; Aamand, J. Groundwater Chemistry Determines the Prokaryotic Community Structure of Waterworks Sand Filters. Environ. Sci. Technol. 2015, 49, 839–846. [CrossRef] [PubMed] Water 2018, 10, 1065 16 of 17 16 of 17 19. Siripattanakul, S.; Wirojanagud, W.; McEvoy, J.M.; Casey, F.X.M.; Khan, E. Atrazine removal in agricultural inﬁltrate by bioaugmented polyvinyl alcohol immobilized and free Agrobacterium radiobacter J14a: A sand column study. Chemosphere 2009, 74, 308–313. [CrossRef] [PubMed] 20. Bonnin, G.M.; Martin, D.; Bingzhang, L.; Parzybok, T.; Yekta, M.; Riley, D. Precipitation-Frequency Atl United States, Volume 2, Version 3.0; National Weather Service: Silver Spring, MD, USA, 2004. 21. Department of Environmental Resources. Design Manual for Use of Bioretention in Stormwater Management; Department of Environmental Protection, Watershed Protection Branch: Prince George’s County, MD, USA, 1993. 22. Mohanty, S.K.; Torkelson, A.A.; Dodd, H.; Nelson, K.L.; Boehm, A.B. Engineering Solutions to Improve the Removal of Fecal Indicator Bacteria by Bioinﬁltration Systems during Intermittent Flow of Stormwater. Environ. Sci. Technol. 2013, 47, 10791–10798. [CrossRef] [PubMed] 23. Page, D.; Vanderzalm, J.; Miotlinski, K.; Barry, K.; Dillon, P.; Lawrie, K.; Brodie, R.S. Determining treatment requirements for turbid river water to avoid clogging of aquifer storage and recovery wells in siliceous alluvium. Water Res. 2014, 66, 99–110. [CrossRef] [PubMed] 24. Lane, D.J. 16S/23S rRNA sequencing. In Nucleic Acid Techniques in Bacterial Systematics; Stackebrandt, E., Goodfellow, M., Eds.; John Wiley & Sons: Chicheester, UL, USA, 1991; pp. 115–147. 25. Preheim, S.P.; Olesen, S.W.; Spencer, S.J.; Materna, A.; Varadharajan, C.; Blackburn, M.; Friedman, J.; Rodríguez, J.; Hemond, H.; Alm, E.J. Surveys, simulation and single-cell assays relate function and phylogeny in a lake ecosystem. In Nature Microbiology; Macmillan Publishers Limited: Basingstoke, UK, 2016; Volume 1, p. 16130. 26. 38. Assih, E.A.; Ouattara, A.S.; Thierry, S.; Cayol, J.L.; Labat, M.; Macarie, H. Stenotrophomonas acidaminiphila sp. nov., a strictly aerobic bacterium isolated from an upﬂow anaerobic sludge blanket (UASB) reactor. Int. J. Syst. Evol. Microbiol. 2002, 52, 559–568. [CrossRef] [PubMed] References Preheim, S.P.; Perrotta, A.R.; Martin-Platero, A.M.; Gupta, A.; Alm, E.J. Distribution-Based Clustering: Using Ecology To Reﬁne the Operational Taxonomic Unit. Appl. Environ. Microbiol. 2013, 79, 6593–6603. [CrossRef] [PubMed] 27. Caporaso, J.G.; Kuczynski, J.; Stombaugh, J.; Bittinger, K.; Bushman, F.D.; Costello, E.K.; Fierer, N.; Pena, A.G.; Goodrich, J.K.; Gordon, J.I.; et al. QIIME allows analysis of high-throughput community sequencing data. Nat. Methods 2010, 7, 335–336. [CrossRef] [PubMed] 28. Callahan, B.J.; McMurdie, P.J.; Rosen, M.J.; Han, A.W.; Johnson, A.J.A.; Holmes, S.P. DADA2: High-resolution sample inference from Illumina amplicon data. Nat. Methods 2016, 13, 581–583. [CrossRef] [PubMed] 29. Schloss, P.D.; Westcott, S.L.; Ryabin, T.; Hall, J.R.; Hartmann, M.; Hollister, E.B.; Lesniewski, R.A.; Oakley, B.B.; Parks, D.H.; Robinson, C.J.; et al. Introducing mothur: Open-Source, Platform-Independent, Community-Supported Software for Describing and Comparing Microbial Communities. Appl. Environ. Microbiol. 2009, 75, 7537–7541. [CrossRef] [PubMed] 30. Quast, C.; Pruesse, E.; Yilmaz, P.; Gerken, J.; Schweer, T.; Yarza, P.; Peplies, J.; Glockner, F.O. The SILVA ribosomal RNA gene database project: Improved data processing and web-based tools. Nucleic Acids Res. 2013, 41, D590–D596. [CrossRef] [PubMed] 31. Olesen, S.W.; Duvallet, C.; Alm, E.J. dbOTU3: A new implementation of distribution-based OTU calling. PLoS ONE 2017, 12, e0176335. [CrossRef] [PubMed] 32. Katoh, K.; Standley, D.M. MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability. Mol. Biol. Evol. 2013, 30, 772–780. [CrossRef] [PubMed] 33. Price, M.N.; Dehal, P.S.; Arkin, A.P. FastTree 2-Approximately Maximum-Likelihood Trees for Large Alignments. PLoS ONE 2010, 5, e9490. [CrossRef] [PubMed] 34. Vazquez-Baeza, Y.; Pirrung, M.; Gonzalez, A.; Knight, R. EMPeror: A tool for visualizing high-throughput microbial community data. Gigascience 2013, 2, 16. [CrossRef] [PubMed] 35. Louca, S.; Parfrey, L.W.; Doebeli, M. Decoupling function and taxonomy in the global ocean microbiome. Science 2016, 353, 1272–1277. [CrossRef] [PubMed] 36. Anderson, M.J. A new method for non-parametric multivariate analysis of variance. Austral Ecol. 2001, 26, 32–46. 37. R Core Team. R: A Language and Environment for Statistical Computing; R Foundation for Statistical Computing: Vienna, Austria, 2015. 38. Assih, E.A.; Ouattara, A.S.; Thierry, S.; Cayol, J.L.; Labat, M.; Macarie, H. Stenotrophomonas acidaminiphila sp. nov., a strictly aerobic bacterium isolated from an upﬂow anaerobic sludge blanket (UASB) reactor. Int. J. Syst. Evol. Microbiol. 2002, 52, 559–568. [CrossRef] [PubMed] Water 2018, 10, 1065 17 of 17 17 of 17 39. Denton, M.; Kerr, K.G. Microbiological and clinical aspects of infection associated with Stenotrophomonas maltophilia. Clin. Microbiol. Rev. 1998, 11, 57–80. [PubMed] 40. References Drancourt, M.; Bollet, C.; Raoult, D. Stenotrophomonas africana sp nov, an opportunistic human pathogen in Africa. Int. J. Syst. Bacteriol. 1997, 47, 160–163. [CrossRef] [PubMed] 41. Guardabassi, L.; Dalsgaard, A.; Olsen, J.E. Phenotypic characterization and antibiotic resistance of Acinetobacter spp. isolated from aquatic sources. J. Appl. Microbiol. 1999, 87, 659–667. [CrossRef] [PubMed] 42. Seifert, H.; Dijkshoorn, L.; GernerSmidt, P.; Pelzer, N.; Tjernberg, I.; Vaneechoutte, M. Distribution of Acinetobacter species on human skin: Comparison of phenotypic and genotypic identiﬁcation methods. J. Clin. Microbiol. 1997, 35, 2819–2825. [PubMed] 43. Rodriguez, C.H.; Nastro, M.; Dabos, L.; Barberis, C.; Vay, C.; Famiglietti, A. First isolation of Acinetobacter johnsonii co-producing PER-2 and OXA-58 beta-lactamases. Diagn. Microbiol. Infect. Dis. 2014, 80, 341–342. [CrossRef] [PubMed] 44. Seifert, H.; Strate, A.; Schulze, A.; Pulverer, G. Vascular catheter-related blood-stream infection due to Acinetobacter-johnsonii (formerly Acinetobacter calcoaceticus var iwofﬁi)—Report of 13 cases. Clin. Infect. Dis. 1993, 17, 632–636. [CrossRef] [PubMed] 45. Haig, S.J.; Quince, C.; Davies, R.L.; Dorea, C.C.; Collins, G. Replicating the microbial community and water quality performance of full-scale slow sand ﬁlters in laboratory-scale ﬁlters. Water Res. 2014, 61, 141–151. [CrossRef] [PubMed] 46. Albers, C.N.; Ellegaard-Jensen, L.; Hansen, L.H.; Sorensen, S.R. Bioaugmentation of rapid sand ﬁlters by microbiome priming with a nitrifying consortium will optimize production of drinking water from groundwater. Water Res. 2018, 129, 1–10. [CrossRef] [PubMed] 47. Bowen, J.L.; Babbin, A.R.; Kearns, P.J.; Ward, B.B. Connecting the dots: Linking nitrogen cycle gene expression to nitrogen ﬂuxes in marine sediment mesocosms. Front. Microbiol. 2014, 5, 429. [CrossRef] [PubMed] 48. Tomasek, A.; Kozarek, J.L.; Hondzo, M.; Lurndahl, N.; Sadowsky, M.J.; Wang, P.; Staley, C. Environmental drivers of denitriﬁcation rates and denitrifying gene abundances in channels and riparian areas. Water Resour. Res. 2017, 53, 6523–6538. [CrossRef] 49. Martiny, A.C.; Treseder, K.; Pusch, G. Phylogenetic conservatism of functional traits in microorganisms. ISME J. 2013, 7, 830–838. [CrossRef] [PubMed] © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W4286697542	https://digibug.ugr.es/bitstream/10481/76742/1/fpsyt-13-903361.pdf	English	null	Masturbation parameters related to orgasm satisfaction in sexual relationships: Differences between men and women	Frontiers in psychiatry	2,022	cc-by	7,261	Masturbation parameters related to orgasm satisfaction in sexual relationships: Diferences between men and women OPEN ACCESS EDITED BY Patrik Roser, University of Duisburg-Essen, Germany REVIEWED BY Markus Zedler, Hannover Medical School, Germany Carlos Miguel Rios-González, National University of Caaguazú, Paraguay CORRESPONDENCE Juan Carlos Sierra jcsierra@ugr.es SPECIALTY SECTION This article was submitted to Public Mental Health, a section of the journal Frontiers in Psychiatry RECEIVED 24 March 2022 ACCEPTED 29 June 2022 PUBLISHED 22 July 2022 CITATION Cervilla O and Sierra JC (2022) Masturbation parameters related to orgasm satisfaction in sexual relationships: Diferences between men and women. Front. Psychiatry 13:903361. doi: 10.3389/fpsyt.2022.903361 COPYRIGHT OPEN ACCESS EDITED BY Patrik Roser, University of Duisburg-Essen, Germany REVIEWED BY Markus Zedler, Hannover Medical School, Germany Carlos Miguel Rios-González, National University of Caaguazú, Paraguay CORRESPONDENCE Juan Carlos Sierra jcsierra@ugr.es SPECIALTY SECTION This article was submitted to Public Mental Health, a section of the journal Frontiers in Psychiatry RECEIVED 24 March 2022 ACCEPTED 29 June 2022 PUBLISHED 22 July 2022 CITATION Cervilla O and Sierra JC (2022) Masturbation parameters related to orgasm satisfaction in sexual relationships: Diferences between men and women. Front. Psychiatry 13:903361. doi: 10.3389/fpsyt.2022.903361 OPEN ACCESS EDITED BY Patrik Roser, University of Duisburg-Essen, Germany REVIEWED BY Markus Zedler, Hannover Medical School, Germany Carlos Miguel Rios-González, National University of Caaguazú, Paraguay CORRESPONDENCE Juan Carlos Sierra jcsierra@ugr.es SPECIALTY SECTION This article was submitted to Public Mental Health, a section of the journal Frontiers in Psychiatry RECEIVED 24 March 2022 ACCEPTED 29 June 2022 PUBLISHED 22 July 2022 CITATION Cervilla O and Sierra JC (2022) Masturbation parameters related to orgasm satisfaction in sexual relationships: Diferences between d Oscar Cervilla and Juan Carlos Sierra Mind, Brain, and Behavior Research Center (CIMCYC), University of Granada, Granada, Spain Mind, Brain, and Behavior Research Center (CIMCYC), University of Granada, Granada, Spain Objective: Masturbation is a behavior that can enhance sexual functioning. This study aims to analyze diferences between men and women in diferent masturbation parameters, and to examine their relation with orgasm satisfaction in sexual relationships. Method: One thousand three hundred and thirty-ﬁfth men and women from the Spanish population aged 18–83 years (M = 36.91; SD = 11.86) participated in an online survey. A questionnaire was used to collect socio-demographic. Sexual history data, negative attitude toward masturbation, solitary sexual desire and orgasm subjective experience upon masturbation were assessed. Given the diferences between men and women, independent regression models are proposed to explain orgasm satisfaction in the sexual relationships context. TYPE Original Research PUBLISHED 22 July 2022 DOI 10.3389/fpsyt.2022.903361 TYPE Original Research PUBLISHED 22 July 2022 DOI 10.3389/fpsyt.2022.903361 TYPE Original Research PUBLISHED 22 July 2022 DOI 10.3389/fpsyt.2022.903361 Introduction and intensity of the subjective orgasm experience obtained by masturbation stand out. Taking a negative attitude toward masturbation has been associated with feeling guilty and ashamed (22, 23), and also with negative sexual experiences (24). Moreover, lower masturbation frequency, more diﬃculty to have an orgasm and lower orgasm satisfaction have been observed in those with a more negative attitude toward masturbation (25). Solitary sexual desire (i.e., interest in solitary sexual activity) has been associated with high sexual satisfaction and self-esteem levels in women (2, 4), and has been related to both sexual satisfaction and unsatisfactory sexual functioning in men (26– 28). In light of all this, a positive relation between solitary sexual desire and the intensity of the subjective orgasm experience in the solitary masturbation context has been found in a sample made up of men and women (29). Subjective orgasm experience in masturbation has been shown to be capable of distinguishing people with and without diﬃculties in having an orgasm during sexual relationships (29). Sierra et al. (30) recently observed that masturbation frequency, negative attitude toward masturbation and the subjective orgasmic experience in masturbation are associated with orgasm satisfaction in sexual relationships in people aged over 50 years. Masturbation is a relatively frequent behavior that is positively associated with sexual health (1–5). More importance has been attached to study it in recent decades, and its capacity to promote sexual self-knowledge and to elicit positive sexual responses has been underlined (6, 7). Among these good points, its usefulness in sexual therapy to improve sexual functioning has been stressed (8). Very little evidence exists for the relation between masturbation and sexual relationships (9). The association between both sexual behaviors has been described by two models: compensatory vs. complementary. The former proposes practicing masturbation to replace desired sexual relationships that do not take place (10, 11). The fact that higher masturbation frequency is related to lower sexual satisfaction, and has been found for women, favors this model (12), while higher masturbation frequency for those who less enjoy their sexual relationships has been described for men (13). The complementary model hypothesizes that a direct positive relation exists between both these sexual activities, and increasing the practice of one would be associated with an increase in the other (14). Masturbation parameters related to orgasm satisfaction in sexual relationships: Diferences between men and women Findings: Men, compared to women, masturbated at a younger age (p < 0.001), and reported higher current masturbation frequency (p < 0.001) and more solitary sexual desire (p < 0.001). Women reported greater intensity in the subjective orgasm experience on its Afective (p < 0.001), Sensory (p < 0.001) and Intimacy (p < 0.001) dimensions. Regarding regression models, the Afective dimension of orgasm was a common parameter in men (β = 0.36; p < 0.001) and women (β = 0.24) to explain orgasm satisfaction during sexual relationships. In men, solitary masturbation frequency (β = −0.10; p = 0.027) acquired a signiﬁcant role. In women, the model also included age (β = 0.09; p = 0.038), negative attitude toward masturbation (β = −0.12; p = 0.005) and solitary sexual desire (β = −0.19; p = 0.001). © 2022 Cervilla and Sierra. 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. Conclusion: When dealing with men and women’s orgasm difculties in the sexual relationships context, it is important to consider the role of masturbation. In men and women, the Afective dimension of the orgasm experience explain the orgasm satisfaction in sexual relationship. Also, in men, the solitary masturbation frequency is negatively related with orgasm satisfaction in sexual relationship, supporting the compensatory hypothesis of masturbation. In women, in addition to the Afective dimension, the orgasm satisfaction in sexual relationship is explained, negatively, by the negative 01 Frontiers in Psychiatry Frontiers in Psychiatry frontiersin.org Cervilla and Sierra 10.3389/fpsyt.2022.903361 10.3389/fpsyt.2022.903361 attitude toward masturbation, and positively, by the solitary sexual desire, which could be associated with more sexual self-knowledge. The relevance of masturbation in understanding sexual functioning is highlighted. orgasm satisfaction, partnered sex, masturbation, subjective orgasm experience, attitude toward masturbation, sex diferences Introduction Bearing in mind the relevance of masturbation for sexual health, and its usefulness in the therapeutic context to improve sexual functioning, this study aims to: analyze diﬀerences between men and women in diﬀerent masturbation parameters (i.e., ﬁrst masturbation experience, current solitary masturbation frequency, negative attitude toward masturbation, solitary sexual desire and subjective orgasm experience); examine their relation, along with age, to orgasm satisfaction in the sexual relationships context. To do so, the following hypotheses are proposed: (1) diﬀerences are expected in masturbation parameters between men and women; (2) orgasm satisfaction in sexual relationships is expected to be linked with masturbation parameters (30). Some works suggest that masturbation does not oﬀer a clear advantage for sexual relationships (15–17). Nonetheless, it has been found that women who masturbate are more likely to have an orgasm during sexual relationships (18), and those who masturbate more frequently describe better sexual experiences in couples and less sexual inhibition (2, 3). Techniques like Directed Masturbation can boost pleasurable stimulation from knowing pleasure points, which improves women’s orgasm facility while couples practice sex (19). Therefore, learning to have orgasms by masturbation allows women to adjust and generalize this orgasm response in sexual relationships in couples (20). These results sustain the usefulness of masturbation as the ﬁrst line of treatment for the Female Orgasmic Disorder (20, 21). Methods Despite some ﬁndings that favor practicing masturbation to improve orgasm capacity, very little evidence exists for the role that the diﬀerent parameters related to this behavior play in orgasms in the sexual relationships context. Of these parameters, attitude toward masturbation, solitary sexual desire Frontiers in Psychiatry Cervilla and Sierra Participants The sample comprised 1,335 Spanish adults (738 men, 597 women) aged 18–83 years (M = 36.91; SD = 11.86). The Frontiers in Psychiatry 02 frontiersin.org frontiersin.org 10.3389/fpsyt.2022.903361 10.3389/fpsyt.2022.903361 Cervilla and Sierra TABLE 1 Sociodemographic characteristics of the participants. Variables Total N = 1,335 Men n = 738 Women n = 597 Age M (SD) 36.91 (11.86) 37.62 (12.43) 36.04 (11.07) Level of education n (%) Primary education 53 (4.0) 26 (3.5) 27 (4.5) Secondary education 390 (29.2) 231 (31.3) 159 (26.6) University degree (ongoing or completed) 892 (66.8) 481 (65.2) 411 (68.9) Currently have a partner n (%) Yes 988 (74.0) 524 (71.0) 464 (77.7) No 347 (26.0) 214 (29.0) 133 (22.3) Praying frequency n (%) Never 989 (74.1) 523 (70.90) 466 (78.1) Less than once a month 123 (9.2) 67 (9.1) 56 (9.4) Once a month 7 (0.5) 6 (0.8) 1 (0.2) A few times a month 54 (4.0) 32 (4.3) 22 (3.7) Once a week 8 (0.6) 3 (0.4) 5 (0.8) A few times a week 57 (4.3) 34 (4.6) 23 (3.9) Once a day 60 (4.5) 41 (5.6) 19 (3.2) More than once a day 37 (2.8) 32 (4.3) 5 (0.8) TABLE 1 Sociodemographic characteristics of the participants. activity using diﬀerent Likert response scales depending on the item (e.g., from 0 = No desire to 8 = Strong desire). Higher scores show more solitary desire. It presents good internal consistency (Cronbach’s α higher than 0.90) and evidence for external validity. Cronbach’s alpha in the present study was 0.91. activity using diﬀerent Likert response scales depending on the item (e.g., from 0 = No desire to 8 = Strong desire). Higher scores show more solitary desire. It presents good internal consistency (Cronbach’s α higher than 0.90) and evidence for external validity. Cronbach’s alpha in the present study was 0.91. The Spanish version of the Orgasm Rating Scale (ORS) (33) adapted to the solitary masturbation context by Cervilla et al. (29). It assesses the subjective orgasm experience in the solitary masturbation context (during any sexual activity performed alone) with 25 adjectives distributed on four dimensions: Aﬀective, Sensory, Intimacy, and Rewards. Items are answered on a 6-point Likert-type scale: 0 (Does not describe it at all) to 5 (Describes it perfectly). Higher scores indicate more intensity in the subjective orgasm experience. Its internal consistency reliability is good and ranges from 0.71 to 0.95. Participants It adequately evidences validity, provided by its measures. In our study, the ordinal alphas for the diﬀerent subscales were: 0.93 for Aﬀective, 0.94 for Sensory, 0.72 for Intimacy and 0.89 for Rewards. The Spanish version of the Arizona Sexual Experience Scale (ASEX) (34) of Sánchez-Fuentes et al. (35). It consists of ﬁve items that assess general sexual functioning (sexual desire, arousal, erection for men/lubrication for women, orgasm, and orgasm satisfaction) in the last 7 days in the sexual relationship context. It uses a Likert-type scale from 1 (hypofunction) to 6 (hyperfunction). It presents good internal consistency (Cronbach’s alpha of 0.81 in men, 0.79 in women) and evidences validity. The orgasm-related item referring to orgasm satisfaction was taken into account. Its score was inverted, so higher scores evidenced more orgasm satisfaction. inclusion criteria were: (a) having Spanish nationality; (b) being heterosexual; (c) currently engaging in sexual relationships; (d) having solitary masturbation experience. Table 1 shows the samples’ socio-demographic characteristics. Frontiers in Psychiatry Regression models For men, a signiﬁcant model was obtained that explained orgasm satisfaction in sexual relationships [F (9, 728) = 13.01; p < 0.001]. Current solitary masturbation frequency (β = −0.10) and the Aﬀective dimension of orgasm (β = 0.36) explained 13% of variance (See Table 3). The model was also signiﬁcant for women [F (9, 587) = 8.88; p < 0.001] and explained 11% of orgasm satisfaction from age (β = 0.09), negative attitude toward masturbation (β = −0.12), solitary sexual desire (β = 0.19) and the Aﬀective dimension of orgasm (β = 0.24) (See Table 4). Procedure Variables M (DT) Males n = 738 Females n = 597 F(1, 1,329) p Cohen’s d First masturbation experience 12.60 (2.03) 15.13 (5.92) 140.51 <0.001 0.60 Current frequency of solitary masturbation 3.17 (0.94) 2.50 (0.96) 185.11 <0.001 0.70 Negative attitude toward masturbation 13.03 (3.09) 13.13 (2.32) 1.91 0.167 - Solitary sexual desire 21.59 (5.59) 19.75 (6.28) 31.96 <0.001 0.31 Subjective orgasm experience-Aﬀective 25.66 (4.84) 27.05 (3.85) 31.69 <0.001 0.31 Subjective orgasm experience-Sensory 33.65 (15.74) 39.17 (15.15) 47.75 <0.001 0.36 Subjective orgasm experience-Intimacy 7.62 (3.64) 8.19 (3.71) 12.48 <0.001 0.15 Subjective orgasm experience-Rewards 11.34 (3.36) 11.56 (3.59) 1.82 0.177 - Statistical analysis the masturbation parameters [Wilk’s lambda = 0.77; F(8, 1,322) = 48.91, p < 0.001; η2 = 0.23]. The intersubject eﬀect on these indicators is shown in Table 2. A cross-sectional correlational study is proposed. First, missing values were imputed using a random forest algorithm by considering the associated variables. To examine diﬀerences in the masturbation parameters between men and women, a MANCOVA was applied for ﬁrst masturbation experience (age), current solitary masturbation frequency (“Never,” “Less than once a month,” “Once a month,” “A few times a month,” “Once a week,” “A few times a week,” “Once a day” and “More than once a day”), negative attitude toward masturbation, solitary sexual desire and subjective orgasm experience caused by masturbation, and by taking into account these covariates: age, level of education (“Primary Education,” Secondary Education” and “University degree–ongoing or completed-”), having a partner (yes or no) and frequency of prayer (similar to the masturbation frequency). Considering the diﬀerences found by sex, the subsequent analyses were presented separately for men and women. The capacity of the masturbation parameters to explain orgasm satisfaction was examined by multiple linear regression using the enter method. Procedure Background questionnaire. This instrument collects data about sex, age, level of education, nationality, sexual orientation, partner relationship, frequency of prayer, age when the ﬁrst masturbation experience occurred and masturbation frequency. Data collection was conducted by distributing a survey using LimeSurvey, which was promoted by paying to Facebook (900e) from 23 December 2019 to 15 March 2020 by adults from Spain. In order to improve the representativeness of the sample, the promotion targeted both men and women from diﬀerent age groups. Online assessments are normally used to evaluate sexual behaviors (1, 36, 37). Previous studies have conﬁrmed that there are no diﬀerences between online and paper-and-pen methods (38, 39). To avoid automatic or fraudulent responses, IP was controlled and a CAPTCHA was used. In addition, responses were carefully examined to rule out non conclusive or abnormal cases. Participation was voluntary, and both anonymity and conﬁdentiality of responses were guaranteed. There was no compensation for taking part in the study. All the participants received informed consent with the study aim before responding. This research was approved by the Ethics Committee of Human Research of the University of Granada. The Spanish version of the Negative Attitudes Toward Masturbation Inventory (NATMI) (25, 31). It evaluates negative attitudes toward masturbation with 10 items (e.g., “I feel guilty about masturbating”) answered on a 5-point Likert-type scale: 1 (Not at all true for me) to 5 (Extremely true for me). Higher scores indicate a more negative attitude toward masturbation. It has a high internal consistency (alpha ordinal) of 0.95, and presents suitable evidence for construct and discriminant validity with other psychosexual variables and sexual functioning. In this sample, the ordinal alpha coeﬃcient was 0.91. The Solitary Sexual Desire subscale from the Spanish version of the Sexual Desire Inventory (SDI) (28, 32). It consists of four items (e.g., “How strong is your desire to engage in sexual behavior by yourself?”) and measures interest in solitary sexual Frontiers in Psychiatry 03 frontiersin.org 10.3389/fpsyt.2022.903361 Cervilla and Sierra TABLE 2 Efects of sex on masturbation-related indicators. TABLE 2 Efects of sex on masturbation-related indicators. Discussion Masturbation is a sexual behavior that is contemplated to deal with sexual dysfunctions, especially orgasm diﬃculties (44–46). Justifying the use of masturbation in sexual therapy lies in the relation between this behavior and orgasm in sexual relationships. This is why the present study analyzes the relation between diﬀerent masturbation parameters in men and women (i.e., ﬁrst masturbation experience, current solitary masturbation frequency, negative attitude toward masturbation, solitary sexual desire and subjective orgasm experience) with orgasm satisfaction in sexual relationships. The results show diﬀerences between men and women in the masturbation parameters, and also in the role that these parameters play in explaining orgasm satisfaction in the sexual relationships context. The R R⃝environment was employed (version 3.6.3) (40) with its RStudio R⃝interface (version 1.2.5042) (41). For missing value imputations, the missForest package was used (version 1.4) (42). For the ordinal alpha, the Psych package was applied (version 1.9.12.31) (43). The other statistical analyses were performed with SPSS v.22. Sex diferences in the masturbation parameters Predictors B SE β 95% CI t p R2 VIF Orgasmic satisfaction 0.11 Age 0.01 0.00 0.09 0.00, 0.02 2.08 0.038 1.21 First masturbation experience 0.01 0.01 0.04 −0.01, 0.02 1.01 0.314 1.10 Current frequency of solitary masturbation −0.07 0.06 −0.07 −0.18, 0.04 −1.27 0.203 1.88 Negative attitude toward masturbation −0.05 0.02 −0.12 −0.09, −0.01 −2.80 0.005 1.15 Solitary sexual desire 0.03 0.01 0.19 0.01, 0.05 3.43 0.001 2.11 Subjective orgasm experience-Aﬀective 0.06 0.01 0.24 0.04, 0.09 4.91 <0.001 1.65 Subjective orgasm experience-Sensory −0.00 0.00 −0.03 −0.01, 0.00 −0.52 0.606 1.77 Subjective orgasm experience-Intimacy 0.00 0.01 0.01 −0.03, 0.03 0.12 0.906 1.68 Subjective orgasm experience-Rewards −0.02 0.01 −0.08 −0.05, 0.00 −1.70 0.090 1.46 B, non-estandardized beta; SE, standard error; β, standardized beta; 95% CI, 95% conﬁdence interval; VIF, variance inﬂation factor. indicate that men older than 50 years take a more negative attitude toward masturbation than women of a similar age. This could indicate younger generations’ positive attitude toward masturbation. This question reﬂects the need to further study in-depth attitudes toward masturbation and the factors related to it to better understand this matter (25). Regarding diﬀerences in solitary sexual desire, the highest level found for men is consistent with previous works that report similar results (27, 28, 53, 54). This is congruent with those studies showing a close association between masturbation and solitary sexual desire (55). indicate that men older than 50 years take a more negative attitude toward masturbation than women of a similar age. This could indicate younger generations’ positive attitude toward masturbation. This question reﬂects the need to further study in-depth attitudes toward masturbation and the factors related to it to better understand this matter (25). Regarding diﬀerences in solitary sexual desire, the highest level found for men is consistent with previous works that report similar results (27, 28, 53, 54). This is congruent with those studies showing a close association between masturbation and solitary sexual desire (55). Traditional sexual socialization could favor more permissiveness in men and more guilty feelings associated with women practicing masturbation (48). In turn, the diﬀerences found in solitary masturbation frequency coincide with previous works in the literature, and a more frequent masturbation frequency observed for men (25, 49, 50). Sex diferences in the masturbation parameters Predictors B SE β 95% CI t p R2 VIF Orgasmic satisfaction 0.11 Age 0.01 0.00 0.09 0.00, 0.02 2.08 0.038 1.21 First masturbation experience 0.01 0.01 0.04 −0.01, 0.02 1.01 0.314 1.10 Current frequency of solitary masturbation −0.07 0.06 −0.07 −0.18, 0.04 −1.27 0.203 1.88 Negative attitude toward masturbation −0.05 0.02 −0.12 −0.09, −0.01 −2.80 0.005 1.15 Solitary sexual desire 0.03 0.01 0.19 0.01, 0.05 3.43 0.001 2.11 Subjective orgasm experience-Aﬀective 0.06 0.01 0.24 0.04, 0.09 4.91 <0.001 1.65 Subjective orgasm experience-Sensory −0.00 0.00 −0.03 −0.01, 0.00 −0.52 0.606 1.77 Subjective orgasm experience-Intimacy 0.00 0.01 0.01 −0.03, 0.03 0.12 0.906 1.68 Subjective orgasm experience-Rewards −0.02 0.01 −0.08 −0.05, 0.00 −1.70 0.090 1.46 B, non-estandardized beta; SE, standard error; β, standardized beta; 95% CI, 95% conﬁdence interval; VIF, variance inﬂation factor. TABLE 3 Multiple regression models for orgasmic satisfaction in men. Predictors B SE β 95% CI t p R2 VIF Orgasmic satisfaction 0.13 Age 0.00 0.00 0.01 −0.00, 0.01 0.03 0.974 1.23 First masturbation experience −0.01 0.02 −0.03 −0.05, 0.02 −0.88 0.379 1.08 Current frequency of solitary masturbation −0.10 0.04 −0.10 −0.19, −0.01 −2.22 0.027 1.79 Negative attitude toward masturbation −0.01 0.01 −0.05 −0.04, 0.01 −1.31 0.191 1.15 Solitary sexual desire 0.01 0.01 0.08 −0.00, 0.03 1.77 0.076 1.92 Subjective orgasm experience-Aﬀective 0.07 0.01 0.36 0.05, 0.08 7.70 <0.001 1.85 Subjective orgasm experience-Sensory −0.00 0.00 −0.05 −0.01, 0.00 −1.09 0.275 1.93 Subjective orgasm experience-Intimacy 0.00 0.01 0.01 −0.02, 0.03 0.32 0.746 1.70 Subjective orgasm experience-Rewards −0.01 0.01 −0.03 −0.03, 0.01 −0.78 0.438 1.40 B, non-estandardized beta; SE, standard error; β, standardized beta; 95% CI, 95% conﬁdence interval; VIF, variance inﬂation factor. non-estandardized beta; SE, standard error; β, standardized beta; 95% CI, 95% conﬁdence interval; VIF, variance inﬂation factor. TABLE 4 Multiple regression models for orgasmic satisfaction in women. Sex diferences in the masturbation parameters The signiﬁcant multivariate covariates were age [Wilk’s lambda = 0.87; F(8, 1,322) = 24.49, p < 0.001; η2 = 0.129], having a partner [Wilk’s lambda = 0.94; F(8, 1,322) = 10.91, p < 0.001; η2 = 0.062] and frequency of prayer [Wilk’s lambda = 0.96; F(8, 1,322) = 7.42, p < 0.001; η2 = 0.04]. Sex had a main eﬀect on The ﬁrst hypothesis is backed by signiﬁcant diﬀerences between men and women in the diﬀerent masturbation parameters. We observe that men’s ﬁrst masturbation experience took place at an earlier age than it did in women, whose ﬁnding coincides with previous studies (1, 2, 25, 30, 47). Frontiers in Psychiatry 04 frontiersin.org frontiersin.org 10.3389/fpsyt.2022.903361 Cervilla and Sierra TABLE 3 Multiple regression models for orgasmic satisfaction in men. TABLE 3 Multiple regression models for orgasmic satisfaction in men. Predictors B SE β 95% CI t p R2 VIF Orgasmic satisfaction 0.13 Age 0.00 0.00 0.01 −0.00, 0.01 0.03 0.974 1.23 First masturbation experience −0.01 0.02 −0.03 −0.05, 0.02 −0.88 0.379 1.08 Current frequency of solitary masturbation −0.10 0.04 −0.10 −0.19, −0.01 −2.22 0.027 1.79 Negative attitude toward masturbation −0.01 0.01 −0.05 −0.04, 0.01 −1.31 0.191 1.15 Solitary sexual desire 0.01 0.01 0.08 −0.00, 0.03 1.77 0.076 1.92 Subjective orgasm experience-Aﬀective 0.07 0.01 0.36 0.05, 0.08 7.70 <0.001 1.85 Subjective orgasm experience-Sensory −0.00 0.00 −0.05 −0.01, 0.00 −1.09 0.275 1.93 Subjective orgasm experience-Intimacy 0.00 0.01 0.01 −0.02, 0.03 0.32 0.746 1.70 Subjective orgasm experience-Rewards −0.01 0.01 −0.03 −0.03, 0.01 −0.78 0.438 1.40 B, non-estandardized beta; SE, standard error; β, standardized beta; 95% CI, 95% conﬁdence interval; VIF, variance inﬂation factor. TABLE 4 Multiple regression models for orgasmic satisfaction in women. Sex diferences in the masturbation parameters Regarding the diﬀerences in their dimensions, not ﬁnding discrepancies would be expected on the Rewards dimension, which is made up of the items “peaceful,” “relaxing,” and “soothing,” because both men and women have pointed out that relaxing is one of the main reasons to masturbate (13, 60, 61). Regarding our second hypothesis, orgasm satisfaction in the sexual relationships context is explained in both men and women by some masturbation parameters. In the model for men and women, the Aﬀective dimension of the subjective orgasm experience during masturbation signiﬁcantly and positively explains orgasm satisfaction in the sexual relationships context. Former ﬁndings stress the importance of the Aﬀective dimension of the subjective orgasm experience for the sexual relationships context, especially for women (62). So it might seem logical to think that this could be the case in the masturbation context where this dimension is more important for explaining orgasm satisfaction in sexual relationships. This study has its limitations, which must be taken into account to generalize its results. The study sample was formed by incidental non probabilistic sampling over social networks and only included the heterosexual population. The cross-sectional correlational experimental design and the performed statistical analyses do not allow for causality relations. So, it may be need longitudinal studies to have a deep approach about the relationship between masturbation and sexual relationships. Diﬀerent parameters of masturbation could be taken into account in future studies, such as the duration of masturbation, the use of erotic toys, the techniques used or the consumption of pornography. Notwithstanding, the ﬁndings are believed relevant for its contribution to the study of masturbation and orgasm satisfaction in the sexual relationships context. Apart from the orgasm Aﬀective dimension in the men’s model, higher solitary masturbation frequency is also associated with lower orgasm satisfaction. These results might appear to contradict works that have described how frequency is associated with more consistent orgasms (7). However, in line with previous studies (30, 52), this association might be explained by the compensatory model of masturbation; that is, masturbation serving as a substitute of unsatisfactory sexual relationships. Therefore, lower orgasm satisfaction in the sexual relationships context might be expected to be compensated by higher masturbation frequency (26, 63). In women, apart from the Aﬀective dimension of orgasm, age and solitary sexual desire are positively associated, and attitude toward masturbation is negatively associated, with orgasm satisfaction in sexual relationships. Sex diferences in the masturbation parameters Attitude to the sexual double standard (i.e., the distinct evaluation made of sexual behavior depending on whether it is practiced by a man or a woman) could explain these diﬀerences given the greater sexual freedom or permissiveness that men have been traditionally conferred than women (38). Alternative considerations have also been applied to explain these diﬀerences in association with hormone levels (51). On subjective orgasm experience in solitary masturbation, and in line with the results obtained by previous studies that have examined the subjective orgasm experience in the heterosexual relationships context (36, 56), women report greater intensity than men, except on the Reward dimension, which has also been shown for the gay population (57). To explain diﬀerences in orgasm intensity between men and women, women have been proposed to better localize orgasms anatomically (56), which would be associated with perceiving greater intensity (58). It has It is worth mentioning that no diﬀerences have been found in negative attitude toward masturbation between men and women. The fact that such diﬀerences are lacking could be related to an increasingly more positive change of attitude in both men and women, as observed in other attitudes like erotophilia (52). These results contradict those recently obtained in older people and reported by Sierra et al. (30), who Frontiers in Psychiatry 05 frontiersin.org 10.3389/fpsyt.2022.903361 10.3389/fpsyt.2022.903361 Cervilla and Sierra with some ﬁndings which reveal that women need time to interiorize a more positive relation with masturbation due to the stigmatization that their engagement in such behavior might imply (2). This suggests that the positive eﬀects of masturbation could increase as women age. Besides, solitary masturbation frequency only has a signiﬁcant eﬀect on men, which falls in line with former results which point out that masturbation frequency in women is not signiﬁcantly associated with orgasm outcomes (18). As higher masturbation frequency in men is associated with lower orgasm satisfaction in sexual relationships, it would be coherent to think that solitary sexual desire plays no relevant role to explain men’s orgasm satisfaction. Finally, the diﬀerences observed in the models of men and women fall in line with the previous literature, which emphasizes how women’s orgasm is associated with more variables than it is for men (56, 57, 67). also been indicated that women could have a bigger repertoire to describe their orgasm sensations (57, 59). Sex diferences in the masturbation parameters The positive association of age would be expected because former works inform about a higher orgasm pleasure level with increasing age (18, 61). Moreover, the positive relation between sexual desire and sexual functioning has been well-described (27, 28, 64). In fact solitary sexual desire is associated with higher masturbation frequency (1, 55), which might imply more self- erotic experiences and sexual self-knowledge (3). Finally, the fact that negative attitude toward masturbation is related to lower orgasm satisfaction is consistent with previous works (25, 65). This attitude has been associated with lower masturbation frequency (25), which might imply fewer opportunities for both sexual response self-knowledge and the associated pleasure points (7, 19). Frontiers in Psychiatry References 1. Burri A, Carvalheira A. Masturbatory behavior in a population sample of German women. J Sex Med. (2019) 16:963–74. doi: 10.1016/j.jsxm.2019.04.015 12. Klapilová K, Brody S, Krejˇcová L, Husárová B, Binter J. Sexual satisfaction, sexual compatibility, and relationship adjustment in couples: the role of sexual behaviors, orgasm, and men’s discernment of women’s intercourse orgasm. J Sex Med. (2015) 12:667–75. doi: 10.1111/jsm.12766 12. Klapilová K, Brody S, Krejˇcová L, Husárová B, Binter J. Sexual satisfaction, sexual compatibility, and relationship adjustment in couples: the role of sexual behaviors, orgasm, and men’s discernment of women’s intercourse orgasm. J Sex Med. (2015) 12:667–75. doi: 10.1111/jsm.12766 2. Carvalheira A, Leal I. Masturbation among women: associated factors and sexual response in a Portuguese community sample. J Sex Marital Ther. (2013) 39:347–67. doi: 10.1080/0092623X.2011.628440 13. Fahs B, Frank E. Notes from the back room: gender, power, and (In)Visibility in women’s experiences of masturbation. J Sex Res. (2014) 51:241–52. doi: 10.1080/00224499.2012.745474 3. Coleman E. Masturbation as a means of achieving sexual health. J Psychol Human Sex. (2003) 14:5–16. doi: 10.1300/J056v14n02_02 3. Coleman E. Masturbation as a means of achieving sexual health. J Psychol Human Sex. (2003) 14:5–16. doi: 10.1300/J056v14n02_02 14. Pinkerton SD, Bogart LM, Cecil H, Abramson PR. Factors associated with masturbation in a collegiate sample. J Psychol Hum Sex. (2003) 14:103–21. doi: 10.1300/J056v14n02_07 4. Kaestle CE, Allen KR. The role of masturbation in healthy sexual development: perceptions of young adults. Arch Sex Behav. (2011) 40:983–94. doi: 10.1007/s10508-010-9722-0 15. Kontula T, Miettinen A. Determinants of female sex orgasm. Socioaﬀect Neurosci Psychol. (2016) 6:1–21. doi: 10.3402/snp.v6.31624 5. Kiliç Onar D, Armstrong H, Graham CA. What does research tell us about women’s experiences, motives and perceptions of masturbation within a relationship context?: a systematic review of qualitative studies. J Sex Marital Ther. (2020) 46:683–716. doi: 10.1080/0092623X.2020.1781722 16. LeﬀJJ, Israel M. The relationship between mode of female masturbation and achievement of orgasm in coitus. Arch Sex Behav. (1983) 12:227–36. doi: 10.1007/BF01542073 6. Driemeyer W. Masturbation and sexual health-a research overview. Z Sexualforsch. (2013) 26:372–83. doi: 10.1055/s-0033-1356159 17. Rowland DL, Sullivan SL, Hevesi K, Hevesi B. Orgasmic latency and related parameters in women during partnered and masturbatory sex. J Sex Med. (2018) 15:1463–71. doi: 10.1016/j.jsxm.2018. 08.003 7. Matsick JL, Conley TD, Moors AC. The science of female orgasms: pleasing female partners in casual and long-term relationships. In: Aumer K, editor. The Psychology of Love and Hate in Intimate Relationships. Switzerland: Springer (2016). Funding promoted in the community context as it can improve the sexual health of the population. promoted in the community context as it can improve the sexual health of the population. This study has been funded by the Ministerio de Ciencia, Innovación y Universidades through the Research Project RTI2018-093317-B-I00 and the Bursary FPU18/03102 for University Professor Training as part of OC’s thesis (Psychological Doctoral Programme B13 56 1; RD 99/2011). Conclusion The obtained results conﬁrm the diﬀerences between men and women in the masturbation parameters and their role to explain orgasm satisfaction in sexual relationships. The Aﬀective dimension of the subjective orgasm experience during solitary masturbation is stressed as a common variable for both men and women to explain orgasm satisfaction in sexual relationships. More masturbation parameters associated with orgasm satisfaction are observed in women than men. These ﬁndings suggest that the relation between solitary masturbation and sexual relationships is a complex one. Masturbation in men could be a substitute for the satisfaction not achieved with orgasm in sexual relationship; in women, the negative attitude toward this behavior would be associated with lower orgasmic satisfaction, and a greater solitary sexual desire could promote more sexual self-knowledge. So it is important to consider these results to look more closely at the association between both sexual behaviors, and to further consolidate the usefulness of solitary masturbation in sexual therapy. Therefore, solitary masturbation is an available resource that should also be Some diﬀerences between the models for men and women are worth stressing. The positive eﬀect of age is only observed in women. This suggests that women beneﬁt from enjoying more orgasm satisfaction as they age to a certain extent. Despite a negative association between age and orgasm capacity having been previously described (36, 66), these results are consistent Frontiers in Psychiatry 06 frontiersin.org 10.3389/fpsyt.2022.903361 10.3389/fpsyt.2022.903361 Cervilla and Sierra Data availability statement The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Author contributions All claims expressed in this article are solely those of the authors and do not necessarily represent those of their aﬃliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. The concept and design: JCS. Acquisition, analysis, interpretation of data, drafting of the manuscript, critical revision of the manuscript, and statistical analysis: JCS and OC. All authors contributed to the article and approved the submitted version. Ethics 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. The studies involving human participants were reviewed and approved by the Ethics Committee of Human Research of the University of Granada. The patients/participants provided their written informed consent to participate in this study. References Hogarth H, Ingham R. Masturbation among young women and associations with sexual health: an exploratory study. J Sex Res. (2009) 46:558–67. doi: 10.1080/00224490902878993 25. Cervilla O, Vallejo-Medina P, Gómez-Berrocal C, Sierra JC. Development of the Spanish short version of negative attitudes toward masturbation inventory. Int J Clin Health Psychol. (2021) 21:100222. doi: 10.1016/j.ijchp.2021.100222 48. Sierra JC, Perla F, Gutiérrez-Quintanilla R. Attitudes toward masturbation in adolescents: psychometric properties of Spanish version of attitudes toward masturbation inventory. Univ Psychol. (2010) 9:531–42. doi: 10.11144/Javeriana.upsy9-2.amap 26. Gerressu M, Mercer CH, Graham CA, Wellings K, Johnson AM. Prevalence of masturbation and associated factors in a British national probability survey. Arch Sex Behav. (2008) 37:266–78. doi: 10.1007/s10508-006-9123-6 49. Malo de. Molina C, Valls Blanco JM, Pérez Gómez A. La conducta sexual de los españoles. Barcelona: Ediciones B (1988). 27. Dosch A, Belayachi S, Van Der Linden M. Implicit and explicit sexual attitudes: how are they related to sexual desire and sexual satisfaction in men and women? J Sex Res. (2016) 53:251–64. doi: 10.1080/00224499.2014.1003361 50. Mercer CH, Tanton C, Prah P, Erens B, Sonnenberg P, Clifton S, et al. Changes in sexual attitudes and lifestyles in Britain through the life course and over time: ﬁndings from the national surveys of sexual attitudes and lifestyles (NATSAL). Lancet. (2013) 382:1781–94. doi: 10.1016/S0140-6736(13)62035-8 28. Moyano N, Vallejo-Medina P, Sierra JC. Sexual desire inventory: two or three dimensions? J Sex Res. (2017) 54:105–16. doi: 10.1080/00224499.2015.1109581 51. Randolph JF, Zheng H, Avis NE, Greendale GA, Harlow SD. Masturbation frequency and sexual function domains are associated with serum reproductive hormone levels across the menopausal transition. J Clin Endocrinol Metab. (2015) 100:258–66. doi: 10.1210/jc.2014-1725 29. Cervilla O, Vallejo-Medina P, Gómez-Berrocal C, de la Torre D, Sierra JC. Validation of the orgasm rating scale in the context of masturbation. Psicothema. (2022) 34:151–59. doi: 10.7334/psicothema2021.223 52. Sierra JC, Gómez-Carranza J, Álvarez-Muelas A, Cervilla O. Association of sexual attitudes with sexual function: General vs. speciﬁc attitudes. Int J Environ Res Public Health. (2021) 18:10390. doi: 10.3390/ijerph181910390 30. Sierra JC, Santamaría J, Cervilla O, Álvarez-Muelas A. Masturbation in middle and late adulthood: its relationship to orgasm. Int J Impotence Res. (2022). doi: 10.1038/s41443-021-00520-w 53. Peixoto MM, Gomes H, Correia A, Pires I, Pereira T, Machado PP. Translation and validation of the Portuguese version of the sexual desire inventory-2: assessing gender diﬀerences. Sex Relatsh Ther. (2020) 35:89–102. doi: 10.1080/14681994.2018.1472374 31. Mosher DL. Negative attitudes toward masturbation. References In: Fisher TD, Davis CM, Yarber WL, Davis SL, editors. Handbook of Sexuality-related Measures. Nueva York, NY: Routledge (2011). p. 487–8. 32. Spector IP, Carey MP, Steinberg L. Sexual desire inventory. In: Davis CM, Yarber WL, Bauserman R, Schreer G, Davis SL, editors. Handbook of Sexuality- related Measures. London, UK: Sage (1998). p. 174–6. 54. Vallejo-Medina P, Rojas-Paoli I, Álvarez-Muelas A. Validation of the sexual desire inventory in Colombia. J Sex Marital Ther. (2020) 46:385–98. doi: 10.1080/0092623X.2020.1739181 33. Mah K, Binik YM. Orgasm rating scale. In: Milhausen RR, Sakaluk JK, Fisher TD, Davis DM, Yarber WL, editors. Handbook of Sexuality-Related Measures. Nueva York, NY: Routledge (2020). p. 503–7. 55. Vowels LM, Vowels MJ, Mark KP. Uncovering the most important factors for predicting sexual desire using explainable machine learning. J Sex Med. (2021) 18:1198–216. doi: 10.1016/j.jsxm.2021.04.010 34. McGahuey CA, Gelenberg AJ, Laukes CA, Moreno FA, Delgado PL, McKnight KM, et al. The arizona sexual experience scale (ASEX): reliability and validity. J Sex Marital Ther. (2000) 26:25–38. doi: 10.1080/009262300278623 56. Arcos-Romero AI, Sierra JC. Factors associated with subjective orgasm experience in heterosexual relationships. J Sex Marital Ther. (2020) 46:314–29. doi: 10.1080/0092623X.2019.1711273 35. Sánchez-Fuentes MM, Moyano N, Granados R, Sierra JC. Validation of the Spanish version of the arizona sexual experience scale (ASEX) using self-reported and psychophysiological measures. Rev Iberoam Psicol Salud. (2019) 10:1–14. doi: 10.23923/j.rips.2019.01.021 57. Mangas P, Granados R, Cervilla O, Sierra JC. Validation of the orgasm rating scale in context of sexual relationships of gay and lesbian adults. Int J Environ Res Public Health. (2022) 19:887. doi: 10.3390/ijerph19020887 58. Mah K, Binik YM. Are orgasms in the mind or the body? psychosocial versus physiological correlates of orgasmic pleasure and satisfaction. J Sex Marital Ther. (2005) 31:187–200. doi: 10.1080/00926230590513401 36. Arcos-Romero AI, Sierra JC. Factorial invariance, diﬀerential item functioning, and norms of the orgasm rating scale. Int J Clin Health Psychol. (2019) 19:57–66. doi: 10.1016/j.ijchp.2018.11.001 59. Arcos-Romero AI, Moyano N, Sierra JC. Psychometric properties of the orgasm rating scale in context of sexual relationship in a Spanish sample. J Sex Med. (2018) 15:741–49. doi: 10.1016/j.jsxm.2018.03.005 37. Calvillo C, del Mar Sánchez-Fuentes M, Parrón-Carreño T, Sierra JC. Validation of the interpersonal exchange model of sexual satisfaction questionnaire in adults with a same-sex partner. Int J Clin Health Psychol. (2020) 20:140–50. doi: 10.1016/j.ijchp.2019.07.005 60. Laumann EO, Gagnon JH, Michael RT, Michaels S. The Social Organization of Sexuality: Sexual Practices in the United States. References p. 47–63. doi: 10.1007/978-3-319-39277-6_4 18. Rowland D, Donarski A, Graves V, Caldwell C, Hevesi B, Hevesi K. The experience of orgasmic pleasure during partnered and masturbatory sex in women with and without orgasmic diﬃculty. J Sex Marital Ther. (2019) 45:550–61. doi: 10.1080/0092623X.2019.1586021 8. Kingsberg SA, Althof S, Simon JA, Bradford A, Bitzer J, Carvalho J, et al. Female sexual dysfunction—medical and psychological treatments, committee 14. J Sex Med. (2017) 14:1463–91. doi: 10.1016/j.jsxm.2017.05.018 19. Komisaruk BR, Beyer-Flores C, Whipple B. The science of orgasm. Baltimore, MD: Hopkins University Press. (2006). 9. Rowland DL, Hevesi K, Conway GR, Kolba TN. Relationship between masturbation and partnered sex in women: does the former facilitate, inhibit, or not aﬀect the latter? J Sex Med. (2020) 17:37–47. doi: 10.1016/j.jsxm.2019.10.012 20. Marchand E. Psychological and behavioral treatment of female orgasmic disorder. Sex Med Rev. (2021) 9:194–211. doi: 10.1016/j.sxmr.2020.07.007 10. Dekker A, Schmidt G. Patterns of masturbatory behaviour: changes between the sixties and the nineties. J Psychol Hum Sex. (2003) 14:35–48. doi: 10.1300/J056v14n02_04 21. Laan E, Rellini AH, Barnes T. Standard operating procedures for female orgasmic disorder: consensus of the international society for sexual medicine. J Sex Med. (2013) 10:74–82. doi: 10.1111/j.1743-6109.2012.0 2880.x 11. Kontula O, Haavio-Mannila E. Masturbation in a generational perspective. J Psychol Hum Sex. (2003) 14:49–83. doi: 10.1300/J056v14n02_05 Frontiers in Psychiatry 07 frontiersin.org Cervilla and Sierra 10.3389/fpsyt.2022.903361 premature ejaculation. Asian J Androl. (2019) 21:631–4. doi: 10.4103/aja.aj a_34_19 22. Castellini G, Fanni E, Corona G, Maseroli E, Ricca V, Maggi M. Psychological, relational, and biological correlates of ego-dystonic masturbation in a clinical setting. Sex Med. (2016) 4:e156–65. doi: 10.1016/j.esxm.2016.03.024 22. Castellini G, Fanni E, Corona G, Maseroli E, Ricca V, Maggi M. Psychological, relational, and biological correlates of ego-dystonic masturbation in a clinical setting. Sex Med. (2016) 4:e156–65. doi: 10.1016/j.esxm.2016.03.024 46. Waldinger MD. Delayed and premature ejaculation. In: Balon R, Taylor Segraves R, editors. Clinical Manual of Sexual Disorders. Nueva York, NY: American Psychiatric Publishing (2009). p. 273–304. 23. Sierra JC, Perla F, Santos-Iglesias P. Sexual guilt in youngsters: the inﬂuence of attitudes and sexual experience. Rev Latinoam Psicol. (2011) 43:73–81. 23. Sierra JC, Perla F, Santos-Iglesias P. Sexual guilt in youngsters: the inﬂuence of attitudes and sexual experience. Rev Latinoam Psicol. (2011) 43:73–81. 47. Driemeyer W, Janssen E, Wiltfang J, Elmerstig E. Masturbation experiences of Swedish senior high school students: gender diﬀerences and similarities. J Sex Res. (2017) 54:631–41. doi: 10.1080/00224499.2016.1167814 24. Cervilla and Sierra 67. Tavares IM, Laan ETM, Nobre PJ. Sexual inhibition is a vulnerability factor for orgasm problems in women. J Sex Med. (2018) 15:361–72. doi: 10.1016/j.jsxm.2017.12.015 References Chicago: University of Chicago press (1994). 38. Álvarez-Muelas A, Gómez-Berrocal C, Sierra JC. Study of sexual satisfaction in diﬀerent typologies of adherence to the sexual double standard. Front Psychol. (2021) 11:609571. doi: 10.3389/fpsyg.2020.609571 61. Rowland DL, Kolba TN, McNabney SM, Uribe D, Hevesi K. Why and how women masturbate, and the relationship to orgasmic response. J Sex Marital Ther. (2020) 46:361–76. doi: 10.1080/0092623X.2020.1717700 39. Sierra JC, Moyano N, Vallejo-Medina P, Gómez-Berrocal C. An abridged Spanish version of sexual double standard scale: factorial structure, reliability and validity evidence. Int J Clin Health Psychol. (2018) 18:69–80. doi: 10.1016/j.ijchp.2017.05.003 62. Mah K, Binik YM. Do all orgasms feel alike? evaluating a two-dimensional model of the orgasm experience across gender and sexual context. J Sex Res. (2002) 39:104–13. doi: 10.1080/00224490209552129 40. R Core Team. R: A Language and Environment For Statistical Computing. Vienna, Austria: R Foundation for Statistical Computing (2020). 63. Regnerus M, Price J, Gordon D. Masturbation and partnered sex: substitutes or complements? Arch Sex Behav. (2017) 46:2111–21. doi: 10.1007/s10508-017-0975-8 41. RStudio Team. RStudio: Integrated Development Environment for R [Computer software]. Boston, MA: RStudio, PBC (2020). 64. Sierra JC, Díaz G, Álvarez-Muelas A, Calvillo C, Granados R, Arcos-Romero AI. Relación del deseo sexual con la excitación sexual objetiva y subjetiva. Rev Psicopatol Psicol Clin. (2019) 24:173–80. doi: 10.5944/rppc.25374 42. Stekhoven DJ, Bühlmann P. Missforest-non-parametric missing value imputation for mixed-type data. Bioinformatics. (2012) 28:112–18. doi: 10.1093/bioinformatics/btr597 65. Kelly MP, Strassberg DS, Kircher JR. Attitudinal and experiential correlates of anorgasmia. Arch Sex Behav. (1990) 19:165–77. doi: 10.1007/BF01542230 43. Revelle W. Psych: Procedures for Personality and Psychological Research. Illinois, USA: Northwestern University, Evanston (2022). Available online at: https://cran.r-project.org/package=psych (accessed October 20, 2021). 66. Sierra JC, Vallejo-Medina P, Santos-Iglesias P, Lameiras Fernández M. Validation of massachusetts general hospital-sexual functioning questionnaire (MGH-SFQ) in a Spanish population. Aten Prim. (2012) 44:516–24. doi: 10.1016/j.aprim.2012.02.004 44. Clayton AH, Hamilton DV. Female orgasmic disorder. In: Balon R, Taylor Segraves R, editors. Clinical Manual of Sexual Disorders. Washington, DC: American Psychiatric Publishing, Inc. (2009). p. 251–71. 67. Tavares IM, Laan ETM, Nobre PJ. Sexual inhibition is a vulnerability factor for orgasm problems in women. J Sex Med. (2018) 15:361–72. doi: 10.1016/j.jsxm.2017.12.015 45. Ma G-, Zou Z-, Lai Y-, Zhang X, Zhang Y. Regular penis-root masturbation, a novel behavioral therapy in the treatment of primary 08 Frontiers in Psychiatry frontiersin.org
https://openalex.org/W2905214370	https://europepmc.org/articles/pmc6295970?pdf=render	English	null	Prevalence of Salmonella in juvenile dogs affected with parvoviral enteritis	Journal of the South African Veterinary Association	2,018	cc-by	5,597	Prevalence of Salmonella in juvenile dogs affected with parvoviral enteritis Read online: Scan this QR code with your smart phone or mobile device to read online. Authors: Willem J. Botha1 Johan P. Schoeman1 Stanley L. Marks2 Zandri Whitehead1 Cornelius H. Annandale3 Affiliations: 1Department of Companion Animal Clinical Studies, University of Pretoria, South Africa 2Department of Medicine and Epidemiology, University of California, United States 3Department of Production Animal Studies, University of Pretoria, South Africa Corresponding author: Willem Botha, wilco.botha@ up.ac.za Dates: Received: 05 Sept. 2018 Accepted: 06 Nov. 2018 Published: 05 Dec. 2018 How to cite this article: Botha, W.J., Schoeman, J.P., Marks, S.L., Whitehead, Z. & Annandale, C.H., 2018, ‘Prevalence of Salmonella in juvenile dogs affected with parvoviral enteritis’, Journal of the South African Veterinary Association 89(0), a1731. https://doi.org/ 10.4102/jsava.v89i0.1731 Copyright: © 2018. The Authors. Licensee: AOSIS. This work is licensed under the Creative Commons Attribution License. Read online: Scan this QR code with your smart phone or mobile device to read online. Authors: Willem J. Botha1 Johan P. Schoeman1 Stanley L. Marks2 Zandri Whitehead1 Cornelius H. Annandale3 Affiliations: 1Department of Companion Animal Clinical Studies, University of Pretoria, South Africa 2Department of Medicine and Epidemiology, University of California, United States 3Department of Production Animal Studies, University of Pretoria, South Africa Corresponding author: Willem Botha, wilco.botha@ up.ac.za Dates: Received: 05 Sept. 2018 Accepted: 06 Nov. 2018 Published: 05 Dec. 2018 How to cite this article: Botha, W.J., Schoeman, J.P., Marks, S.L., Whitehead, Z. & Annandale, C.H., 2018, ‘Prevalence of Salmonella in juvenile dogs affected with parvoviral enteritis’, Journal of the South African Veterinary Association 89(0), a1731. https://doi.org/ 10.4102/jsava.v89i0.1731 Copyright: © 2018. The Authors. Licensee: AOSIS. This work is licensed under the Creative Commons Attribution License. Authors: Willem J. Botha1 Johan P. Schoeman1 Stanley L. Marks2 Zandri Whitehead1 Cornelius H. Annandale3 Salmonellosis is a disease of major zoonotic importance and canine parvovirus is a potentially fatal cause of canine enteritis with a world-wide distribution. Persistent isolation of Salmonella during routine environmental sampling surveys of a hospital ward, reserved for the treatment of dogs with canine parvovirus infection, prompted investigation into a possible source. We hypothesised that dogs affected by canine parvovirus would have a higher prevalence of faecal salmonellae compared to an apparently healthy cohort. Seventy-four client-owned dogs naturally infected with canine parvovirus and 42 apparently healthy client-owned dogs were included in the study. This prospective, longitudinal, observational study was conducted over an 18-month period. Journal of the South African Veterinary Association ISSN: (Online) 2224-9435, (Print) 1019-9128 Page 1 of 6 Original Research Journal of the South African Veterinary Association ISSN: (Online) 2224-9435, (Print) 1019-9128 Page 1 of 6 Original Research Journal of the South African Veterinary Association ISSN: (Online) 2224-9435, (Print) 1019-9128 Page 1 of 6 Original Research Page 1 of 6 Original Research Original Research Page 1 of 6 Introduction Canine parvovirus (CPV) is a universally prevalent and potentially fatal cause of canine enteritis, which is often exacerbated by concurrent enteropathogen infections (Prittie 2004). Salmonellosis is a well-established major zoonotic disease, which is commonly acquired as a foodborne disease in humans mostly through faecally contaminated food (Behravesh et al. 2010; Imanishi et al. 2014). Zoonotic transmission of Salmonella within a veterinary practice and outbreaks of salmonellosis in both large and small animal facilities have also been reported (Cherry et al. 2004; Hartmann et al. 1996; Ketaren et al. 1981). In addition, Salmonella enterica subsp. enterica is also a well-recognised nosocomial pathogen in large-animal veterinary hospitals (Timoney, Neibert & Scott 1978). Animals have been infected with S. enterica via oral exposure under experimental conditions, but the circumstances predisposing to and promoting transmission under natural conditions remain unclear. As a consequence, it is important to evaluate the risk factors that increase the likelihood of infection (Grassl & Finlay 2008). Following an outbreak of salmonellosis in the large-animal section of the Onderstepoort Veterinary Academic Hospital, infection control measures were initiated, and in-hospital environmental niches of Salmonella were identified and controlled. However, the persistent isolation of Salmonella during follow-up of microbiological surveys targeting the isolation ward, dedicated to the treatment of dogs infected with CPV, prompted further investigation into this cohort of patients being a possible source of contamination. Copyright: © 2018. The Authors. Licensee: AOSIS. This work is licensed under the Creative Commons Attribution License. The prevalence of salmonellae amongst diarrhoeic dogs in South Africa is unknown. This study was designed to determine the comparative prevalence of Salmonella shedding in juvenile dogs infected with CPV and a cohort of apparently healthy age-matched control dogs. Prevalence of Salmonella in juvenile dogs affected with parvoviral enteritis Fresh faecal samples were collected from dogs aged 6 weeks to 9 months diagnosed with canine parvovirus infection and admitted for treatment, and from apparently healthy dogs presented for vaccination or routine hospital procedures. Faeces were submitted for the isolation, antimicrobial susceptibility testing and serotyping of salmonellae. The prevalence of faecal Salmonella shedding was 22% and 31% for the affected and apparently healthy dogs, respectively, which was not statistically different. No significant associations between Salmonella status and possible risk factors or continuous variables such as age, body weight and duration of hospitalisation were identified. All the Salmonella isolates (n = 32) were resistant to penicillin G, lincomycin and tylosin. Salmonellae from nine different serotypes were identified. The prevalence of Salmonella shedding in both groups was higher than that commonly reported, yet similar to those in previous reports on young dogs, shelter dogs or dogs fed a raw meat diet. Affiliations: 1Department of Companion Animal Clinical Studies, University of Pretoria, South Africa Culture of Salmonella enterica subsp. enterica Faeces were submitted for the selective isolation of Salmonella using a previously reported technique (Lyle et al. 2015). The recovered isolates were stored at -70 °C in brain–heart infusion broth, pending serotyping using typing antisera at a reference laboratory (General Bacteriology Laboratory, Agricultural Research Council, Onderstepoort Veterinary Institute, Onderstepoort, South Africa). Dogs were included in the affected cohort if they were (1) aged between 6 weeks and 9 months, (2) diagnosed with CPV infection using a commercial quick enzyme- linked immunosorbent assay (IDEXX CPV SNAP, IDEXX Laboratories, Westbrook, ME, United States [US]) which was confirmed by electron microscopic identification of the virus in faeces, with supporting clinical signs such as inappetence, vomiting and diarrhoea, and (3) were admitted for treatment to the isolation ward. The submitted faecal specimens were incubated in an enrichment broth of buffered peptone water at 37 °C for 24 hours. The specimens were then vortexed, and 1 mL incubated in a selective tetrathionate broth with brilliant green (TBG) for a further 24 h at 43 °C. An aliquot (0.1 mL) of vortexed TBG was then transferred to 10 mL of Rappaport-Vassiliadis broth (RV) and incubated for 24 h at 43 °C after which a vortexed sample was plated onto xylose-lysine-tergitol (XLT4) agar. After overnight incubation at 43 °C, suspect colonies (pink colonies with or without black centres) were plated onto Columbia blood agar plates and incubated for 24 h at 37 °C. Salmonella enterica isolation was confirmed by biochemical testing using a commercial kit (API10S, BioMirieux, Marcy I’Etoile, Rhône-Alpes, France). Dogs were included in the apparently healthy cohort when they presented for (1) vaccinations, blood donor screening, ovariohysterectomy and orchiectomy, (2) were aged between 6 weeks and 9 months of age and (3) were deemed clinically healthy based on anamnesis and full clinical examination. Dogs were excluded from the study, in either cohort, if they had received antibiotic therapy at any stage throughout their lives prior to presentation. In addition, dogs from the apparently healthy cohort were excluded from the study if they had any history suggestive of previous or current illness, or if CPV was detected on electron microscopy of faeces. Antimicrobial susceptibility testing was performed using the Kirby-Bauer disc diffusion method (Bauer et al. 1966). Animal information Sample size calculations performed at an approximate prevalence of Salmonella shedding in diarrhoeic dogs of 5% (Marks et al. 2011) with a precision level of 5% and 95% confidence interval suggested that a minimum number of 73 dogs with CPV infection should be collected. As a lower prevalence was expected in apparently healthy dogs, it was aimed to collect approximately one healthy dog for every two CPV-affected dogs. Materials and methods Study overview This was a prospective, longitudinal, observational study performed on client-owned dogs. Juvenile dogs, diagnosed with CPV infection and admitted to the Onderstepoort Veterinary http://www.jsava.co.za Open Access Original Research Page 2 of 6 Veterinary Tropical Diseases and Electron Microscopy Unit, Department of Anatomy and Physiology, Faculty of Veterinary Science, University of Pretoria, in capped 1 millilitre (mL) syringes (diarrhoeic specimens) or Eppendorf tubes (formed stool). Canine parvovirus shedding from the infected cohort (CPV ELISA SNAP positive dogs) was confirmed via negative-staining transmission electron microscopy (Philips CM 10 transmission electron microscope, Philips Electron Optical Division, Eindhoven, The Netherlands). Faecal specimens from the apparently healthy cohort were submitted for negative-staining transmission electron microscopy to exclude faecal shedding of CPV. Canine parvovirus ELISA SNAP tests were not performed on this cohort. Academic Hospital for treatment, were sampled as an affected cohort. Clinically healthy dogs within a matching age-range, presented for routine hospital visits, were sampled as an apparently healthy cohort. Academic Hospital for treatment, were sampled as an affected cohort. Clinically healthy dogs within a matching age-range, presented for routine hospital visits, were sampled as an apparently healthy cohort. The study was conducted over the course of 18 months from October 2015 to March 2017. Dogs were only entered into the study with informed consent from their owners. Culture of Salmonella enterica subsp. enterica Antimicrobial agents used in susceptibility testing included a standardised panel of amikacin, amoxicillin/ ampicillin, doxycycline/oxytetracycline, enrofloxacin, gentamicin, penicillin G, trimethoprim/sulphamethoxazole, chloramphenicol, cephalexin/cephalothin, kanamycin, lincomycin, lincospectin, orbifloxacin, amoxicillin clavulanic acid, tylosin and polymyxin B. Results The study comprised 74 CPV-infected dogs and 42 apparently healthy dogs. The prevalence of Salmonella shedding was 22% (16/74) and 31% (13/42) in infected and apparently healthy dogs, respectively, and the difference was not significant (P = 0.26). The mortality rate (18%; 13/74) in the CPV-infected cohort was similar to that previously reported in the literature in general and from the same institution, in particular (Goddard et al. 2008; Schoeman, Goddard & Leisewitz 2013; Schoeman & Herrtage 2008). Five dogs were euthanised owing to poor prognosis and eight dogs died naturally. The median hospitalisation duration was 5 days (range = 2–11). No significant association between isolation of S. enterica and length of hospitalisation (P = 0.72) or survival (P = 0.328) was identified in the CPV-infected cohort. The infected cohort comprised 45% (33/74) female and 55% (41/74) male dogs and the apparently healthy cohort comprised 62% (26/42) female and 38% (16/42) male dogs. There was no significant difference in sex ratio between the groups (P = 0.07), nor was there any association between sex and the isolation of S. enterica (P = 0.623). The median age of both the infected and apparently healthy cohorts was 3 months (range = 6 weeks to 8 months). The median body weight for the infected and apparently healthy cohorts was 5.9 kg (range = 0.8 kg – 30.8 kg) and 6.3 kg (range = 1.9 kg – 22.0 kg), respectively. Neither age (P = 0.241) nor body weight (P = 0.223) were significantly associated with the isolation of S. enterica. Both cohorts comprised various breeds with the most common being mixed breed (19%, 14/74), American Pitbull terriers (15%, 11/74) and Boerboels (14%, 10/74). The rest of the breeds included six Jack Russell terriers, five Dachshunds, four Belgian Malinois Shepherds, four Staffordshire Bullterriers, three Rottweilers, three Yorkshire terriers, two Labrador Retrievers, and one each of the following breeds: Maltese terrier, Miniature Pinschers, Border Collie, German Shepherd, Golden Retriever, Pekingese, Pomeranian, Pug, Rhodesian Ridgeback, Scottish terrier and Siberian Husky. All S. enterica subsp. enterica isolates (n = 32) were resistant to penicillin G, lincomycin and tylosin. Nine of the isolates were resistant to lincospectin and 21 showed intermediate (n = 20) or complete resistance (n = 1) to doxycycline/oxytetracycline. All the isolates were sensitive to amikacin, amoxicillin/ampicillin, enrofloxacin, gentamicin, trimethoprim sulphamethoxazole, chloramphenicol, cephalexin/cephalothin, orbifloxacin, amoxicillin clavulanic acid and polymixin B. Sampling On presentation, data were collected regarding several historical and clinical variables, that is, the dietary history of the dog, current or recent (< 1 month) antibiotic use by any humans or animals within the household shared by the dog and previous visits of the animal to either a veterinary or human hospital. The diet was recorded as being home- cooked, commercial (store-bought), premium (veterinary retail only) or mixed. The remainder of the questions was only answered as ‘yes’ or ‘no’. Dogs from the infected cohort were treated according to a standard protocol, with adjustments as dictated by the clinical condition. In addition, data regarding the length of hospitalisation and outcome were collected and recorded. Where possible, fresh rectal faecal specimens were collected again at discharge from affected dogs. These faecal samples were also submitted for Salmonella isolation. Fresh faecal specimens from both cohorts were collected on admission by using either a sterile 1 mL syringe or a gloved finger inserted into the rectum. The faecal specimens were submitted to the Bacteriology Laboratory, Department of http://www.jsava.co.za http://www.jsava.co.za Page 3 of 6 Page 3 of 6 Original Research 22% of the apparently healthy cohort reported prior visits to a veterinary practice. The nature of these visits was not recorded for every individual. Of the 74 CPV-infected dogs, 3% (2/74) had three vaccinations, 16% (12/74) had two vaccinations, 35% (26/74) had a single vaccination and 46% (34/74) had never been vaccinated. There was no significant association between the type of diet fed (P = 0.335), antibiotic use in the home environment (P = 0.483), previous hospital visits (P = 0.678) or previous vaccinations (P = 0.177) and the isolation of S. enterica. Statistical analysis 22% of the apparently healthy cohort reported prior visits to a veterinary practice. The nature of these visits was not recorded for every individual. Of the 74 CPV-infected dogs, 3% (2/74) had three vaccinations, 16% (12/74) had two vaccinations, 35% (26/74) had a single vaccination and 46% (34/74) had never been vaccinated. There was no significant association between the type of diet fed (P = 0.335), antibiotic use in the home environment (P = 0.483), previous hospital visits (P = 0.678) or previous vaccinations (P = 0.177) and the isolation of S. enterica. Results were entered into an Excel (Microsoft Excel, Microsoft Corporation, Redmond, WA, US) spreadsheet. Continuous data were assessed for normality using Shapiro-Wilk testing and descriptive statistics was calculated using a statistical software package (IBM SPSS Statistics Version 24, Chicago, IL, US). The Chi-Square or Fisher’s exact tests were performed to test for significance between proportions as required by the specific data sets. A 5% level of significance was considered statistically significant for all comparisons. Thirty-eight per cent (28/74) of the CPV-infected cohort had a follow-up faecal specimen collected at discharge and S. enterica was isolated from 7% of dogs (2/28). One dog was positive for the isolation of S. enterica on both the admission and discharge specimen, and the second dog was positive on the discharge specimen only. In addition, three dogs that were positive for S. enterica at admission were negative at discharge. Ethical considerations The study was approved by the animal ethics committee of the University of Pretoria (V091-15). Discussion This study is the first to report the prevalence of Salmonella shedding in a cohort of CPV-infected dogs, and it identified a prevalence of S. enterica of 22% in CPV-infected dogs and 31% in apparently healthy dogs. The comparative prevalence of S. enterica shedding was not statistically different between the dogs with parvoviral enteritis and an age-matched clinically healthy cohort. The persistent isolation of Salmonella during targeted environmental surveillance of the isolation ward suggested that the population of dogs housed in this environment may be a persistent source of environmental contamination. Contamination of this area was thought to act as a nidus of infection and consequent spread to other parts of the hospital. Moreover, salmonellosis is considered an important nosocomial disease in large-animal veterinary hospitals (Lyle et al. 2015). In this study, S. Heidelberg was the only serotype recovered from the environment in the large-animal section within the same facility (Lyle et al. 2015). This finding may suggest that there was no significant cross contamination between the two sections of the hospital. However, the relatively high prevalence of Salmonella shedding in juvenile dogs may raise concern for possible contamination by this population of patients within the small animal hospital. Further studies are needed to determine the significance of this notion, especially considering that targeted environmental surveillance for Salmonella may not be as stringent as that in large-animal hospitals. The reported prevalence of S. enterica in diarrhoeic dogs is extremely variable, ranging between 0% and 76% (Khan 1970; Seepersadsingh, Adesiyun & Seebaransingh 2004; Stone et al. 1993). However, most non-diarrhoeic dogs ingesting processed commercial diets have a prevalence of S. enterica below 4.4% (Adesiyun, Campbell & Kaminjolo 1997; Seepersadsingh et al. 2004; Shimi, Keyhani & Bolurchi 1976; Timbs et al. 1975). Higher prevalences have been reported in dogs housed in a shelter, stray populations, working dogs used at an abattoir or on farms, hunting dogs and dogs ingesting raw meat diets (Frost et al. 1969; Khan 1970; Shimi et al. 1976; Stucker et al. 1952). One study reported a prevalence of 25% in dogs younger than 6 months of age compared to a prevalence of 5.2% in older dogs (Förster, Holland & Tesfamariam 1974). The prevalence of S. enterica shedding identified in this study supports the notion that juvenile dogs have a higher prevalence of S. enterica but failed to identify or sanction any of the previously reported risk factors. Discussion Murine studies have shown a 100 000-fold decrease in the 50% implantation dose for Salmonella colonisation following the disruption of the intestinal microbiota by streptomycin treatment (Que & Hentges 1985). This would suggest that all juvenile animals may have a greater susceptibility to Salmonella colonisation associated with the lack of a well-established intestinal microbiota (Carter & Quin 2000). Nonetheless, CPV-infected dogs are likely to suffer from a greater degree of dysbiosis compared to healthy individuals, arguing against dysbiosis as a major reason for the higher prevalence of faecal Salmonella in juvenile animals, when compared to adult animals. All S. enterica isolates in this study were resistant to at least three antibiotics and the prevalence of resistance amongst these isolates was higher than that previously reported for isolates from dogs (Seepersadsingh et al. 2004). All the isolates were resistant to tylosin, lincospectin and penicillin G. Resistance to tylosin is unsurprising considering their limited efficacy against gram-negative bacteria (Kim et al. 2014). Lincospectin is not commonly used in small animal practice; hence, resistance to these antibiotics is of little clinical significance. Despite the fact that all isolates were resistant to penicillin G, no resistance was reported to other beta-lactam antibiotics commonly used in practice. In conclusion, none of these antibiotics are routinely used in the empirical treatment of suspected salmonellosis and therefore these resistance patterns are unlikely to have therapeutic implications. In contrast, a few isolates did show intermediate resistance to doxycycline, which may need to be closely monitored in the future. The reported risk factors for the isolation of Salmonella include contact with livestock, multiple-dog households, administration of antibiotics, hospitalisation and the feeding of raw diets or treats including raw meat and eggs (Leonard et al. 2011; Reimscheussel et al. 2017; Uhaa et al. 1988). The transmission of Salmonella is thought to occur most frequently via the faecal-oral route (Tanaka, Katsube & Imaizumi 1976). Interestingly, in our study, most dogs were fed solely commercial or premium pelleted diets and only 28% and 22% of dogs in the CPV-infected and apparently healthy cohorts, respectively, were fed chicken, pet mince or table scraps in addition to their staple diet. One dog was fed a diet of raw meat only and Salmonella was not isolated from this dog. Natural treats and chews have been implicated as a possible source of exposure to both pets and owners (Finley et al. 2006). Results Four serotypes were identified amongst the 32 isolates of S. enterica. The serotype of 16 isolates could not be determined by the reference laboratory and seven could only be partially serotyped. The serotyping results are listed in Table 1. TABLE 1: Serotypes of Salmonella spp. recovered from 32 faecal isolates of juvenile dogs co-infected with canine parvovirus and an apparently healthy cohort of age-matched controls. Serotype Number of isolates S. Heidelberg 4,5:r:1,2 4 Salm II 18:z10:z6 4 S. Chile 6,7:z:1,5 2 S. Cotia 18:-:1,6 2 S. Braenderup 6,7,14:e,h:e,n,z1 1 Salm II 4,5:z:1,5 1 Salm II 16:z:e,n,x 1 Salm II 30:b:z6 1 Salm Poly OMD 16 Note: Salmonella spp. serotypes as identified by a reference laboratory using commercial antisera. Salm Poly OMD isolates could not be completely serotyped and Salm II isolates were only partially identified. TABLE 1: Serotypes of Salmonella spp. recovered from 32 faecal isolates of juvenile dogs co-infected with canine parvovirus and an apparently healthy cohort of age-matched controls. Of the CPV-infected cohort, 3% (2/74) of dogs were fed home-cooked diets, 68% (50/74) commercial diets, 1% (1/74) premium diets and 28% (21/74) mixed diets. None of the dogs in the apparently healthy cohort were fed a home- cooked diet, and 45% (19/42) were fed commercial, store- bought diets, 33% (14/42) premium, veterinary-specific diets and 22% (9/42) mixed diets. Eleven per cent (8/74) of owners in the CPV-infected cohort indicated that antibiotics were being used at home at the time of presentation with none reporting antibiotic use in the apparently healthy cohort. Fifty-nine per cent (44/74) of the CPV-infected cohort and Note: Salmonella spp. serotypes as identified by a reference laboratory using commercial antisera. Salm Poly OMD isolates could not be completely serotyped and Salm II isolates were only partially identified. http://www.jsava.co.za Page 4 of 6 Page 4 of 6 dogs may further have increased exposure via coprophagia, contact with wildlife species and ingestion of carrion, considering their inquisitive nature. Acknowledgements isolated at discharge, antibiotic therapy was in effect unsuccessful in preventing colonisation or clearing the dog from S. enterica, despite sensitive susceptibility patterns being reported to the antibiotics routinely used in the treatment of CPV cases. However, this phenomenon was negated by the three dogs that were positive for isolation of S. enterica at admission and negative for isolation of S. enterica at discharge. Asymptomatic dogs have been reported to intermittently shed salmonellae for up to 6 weeks or longer post-exposure, or up to 2 weeks following ingestion of a single contaminated meal, which suggests that longitudinal studies following juvenile dogs over several weeks with multiple cultures may be needed to truly elucidate the epidemiology of salmonellae in this population (Finley et al. 2007). isolated at discharge, antibiotic therapy was in effect unsuccessful in preventing colonisation or clearing the dog from S. enterica, despite sensitive susceptibility patterns being reported to the antibiotics routinely used in the treatment of CPV cases. However, this phenomenon was negated by the three dogs that were positive for isolation of S. enterica at admission and negative for isolation of S. enterica at discharge. Asymptomatic dogs have been reported to intermittently shed salmonellae for up to 6 weeks or longer post-exposure, or up to 2 weeks following ingestion of a single contaminated meal, which suggests that longitudinal studies following juvenile dogs over several weeks with multiple cultures may be needed to truly elucidate the epidemiology of salmonellae in this population (Finley et al. 2007). The authors are thankful to the Bacteriology Laboratory, Electron Microscopy Unit and Clinical Pathology Laboratory of the Faculty of Veterinary Science for their assistance. Competing interests The authors declare that they have no financial or personal relationships which may have inappropriately influenced them in writing this paper. Discussion Unfortunately, the use of these products in our population was not assessed but may serve as an additional source owing to their frequency of use in puppies. Juvenile Salmonella enterica was identified from two dogs at discharge. One of these two dogs had S. enterica isolated at admission and discharge, whereas the second dog had S. enterica isolated at discharge only. Possible explanations for the negative isolation of S. enterica at admission and positive isolation at discharge include sampling or isolation error or colonisation during the hospitalisation. The expected relatively low sensitivity of single sample versus multiple sample culture for the detection of Salmonella shedding may also contribute to this finding. The use of antimicrobials in the treatment of CPV may aid in the colonisation of S. enterica by transiently disrupting the normal microbiota and weakening the colonisation resistance offered by these microbes. However, in both cases, with positive S. enterica http://www.jsava.co.za http://www.jsava.co.za Open Access Original Research Page 5 of 6 Page 5 of 6 References Adesiyun, A., Campbell, M. & Kaminjolo, J., 1997, ‘Prevalence of bacterial enteropathogens in pet dogs in Trinidad’, Journal of Veterinary Medicine Series B 44(1), 19–27. https://doi.org/10.1111/j.1439-0450.1997.tb00946.x Bauer, A., Kirby, W., Sherris, J.C. & Turck, M., 1966, ‘Antibiotic susceptibility testing by a standardized single disk method’, American Journal of Clinical Pathology 45(4), 493. https://doi.org/10.1093/ajcp/45.4_ts.493 Behravesh, C.B., Ferraro, A., Deasy, M., Dato, V., Moll, M., Sandt, C. et al., 2010, ‘Human Salmonella infections linked to contaminated dry dog and cat food, 2006–2008’, Pediatrics 126(3), 477–483. https://doi.org/10.1542/peds.2009-3273 Carter, M.E. & Quin, P.J., 2000, ‘Salmonella infections in dogs and cats’, in C. Wray & A. Wray (eds.), Salmonella in domestic animals, pp. 57–72; 231–244, CABI, Wallingford. Cherry, B., Burns, A., Johnson, G.S., Pfeifer, H., Dumas, N., Barrett, D. et al., 2004, ‘Salmonella Typhimurium outbreak associated with veterinary clinic’, Emerging Infectious Diseases Journal 10(12), 2249–2251. https://doi.org/10.3201/ eid1012.040714 Finley, R., Reid-Smith, R., Weese, J.S. & Angulo, F.J., 2006, ‘Human health implications of Salmonella-contaminated natural pet treats and raw pet food’, Clinical Infectious Diseases 42(5), 686–691. https://doi.org/10.1086/500211 Finley, R., Ribble, C., Aramini, J., Vandermeer, M., Popa, M., Litman, M. et al., 2007, ‘The risk of salmonellae shedding by dogs fed Salmonella-contaminated commercial raw food diets’, Canadian Veterinary Journal 48(1), 69–75. Förster, D., Holland, U. & Tesfamariam, H., 1974, ‘Occurrence of canine Salmonella infections’, Journal of Veterinary Medicine Series B 21(1), 124. Frost, A., Eaton, N., Gilchrist, D. & Moo, D., 1969, ‘The incidence of Salmonella infection in the dog’, Australian Veterinary Journal 45(3), 109–110. https://doi. org/10.1111/j.1751-0813.1969.tb01890.x Funding information The authors gratefully acknowledge the financial support provided by the Health and Welfare Sector Education and Training Authority, Department of Higher Education and Training, South Africa. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Authors’ contributions W.J.B., J.P.S., S.L.M., Z.W. and C.H.A. were involved in the design of the study and revision of the manuscript. Data were collected by W.J.B. and Z.W. W.J.B. analysed the data and wrote the manuscript. There were several limitations to this study. Firstly, dogs diagnosed with mild CPV infection and treated on an outpatient basis were not included in this study. Therefore, the prevalence of Salmonella shedding identified in this study potentially only reflects the more severely affected dogs and not the whole population of juvenile dogs infected with CPV. In addition, the study of CPV-infected dogs that were hospitalised and treated introduced a potential population bias, as only dogs that were owned by people who were able to afford the costs of hospitalisation and treatment were included in the study. Secondly, the use of a single sample for the isolation of Salmonella on admission may have caused some positive animals to remain undetected, as sensitivity of Salmonella culture is poor and typically 3–5 negative cultures are required to confirm lack of shedding in clinical settings (Marks et al. 2011). Thirdly, PCR or ELISA may have been a better modality than electron microscopy to exclude CPV shedding in the apparently healthy cohort owing to a higher sensitivity of the former tests (Schmitz et al. 2009). However, lack of availability and relatively higher expense precluded its use in this study. Lastly, the relatively small number of dogs in which S. enterica was isolated might have led to a type 2 error, thereby retaining a false null hypothesis, when assessing the risk factors for the shedding of this bacterial enteropathogen. In addition, even though a logistic regression model of statistical analysis may have been more appropriate for the assessment of possible risk factors for the identification of Salmonella, this was not performed owing to the relatively few Salmonella-positive animals and also it was not the primary aim of this study. Conclusion Goddard, A., Leisewitz, A.L., Christopher, M.M., Duncan, N.M. & Becker, P.J., 2008, ‘Prognostic usefulness of blood leukocyte changes in canine parvoviral enteritis’, Journal of Veterinary Internal Medicine 22(2), 309–316. https://doi.org/10.1111/ j.1939-1676.2008.0073.x The prevalence of Salmonella shedding in dogs with CPV infection was not statistically different from that in a healthy cohort. However, the prevalence in both groups was considerably higher than that previously reported (0.0% – 3.6%) for non-diarrhoeic dogs, yet similar to that reported for young dogs, shelter dogs or dogs fed a raw diet (25% – 69%). To the authors’ knowledge, this is the first report of the prevalence of Salmonella shedding in dogs diagnosed with canine parvoviral enteritis. Grassl, G.A. & Finlay, B.B., 2008, ‘Pathogenesis of enteric Salmonella infections’, Current Opinion in Gastroenterology 24(1), 22–26. https://doi.org/10.1097/ MOG.0b013e3282f21388 Hartmann, F.A., Callan, R.J., McGuirk, S.M. & West, S.E., 1996, ‘Control of an outbreak of salmonellosis caused by drug-resistant Salmonella anatum in horses at a veterinary hospital and measures to prevent future infections’, Journal of the American Veterinary Medical Association 209(3), 629–631. Imanishi, M., Rotstein, D.S., Reimscheussel, R., Schwensohn, C.A., Woody, D.H., Jr., Davis, S.W. et al., 2014, ‘Outbreak of Salmonella enterica serotype Infantis infection in humans linked to dry dog food in the United States and Canada, 2012’, Journal of the American Veterinary Medical Association 244(5), 545–553. https:// doi.org/10.2460/javma.244.5.545 http://www.jsava.co.za Open Access Original Research Page 6 of 6 Ketaren, K., Brown, J., Shotts, E.B., Hornsby, P.S. & McClelland, C.L., 1981, ‘Canine salmonellosis in a small animal hospital’, Journal of the American Veterinary Medical Association 179(10), 1017–1018. Schoeman, J.P., Goddard, A. & Leisewitz, A.L., 2013, ‘Biomarkers in canine parvovirus enteritis’, New Zealand Veterinary Journal 61(4), 217–222. https://doi.org/10.108 0/00480169.2013.776451 Khan, A., 1970, ‘Salmonella infections in dogs and cats in the Sudan’, British Veterinary Journal 126(11), 607–612. https://doi.org/10.1016/S0007-1935(17)48076-6 Schoeman, J.P. & Herrtage, M.E., 2008, ‘Serum thyrotropin, thyroxine and free thyroxine concentrations as predictors of mortality in critically ill puppies with parvovirus infection: A model for human paediatric critical illness?’, Microbes and Infection 10(2), 203–207. https://doi.org/10.1016/j.micinf. 2007.11.002 Kim, H.B., Singer, R.S., Borewicz, K., White, B.A., Sreevatsan, S., Johnson, T.J. et al., 2014, ‘Effects of tylosin administration on C-reactive protein concentration and carriage of Salmonella enterica in pigs’, American Journal of Veterinary Research 75(5), 460–467. https://doi.org/10.2460/ajvr.75.5.460 Seepersadsingh, N., Adesiyun, A.A. & Seebaransingh, R., 2004, ‘Prevalence and antimicrobial resistance of Salmonella spp. Conclusion in non-diarrhoeic dogs in Trinidad’, Journal of Veterinary Medicine Series B: Infectious Diseases and Veterinary Public Health 51(7), 337–342. https://doi.org/10.1111/j.1439-0450. 2004.00785.x Leonard, E., Pearl, D., Finley, R., Janecko, N., Peregrine, A., Reid-Smith, R. & Weese, J., 2011, ‘Evaluation of pet-related management factors and the risk of Salmonella spp. carriage in pet dogs from volunteer households in Ontario (2005–2006)’, Zoonoses and Public Health 58(1), 140–149. https://doi.org/10.1111/j.1863-2378.2009.01320.x Shimi, A., Keyhani, M. & Bolurchi, M., 1976, ‘Salmonellosis in apparently healthy dogs’, Veterinary Record 98(6), 110–111. https://doi.org/10.1136/vr.98.6.110 Lyle, C.H., Annandale, C.H., Gouws, J. & Morley, P.S., 2015, ‘Comparison of two culture techniques used to detect environmental contamination with Salmonella enterica in a large-animal hospital’, Journal of the South African Veterinary Association 86(1), 1–5. https://doi.org/10.4102/jsava.v86i1.1292 Stone, G.G., Chengappa, M., Oberts, R.D., Gabbert, N.H., McVey, S., Hennessy, K.J. et al., 1993, ‘Application of polymerase chain reaction for the correlation of Salmonella serovars recovered from greyhound feces with their diet’, Journal of Veterinary Diagnostic Investigation 5(3), 378–385. https://doi.org/10.1177/ 104063879300500313 Marks, S.L., Rankin, S.C., Byrne, B.A. & Weese, J.S., 2011, ‘Enteropathogenic bacteria in dogs and cats: Diagnosis, epidemiology, treatment, and control’, Journal of Veterinary Internal Medicine 25(6), 1195–1208. https://doi.org/10.1111/j.1939- 1676.2011.00821.x Stucker, C.L., Galton, M.M., Cowdery, J. & Hardy, A.V., 1952, ‘Salmonellosis in dogs: II. Prevalence and distribution in Greyhounds in Florida’, Journal of Infectious Diseases 91(1), 6–11. https://doi.org/10.1093/infdis/91.1.6 Prittie, J., 2004, ‘Canine parvoviral enteritis: A review of diagnosis, management, and prevention’, Journal of Veterinary Emergency and Critical Care 14(3), 167–176. https://doi.org/10.1111/j.1534-6935.2004.04020.x Tanaka, Y., Katsube, Y. & Imaizumi, K., 1976, ‘Experimental carrier in dogs produced by oral administration of Salmonella typhimurium’, Japanese Journal of Veterinary Science 38(6), 569–578. https://doi.org/10.1292/jvms1939.38.569 Que, J.U. & Hentges, D., 1985, ‘Effect of streptomycin administration on colonization resistance to Salmonella typhimurium in mice’, Infection and Immunity 48(1), 169–174. Timbs, D., Carter, M.E., Davis, G. & Carman, M.G., 1975, ‘The Salmonella excretor incidence of dogs in Hawke’s Bay’, New Zealand Veterinary Journal 23(4), 54–56. https://doi.org/10.1080/00480169.1975.34193 Reimscheussel, R., Grabenstein, M., Guag, J., Nemser, S.M., Song, K., Qiu, J. et al., 2017, ‘Multilaboratory survey to evaluate Salmonella prevalence in diarrheic and nondiarrheic dogs and cats in the United States between 2012 and 2014’, Journal of Clinical Microbiology 55(5), 1350–1368. https://doi.org/10.1128/JCM.02137-16 Timoney, J.F., Neibert, H.C. & Scott, F.W., 1978, ‘Feline salmonellosis. A nosocomial outbreak and experimental studies’, Cornell Veterinarian 68(2), 211–219. Schmitz, S., Coenen, C., Heinz-Jürgen, T. Conclusion & Neiger, R., 2009, ‘Comparison of three rapid commercial canine parvovirus antigen detection tests with electron microscopy and polymerase chain reaction’, Journal of Veterinary Diagnostic Investigation 21(3), 344–345. https://doi.org/10.1177/104063870902100306 Uhaa, I., Hird, D., Hirsh, D. & Jang, S., 1988, ‘Case-control study of risk factors associated with nosocomial Salmonella krefeld infection in dogs’, American Journal of Veterinary Research 49(9), 1501–1505. http://www.jsava.co.za Open Access
https://openalex.org/W3156881805	https://www.researchsquare.com/article/rs-50382/latest.pdf	English	null	Analyzing the communication in social media of the main sustainable brands during COVID-19 crisis: the Spanish vs. Italian cases	Research Square (Research Square)	2,020	cc-by	11,504	Carmen Zarco (  carmen.zarco@unir.net ) Universidad de Granada https://orcid.org/0000-0001-5112-5629 Universidad de Granada https://orcid.org/0000-0001-5112-5629 Analyzing the communication in social media of the main sustainable brands during COVID-19 crisis: the Spanish vs. Italian cases Carmen Zarco (  carmen.zarco@unir.net ) Abstract. Communication is now a valuable element to demonstrate the degree of commitment and transparency that organizations have towards Sustainability. In a critical moment such that experienced with the COVID-19 crisis, companies recognized facing the challenge of continuous and committed communication on social networks as a sustainable activity. All the latter raises the question whether the brands recognized as the most sustainable have actually succeed on being aware of communicating at the most critical moments of the COVID-19 crisis. In this context, an analysis is made of the activity maintained by the main sustainable companies, both Spanish and Italian, within two of the most popular social networks, Twitter and Instagram, at the very early stages of the crisis in both countries. Our results show that most companies have managed to rise to the occasion and show their commitment to the population through social networks. Keywords: COVID-19 crisis, Sustainable brands, Communication, Social Networks, Spain, Italy. p g Carmen Zarco Faculty of Business and Communication. Universidad Internacional de La Rioja, Avda. de la Paz nº 137 26006, Logroño, Spain; carmen.zarco@unir.net Analyzing the communication in social media of the main sustainable brands during COVID-19 crisis: the Spanish vs. Italian cases Carmen Zarco1** and Oscar Cordón 2 1 Faculty of Business and Communication. Universidad Internacional de La Rioja, Avda. de la Paz nº 137. 26006 - Logroño, Spain; carmen.zarco@unir.net 2 Instituto Andaluz Interuniversitario de Ciencia de Datos e Inteligencia Computacional (DaSCI). University of Granada, C/ Daniel Saucedo Aranda, s/n. 18071 - Granada, Spain; ocordon@decsai.ugr.es Research Article Keywords: COVID-19 crisis, Sustainable brands, Communication, Social Networks, Spain, Italy Posted Date: July 30th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-50382/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License * Corresponding author: 1. Introduction From the distance, when 2019 had not yet ended in some countries and the New Year was just starting in others, China alerted of the existence of an unknown condition originated in Wuhan that was spreading dizzily among its citizens (Güell, 2020). Even before its name was known, COVID-19 or coronavirus (as it is popularly known), the media already predicted the effects on the global economy. Thus, if the worst omens were fulfilled, how would the coronavirus crisis affect brand marketing and communication? The closure of production chains of many companies, such as automobile brands, located in the area were quickly followed by the quarantines and travel restrictions in the Asian continent. That break coincided with the Chinese New Year festivities, a time of large sales figures that could plunge a company into a deep crisis (considering that the Chinese market has become an important source of income for many brands). The scene that shook the first tab of that brand domino making events to be postponed or contingency plans to be activated and communicated was the Mobile World Congress (MWC) (García Muntané, 2020) in Barcelona. It was the first major event shaken by the health alarm and the first time that brands had to decide whether to put security before their own economic interests. The frightening of the brands of the MWC was not in vain. Weeks later the cases spread throughout the world, splashing mainly into Italy and Spain. What was the reaction then? Cancellations in blocks of massive events such as the Geneva Motor Show or any other agglomeration greater than 1,000 people as announced by the Swiss government. Security had become a priority and putting it at risk due to a marketing campaign that does not know how to adapt to the prevailing circumstances would be a critical error. p p g Some brands chosed not to lower their activity by increasing security measures even though they lessen the impact of their actions. A clear example is that of the fashion firm Armani who, in the middle of the Milan fashion week and at the most critical moment of contagion spread in the city, decided to carry out their show behind closed doors without media or buyers but broadcasted via streaming (Michel, 2020). That decision was accompanied by issuing a press release expressing their concern without generating more alarm than the situation itself. 1. Introduction Like the Italian firm, other brands have positioned themselves as the technological Huawei that had planned to present its folding smartphone model at the MWC, but alternatively chose to do so via streaming with small private presentations in different cities. Likewise, Facebook canceled the most important annual developer event, the F8, keeping the sessions online and donating all the money raised from ticket sales. The COVID-19 crisis is testing the ability of organizations to respond to an unprecedented health emergency caused by a global pandemic but also to impending problems in employment, human resource management, risk management at all levels, and solidarity. The goal of the current study is to shed some light on the communication strategy of brands in social networks during these difficult times. Our aim is to empirically test the commitment of a subset of brands, considered as the most sustainable in Spain and Italy, through an analysis of the communication established with their followers in social networks at the early stages of the pandemic. As said, we have selected these two countries since they were the most affected ones when the COVID-19 started to spread in Europe. We focused on the use of hashtags during the analyzed period as they are the most specific information units that can be identified in the brands’ communication strategies in social networks as well as they allow the brands to enhance the message they wanted to provide. We thus collected data from Twitter and Instagram, two social media commonly used by brands that make an extended use of hashtags in their posts. By means of social network analysis and visualization techniques (Wasserman & Faust, 1994) (Scott, 1988) (Kobourov, 2012), we manage to uncover common and differential communication approaches followed by the brands analyzed in both countries and in the two social networks. This allows us to determine which of those sustainable brands have been the most successful when communicating in social networks at the most critical moments of the COVID-19 crisis. The structure of this contribution is as follows. First we will briefly introduce a background about brands and social media, describing how they have progressively recognized the importance of social networks to get in touch with consumers. We will also refer to the importance of sustainability and its role nowadays as well as to how COVID-19 has extended its radius of action to all fields of society, including brands. 1. Introduction Next, we will explain the methodology applied in our research study. The next and main section is devoted to analyze the collected data using social network analysis and visualization techniques to draw conclusions from it. Finally, some concluding remarks will be presented. 2.1. Brands and Social Media Social networks are considered as a “set of Internet-based applications that rely on the ideological and technological foundations of Web 2.0, and that allows the creation and exchange of user-generated content” (Kaplan & Haenlein, 2010) (Gomez, et al., 2019). Social networks facilitate interaction and participation between users and companies or brands (Leung, et al., 2017) (Mangold & Faulds, 2009). They allow information to flow bi-directionally, which makes users not only receivers of content but also creators of brand information and therefore influencers (Tsai & Men, 2013) (Fournier & Avery, 2011). ( ) ( y ) We must understand that social networks give us the opportunity for brand building and brand value creation, including brand image and brand loyalty and brand management (Gomez, et al., 2019). Furthermore, experts recognize challenges such as measuring the influence of social media marketing activities on brand success, “dealing with the growing amount of customer brand information” (Harrigan, et al., 2017) and “identification mechanisms to improve brand pages to attract consumers and improve consumer-brand relationships” (De Vries & Carlson, 2014). Brand communication has been recognized as one of the most important elements in brand value (Simon & Sullivan, 1993) (Yoo, et al., 2000). Some recent studies recognize the impact of social networks on brand value. For example, while (Laroche, et al., 2012) evaluates the effect of brand communities on social networks in brand loyalty, (Schivinski & Dabrowski, 2015) analyzes the influence of brand communication on brand value through Facebook. Among the existing social networks, Twitter and Instagram are especially distinguished by using a very common element nowadays in their form of communication. This element is the hashtag, based on describing contents, pictures and videos using a short text preceded by the # symbol (Hu, et al., 2014). Twitter is recognized for being a long-known platform characterized by its immediacy (Zarco, et al., 2019). Meanwhile, Instagram is a relatively young social network allowing to share content through photos and retouching them with filters. It has experienced a rapid growth since it was launched in October 2010, resulting in an increase of brands’ attention focus in recent years (Hu, et al., 2014). Nevertheless, user behavior is different for each social network. Twitter and Instagram, like others as Facebook, have different purposes and their audiences use each of them for different purposes. 2.1. Brands and Social Media For example, people usually share a maximum of two hashtags on Twitter as it only allows to write 280 characters. On the opposite, Instagram users write an average of four hashtags per post (McCune & Thompson, 2011). 3. Methodology To carry out this study of the communication in social media of the main sustainable Spanish and Italian brands during COVID-19 crisis, we have compiled data related to their presence in Twitter and Instagram. First, we identified the brands considered to be the most sustainable in both countries according to the global study Dow Jones Sustainability World Index (ROBECOSAM AG, 2019) In this report, the companies perceived as the most sustainable on the planet are listed, organized by countries. In the case of Spain there are a total of 15 companies: Amadeus IT Group SA and Indra (Software & Services); BBVA, Banco Santander, Bankinter and CaixaBank (Banks); Enagas (Energy); Endesa, Naturgy Energy Group, Iberdrola and Red Eléctrica (Utilities); Inditex (Retailing); Mapfre (Insurance); and ACS and Ferrovial (Capital Goods). For Italy, the index included only 12: Pirelli & C (Automobiles & Components); Intesa Sanpaolo (Banks); Leonardo and Prysmian (Capital Goods); Moncler (Consumer Durables &Apparel); Saipem and Sna (Energy); Assicurazioni Generali and Poste Italiane (Insurance); Enel, Italgas and Terna Rete Elettrica Nazionale (Utilities). The information was collected between January 30 and April 8 2020. These specific dates were selected because they include the key moments in the spread of the pandemic in both countries. That is, the day where the first infection cases were jointly reported in both countries as well as those weeks with highest spikes in infections and, therefore, in uncertainty. During these days that coincide with the state of alarm and the confinement of the population decreed by Spanish and Italian governments, it is when the users of social networks are most active. They wanted immediate information and companies tried to react to everything that was happening. To scrape information from Twitter, we used the Tweepy Python library (https://www.tweepy.org/). For Instagram, we used an Apify scrapper (https://apify.com/jaroslavhejlek/instagram-scraper). Both tools allowed us to download the required data in a fast and orderly manner. The basis of our study will be certain techniques of social network analysis (SNA) (Wasserman & Faust, 1994) (Scott, 1988) and visualization (Kobourov, 2012). These allow the design of bipartite social networks of brands and hashtags that show the characteristics of the Twitter and Instagram communication models of these companies. The use of SNA techniques has demonstrated its ability to analyze complex social systems and to generate high-quality schematic visualizations of network-based representations in various fields of knowledge (Dearholt & Schvaneveldt, 1990). 2.3 COVID-19 and Sustainable Brands The scale of the COVID-19 public health emergency is unmatched in our lives and will have serious social and economic consequences (European Council, 2020) (United Nations, 2020). All of this becomes vitally important when a recent study conducted at Harvard University argues that there is an important correlation between air pollution and COVID-19 lethality (Wu, et al., 2020). While air pollution is already at a minimum thanks to widespread closures, scientists stress that ensuring cleaner air in the future will help reduce deaths from COVID-19. These studies and their conclusions help us understand the importance of sustainability awareness in companies and how it will help improve our quality of life, which has been altered by the pandemic. This moment is thus very important for brands, which have the opportunity to regain or not lose the trust they have achieved with users, demonstrating their values, purposes and CSR (Corporate Social Responsibility) policies. 2.2. Sustainability The traditional, market-driven approach to develop strong brand relationships. It has been challenged by broader performance frameworks that include social responsibility, ethics, and sustainability (Mena, et al., 2019). This is also due to the increasing attention that customers have begun to pay to sustainable/ecological products and to corporate social responsibility. Brand values significantly affect company stock prices and long-term sustainability. As a prototypical example, the recent debacle of the Volkswagen’s emissions scandal showed that social responsibility and (un) ethical actions of a company can have a significant impact on the value and classification of its brand (Harjoto & Salas, 2017) . j Sustainability is already a decisive factor when shopping. This is indicated by the results of the AECOC Shopperview study “How sustainability affects purchasing habits” (AECOC, 2020), presented during the celebration of the Second Congress of Sustainable Development, co- organized by AECOC (Association of Manufacturers and Distributors Companies) ¾one of the largest business organizations in Spain¾ and FIAB (Spanish Federation of Food and Beverage Industries). The report indicates that 44% of the surveyed consumers affirm that they have stopped buying products of those brands that they do not consider sustainable, with a particularly accentuated figure among those under 34 years of age. Brands, also changing entities and sensitive to external circumstances, are currently immersed in the process of going backwards and then moving faster. One of the predictions in Kantar’s Media Trends & Predictions report for 2020 (Kantar, 2020) was that brands must take the step of supporting great social causes. Therefore, brands must work to accelerate the process, adapt to this new situation and succeed, and even reinforce or consolidate their value proposition. the step of supporting great social causes. Therefore, brands must work to accelerate the process, adapt to this new situation and succeed, and even reinforce or consolidate their value proposition. 2.3 COVID-19 and Sustainable Brands 3. Methodology For example, (Jun & Park, 2017) considered them to analyze brand positioning by establishing relationships among brands as well as among brand and product attributes based on the structure of online web searchers developed by the users. Besides, (Zarco, et al., 2019) proposes using these kinds of network-based visual representations to identify the communication model followed by Spanish wineries in Twitter. In our case, the analysis of the bipartite social networks as well as their projections on the brands networks, on the one hand, and the hashtag networks, on the other hand, allows us to identify the communication models, uncover common and different patterns, and determine the most and less active actors in the complex systems. Bipartite networks are a very usual network model for SNA (Barabási, 2016). In these kinds of networks, the nodes belong to two different sets, A and B, and the undirected links can only connect nodes from a different set, i.e. a node from A with a node from B (see the central part of Figure 1). Therefore, they are very well adapted to model collaboration activities in complex social systems as each link represents a relation between two nodes, one from each node set. A typical example of a bipartite social network is the Hollywood actor network, generated from the data in IMDb.com (Barabási & Frangos, 2002), in which actors compose the first node set A and movies compose the second B. A link between an actor ai and a movie bk represents that actor ai played on movie bk. Figure 1: Example of a biparte network and its two projections Figure 1: Example of a biparte network and its two projections Notice that, two projections can be obtained from a bipartite network (see the left and hand side of Figure 1). The first projected network is only composed of nodes from the set A and reflects the interactions among them. There will be a link between two nodes ai and aj if both of them are linked to the same node from the B set in the original bipartite network. For example, in the Hollywood actor network this projection would correspond to the actor network and would correspond to a collaboration network: two actors would be linked only when they have played in at least one movie together. 3. Methodology Reciprocally we obtain the projected network for B, which in the Hollywood actor network would correspond to the movie network. Two movies would be linked in case they have at least one actor in common in their respective casts. In our case, the first node set comprises the selected sustainable brands while the second corresponds to the hashtags used in any post made by those companies in the social network during the analyzed period. We thus build four different bipartite networks, to analyze the behavior of the brands for two different countries, Spain and Italy, in two different social media, Twitter and Instagram: the Twitter Spain, Twitter Italy, Instagram Spain, and Instagram Italy bipartite networks. To become more informative, we consider weighted links. The link weight is a normalized count of the number of times a brand has posted the specific hashtag during the analyzed period. In this way, we can not only account that a hashtag has been used by a specific brand in its communication strategy but also how actively has it been used. Of course, brands which have not showed any activity in the respective social network during the analyzed period are not included in the brand node set and thus on the network. As a consequence, each of the four bipartite networks can show a variable number of brands, depending on whether they have activity or not on the social network, and of hashtags, depending on the different hashtags used by every brand in their posts in that social network during the considered period. Each of this bipartite networks is then projected into a brand network and a hashtag network, thus obtaining eight different networks: Twitter Spain brands, Twitter Italy brands, Instagram Spain brands, and Instagram Italy brands, on the one hand, and Twitter Spain hashtags, Twitter Italy hashtags, Instagram Spain hashtags, and Instagram Italy hashtags, on the other hand. These eight networks are no longer bipartite but weighed networks with a single node set. The weights in the links are computed by accumulating the normalized weights of the original bipartite network from which they are generated. All the latter networks are processed and visualized using the Gephi tool (Bastian, et al., 2009). 3. Methodology The usual network metrics in SNA as density, degree distribution, average degree, average weighted degree, and average clustering coefficient (Wasserman & Faust, 1994) (Scott, 1988) (Barabási, 2016) are computed and analyzed to understand the behavior of the complex social system represented by the four bipartite networks. The most important brands and hashtags are identified using the weighted degree, the measure best representing the activity for our case study. The betweenness centrality and eigenvector centrality measures are also considered eventually to identify prominent nodes for the case of the projected networks. Finally, network layouts are also provided to make the analysis easier. Among the existing layout algorithms, we make use of force-based methods (Kobourov, 2012) as they provide visualizations with interesting characteristics for the analysis. Due to its operation, the most important and most related nodes from a global perspective are located in the center of the layout while the less important ones are located in the periphery (Borgatti & Everett, 2000). In particular, we consider some variants of the classical Kamada–Kawai method (Kamada & Kawai, 1989) available in Gephi, the Event graph layout plugin (see https://www.wouterspekkink.org/software/) for the bipartite networks and the Force Atlas 2 algorithm (Jacomy, et al., 2014). To make the network layout more representative, node sizes and link widths are scaled according to their weighted degree and weight respectively in every case. Brand nodes are always colored in pink while hashtag nodes are colored in red. 4.1. Analysis of the Bipartite Networks 4.1. Analysis of the Bipartite Networks 4.1.1. Twitter Spain 4 Analysis of the Networks Generated The current section is devoted to analyze the networks generated following the methodology introduced in Section 3. The first subsection focuses on the four bipartite networks while the second deals with the eight projected networks. 4.1.1. Twitter Spain Figure 2: Twitter Spain bipartite network Figure 2: Twitter Spain bipartite network Figure 2: Twitter Spain bipartite network The Twitter Spain bipartite network includes 13 brands and 13 hashtags. In this way, the maximum number of possible links is 13·13=169. The network shows 67 of those 169 links, thus having a density of 0.3965. This can be seen as a rather high value, meaning an active overall use of the hashtags by all the brands. Concerning the brands, the node degree ranges from 1 to 10. The highest degree node is that of the BBVA brand with 10 links. This brand thus shows the highest diversity in its communication strategy, as it makes use of 10 of the 13 existing hashtags. Apart from a large use of the most general ones, #COVID-19 and #coronavirus (see the thick links in Fig. 2), it also uses 8 more. The new two brands with the most diverse communication strategy are Iberdrola and Mapfre with 7 hashtags each. On the opposite, the lowest value corresponds to Amadeus, that only used the generic #COVID-19 hashtag and just a small number of times, as illustrated by the very thin link showed in the figure. The next two “less diverse” brands are Enagas and Endesa, which at least use 3 different hashtags. Overall, the average degree of the network is 5.1538, showing that the brands use around 5 different hashtags in average, while the standard deviation is 2.2303. As regards the hashtags, the degree distribution ranges from 1 to 12. The highest value is for #COVID-19, thus being used by 12 of the 13 brands, followed by #EsteVirusloParamosUnidos, used by 11. Notice that, the next two are #coronavirus and #teletrabajo, both used by 8 brands. The less general hashtags are #coronabonos, #Aplausocolectivo, and #StayHome, all of them used by only one brand. While the average degree is of course the same, 5.1538, the standard deviation is higher than for the brands nodes, 3.8045, showing a higher dispersion on the use of the hashtags. 4 Analysis of the Networks Generated The last metric to be analyzed for this network is the weighted degree, which allows us to analyze the overall brand activity and the overall hashtag use. The average is 0.5283 (remember that the link weights have been normalized). The standard deviation is 0.5742 for the brands and 0.8116 for the hashtags, showing the same behavior as the degree, higher dispersion for the latter ones. The weighted degree values allows us to stress the activity differences in both cases. For the brands, while BBVA has the highest weighted degree of 2.1371, the second most active brand is Bankinter (and not Mapfre neither Iberdrola, which showed the second position in the degree measure with 7) with 0.9239 and only a degree of 5. In fact, although Iberdrola is using 7 different hashtags, its weighted degree is only 0.3401, one of the lowest values, showing a small activity. These differences are also observed for the hashtags: #COVID-19 is not only the one most used by the brands (degree=12) but also the one most twitted (weighted degree=2.6701). However, although #EsteVirusLoParamosUnidos is used by 11 brands, it is significantly less twitted (weighted degree=1.0406). In fact, #coronavirus is in between both of them, with the second highest weighted degree (1.6751) but with a lower degree, 8. This means that although it is used by less brands, it is more actively twitted. The weighted degrees of the remaining hashtags are significantly lower. The case of the next two hashtags with the higher degrees is clear, #teletrabajo with degree=8 and #QuédateEnCasa with degree=7, are used by more than the half of the brands but weakly tweeted as both weighted degrees are under 0.25. The following two tables report the values of both degree measures for every brand and hashtag node, respectively. The cells in each column follow a color scale where the lowest values start from red and move to green while increasing. 4 Analysis of the Networks Generated Brand Weighted degree Degree Amadeus IT Group SA 0.0203 1 BBVA 2.1371 10 Banco Santander 0.1472 6 Bankinter 0.9239 5 CaixaBank 0.2132 5 Enagas 0.0863 3 Endesa 0.6091 3 Ferrovial 0.5685 5 Naturgy Energy Group 0.6802 6 Iberdrola 0.3401 7 Indra 0.0609 4 Mapfre 0.8782 7 Red Eléctrica 0.2030 5 Hashtag Weighted degree Degree #EsteVirusLoParamosUnidos 1.0406 11 #COVID-19 2.6701 12 #coronavirus 1.6751 8 #EsteVirusLoParamosEntreTodos 0.0508 2 #teletrabajo 0.2538 8 #phishing 0.5685 4 #StayHome 0.0102 1 #YoMeQuedoEnCasa 0.2437 5 #QuédateEnCasa 0.2487 7 #autónomos 0.0558 5 #DíaDelPadre 0.0203 2 #AplausoColectivo 0.0102 1 #coronabonos 0.0203 1 4.1.2. Instagram Spain Brand Weighted degree Degree Amadeus IT Group SA 0.0203 1 BBVA 2.1371 10 Banco Santander 0.1472 6 Bankinter 0.9239 5 CaixaBank 0.2132 5 Enagas 0.0863 3 Endesa 0.6091 3 Ferrovial 0.5685 5 Naturgy Energy Group 0.6802 6 Iberdrola 0.3401 7 Indra 0.0609 4 Mapfre 0.8782 7 Red Eléctrica 0.2030 5 Hashtag Weighted degree Degree #EsteVirusLoParamosUnidos 1.0406 11 #COVID-19 2.6701 12 #coronavirus 1.6751 8 #EsteVirusLoParamosEntreTodos 0.0508 2 #teletrabajo 0.2538 8 #phishing 0.5685 4 #StayHome 0.0102 1 #YoMeQuedoEnCasa 0.2437 5 #QuédateEnCasa 0.2487 7 #autónomos 0.0558 5 #DíaDelPadre 0.0203 2 #AplausoColectivo 0.0102 1 #coronabonos 0.0203 1 4.1.2. Instagram Spain 4.1.2. Instagram Spain Figure 3: Instagram Spain bipartite network Figure 3: Instagram Spain bipartite network Figure 3: Instagram Spain bipartite network The Instagram Spain bipartite network is comprised by a lower number of brands and hashtags than the Twitter one, 8 brands and 12 hashtags. The network has 42 of the 96 possible links, showing a density of 0.4375, a similar value to the former. Again, this an important overall activity. In this case we find five companies less than on Twitter: Amadeus IT Group S.A., CaixaBank, Enagas, Indra and Red Eléctrica, although the number of hashtags used are practically the same. This clearly show that Spanish brands are less interested on using Instagram in their communication strategy than on using Twitter. The brands show a homogeneous behavior, with an average degree of 5.25 and a small standard deviation (1.8323). BBVA is again the most diverse brand in hashtag use with 8 links (but this time it is not using other 4), followed by Banco Santander and Iberdrola with 7 links each. Ferrovial is the lowest degree brand but at least it uses 3 hashtags, followed by other three brands with 4. The average degree of the hashtag nodes is lower (3.5) but its standard deviation is higher (2.0671), showing again a high dispersion on the use of the hashtags. Four different hashtags are used by 6 of the 8 brands: #QuédateEnCasa, #Covid19, #coronavirus, and #EsteVirusLoParamosUnidos. On the opposite, other three are only used by a single brand: #homeoffice, #remotework, and #AplausoColectivo. Concerning the weighted degrees, their values are higher than for the Twitter network. The brand nodes show an average of 1.2917 (std. dev. of 0.98) and the hashtag nodes have an average of 0.8611 (std. dev. of 0.7515). This measure allows us to uncover interesting behaviors. Iberdrola is the most active brand, showing a higher weighted degree than BBVA (2.5833 vs. 2.25) even if it uses one hashtag less. In fact, Naturgy Energy Group also shows a higher value than BBVA with only 5 hashtags but a larger use of them (weighted degree of 2.5). On the opposite, Banco Santander arises as a low activity brand (weighted degree of 0.6667) even if it uses a high number of hashtags (7). All the latter allows us to conclude that companies destined for public services (utilities) such as Naturgy and Iberdrola have preferred to enhance their communication on Instagram rather than on Twitter, thus trying to connect with a younger audience that is committed to the environment and corporate social responsibility. Figure 3: Instagram Spain bipartite network This is just the opposite situation we identify for the banks, that are more active in Twitter overall. Besides, their activity profile in Instagram is characterized by a few posts that jointly include several hashtags. The only exception is BBVA, which maintains a certain coherence in its communication policy in both social networks. This behavior is not observed for the case of the hashtags. The degrees and the average degrees are pretty correlated and the same nodes have the highest values in both measures. This means that the hashtags used by more brands are also those more actively posted, while those used by less brands are less posted. Notice that, this is a different behavior than the one uncovered in the Twitter network. The following two tables report the values of both degree measures for every brand and hashtag node, respectively. Brand Weighted degree Degree BBVA 2.2500 8 Banco Santander 0.6667 7 Bankinter 0.4167 4 Endesa 0.9167 4 Ferrovial 0.2500 3 Naturgy Energy Group 2.5000 5 Iberdrola 2.5833 7 Mapfre 0.7500 4 Hashtag Weighted degree Degree #YoMeQuedoEnCasa 1.1667 4 #QuédateEnCasa 2.3333 6 #Covid19 1.2500 6 #coronavirus 1.2500 6 #teletrabajo 0.5833 3 #EsteVirusLoParamosUnidos 2.0000 6 #homeoffice 0.4167 1 #remotework 0.0833 1 #AplausoColectivo 0.0833 1 #AplausoSanitario 0.1667 2 #empleos 0.2500 3 #FrenarLaCurva 0.7500 3 4.1.3. Twitter Italy Figure 4: Twitter Italy bipartite network 4.1.3. Twitter Italy 4.1.3. Twitter Italy Figure 4: Twitter Italy bipartite network The Twitter Italy bipartite network is comprised by a lower number of brands and hashtags than the two Spain bipartite networks. There are only 8 brands with presence in Twitter out of the 12 initial ones and they have twitted only 3 hashtags. The network has 14 of the 24 possible links, showing a density of 0.5833, which is higher than the previous two networks. This can be seen as a fairly high value, which means an active general use of the hashtags by all the brands, and can be understood in view of the low number of hashtags. Brands have a quite similar behavior, with an average degree of 1.75 and a small standard deviation (0.7071). The highest degree node is that of the Poste Italiane SpA brand with 3 links. This is the only brand whose publications during this period of confinement used the three hashtags, which is considered as strategy to convey more impact. Figure 3: Instagram Spain bipartite network Pirelli & C SpA, Intesa Sanpaolo SpA, Snam SpA and Terna Rete Elettrica Nazionale SpA have 2 links and Assicurazioni Generali SpA, Enel SpA and Italgas SpA are the lowest degree brands with a single link. The average degree of the hashtag nodes is of course higher (4.6667) as well as its standard deviation (3.0551), showing a high dispersion on the use of the hashtags. There is only one hashtag, #Covid19, which is used by every brand. The hashtag #coronavirus, despite having a significant impact by its own name, is not as widely used as the previous one, and only half of the brands have twitted it. The third one, #iorestoacasa, is only used by two of them. Concerning the weighted degrees, the brands show an average of 0.68 (std. dev. of 0.37) while the hashtags an average of 1.8148 (std. dev. of 1.8428). In this case, Terna Rete Elettrica Nazionale SpA is the most active brand, showing a significantly higher weighted degree than the highest degree brand, Poste Italiane SpA (almost four times higher, 1.1667 vs. 0.3333). This means that although this brand has used less hashtags, it has applied an active communication strategy focusing its attention on specific messages. Finally, due to the small number of existing hashtags, their weighted degrees are much related to their degree, thus not providing any additional information. This means that the hashtags used by more brands are also those more actively posted, while those used by less brands are less posted. The following two tables report the values of both degree measures for every brand and hashtag node, respectively. The following two tables report the values of both degree measures for every brand and hashtag node, respectively. Brand Weighted degree Degree Pirelli & C SpA 0.2222 2 Intesa Sanpaolo SpA 0.7222 2 Snam SpA 1.1111 2 Assicurazioni Generali SpA 1.0000 1 Poste Italiane SpA 0.3333 3 Enel SpA 0.3333 1 Italgas SpA 0.5556 1 Terna Rete Elettrica Nazionale SpA 1.1667 2 Brand Weighted degree Degree Pirelli & C SpA 0.2222 2 Intesa Sanpaolo SpA 0.7222 2 Snam SpA 1.1111 2 Assicurazioni Generali SpA 1.0000 1 Poste Italiane SpA 0.3333 3 Enel SpA 0.3333 1 Italgas SpA 0.5556 1 Terna Rete Elettrica Nazionale SpA 1.1667 2 Hashtag Weighted degree Degree #iorestoacasa 0.2222 2 #Covid19 3.8333 8 #coronavirus 1.3889 4 4.1.4. Figure 3: Instagram Spain bipartite network Instagram Italy Figure 5: Instagram Italy bipartite network The Instagram Italy bipartite network is comprised by the same number of brands than the Twitter one, 8 brands, but more hashtags in this case, 6. The 3 hashtags used in Twitter are also posted by the brands in Instagram, showing their very general nature, but there are 3 new ones which are also very generic (#Grazie, #sicurezza, and #emergenza). For the case of the brands, 6 of them ha e considered both social media in their communication strategies but the other two Brand 4.1.4. Instagram Italy Figure 5: Instagram Italy bipartite network Figure 5: Instagram Italy bipartite network Figure 5: Instagram Italy bipartite network Figure 5: Instagram Italy bipartite network The Instagram Italy bipartite network is comprised by the same number of brands than the Twitter one, 8 brands, but more hashtags in this case, 6. The 3 hashtags used in Twitter are also posted by the brands in Instagram, showing their very general nature, but there are 3 new ones, which are also very generic (#Grazie, #sicurezza, and #emergenza). For the case of the brands, 6 of them have considered both social media in their communication strategies but the other two have opted by a single one: Assicurazioni Generali SpA and Enel SpA for Twitter while Leonardo SpA and Saipem SpA for Instagram. The current network has 19 of the 48 possible links, showing a density of 0.3958, a little bit lower value than in the former network. The brand activity is thus more distributed and it is not as concentrated as in the previous case, probably as a consequence of having the chance to provide different messages by means a higher number of hashtags. The average degree of the brand nodes is not very high (2.3750) and the standard deviation is a rather small (1.1877). The highest degree nodes are those of the Snam SpA and Italgas SpA with 4 links, showing that there is no brand posting the 6 hashtags. Terna Rete Elettrica Nazionale SpA have 3 links, Leonardo SpA, Saipem SpA and Poste Italiane SpA have 2 links, and Pirelli & C SpA and Intesa Sanpaolo SpA are the lowest degree brands with only 1 link. Overall, this represents a cohesive behavior in the number of hashtags used but also a pretty high diversity in their choice. Figure 3: Instagram Spain bipartite network For the case of hashtags, the average degree is 3.1667 and its standard deviation is 1.3292, which shows a considerable distribution in the use of the hashtags by the brands. In this case, only one hashtag is used once, #emergenza. The rest has been used a fairly homogeneous number of times. The general #covid-19 is the highest degree hashtag but has only been used by 5 of the 8 brands, showing again the diversity in the communication strategies in this social media. As for the weighted degrees, the brands show an average of 0.4479 (std. dev. of 0.5472) and the hashtags an average of 0.5972 (std. dev. of 0.3779). Both values are pretty low, showing a small overall activity as reflected in the thin links in Figure 5. Snam SpA is the most active brand, showing a very higher weighted degree than Italgas SpAm, which is next (almost three times higher, 1.75 vs. 0.5833). Both brands showed also the highest degree, with 4 links. That leads us to conclude that Snam SpA has been very consistent in the publication of content, enhancing his message with the use of the different hashtags considered. The weighted degrees of the hashtags allows us to identify an interesting behavior. We can see how #sicurezza has the highest value (1.25), which is well above the next one that corresponds to #COVID-19 (0.75). However, the former has a degree of 3 while the latter has a degree of 5. This means that 3 brands have actively posted the #sicurezza hashtag. In particular, the network layout clearly shows that the link with the Snam Spa brand has clearly the highest weight in the network, while the remainder maintain a relatively similar weight. This gives us to understand the importance that this brand has given to this hashtag in its publications. The following two tables report the values of both degree measures for every brand and hashtag node, respectively. Figure 3: Instagram Spain bipartite network Brand Weighted degree Degree Pirelli & C SpA 0.0833 1 Intesa Sanpaolo SpA 0.2500 1 Leonardo SpA 0.1667 2 Saipem SpA 0.1667 2 Snam SpA 1.7500 4 Poste Italiane SpA 0.3333 2 Italgas SpA 0.5833 4 Terna Rete Elettrica Nazionale SpA 0.2500 3 Hashtag Weighted degree Degree #iorestoacasa 0.5000 3 #covid-19 0.7500 5 #coronavirus 0.5833 4 #emergenza 0.1667 1 #Grazie 0.3333 3 #sicurezza 1.2500 3 Brand Weighted degree Degree Pirelli & C SpA 0.0833 1 Intesa Sanpaolo SpA 0.2500 1 Leonardo SpA 0.1667 2 Saipem SpA 0.1667 2 Snam SpA 1.7500 4 Poste Italiane SpA 0.3333 2 Italgas SpA 0.5833 4 Terna Rete Elettrica Nazionale SpA 0.2500 3 Brand Weighted degree Degree Pirelli & C SpA 0.0833 1 Intesa Sanpaolo SpA 0.2500 1 Leonardo SpA 0.1667 2 Saipem SpA 0.1667 2 Snam SpA 1.7500 4 Poste Italiane SpA 0.3333 2 Italgas SpA 0.5833 4 Terna Rete Elettrica Nazionale SpA 0.2500 3 Brand 4.2. Analysis of the Projected Networks 4.2.1. Twitter Spain Brands: Figure 6: Twitter Spain brand network 4.2. Analysis of the Projected Networks 4.2.1. Twitter Spain 4.2.1. Twitter Spain Brands: Figure 6: Twitter Spain brand network The Twitter Spain brand projected network has 13 nodes, corresponding to the brands showing activity on Twitter during the analyzed period, and shows a high average degree (11.846) and density (0.987). This means that their communication strategies are strongly related and almost all of them share at least a single hashtag used in their tweets. As a consequence, the local density is also high, the clustering coefficient is 0.987 as well. However, the average weighted degree is significantly low, 0.674, stressing the differences in the communication strategies applied by the different brands. This means that they have not tweeted a large number of common hashtags overall. The center-periphery effect resulting from the use of the Force Atlas 2 layout is especially relevant in our case, easing the network analysis at first sight. The same happens for the node sizes and link widths. We should remember that a thick link between two brands in the projected network means that they have tweeted several hashtags in common, i.e., they have a common communication strategy in Twitter. Besides, the closeness of several nodes in the graphical representation also means a high use of common hashtags and every path in the network reflects a sequence of relations. Figure 3: Instagram Spain bipartite network That is, a path ai – aj - ak means that brand ai has some hashtags in common with brand aj while brand aj has some hashtags in common with brand ak, with the similarity degree reflected by the link weight. The appearance of triangles or cliques mean a global relation between three or more brands. In view of the latter, the layout easily allows us to uncover the four most relevant Spanish brands according to their communication strategies on Twitter. They are the four ones located in the center: BBVA, Bankinter, Mapfre, and Ferrovial. This means that they have tweeted many common hashtags with the rest of the brands (they show a high similarity in their communication strategy overall). Their ranking of importance is thus reflected by their node sizes, scaled with respect to the weighted degree. Two strong triangles can be identified: BBVA-Bankinter-Ferrovial and BBVA-Bankinter-Mapfre, uncovering two different subsets of hashtags used. The former triangle shares the hashtags #coronavirus, #COVID-19, and #QuédateEnCasa while the latter shares the hashtags #coronavirus, #QuédateEnCasa, and #autónomos, thus uncovering two different communication strategies. The brands on the left side of the layout will be related to the first communication strategy while those on the right side to the second. gy g Finally, we can quickly identify the less active brands as those located in the periphery of the representation: Amadeus IT Group SA, Enagas, Red Eléctrica, Indra, and Banco Santander (listed clockwise). They have a small activity in the use of different hashtags, as also seen in the analysis of the bipartite networks, and a low interaction with other brands using more hashtags and in a more active way. That is, their communication strategies in Twitter are poor during the analyzed period. Hashtags: Hashtags: Figure 7: Twitter Spain hashtag network Figure 7: Twitter Spain hashtag network The Twitter Spain hashtag projected network also has 13 nodes, corresponding to the different hashtags tweeted by the 13 considered brands. It also shows a high average degree (9.385) and density (0.782), lower than those for the brand network as well. This means that some of the hashtags are not jointly used by any brand. Even so, the local density stays very high (0.892), showing a joint use of triplets of hashtags in many cases. However, the average weighted degree is extremely low (0.371), stressing the strong differences in the communication strategies of the different brands, reflected in this case by the lack of a joint use of a large number of hashtags, as already mentioned. The center of the layout is occupied by a single strong triangle, comprised by the #COVID- 19, #coronavirus, and #EsteVirusLoParamosUnidos hashtags, following that ranking of importance. This means that these three hashtags have been jointly tweeted by many brands, especially the former two. Looking around that salient triangle in each direction, we can identify the hashtags that have been more jointly used with those three vertices of the triangle. For example, #QuédateEnCasa and #teletrabajo have been mainly jointly used with #COVID-19 and #coronavirus by the brands. Meanwhile, #YoMeQuedoEnCasa and #phishing have been mainly jointly used with #COVID-19 and #EsteVirusLoParamosUnidos. #coronavirus by the brands. Meanwhile, #YoMeQuedoEnCasa and #phishing have been mainly jointly used with #COVID-19 and #EsteVirusLoParamosUnidos. Again, the less used hashtags are in the periphery of the layout. The most relevant conclusion is that #AplausoColectivo and #EsteVirusLoParamosEntreTodos are the farthest ones. Even if they have a relevant degree, 5 and 8 respectively, their weighted degree is very low, 0.0014 and 0.008, thus showing the brands are only using them very occasionally. 4.2.2. Instagram Spain Brands: Figure 8: Instagram Spain brand network 4.2.2. Instagram Spain Brands: Figure 8: Instagram Spain brand network Figure 8: Instagram Spain brand network Figure 8: Instagram Spain brand network The Instagram Spain brand projected network has 8 nodes, corresponding to the brands showing activity on Instagram during the analyzed period, and shows a significantly high average degree (6.5) and density (0.929). The local density also stays very high (0.94). The average weighted degree is extremely high as well (1.307). The center of the layout is occupied by a strong triangle comprised by the Iberdrola, Naturgy Energy Group and BBVA brands. This means that they have made their posts with common hashtags with the rest of the brands. Its importance can be drawn by the size of its nodes. The hashtags shared by these brands have been #QuédateEnCasa, #coronavirus and #EsteVirusLoParamosUnidos, some labels that appeal to the sense of protection in the community. The farthest node is that of Ferrovial, which also shows the lowest average degree and is mainly linked to the central triangle but with very thin links showing the low activity of its communication strategy in this social media. Hashtags: Figure 9: Instagram Spain hashtag network Figure 9: Instagram Spain hashtag network The Instagram Spain hashtag projected network has 12 nodes and its layout is very spread, showing a higher diversity than in the previous cases. It has a low average degree (3.2) and density (0.221), values that also corroborate the latter assumption. Even so, the local density stays very high (0.868), thus meaning that many different triplets of hashtags are shared by the brands in their diverse communication strategies. The average weighted degree is high (1.033), representing an important activity, but the different node sizes allows us to conclude this activity level is also very diverse among the different brands. The center of the layout is occupied by a strong triangle, comprised by the #Quedateencasa, #EsteVirusLoParamosUnidos and #YoMeQuedoEnCasa hashtags, following that ranking of importance. Nevertheless, on the opposite of the Instagram brand network, which was dominated by a single, strong, central triangle, in this case it is surrounded by some other important triangles. That is another proof of the diversity in the communication strategies applied by the brands in this social media. 4.2.3. Twitter Italy Brands: Comparing the brand messages with those made in Twitter, the meaning of the hashtags on Instagram is somewhat different since those that are more widespread here refer to the protection of people, and the sense of community and of union. For the Twitter case, they appealed more to the alarm situation, information about the disease, and the seriousness of the situation. Besides, the less used hashtags are clearly identified thanks to their peripheral location: #remotework and #empleos, and especially #AplausoColectivo and #AplausoSanitario. 4.2.3. Twitter Italy Brands: Figure 10: Twitter Italy brand network Brands: Figure 10: Twitter Italy brand network The Twitter Italy brand projected network includes 8 brands and has the maximum values for the average degree (7) and density (1). The average weighted degree is also extremely high (1.691) and the layout clearly shows a low dispersion for this measure in 5-6 of the 8 nodes. The Twitter Italy brand projected network includes 8 brands and has the maximum values for the average degree (7) and density (1). The average weighted degree is also extremely high (1.691) and the layout clearly shows a low dispersion for this measure in 5-6 of the 8 nodes. The center of the layout is again dominated by a single strong triangle comprised by the Assicurazioni Generali SpA, Snam SpA and Terna Rete Elettrica Nazionale SpA brands. This means that they have made their posts with common hashtags with the rest of the brands. Its importance can be deduced by the size of its nodes. The novelty is that we can also identify at least two other, less strong but still very strong, triangles: Italgas SpA - Assicurazioni Generali SpA - Terna Rete Elettrica Nazionale SpA and Intesa Sanpaolo SpA- Snam SpA - Terna Rete Elettrica Nazionale SpA. This leads us to conclude this complex social system has a more cohesive behavior as the hashtags commonly used and the global activity is very similar. We should remember that only three hashtags were twitted in this case, what reinforces our conclusion as the diversity of possible communication strategies is clearly reduced. Poste Italiane SpA and Pirelli & C SpA can be clearly identified as the most differential brands, but in this case due to their lower activity. Hashtags: Hashtags: Figure 11: Twitter Italy hashtag network Figure 11: Twitter Italy hashtag network The Twitter Italy hashtag projected network is very simple. It has only 3 nodes and it is fully connected, thus being composed of a single triangle. Even so, the average weighted degree has a low value (0.519) as two of the three links have a very low weight. In this network, having so few nodes, it is easier to see the behavior of the links and understand the values obtained. All the hashtags are related and all of them have been used by the brands considered. However, there are two (#Covid19 and #coronavirus) that have a very strong relationship between them, much greater than with the third node (#iorestoacasa). 4.2.4. Instagram Italy 4.2.4. Instagram Italy Brands: Brands: Figure 12: Instagram Italy brand network Figure 12: Instagram Italy brand network Figure 12: Instagram Italy brand network The Instagram Italy brand projected network has 8 nodes. I shows a low degree (3.75) and a medium density value (0.536). The average clustering coefficient stays very high (0.842), showing a high local density. On the opposite, the average weighted degree is very low, only 0.174, and the network sizes clearly show us it has a very low dispersion. All the latter results in a high diversity of behavior patterns of the Italian brand communication strategies in this social media. If view of the network layout, we can identify two different groups can be seen united by the Poste Italiane SpA, Leonardo SpA, and Italgas SpA brands. In fact, these three nodes show the highest betweenness centrality values in the network, a SNA metric that measures the brokerage capability of a node. The group on the right has a strong relationship between Italgas SpA, Snam SpA and Terna Rete Elettrica Nazionale SpA. They are brands that have shared the same hashtags in their publications, showing a more consistent communication. Meanwhile, we can identify another, very isolated group of other three brands on the left: Intesa Sanpaolo SpA, Poste Italiane SpA and Pirelli & C SpA, that are more distant and with slightly weaker links. These brands have shared a hashtag in their communications but in a very subtle way, with hardly any mention. The eigenvector centrality measure has been computed in this case to identify the most relevant brands in the communication strategies applied and Leonardo SpA and Italgas SpA brands have both shown the maximum value. This makes full sense according to their central location and their high degrees, showing a large similarity with the communication strategies applied by the other brands. 5. Discussion and Conclusions The arrival of social media has introduced new channels of brand communication and has leaded to the application of online tools to engage with consumers (Gomez, et al., 2019). This study contributes to the general analysis of brand engagement on social media through the analysis of brand participation and communication in social networks (Bruhn, et al., 2012) (Pentina, et al., 2013) as well as commitment of brands in complex situations such as that the COVID-19 crisis recently experienced (Petts, et al., 2010) (Smit, et al., 2007). To do so, we have selected a set of Spanish and Italian brands considered as the most sustainable and have analyzed the communication activity and the communication strategies followed (represented in terms of the considered hashtags) in two different social media, Twitter and Instagram, during the early stages of the pandemic in their respective countries. Regarding the results obtained, we observe that of the15Spanish brands considered, all but one (ACS) have a Twitter profile, but only 12 of them have maintained a constant activity during the analyzed period. Both Amadeus IT Group and Inditex have their profiles opened but they did not show any activity. In the case of Spanish brands on Instagram, we recognized that only 9 brands had an active profile and all of them also made publications related to COVID-19. If we go into more detail, we can see how most of the publications made on both networks by Spanish brands had a common message: transmitting empathy and strength to those affected by COVID-19. Of course, some of the brands that did not show any activity on the social networks analyzed have alternatively acted in other contexts, such as Inditex which has shown its commitment to its country acting in solidarity and buying medical supplies to donate to hospitals. It is noteworthy that two brands, Ferrovial and Caixa Bank, which have a presence on Twitter and Instagram, opted for not using both social networks as communication tools in relation to the pandemic. Both of them only focused their communication strategies on Twitter. Leaving this detail apart, in general terms we can conclude that all brands shared the main hashtags that were used to unify the message of support they wanted to transmit. The most used hashtags were #COVID-19, #coronavirus, and #EsteVirusLoParamosUnidos. Hashtags Figure 13: Instagram Italy hashtag network Figure 13: Instagram Italy hashtag network The Instagram Italy hashtag projected network has 6 nodes. It shows medium values for the average degree (3.33) and density (0.667). The clustering coefficient stays high (0.8), showing again a high local density. The average weighted degree is low (0.373). All the latter makes us conclude we can find again an important diversity in the communication strategies applied, as in the previous network. A group of hashtags are clustered in the lower part of the graph, #coronavirus, #COVID-19, #sicurezza, and #emergenza, while in the highest and most distant part we find two: #iorestoacasa and #Grazie. We see that there is a strong relationship between three of these nodes: #coronavirus, #COVID-19, and #sicurezza, which suggests that they have been used together in the same publications. Actually, these three nodes have the highest value in the eigenvector centrality. There is another strong triangle sharing two nodes with the latter one: #coronavirus, #sicurezza, and #emergenza. Focusing on the two most distant nodes, #Grazie plays the gatekeeper role between the group at the bottom and the one at the top. This is also clearly reflected by it high betweenness centrality value, 7 times higher than that of the next three nodes. Its location at the top of the layout implies that it has been jointly used with #iorestoacasa in some posts. It has also jointly used with the three hashtags in the main triangle several times, which is reflected in a high eigenvector centrality value. layout implies that it has been jointly used with #iorestoacasa in some posts. It has also jointly used with the three hashtags in the main triangle several times, which is reflected in a high eigenvector centrality value. 5. Discussion and Conclusions In short, focusing a little more on the particularities of brands on social networks in this period analyzed, we see in the Spanish case that the brands that have been on Twitter BBVA, which is a brand of a bank and is characterized by being very active on this social network; and on Instagram Iberdrola and Narturgy, which are more linked to the energy and public services sector, which makes them companies that, in theory, are more linked to the environment and its protection. On the contrary, the least active in the networks have been Amadeus in the case of Twitter and Ferrovial in the case of Instagram. Regarding the most used hashtags globally, we have seen that on Twitter it has been #Covid- 19, something that seems consistent due to the main characteristics of this network: immediacy and real and direct information; while in Instagram they have been #QuédateEnCasa and #EsteVirusLoParamosUnidos, two labels that more than mention the problem, invite us to empathize and be responsible with our peers. In the case of Italian brands, we see that all the 12 sustainable brands but one, Saipem, had a profile on Twitter, although Moncler did not show any type of activity during the period. Meanwhile, if we look at the Italian brands on Instagram, we see that the 12 brands actually had a profile on the network and that they also maintain a very continuous activity, although not all of them referred to the current situation of the pandemic. It is worth noting that in the case of Italian brands, unlike the Spanish ones, there was not a great diversity of hashtags. In most cases, the use was limited to a maximum of 6 while the Spanish ones considered up to 13. This clearly showed a most homogeneous communication strategy in terms of the broadcasted message for the case of Italy. Compared to the Spanish case, the activity of Italian brands was much lower on both networks. In this case, there are companies that have very active profiles (of the 10 brands, 6 use the two networks, 2 use only Twitter and 2 only Instagram), but on Twitter they used less variety of hashtags than on Instagram, which suggests a different communication policy on one network and another. 5. Discussion and Conclusions That was the case of Leonardo SpA, who made no mention to the pandemic in his tweets but did so in the posts made in his Instagram profile. The case of Assicurazioni Generali SpA is quite the opposite. He mentioned the pandemic on Twitter but did not post anything related to COVID-19 on Instagram, but there was content related to the pandemic in the "Stories" (content that allows users to share moments and then personalize them with text, drawings and emojis, and pin them to do not lose them as they have the characteristic of being deleted after 24 hours) in the profiles of this network during the analyzed period. This decision may be due to the fact that they wanted to give more visibility to the publications related to the pandemic by giving them a prominent place as are the stories in their profile. p As for the most widespread terms among Italian brands to refer to the current health crisis, the hashtags were #coronavirus, #iorestoacasa, and #COVID-19. If we look at the particularities of the Italian brands in the networks considered, we see that the most active on Twitter have been Poste and Terna Rete Elettrica, from the insurance and utilities sectors respectively. On Instagram it is Snam who leads the way, which belongs to the energy sector. By contrast, the least active on Twitter has been Enel and in the case of Instagram it is Pirelli & C. In the case of the hashtags used, we have seen that on Twitter # Covid19 is the most numerous in all cases, while on Instagram #sicurezza and # Covid19 have been very similar in their use. In short, this study has allowed us to analyze the behavior of the brands considered to be the most sustainable in Spain and Italy on two of the most extended social networks during the early stages of the COVID-19 crises in their respective countries. The commitment of these brands to the current situation and their presence in social media can be considered very relevant and will permit them to strengthen their solidarity character. We have been able to verify that Spanish brands have more activity on Twitter than on Instagram, therefore targeting a more mature audience, although not less committed to sustainable actions. 5. Discussion and Conclusions We also uncovered that the Italian brands had a more prominent presence on Instagram than on Twitter, revealing an intention to communicate with a younger audience which is usually more committed to social causes, social responsibility corporate and sustainability-oriented actions. Acknowledgements This work is supported by the Spanish Agencia Estatal de Investigación, the Andalusian Government, the University of Granada, and European Regional Development Funds (ERDF) under grants EXASOCO (PGC2018-101216-B-I00) and AIMAR (A-TIC-284-UGR18). References AECOC, 2020. Cómo la sostenibilidad afecta los hábitos de compra, Madrid: s.n. Barabási, A., 2016. Network science. s.l.:Cambridge University Press. Barabási, . A. L. & Frangos, J., 2002. Linked: the new science of networks science of networks. Bas Books. Science, and Everyday Life ed. s.l.:Hachette UK. Bastian, M., Heymann, . S. & Jacomy, M., 2009. Gephi: an open source software for exploring an manipulating networks. s.l., s.n. Borgatti, S. & Everett, M., 2000. Models of core/periphery structures. Social networks, 21(4), p. 375-395. Bruhn, M., Schoenmueller, V. & Schäfer, D., 2012. Are social media replacing traditional media in terms of brand equity creation?. Management Research Review, 35(9), p. 770–790. De Vries, N. & Carlson, J., 2014. Examining the drivers and brand performance implications of customer engagement with brands in the social media environment. Journal of Brand Management, 21(6), pp. 495-515. pp Dearholt, D. & Schvaneveldt, R., 1990. Properties of Pathfinder networks. In: Pathfinder associative networks: Studies in knowledge organization Westport. Connecticut: Ablex Publishing, pp. 1-30. European Council, 2020. Report on the comprehensive economic policy response to the COVID-19 pandemic, s.l.: s.n. Fournier, S. & Avery, J., 2011. The uninvited brand. Business horizons, 54(3), pp. 193-207. Fournier, S. & Avery, J., 2011. The uninvited brand. Business horizons, 54(3), pp. 193-207. García Muntané, D., 2020. Consecuencias de la cancelación del 'Mobile World Congress': Fournier, S. & Avery, J., 2011. The uninvited brand. Business horizons, 54(3), pp. 193-207. García Muntané, D., 2020. Consecuencias de la cancelación del 'Mobile World Congress': perspectiva pericial. Cinco Días, 3 Marzo. García Muntané, D., 2020. Consecuencias de la cancelación del 'Mobile World Congress': perspectiva pericial. Cinco Días, 3 Marzo. Gomez, M., Lopez, C. & Molina, A., 2019. An integrated model of social media brand engagement. Computers in Human Behavior, Volume 96, pp. 196-206. Güell, O., 2020. Origen, síntomas, letalidad... Lo que se sabe del nuevo virus de China. El País, 31 Enero. Harjoto, M. & Salas, J., 2017. Strategic and institutional sustainability: corporate social responsibility, brand value, and Interbrand listing. Journal of Product & Brand Management, 26(6), pp. 545-558. pp Harrigan, P., Evers, U., Miles, M. & Daly, T., 2017. Customer engagement with tourism social media brands. Tourism management, Volume 59, pp. 597-609. Hu, Y., Manikonda, L. Acknowledgements & Kambhampati, S., 2014. What we instagram: A first analysis of instagram photo content and user types. s.l., In Eighth International AAAI conference on weblogs and social media, pp. 595-498. pp Jacomy, M., Venturini, T., Heymann, S. & Bastian, M., 2014. ForceAtlas2, a continuous graph layout algorithm for handy network visualization designed for the Gephi software. PloS one, 9(6). Jun, S. & Park, D., 2017. Visualization of brand positioning based on consumer web search information: Using social network analysis. Internet Research, 27(2), pp. 381-407. Kamada, T. & Kawai, S., 1989. An algorithm for drawing general undirected graphs. Information processing letters, 31(1), pp. 7-15. Kantar, 2020. Media Trends & Predictions, s.l.: Kantar. Kantar, 2020. Media Trends & Predictions, s.l.: Kantar. Kaplan A & Haenlein M 2010 Users of the world u Kantar, 2020. Media Trends & Predictions, s.l.: Kantar. Kaplan, A. & Haenlein, M., 2010. Users of the world, unite! The challenges and opportunities of Social Media. Business horizons, 53(1), pp. 59-68. Kobourov, S., 2012. Force-directed drawing algorithms. Arizona: University of Arizona. Kaplan, A. & Haenlein, M., 2010. Users of the world, unite! The challenges and opportunities of Social Media. Business horizons, 53(1), pp. 59-68. of Social Media. Business horizons, 53(1), pp. 59-68. Kobourov, S., 2012. Force-directed drawing algorithms. Arizona: University of Arizona. Laroche, M., Habibi, M. R., Richard, M. & Sankaranarayanan, R., 2012. The effects of social media based brand communities on brand community markers, value creation practices, brand trust and brand loyalty. Computers in Human Behavior, 28(5), pp. 1755-1767. Leung, X., Bai, B. & Erdem, M., 2017. Hotel social media marketing: a study on message strategy and its effectiveness. Journal of Hospitality and Tourism Technology, 8(2), pp. 239-255. Mangold, W. & Faulds, D. J., 2009. Social media: The new hybrid element of the promotion mix. Business horizons, 52(4), pp. 357-365. McCune, Z. & Thompson, J., 2011. Consumer production in social media networks: A case study of the ‘Instagram’iPhone app, s.l.: University of Cambridge. Mena, J., Hult, G., Ferrell, O. & Zhang, Y., 2019. Competing assessments of market-driven, sustainability-centered, and stakeholder-focused approaches to the customer-brand relationships and performance. Journal of Business Research, Volume 95, pp. 531-543. Michel, M., 2020. Armani celebra su desfile en Milán a puerta cerrada, sin invitados, por el coronavirus. El Mundo, 20 Febrero. Pentina, I., Gammoh, B., Zhang, L. & Mallin, M., 2013. Acknowledgements Drivers and outcomes of brand relationship quality in the context of online social networks. International Journal of Electronic Commerce, 17(3), p. 63–86. Petts, J., Draper, H., Ives, J. & Damery, S., 2010. Risk communication and pandemic influenza. In: Risk communication and public health. s.l.: University of Glasgow, pp. 147-163. ROBECOSAM AG, 2019. Dow Jones Sustainability World Index, Switzerland: s.n. S hi i ki B & D b ki D 2015 Th i t f b d i ti b ROBECOSAM AG, 2019. Dow Jones Sustainability World Index, Switzerland: s.n. Schivinski, B. & Dabrowski, D., 2015. The impact of brand communication on brand. ROBECOSAM AG, 2019. Dow Jones Sustainability World Index, Switzerland: s.n. Schivinski, B. & Dabrowski, D., 2015. The impact of brand communication on brand. T Schivinski, B. & Dabrowski, D., 2015. The impact of brand communication on brand. The l f R h i I di M di i 9(1) 31 53 Schivinski, B. & Dabrowski, D., 2015. The impact of brand communication on brand. The Journal of Research in Indian Medicine, 9(1), p. 31–53. Journal of Research in Indian Medicine, 9(1), p. 31–53. Scott, J., 1988. Social network analysis. Sociology, 22(1), pp. 109-127. Journal of Research in Indian Medicine, 9(1), p. 31 53. Scott, J., 1988. Social network analysis. Sociology, 22(1), pp. 109-127. Simon, C. & Sullivan, M., 1993. The measurement and determinants of brand equity: A financial approach. Marketing science, 12(1), pp. 28-52. Simon, C. & Sullivan, M., 1993. The measurement and determinants of brand equity: A financial approach. Marketing science, 12(1), pp. 28-52. Smit, E., Bronner, F. & Tolboom, M., 2007. Brand relationship quality and its value for personal contact. Journal of Business Research, 60(6), p. 627–633. p J f ( ) p Tsai, W. H. S. & Men, L. R., 2013. Motivations and antecedents of consumer engagement with brand pages on social networking sites. Journal of Interactive Advertising, 13(2), pp. 76-87. United Nations, 2020. Everyone Included: Social Impact of COVID-19, s.l.: s.n. Wasserman, S. & Faust, K., 1994. Social network analysis: Methods and applications. s.l.:s.n. Wu, X. et al., 2020. Exposure to air pollution and COVID-19 mortality in the United States. Tsai, W. H. S. & Men, L. R., 2013. Motivations and antecedents of consumer engagement with brand pages on social networking sites. Journal of Interactive Advertising, 13(2), pp. 76-87. United Nations, 2020. Everyone Included: Social Impact of COVID-19, s.l.: s.n. Wasserman, S. Acknowledgements & Faust, K., 1994. Social network analysis: Methods and applications. s.l.:s.n. Wasserman, S. & Faust, K., 1994. Social network analysis: Methods and applications. s.l.:s.n. Wu, X. et al., 2020. Exposure to air pollution and COVID-19 mortality in the United States. medRxiv, p. In Press. p Yoo, B., Donthu, N. & Lee, S., 2000. An examination of selected marketing mix elements and brand equity. Journal of the academy of marketing science, 28(2), pp. 195-211. Zarco, C., Santos, E. & Cordón, O., 2019. Advanced visualization of Twitter data for its analysis as a communication channel in traditional companies. Progress in Artificial Intelligence, 8(3), pp. 307-323. Zarco, C., Santos, E. & Cordón, O., 2019. Advanced visualization of Twitter data for its analysis as a communication channel in traditional companies. Progress in Artificial Intelligence, 8(3), pp. 307-323. Figures Figures Figure 1 Example of a biparte network and its two projections Figure 1 Example of a biparte network and its two projections Figure 1 Example of a biparte network and its two projections Figure 2 Figure 2 Twitter Spain bipartite network Figure 2 Figure 2 Twitter Spain bipartite network Figure 3 Instagram Spain bipartite network Instagram Spain bipartite network Figure 4 Twitter Italy bipartite network Figure 5 Figure 5 Instagram Italy bipartite network Figure 6 Instagram Italy bipartite network Instagram Italy bipartite network Instagram Italy bipartite network Figure 6 Twitter Spain brand network Figure 4 Twitter Italy bipartite network Figure 5 Instagram Italy bipartite network Figure 6 Twitter Spain brand network Figure 6 Twitter Spain brand network Figure 7 Twitter Spain hashtag network Figure 8 Instagram Spain brand network Figure 8 Instagram Spain brand network Figure 8 Instagram Spain brand network Instagram Spain brand network Figure 9 Instagram Spain hashtag network Figure 9 Instagram Spain hashtag network Figure 10 Figure 11 Twitter Italy hashtag network Figure 11 Twitter Italy hashtag network Figure 11 Twitter Italy hashtag network Twitter Italy hashtag network Figure 12 Figure 10 Twitter Italy brand network Figure 12 Figure 12 Instagram Italy brand network Figure 13 Instagram Italy hashtag network
W597301671.txt	https://archive.org/download/fredericmistral00downiala/fredericmistral00downiala_bw.pdf	fr		Frédéric Mistral. Poet and Leader in Provence	Columbia University Press eBooks	1,901	public-domain	50,924	AX J?- l^^fflj ?^% f / 30; Digitized by the Internet Arciiive in 2007 with funding from IVIicrosoft Corporation http://www.archive.org/details/fredericmistralOOdowniala Columbia toibersitg STUDIES IN ROMANCE PHILOLOGY AND LITERA rURE FREDERIC MISTRAL POET AND LEADER IN PROVENCE y^y^ FRl:DfeRIC MISTRAL FREDERIC MISTRAL POET AND LEADER IN PROVENCE BY CHARLES ALFRED DOWNER ASSISTANT PROFESSOR IN THE FRENCH LANGUAOS AND LITERATURE IN THE COLLEQB OF THB CITT OF NEW YORK "Ntta gorfe THE COLUMBIA UNIVERSITY PRESS THE MACMILLAN COMPANY, Agents 66 Fifth Atbnub 1901 AU rigkU rttervtd COPTBIOBT, 1901, Bi THE MACMILLAN COMPANY. J. 8- Cnihing It Co. — Berwick k Smith Norwood Mms. VS.A. PREFACE This study of the poetry and life-work of the leader of the modern Provencal renaissance was submitted in partial fulfilment of the requirements for the degree of Doctor of Philoso- phy at Columbia University. My interest in Mistral was first awakened by an article from the pen of the great Romance philologist, Gaston Paris, which appeared in the Revue de Paris in October, 1894. The idea of writing the book came to me during a visit to Provence in 1897. Two years later I visited the south of France again, and had the pleasure of seeing Mistral in his own home. It is my pleasant duty to express here once again my gratitude for his kindly hospitality and for his sugges- works upon the history of Not often does he who studies tions in regard to the Felibrige. the works of a poet in a foreign tongue enjoy as I did the privilege of hearing the verse from the poet's own lips. It was an hour not to be PREFACE vi forgotten, and the beauty of the language has been for me since then as real as that of music finely rendered, and the force of the poet's per- sonality was impressed upon me as it scarcely could have been even from a most sympathetic and searching perusal of his works. His great influence in southern France and his great per- sonal popularity are not difficult to understand when one has seen the man. As the striking fact in the works of this Frenchman is that they are not written in French, but in Provencal, a considerable portion of the present essay language itself. is devoted to the But it did not appear fitting that too much space should be devoted to the purely linguistic side of the subject. There is a field here for a great deal of special study, and the results of such investigations will be embodied in special works by those who make philological studies their special province. In the first division of the present work, however, along with the life of the poet and the history of the Felibrige, a description of the language is given, which is distinctive features. an account at least of its A short chapter will be found devoted to the subject of the versifica- PREFACE vii tion of the poets who write in the new speech. This subject is not treated in Koschwitz's admirable grammar of the language. The second division is devoted to the poems. The epics of Mistral, if we may venture to use the term, are, with the exception of Lamartine's Jocelyn^ the most remarkable long narrative poems that have been produced in France in At least one of them would modern times. appear to be a work of the highest rank and Among the short poems that destined to live. constitute the volume called Lis Isclo d' Or are a number of masterpieces. This book aims to present all the essential facts in the history of this astonishing revival of a language, and to bring out the chief aspects of Mistral's life-work. In our conclusions we have not yielded to the temptation to prophesy. The conflicting tendencies of cosmopolitanism and nationalism abroad in the world to-day give rise to fascinating speculations as to the future. In the Felibrean movement we have a very interesting problem of this kind, and no one can terminate a study of the subject without asking himself the question, "What is going to come out of it all ? " No one can tell, and PREFACE viii SO we have not ventured beyond the attempt to present the case as it actually exists. Let me here also offer an expression of gratitude to Professor Adolphe Cohn and to Professor Henry A. Todd of Columbia University for their advice six years. and guidance during the past Their kindness and the inspiration of their example must be reckoned among those things that cannot be repaid. New Yobk, March, 1901. CONTENTS PART FIRST The Revival of the Provencal Language ... CHAPTER I. Introduction. II. The Fdlibrige Life of Mistral 3 24 III. The Modern Proven9al, or, more accurately, rV. The Language of the Felibres The Versification of the Felibres V. Mistral's guage. PA6B ... ... 43 75 Dictionary of the Proven9al Lan- (Lou Tresor ddu Felibrige) . . 92 PART SECOND The Poetical Works of Mistral L The Four Longer Poems 1. Mireio 2. Calendau 99 . . . . . 3. Nerto 4. Lou Poufemo ddu Rose . 99 127 151 n. Lis Isclo d'Or III. The Tragedy, La Reino Jano 159 181 . ix 212 CONTENTS X PART THIRD PAOB Conclusions 237 Appendix. 253 Translation of the Fsalm of Penitence Bibliography 259 Index 265 PART FIRST THE BEVIVAL OF THE PROVENgAL LANGUAGE FREDERIC MISTRAL CHAPTER I INTRODUCTION The present century has witnessed a remarkable literary phenomenon in the south of France, a remarkable rebirth of local patriotism. A language has been born again, so to speak, and once more, after a sleep of many hundred years, the sunny land that was the cradle of modern literature, offers us a new efflorescence of poetry, embodied in the musical tongue that never has ceased to be spoken on the soil where the Troubadours sang of love. Those who began this movement knew not whither they were tending. From small beginnings, out of a kindly desire to give the humbler folk a simple, homely literature in the their firesides, there language of grew a higher ambition. The ProveuQal language put forth claims to exist coequally with the 3 French tongue on 4 frI:d6ric mistral French soil. Memories of the former glories of the southern regions of France began to the modern poets and They began to chafe under the strong stir within the hearts of leaders. political and intellectual centralization that pre- vails in France, change. and to seek to bring about a The movement has passed through numerous phases, has been frequently misinterpreted and misunderstood, and may now, after it has attained to tangible results, be defined as an aim, on the part of its leaders, make the south intellectually independent of Paris. It is an attempt to restore among to the people of the Rhone region a love of their ancient customs, language, and traditions, an effort to raise a sort of dam against the flood modern tendencies that threaten to overwhelm local life. These men seek to avoid of that dead level of uniformity to which the national life of France appears to them in danger of sinking. leaders of this at Paris In the earlier days, the movement were often accused of a spirit of political separatism; they were actually mistrusted as secessionists, and certain it is that among them have been several champions of the idea of decentraliza- INTRODUCTION 6 To-day there are found in their ranks a few who advocate the federal idea in the politition. cal organization of France. However, there seems never to have been a time when the movement promised seriously to bring about practical political changes; and whatever po- may have to-day goes no farther than what may be contained in germ in litical significance it the effort at an intense local life. The land of the Troubadours is now the land of the Felibres these modern singers do not ; forget, nor will they allow the people of the south to forget, that the union of France with Provence was that of an equal with an equal, not of a principal with a subordinate. Patriots they are, however, ardent lovers of France, and proofs of their strong affection for their country are not wanting. To-day, amid all their activity and demonstrations in behalf of what they often call " la petite patrie" no enemies or doubters are found to question their loyalty to the greater fatherland. The movement began in the revival of the Provengal language, and was at first a very modest attempt to make it serve merely better purposes than it had done after the eclipse that fr6d6ric mistral 6 For a long time followed the Albigensian war. the linguistic and literary aspect of all this was the only one that attracted any activity attention in the rest of France or in Provence itself. Not that the Provengal language had ever quite died out even as a written language. Since the days of the Troubadours there had been a continuous succession of writers in the various dialects of southern Fjance, but very few of them were men of power and talent. Among the immediate predecessors of the Felibres must be mentioned Saboly, whose Noels, or Christmas songs, are to-day known all over the and Jasmin, who, however, wrote in a Jasmin's fame extended far different dialect. beyond the limited audience for which he wrote region, ; his work came to the attention of the cultured through the enthusiastic praise of Sainte-Beuve, and he is to-day very widely known. The English-speaking world became acquainted with him chiefly through the translations of LongJasmin, however, looked upon himself fellow. as the last of a line, and when, in his later years, he heard of the growing fame of the new poets of the Rhone country, it is said he looked upon them with disfavor, if not jealousy. INTRODUCTION 7 Strange to say, he was, in the early days, un- known to those whose works, like his, have now attained well-nigh world-wide celebrity. The man who must justly be looked upon as the father of the present movement was Joseph Roumanille. He was born in 1818, in the little town of Saint-Remy, a quaint old place, proud of some remarkable Roman remains, situated Roumanille was far to the south of Avignon. from foreseeing the consequences of the impulse he had given in arousing interest in the old dialect, and, until he beheld the astonislihig successes of Mistral, strongly disapproved the ambitions of a number of his fellow-poets to seek an audience foy their productions outside of the immediate region. He had no more ambi- tious aim than to raise the patois of Saint-Remy out of the veritable mire into which it had sunk ; it pained him to see that the speech of his fire- side was never used in writing except for trifles Of him is told the touching story that one day, while reciting in his home before a company of friends some poems in and obscenities. French that he had written, he observed tears in his mother's eyes. She could not understand the poetry his friends so much admired. Rou- FRfeDfiRIC 8 MISTRAL manille, much moved, resolved to write no verses that his mother could not enjoy, and henceforth devoted himself ardently to the task of purifying and perfecting the dialect of Saint-Remy. It has been said, no less truthfully than poetically, that from a mother's tear was born the new Provengal poetry, destined to so splendid a career. We of the English-speaking race are apt to wonder at this love of a local dialect. This vigorous attempt to create a first-rate literature, alongside and independent of the national literature, seems strange or unnatural. We are accus- tomed to one language, spoken over immense areas, and we rejoice to see it grow and spread, With all more and more perfectly unified. their local color, in spite of their expression provincial or colonial the writings of of life, a Kipling are read and enjoyed wherever the English language has penetrated. find patriots In Italy we and writers working with utmost energy to bring into being a really national language. Nearly all the governments of Europe seek to impose the language of the capital upon the schools. Unification of language seems a most desirable thing, and, superficially consid- INTRODUCTION 9 ered, the tendency would appear to be in that direction. But the truth is that there exists all over Europe a war of tongues. The Welsh, the Basques, the Norwegians, the Bohemians, the Finns, the Hungarians, are of one mind with Daudet and Mistral, who both express the sentiment, " He who holds to his language, holds the key of his prison." So Roumanille loved and cherished the melodious speech of the Rhone valley. He hoped to see the langue d'oc saved from destruction, he strove against the invasion of the northern speech that threatened to overwhelm it. He wrote sweet verses and preached the gospel of the home-speech. One day he discovered a boy whom he calls " I'enfant sublime," and the pupil soon carried his dreams to a realization far Not Roumanille, but Frederic Mistral has made the new Provengal literature what it is. In him were combined beyond his fondest hopes. all the qualities, all the powers requisite for the task, and the task grew with time. It became more than a question of language. Mistral soon came to seek not only the creation of an independent literature, he aimed at nothing less than a complete revolution, or rather a com- FRfeDfjRIC 10 plete rebirth, of France. ality MISTRAL the mental southern life of Provence was to save her individuGeographically at the central entire. point of the lands inhabited by the so-called Latin races, she was to regain her ancient prominence, and cause the eyes of her sisters to turn her way once more with admiration and affec- The tion. patois of Saint- Remy has been developed and expanded into a beautiful erary language. lit- The inertia of the Provencals themselves has been overcome. There is un- doubtedly a new intellectual life in tlie Rhone valley, and the fame of the Felibres great work has gone abroad and their into distant lands. The purpose, then, of the present dissertation, will be to give an account of the language of the Felibres, and to literary examine critically the work of their acknowledged chief and guiding spirit, Frederic Mistral. The story of his life he himself has told most admirably in the preface to the first edition of Ids- Isclo d' Or, published at Avignon in 1874. He was born in 1830, on the 8th day of September, at Maillane. Maillane is a village, near Saint-Remy, situated in the centre of a broad INTRODUCTION 11 plain that lies at the foot of the Alpilles, the westernmost rocky heights of the Alps. Here the poet is still living, and here he has passed his life almost uninterruptedly. His father's home was a little way out of the village, and the boy was brought up at the mas,^ amid farm- hands and shepherds. His father had married a second time at the age of fifty-five, and our poet was the only child of this second marriage. The story of the first meeting of his parents is thus told by the poet : — "One year, on St. John's day, Maitre Fran^ Qois Mistral was in the midst of his wheat, which a company of harvesters were reaping. A throng of young girls, gleaning, followed the reapers and raked up the ears that fell. Maitre Frangois (Meste Frances in Provencal), father, noticed a beautiful girl that behind as if my remained she were ashamed to glean like — the others. He drew near and said to her " ' My child, whose daughter are you? What : is your name ? ^The word mas, which is kin with the English manse and mansion, signifies the home in the country with numerous outbuildings grouped closely about it. FR^D^RIC MISTRAL 12 " The young girl replied, ' I am the daughter of Etienne Poulinet, Maire of Maillane. name is Delaide.' "What! the daughter Maillane gleaning the of My Maire of ! " ' Maitre,' she replied, ' our family is large, six girls and two boys, and although our father well to do, as you know, when we ask him for money to dress with, he answers, " Girls, And that is why if you want finery, earn it " I came to glean.' "Six months after this meeting, which reminds one of the ancient scene of Ruth and is pretty ! Boaz, Maitre Francois asked Maitre Poulinet for the hand of Delaide, and I was born of that marriage." His father's lands were extensive, and a great number of men were required to work them. The poem, Mireio^ is filled with pictures of the Of sort of life led in the country of Maillane. his father he says that he towered above them all, in stature, in wisdom, and in nobleness of bearing. He was a handsome old man, digni- fied in language, firm in command, kind to the poor about him, austere with himself alone. The same may be said of the poet to-day. He INTRODUCTION is 13 a strikingly handsome man, vigorous and active, exceedingly manner. gracious and simple in His utter lack of affectation is the more remarkable, in view of the fact that he has been for years an object of adulation, and lives in constant and close contact with a population of peasants. His schooling began at the age of nine, but the boy played truant so frequently that he was sent to boarding-school in Avignon. Here he had a sad time of it, and seems especially to have felt the difference of language. and pupils alike made fun of Teachers his patois, for which he had a strong attachment, because of the charm of the songs his mother sung to Later he studied well, however, and became filled with a love of Virgil and Homer. In them he found pictures of life that recalled vividly the labors, the ways, and the ideas of him. the Maillanais. At this time, too, he attempted a translation, in Proven 9al, of the first eclogue and confided his efforts to a schoolwho became his lifelong friend and one of the most active among of Virgil, mate, Anselme Mathieu, the Felibres. It was at this school, in 1845, that he formed FRfeDfiRIC MISTRAJ. 14 his friendship with Roumanille, who there as a teacher. It is that the revival of grew out of this had come not too much to say the Provencal meeting. language Roumanille had already written his poems, Li Margarideto (The Daisies). "Scarcely had he shown me," says Mistral, "in their spring-time freshness, these lovely field-flowers, when a thrill ran through my being and I exclaimed, 'This is the dawn my soul awaited to awaken to the light ! ' Mistral had read some Provengal, but at that time the dialect was employed merely in derision ; the writers used the speech itself as the chief comic element in their productions. The poems of Jasmin were as yet unknown to him. Roumanille was the first in the Rhone country to sing the poetry of the heart. Master and pupil became firm friends and worked together for years to raise the home-speech to the dignity of a literary language. At seventeen Mistral returned home, and began a poem in four cantos, that he has never published ; though portions of it are among the poems of Lis Isclo d'Or and in the notes of This poem is called Li Meissoun (Har- Mireio. vest). His family, seeing his intellectual supe- INTRODUCTION riority, sent 16 him to Aix to study law. Here he again met Mathieu, and they made up for the aridity of the Code by devoting Civil themselves to poetry in Provengal. In 1851 the young man returned to the wa«, a licenciS en droits and his father said to him " Now, my dear son, I have done my duty ; you know more than ever I learned. Choose your And the poet tells career; I leave you free." us he threw his lawyer's gown to the winds and gave himself up to the contemplation of what he so loved, — the splendor of his native Provence. Through Roumanille he came Aubanel, Croustillat, and others. at to know They met Avignon, full of youthful enthusiasm, and during this period Mistral, encouraged by his friends, worked upon his greatest poem, Mireio. In 1854, on the 21st of May, the Felibrige was founded by the seven poets, nille, — Joseph Rouma- Paul Giera, Theodore Aubanel, Eugene Garcin, Anselme Mathieu, Frederic Alphonse Tavan. a violent attack Mistral, In 1868, Garcin published upon the Felibres, accusing them, in the strongest language, of seeking to bring about a political separation of southern France from the rest of the country. This FRfeDfeRIC 16 MISTRAL apostasy was a cause of great grief to the and Garcin's name was stricken from others, the official list of the founders of the Felibrige, and replaced by that of Jean Brunet. Mistral, in the sixth canto of Mireio, addresses in elo- quent verse his comrades and there we Pleiade, still in the Provengal find the name of Garcin. Tu' nfin, de quau un vent de flamo Ventoulo, emporto e fouito I'amo Garcin, o fieu ardent ddu manescau d' Alen I (And finally, thou whose soul is stirred and swept and whipped by a wind of flame, Garcin, ardent son of the smith of Alleins.) This attack upon the Felibrige was the first of the kind ever made. Many years later, Gar- cin became reconciled to his former friends and in 1897 he was vice-president of the Felibrige de Paris. The number seven and the task undertaken by these poets and literary reformers remind us instantly of the Pleiade, whose work in the sixteenth century in attempting to perfect the French language was of a very similar character. It is certain, however, that the seven INTRODUCTION 17 poets who inaugurated their work at the Cha- teau of Font-Segugne, had no thought of imitating the Pleiade either in the choice of the number seven or in the reformation they were about to undertake. They began their propaganda by founding an annual publication called the Armaria Prouvengau^ which has appeared regularly since 1855, and many of their writings were first printed in Of the seven, Aubanel this official magazine. alone besides Mistral has attained celebrity as a poet, and these two with Roumanille have been usually associated in the minds of all who have followed the movement with interest as its three leaders. The poem Mistral completed Mireio in 1859. was presented by Adolphe Dumas and Jean Reboul to Lamartine, who devoted to it one of the " Entretiens " of his Cours familier de This article of Lamartine, and his littSrature. personal efforts on behalf of Mistral, contrib- uted greatly to the success of the poem. " Lam- A great artine wrote among other things epic poet born A true Homeric poet in is our own time ; ! : a poet, born like the men of Deucalion, from a stone on the Crau, a primi- FR^D^RIC MISTRAL 18 tive poet in our decadent age ; a Greek poet at Avignon ; a poet who has created a language out of a dialect, as Petrarch created Italian ; one who, out of a vulgar patois^ has made a language full of imagery and harmony delighting the imagination and the ear. might say of the that, Archipelago, parted from its . . . We during the night, an island a Delos, floating has group of Greek or Ionian islands and come silently to join the mainland of sweet-scented Provence, bringing along one of the divine singers of the family of the Mele- sigenes." Mistral went to Paris, where for a time he was The French Academy crowned his poem, and Gounod comthe lion of the literary world. posed the opera Mireille, which was performed for the first time in 1864, in Paris. The poet did not remain long in the capital. He doubtless realized that he was not destined to join the galaxy of Parisian writers, and it is certain that if he had remained there his life and his influence would have been utterly different. He returned home and immediately in another set to work upon a second epic ; seven years he completed Calendau, published INTRODUCTION in Avignon in 1866. 19 The success of this poem was decidedly less than that of Miriio. During these years he published many of the shorter poems that appeared in one volume in (The Meanwhile the idea of the 1875, under the title of Lis Isclo d'Or Golden Islands). The language now a fixed orthography and definite grammatical form. The appearFelibrige made great progress. of the Felibres had ance of a master-work had given a wonderful impulse. The exuberance of the southern tem- perament responded quickly to the call for a manifestation of patriotic enthusiasm. The Catalan poets joined their brothers beyond the The games were founded. The Felibrige passed westward beyond the Rhone and found adherents in all south France. Pyrenees. Floral The centenary of Petrarch celebrated at Avignon in 1874 tended to emphasize the importance and the glory of the new literature. The definite organization of the Felibrige into a great society with its hierarchy of officers took place in 1876, with Mistral as CapouliS (Chief or President). In this same year also the poet married Mdlle. Marie Riviere of Dijon, and this lady, who was named first Queen of the Felibrige FRlfeD^RIC aO MISTRAL by Albert de Quintana of Catalonia, the poetlaureate of the year 1878 at the great Floral Games held in Montpellier, has become at heart and in speech a Provengale. A third poem, Nerto^ appeared in 1884, and showed the poet in a new light his admirers now compared him to Ariosto. This same year he made a second journey to Paris, and was ; again the lion of the hour. Cigale, which The SociStS de la had been founded in 1876, as a Paris branch of the Felibrige, and which later became the SoeiitS des FSlibres de Paris, organized banquets and festivities in his honor, and celebrated the Floral Games at Sceaux to commemorate the four hundredth anniversary of the day when Provence became united, of her own free-will, with France. Mistral was received with distinction by President Grevy and by the Count of Paris, and his numerous Parisian friends vied in bidding him welcome to the capi- His new poem was crowned by the French Academy, receiving the Prix Vitet, the presentation address being delivered by Legouve. Four years later, Lou Tresor d6u Felibrige, a tal. great dictionary of all the dialects of the langue d'oc, was completed, and in 1890 appeared his INTRODUCTION only dramatic work, Joanna). 21 La Reino Jano (Queen In 1897 he produced his last long poem, epic in form, Lou Pouemo d6u Rose (the Poem of the Rhone). At present he is engaged upon his Memoirs. Aside from his rare journeys to Paris, a visit to Switzerland, and another to Italy, Mistral has rarely gone beyond the borders of his beloved He is still living quietly in the little region. village of Maillane, in a simple but beautiful home, surrounded with works of art inspired He has survived by the Felibrean movement. many of his distinguished friends. Rouma- Aubanel, Daudet, and Paul Arene have all passed away a new generation is about him. But his activity knows no nille, Mathieu, ; rest. The Felibrean festivities continue, the numerous publications in the Proven9al tongue still have in him a constant contributor. In 1899 the Museon Arlaten (the Museum of Aries) was inaugurated, and of the constant energy poet. is another proof and enthusiasm of the He is to-day the greatest man in the south of France, universally beloved and revered. His life after all has been less a literary life 22 FRiJDfeRIC than one of direct MISTRAL and unceasing personal action upon the population about him. The resurrection of the language, the publication of poeras, magazines, and newspapers, are only part of a programme tending to raise the people of the south to a conception of their individuality as a race. He has striven untiringly to communicate to them his own glowing enthusi- asm for the past glories of Provence, to fire them with his dream of a great rebirth of the Latin races, to lay the foundation of a great ideal Latin union. Wonderful is his optimism. Some of the Felibres about him are somewhat discouraged, many of them have never set their aspirations as high as he has done, look upon his dreams as Utopian. and some Whatever be the future of the movement he has founded. Mistral's life in its simple oneness, and in its astonishing success, is indeed most remarkable. Provence, the land that first gave the world a literature after the decay of the classic tongues, has awakened again under his magic touch to an active mental life. A second literature is in active being on the soil of France, a second literary language is there a reality. permanent or evanescent, this Whether glorification of INTRODUCTION 28 poetry, this ardent love of the beautiful the ideal, is a noble and inspiring and spectacle amid the turmoil and strife of this age of material progress. CHAPTER II THE FELIBRIGE The history of the Felibrige, from its beginning, in 1854, down to the year 1896, has been admirably written by G. Jourdanne.^ The work is quite exhaustive, containing, in addition to the excellently written narrative, an engraving of the famous cup, portraits of all the most noted Felibres, a series of elaborately written notes that discuss or set forth many questions relating to the general theme, a very large bibliography of the subject, comprising long lists of works that have been written in the dialect or that have appeared in France and in other countries concerning the Felibres, a copy of the constitution of the society and of various statutes relating to 1 Histoire it. It not only du Felibrige, par G. Jourdanne, Librairie Bou- manille, Avignon, 1897. 24 THE F^LIBRIGE contains all the material that the study of the Felibrige, but 26 is necessary for it is worthy of the highest praise for the spirit in which it is an honest attempt to explain the Felibrige, and to present fairly and fully all written. It is the problems that so remarkable a movement has A perusal of the book makes it evi- created. dent that the author believes in future political consequences, and while well aware that it is unsafe to prophesy, he has a chapter on the future of the movement. His history endeavors to show that the Felibrean renaissance was not a spontaneous springing into existence. however, it On the purely literary side, certainly bears the character of a creation; as writers, the Provengal poets may scarcely be said to continue any preceding school or to be closely linked with any literary past. In its inception it was a mere attempt to write pleasing, popular verse of a better kind in the dialect of the fireside. But the movement developed rapidly into the ambition to endow the whole region with a real literature, to awaken a consciousness of race in the men of the south; these aims have been realized, and a change has come over the life of Provence and FRfeD]fcRIC 26 MISTRAL the land of the langue d'oc in general. The author believes and adduces evidences to show that all this could not have come about had the seed not fallen upon a soil that was ready. The Felibrige dates from the year 1854, but the idea that lies at the bottom of it must be traced back to the determination of Roumanille to write in Provencal rather than in French. He produced his Margarideto in 1847 and the Sounjarello in 1851. In collaboration with Mistral and Anselme Mathieu, he edited a col- poems by living writers under the During these years, too, there were meetings of Provengal writers for lection of title lA Prouvenpalo. the purpose of discussing questions of grammar and spelling. These meetings, including even May 21, 1854, were, howmore than friendly, social gatherings, where a number of enthusiastic They had friends sang songs and made merry. the historic one of ever, really little none of the solemnity of a conclave, or the dignity of literary assemblies. There was no formal organization. Those writers who were zealously interested in the rehabilitation of the Provengal speech and connected themselves with Mistral and his friends were the Feli- THE FJfcLIBRIGE Not bres. until 27 1876 was there a Felibrige with a formal constitution and an elaborate organization. The word FSlibre was furnished by Mistral, who had come upon it in an old hymn wherein occurs the expression that the Virgin met Jesus in the temple among "the seven Felibres of The origin and etymology of this the law." word have given rise to various explanations. The Greek philabros, lover of the beautiful; philebraios, lover of Hebrew, hence, among the Jews, teacher; felibris, nursling, according to Ducange; the Irish Jilea, bard, and ber, chief, Jeanroy (in Romania, have been proposed. XIII, p. 463) offers the etymology : Spanish feligres, filii Ucclesice, sons of the church, parish- ioners. None of these is certain. Seven poets were present at this first meeting, and as the day happened to be that of St. emblem of a seven-pointed star was adopted. Very fond of the number seven are these Felibres; they tell you of the seven Estelle, the chief churches of Avignon, its seven gates, seven colleges, seven hospitals, seven popes who were there seventy years; the word FSlibre has seven letters, so has Mistral's name, FRfeDlfeRIC 28 MISTRAL and he spent seven years in writing each of his epics. The task that twofold they had : was prune and lay before these poets not only to purify their dialect and produce verses, they had also to find readers, to create a public, to begin a propaganda. The first means adopted was the publication of the Armana prouvengau^ already referred to. In 1855, five hundred copies were issued, in 1894, twelve thousand. For four years this magazine was destined for Provence alone of Mireio, it ; in 1860, after the appearance was addressed to all the dwell- ers in southern France. The great success of Mireio began a new period in the history of the Mistral himself and the poets about Felibrige. him now took an entirely new view of their The uplifting of the people, the creamission. tion of a literature that should be admired abroad as well as at home, the complete expression of the aspects, past and present, escape from the im- placable life of centralization Provence, in that all its tends to destroy — and originality such were the higher aims toward which they now bent their all initiative efforts. The attention of Paris was turned in THE F^LIBRIGE their direction. 29 Jasmin had already shown the Parisians that real poetry of a high order could Lamartine and Villemain welcomed the new literature most cordially, and the latter declared that " France is rich enough to have two literatures." But the student of this history must not lose be written in a patois. sight of the fact that the Provencal poets are not first of all litterateurs ; they are not men devoting themselves to literature for a livelihood, or even primarily for fame. patriots before they are poets. They are The choice of subjects and the intense love of their native land that breathes through all their writings, are ample proof of this. They meet to sing songs and to speak always of Provence ; it is that they sing and speak. are men who ply some trade, hardly one lives by his pen alone. cial Almost all of them This fact gives a very spe- character to their whole production. The Felibrean movement is more than an astonish- ing literary phenomenon. The idea from this time on acquired more and more adherents. Scores of writers appeared, and volumes whose titles filled many pages swelled the output of Provencal verse. MISTRAL FRfeDfiRIC 80 These new aims were due to the success Mireio ; of but it must not be forgotten that Mis- poem and in the shorter poems of the same period, gave distinct exprestral himself, in that sion to the new order of ideas, so that we are constantly led back to him, in all our study of the matter, as the creator, the continuer, and the ever present inspirer of the Felibrige. Whatever it is, it is through him primarily. Roumanille must be classed as one of those precursors who are unconscious of what they do. To him the Felibres owe two things: first of the idea of writing in the dialect all, works of literary merit ; and, secondly, the dis- covery of Frederic Mistral. Among these new ideas, one that dominates henceforth in the story of the Felibrige, is the idea of race. Mistral is well aware that there is no Latin race, in the sense of blood relation- ship, of physical descent ; he knows that the so-called Latin race has, for the base of its unity, a common history, a common tradition, a com- mon religion, a common language. But he dionale believes that there is a race mSri- that has been developed into a kind of unity out of the various elements that com- THE FfeLIBRIGE 31 pose it, through their being mingled together, and accumulating during many centuries common memories, ideas, customs, and interests. So Mistral has devoted himself to promoting knowledge of its history, traditions, language, and religion. As the Felibrige grew, and as Mistral felt his power as a poet grow, he sought a larger public to the peoples ; he turned naturally most closely related to his own, and Italy and Spain were embraced in his sympathies. The Felibrige spread beyond the limits of France first into Spain. Victor Balaguer, ex- iled from his native country, was received with William Bona- open arms by the Provengals. parte-Wyse, an Irishman and a grand-nephew of the first Napoleon, while on a journey through Provence, had become converted to the Felibrean doctrines, and became an active spirit among these poets and orators. He organized a festival in honor of Balaguer, and when, later, the Catalan poet was permitted to return home, the Catalans sent the famous cup to For the their friends in Provence. Felibres this cup is an emblem of the idea of a Latin federation, and as to hand and from it passes from lip to lip at hand the Felibrean FRfeDf:RIC 32 MISTRAL banquets, the scene is not unlike that wherein the Holy Graal passes about among the Knights of the Round Table. Celebrations of this kind have become a regular institution in southern France. Since the day in 1862 when the town of Apt received the Felibres officially, organizing Floral Games, in which prizes were offered for the best poems people have become accustomed to the sight of these triumphal entries of in Provengal, the Reports of these the poets into their cities. brilliant festivities lands. have gone abroad into all If the love of noise and show that char- acterizes the southern temperament has caused somewhat unfavorably on the other hand the has genuine, and the results enthusiasm been these reunions to be criticised as theatrical, 1 The stem of the cup has the form of a palm tree, under which two female figures, representing Catalonia and Provence, stand in a graceful embrace. Below the figures are engraved the two following inscriptions : Morta la diuhen qu'es, Mes jo la crech viva. Ah Ah ! se me sabien entfendre ! ! se me voulien segui (V. Balaguer.) (They say she is dead, but I believe she lives.) — (F. Mistral.) (Ah, if they could understand me Ah, if they would follow me I) ! THE Ff:LIBRIGE real and lasting. 33 The FSlihrSes^ so they are called, have not all taken place in France. In 1868, Mistral, Roumieux, Bonaparte-Wyse, and Paul Meyer went to Barcelona, where they were received with great pomp and ceremony. Men eminent in literary and philological circles in Paris have often accepted invitations to these festivities. In 1876, a Felibrean club, "La was founded in the capital its first president was Henri de Bornier, author of La Professors and students of Mile de Roland. literature and philology in France and in other countries began to interest themselves in the Felibres, and the Felibrige to-day counts among its members men of science as well as men of Cigale," ; letters. In 1874 one of the most remarkable of the celebrations, due to the initiative of M. de Berluc-Perussis, was held at Vaucluse to celebrate the fifth centenary of the death of Petrarch. At this Felihree the Italians first became affiliated to the idea^ and the Italian ambassador. Nigra, the president of the Accade- mia della Crusca, Signor Conti, and Professor Minich, from the University of Padua, were the delegates. The Institute of France was repre- FR^D^RIC MISTRAL S4 This celebration was sented for the first time. highly important and significant, and the scenes of Petrarch's inspirations and the memories of the founder of the Renaissance must have awak- ened responsive echoes in the hearts of the poets who aimed at a second rebirth of poetry and learning in the same region. The following year the SociSti des langues romanes at Montpellier offered prizes for philological as well as purely literary works, and for the first time other dialects than the Provengal proper were admitted in the competitions. The Languedocian, the Gascon, the Limousin, the Bearnais, and the Catalan dialects were thus The members of the jury were men included. of the greatest note, Gaston Paris, Michel Breal, Mila y Fontanals, being of their number. Finally, in 1876, on the 21st of May, the statutes of the Felibrige were adopted. them we quote the following — From : "The Felibrige is gether and encourage established to bring toall those who, by their works, preserve the language of the land of oc, men of science and the artists who study and work in the interest of this as well as the country." THE FijLIBRIGE "Political 86 and religious discussions are for- bidden in the Felibrean meetings." The organization is interesting. The Feli- bres are divided into Majoraux and Mainteneurs. The former are limited to fifty in number, and form the Consistory, which elects its own members; new members are received on the feast of St. Estelle. The Consistory is presided over by a Capoulie, who wears as the emblem of his office a seven-pointed golden star, the other Majoraux, a golden grasshopper. The other Felibres are unlimited in number. Any seven Felibres dwelling in the same place may ask the Maintenance to form them into a school. The schools administer their own affairs. Every seven years the Floral Games are held, at which prizes are distributed; every year, on the feast of St. Estelle, a general meeting of the Felibrige takes place. Each Maintenance must meet once a year. At the Floral Games he who is crowned poetlaureate chooses the Queen, and she crowns him with a wreath of olive leaves. To-day there are three Maintenances within FR^DfeRIC MISTRAL 36 the limits of French soil, Provence, Languedoc, Aquitaine. Among other facts that should doubtless be reported here have been is, the Mistral Capoulies. list of They (1876-1888), Roumanille (1888-1891), and Felix Gras ; the Queens have been Madame Mistral, Mile. Therese Rouma- nille, Mile. Marie Girard, and the Comtesse Marie-Therese de Chevigne, who is descended upon her mother's side from Laura de Sade, generally believed to be Petrarch's Laura. Since the organization went into effect the Felibrige has expanded in many ways, its influ- ence has continually grown, new questions have arisen. Among these last have been burning questions of religion and politics, for although discussions of them are banished from Felibrean meetings, opinions of the most various kind exist among the Felibres, have found expres- sion, and have well-nigh resulted in difficulties. Until 1876 these questions slept. Mistral is a Catholic, but has managed to hold more or less aloof from political matters. Aubanel was a zealous Catholic, and had the title by inheritance of Printer to his Holiness. Catholic, Roumanille was a and an ardent Royalist. When the THE F^LIBRIGE 37 came to extend its limits over into Languedoc, the poet Auguste Foures and his fellows proclaimed a different doctrine, and called up memories of the past with a different view. They affirmed their adherence to the Renaissance meridionale, and claimed equal Felibrige rights for the Languedocian dialect. They was and protested vigorously against asserted, however, that the true tradition republican, the clerical and monarchical parties, which, in their opinion, had always been for Languedoc a cause of disaster, servitude, and misery. The memory of the terrible crusade in the thirteenth century inspired fiery poems among them. Hatred of Simon de Montfort and of the invaders who followed him, federalism free-thought, and found vigorous expression in their productions. all In Provence, too, there have been opinions differing widely from those of the original founders, and the third Capoulie, Felix Gras, was a Protestant. danne writes — Of him M. Jour- : "Finally, in 1891, after the death of Roumanille, the highest office in the Felibrige was taken by a man who could rally about him the two elements that we have seen manifested, 88 fr6d6ric mistral sufficiently Republican satisfy the to most ardent in the extreme Left, sufficiently steady not to alarm the Royalists, a great enough poet to deserve without any dispute the first place in an assembly of poets." He, like Mistral, wrote epics in twelve cantos. His first work, Li CarhouniS^ has on its title- page three remarkable lines : — " I love my village more than thy village, I love my Provence more than thy province, I love France more than all." Possibly no other three lines could express as well the whole spirit of the Felibrige. Our subject being Mistral and not Felix Gras, a passing mention must suffice. One of his remarkable works is called Toloza, and recounts the crusade of the Albigenses, and his novel, The Reds of the Midi, first published in New York in the English translation of Mrs. Thomas A. Janvier, is probably the most remarkable prose work that has been written in Provengal.^ Only the future can tell whether the Provengal will pass cycle, 1 through a prose cycle after its poetic in the manner of all literatures. To In 1899, F^lix Gras published a novel called The White Terror. His death occurred early in 1901. THE FfeLIBRIGE many serious thinkers the 39 attempt to create a complete literature seems of very doubtful success. The problems, then, which confront the Felibres are numerous. Can they, with any assurance of permanence, maintain languages in the same region? two literary It is scarcely necessary to state, of course, that no one dreams of supplanting the French language on French soil. What attitude anywhere shall assume toward the " patoisants," that is, they those who insist on using the local dialect, and refuse to conform to the usage of the Felibres ? Is it not useless, after all, to hope for a more perfect unification of the dialects of the langue cToc^ and, if unification is the aim, does not logical rea- soning lead to the conclusion that the French language already exists, perfectly unified, and absolutely necessary? In the matter of poli- tics, the most serious questions may arise if the desires of some find more general favor. Shall the Felibres aim at local self-government, at a confederation something like that of the Swiss cantons ? Shall they advocate the idea of inde- pendent universities ? As a matter of fact, none of these problems frI}d6ric mistral 40 are solved, and they will only be solved by the The attitude of the natural march of events. leaders toward all these differing views has be- come one of easy toleration. If the language of the Felibres tends already to dominate the other dialects, if its influence is already plainly felt far beyond Provence itself, this is due sheer superiority of their their literature to the literary work. If had the conventional character of that of the Troubadours, if it were addressed exclusively to a certain elite, then their lan- guage might have been adopted by the poets of other regions, just as in the days of the Troubadours the masters of the art of " trobar But the movement has pre- preferred to use the Limousin dialect. popular character of the vented preached the love of the this. village, and It has each locality, as fast as the Feli- brean idea gained ground, has shown greater affection for its own dialect. work has often been compared to But Dante did not impose his lanDante's. guage upon Italy by the sole superiority of his Mistral's great poem. social, little All sorts of events, political and contributed to the result, and there is reason to expect the same future for the THE F^LIBRIGE work of Mistral. 41 from the linguistic point of view; likely that poets. is made it is This comparison not any one will compare the two as At most, it may be said that if Dante gave expression to the whole spirit of his age, Mistral has given complete expression to the spirit of his little patrie. Should the trend of events lead to a further unification of the dialects of southern France, there is no doubt that the Felibrean dialect has by far the greatest chance of success. The people of Provence owe a great debt to the Felibres, who have endowed them with a literature that comes closer to their sympathies than the classic literature of France can ever come ; they have been raised in their own esteem, and there has been undoubtedly a great awakening in their mental life. The Felibrige has given expression to all that is noblest and best in the race, and has invariably led onward and upward. Its mission has been one that commands respect and admiration, and the Felibres to-day are in a position to point with pride to the great people. " work accomplished among their Arsene Darmesteter has well said A nation needs poetry ; it lives : — not by FR^DfeRIC MISTRAL 42 bread alone, but in the ideal as well. Religious beliefs are weakening ; and if the sense of poetic ideals dies along with the religious sentiment, there will remain nothing among the lower classes but material and brutal instincts. " Whether the Felibres were conscious of this danger, or met this popular need instinctively, I cannot say. At any rate, their work is a There still good one and a wholesome one. circulates, down to the lowest stratum of the people, a stream of poetry, often obscure, until now looked upon with disdain by all except scholars. I mean folklore, beliefs, traditions, legends, and popular tales. Before this source of poetry could disappear completely, the Feli- bres had the happy idea of taking it up, giving it a new literary form, thus giving back to the people, clothed in the brilliant colors of poetry, the creation of the people themselves." And again of : " As for this general renovation popular poetry, I would give it no other name than that of the Felibrige. To the Feliit is bres is due the honor of the movement ; their ardor and their faith that have developed and strengthened it." CHAPTER III THE MODERN PROVENgAL LANGUAGE The language of the Felibres is based upon the dialect spoken in the plain of Maillane, in and about the town of Saint-Remy. This dialect is one of the numerous divisions of the langue d'oc^ which Mistral claims is spoken by nearly twelve millions of people. The literary history of these patois has been written by B. Noulet, and shows that at the close of the terrible struggles of the Albigenses the seemed dead. language In 1324 seven poets attempted to found at Toulouse the competitions of the Grai Savoir, and so to revive the ancient and the ancient language. poetry Their attempt failed. There was literary production of varying degree two or three centuries but until the time of Jasmin no writer attracted any attention beyond his immediate vicinity; and it is significant that the Felibres themselves were long in ignorance of Jasmin. It is of merit throughout 43 FRfeD^RIC MISTRAL 44 then not difficult to demonstrate that the Felibrige revival bears more the character of a creation than of an evolution. It is not at all an evolution of the literature of the Troubadours ; it is in no way like it. The language of the Felibres is not even the descendant of the special dialect that dominated as a literary- language in the days of the Troubadours; for was the speech of Limousin that formed the of that language, and only two of the greater poets among the Troubadours, Raimond it basis de Vaqueiras and Fouquet de Marseille, were natives of Provence proper. The dialect of Saint-Remy is simply one of countless ramifications of the dialects descended from the Latin. Mistral and his associates have made their literary language out of this dialect as they found it, and not out of the language of the Troubadours. They have regularized the spelling, and have deliberately eliminated as far as possible words and forms that appeared to them to be due to French influence, substituting older and more genuine forms forms that appeared more in accord with the genius of the — langue d'oc as contrasted with the langue d'oil. Thus, gloria istori^ paire, replace gloaro, istouero^ THE MODERN PROVENQAL LANGUAGE 45 j3ero, which are often heard among the people. The second step taken arose from the necessity of making this speech This was the first step. of the illiterate capable of elevated expression. Mistral claims to have used no word unknown to the people or unintelligible to them, with the exception that he has used freely of the stock of learned words common to the whole Romance family of languages. These words, too, he transforms more or less, keeping them in har- mony with the forms peculiar d'oe. to the langue Hence, it is true that the language of the Felibres is a conventional, literary language, that does not represent exactly the speech of any section of France, and is related to the popular speech more or less as any official language is to the dialects that underlie it. As the Felibres themselves have received all their instruction and literary culture in the French lan- guage, they use it among themselves, and their prose especially shows the influence of the French to the extent that it may be said that the Provengal sentence, in prose, appears to be a word-for-word translation of an underlying French sentence. Phonetically, the dialect offers certain marked FRfeDfiRIC 46 MISTRAL differences when contrasted with French. First of all is the forceful utterance of the stressed syllable ; the Provencal has post-tonic syllables, unlike the sister-speech. to Here it may be said occupy a sort of middle position between Italian and Spanish on the one hand, and French on the other ; for in the former languages the accent is found in all parts of the word, in French practically only upon the final, and then it is generally weak, so that the notion of a stress is almost lost. is The stress in Provengal placed upon one of the last two syllables only, and only three vowels, e, i, o, may follow the tonic syllable. The language, therefore, has a cadence that affects the ear differently from the French, and that resembles more that of the Italian or Spanish languages. The nasal vowels are again unlike those of The vowel affected by the following nasal consonant preserves its own the French language. quality of sound, and the consonant is pro- nounced ; at the end of a word both m and n are pronounced as w^ in the English word ring. The Provengal utterance of matin, terns, is therefore quite unlike that of the French matin, temps. This change of the nasal consonants THE MODERN PROVENQAL LANGUAGE into the ng sound whenever they 47 become final occurs also in the dialects of northern Italy and northern Spain. This pronunciation of the nasal vowels in French is, as is well known, an important factor in the famous "accent du Midi." The oral French. vowels are in general like the It is curious that the close o is heard only in the infrequent diphthong du^ or as an obscured, unaccented final. close This absence of the in the modern language has led Mistral to believe that the close o of Old Provencal was pronounced like ou in the modern dialect, which regularly represents it. A second element of the "accent du Midi" just referred to is the substitution of an open for a close o. The vowel sound of the word peur is not distin- guished from the close sound in peu. In the orthography of the Felibres the diagraph ue is used as we find it in Old French to represent this vowel. Probably the most striking feature of the pronunciation is the unusual number of diphthongs and triphthongs, both ascending and descending. Each vowel preserves its proper sound, and the component vowels seem to be pronounced more slowly and separately than in FRfeD^RIC MISTRAL 48 many languages. It is to be noted that w in a diphthong has the Italian sound, whereas when single it sounds as in French. e represents The unmarked the French ^, as the e mute is un- known to the Provencal. The e has come to sound like « before e and i, Ch and j represent the sounds t% and dz respectively, and g before e and i has as in French. the latter sound. voiced. The The « between vowels is There is no aspirate h. r is generally uvular. Only Z, r, «, and n are pronounced as final consonants, I being extremely rare. tral has preserved or restored other final con- Mis- sonants in order to show the etymology, but they are silent except in liaison in the elevated style of reading. The language is richer in vowel variety than Italian or Spanish, and the proportion of vowel to consonant probably greater than in either. Fortunately for the student, the spelling represents the pronunciation very faithfully. final A consonant preceded by another is mute among single final consonants only I, ; w, n, r, s are sounded ; otherwise all the letters written The stressed syllable is indiwhen not normal, by the application of are pronounced. cated, THE MODERN PROVENgAL LANGUAGE practically the 49 same principles that determine the marking of the accent in Spanish. The pronunciation of the Felibres is heard among the people at Maillane and round about. Variations begin as near as Avignon.^ Koschwitz' ^ Grammar treats the language The edition of Mireio published by Lemerre in 1886 con- tains an Avis sur laprononciation provenqale wherein numer- ous errors are to be noted. Here the statement is made that all the letters are pronounced ; that ch is pronounced ts, as in the Spanish word muchacho. The fact about the pro- nunciation of the ch is that it varies in different places, having at Maillane the sound ts, sound in the English chin. It is stated further on that fer- at Avignon, for instance, the ramento, capello, febre, are pronounced exactly like the Italian words ferramento, capello, febbre. The truth is that they are each pronounced somewhat differently from the Italian words. Provencal knows nothing of double consonants in pronunciation, and the vowels are not precisely alike in each pair of words. Later this sentence occurs : " Dans les triphthongues, comme biais, piei, vuei, nine, la voix doit dominer sur la voyelle interm^diaire, tout en faisant sentir les autres." Only the first two of these four words contain a triphthong. Vuei is a descending diphthong, the ue representing the Niue offers the same two vowel sounds inverted, French eu. with the stress on the second. Lastly, the example is given of the name J^use. It is spelled without the accent mark, and the reader is led to init is pronounced as though it were a French name. Here the eu is a diphthong. The first vowel is the French e, fer that the second the Italian u. The stress is on the first vowel. FR^DifeRIC 60 MISTRAL and renders unnecessary here the historically, presentation of more than peculiarities. its most striking Of these, one that evokes sur- upon first acquaintance with the dialect is the fact that final o marks the feminine of nouns, adjectives, and participles. It is a close 0, somewhat weakly and obscurely pronounced, prise as compared, for instance, with the final o in Italian. In this respect anomalous among Provencal Romance is languages. quite In some regions of the Alps, at Nice, at Montpellier, at Le Velay, in Haute-Auvergne, in Roussillon, and in Catalonia the Latin final a is preserved, as in Italian and Spanish. The noun has but one form for the singular and plural. The distinction of plural and singular depends upon the article, or upon the demonstrative or possessive adjective accom- panying the noun. « In as a plural sign. Provengal liaison adjectives take So that, for the ear, the and French languages alike in regard to this matter. are quite The Provengal has not even the formal distinction of the nouns in aZ, which in French make their plural in aux. Cheval in Provencal is chivau, and the plural is like the singular. A curious fact is the use of THE MODERN PROVENgAL LANGUAGE 51 uni or unis, the plural of the indefinite article, as a sign of the dual number and this is its ; exclusive use. The subject pronoun, when unemphatic, is not expressed, but understood from the termi- ISu (je), tu (tu), and Su used as disjunctive forms, in contrast nation of the verb. (il) are The with the French. possessive adjective leur is represented by si; and the reflective se used for the plural as well as for the is first third singular and third plural. The moods and tenses correspond exactly to those of the French, and the famous rule of the past participle is identical with the one that prevails in the sister language. Aside from the omission of the pronoun subject, and the use of one or two constructions not unknown to French, but not admitted to use in the literary language, the syntax of the Provencal is identical with that of the French. The inversions of poetry may disguise this fact a little, but the lack of individuality in the sentence construction is obvious in prose. lation of Provengal prose into Trans- French prose is practically mere word substitution. Instances of the constructions just mentioned FRtDtRIC MISTRAL 62 are the following. is often repeated the verb has The relative object pronoun as a personal pronoun, so that its object The expressed twice. French continually offers redundancy of subject or complement, but not with the relative. " Estre, i^u, lou marran que tduti L'estrangisson I Estre, idu, I'estrangie que tduti lou f ugisson 1 " £!tre, moi, le paria, que tous rebutent £!tre, moi, I'^tranger que tout le monde fuit " ! (Za Reino Jano, Act I, Scene iii.) The particle ti is added to a verb to make it interrogative. E.g. soun-ti? fero4ii? sont-ils? Petrarco ignoro-ti ? ^tait-il? Petrarque ignore-t-il ? This is the regular form of interrogative in the third person. It is, of course, entirely due to the influence of colloquial French. The French indefinite statement with the pronoun on may be represented in Provencal by the third plural of the verb; on m'a demands is translated Tn'an demanda, or on vCa demanda. The negative ne is often suppressed, even with the correlative que. The verb estre is conjugated with itself, as in Italian. THE MODERN PROVENQAL LANGUAGE 53 The Provengal speech is, therefore, not at all what it would have been if it had had an independent literary existence since the days of the The Troubadours. influence has been overwhelming, as is of the French naturally to be A great number of idioms, that expected. seem to be pure gallicisms, are found, in spite of the deliberate effort, referred to above, to In La Reino Jano^ Act III, Scene iv, we find 14 vai de nostis os, eliminate French forms. II y va de nos os. — Vejan, voi/ons, is used as a The parti- sort of interjection, as in French. tive article is used precisely as in French. meet the narrative infinitive with de. We In short, the French reader feels at home in the Provengal sentence ; it is the same syntax and, to a great degree, the same rhetoric. Only in the vocabulary does he feel himself in a strange atmosphere. The strength, the originality, the true ranon d'Stre of the Provengal speech resides in rich vocabulary. It contains a great of terms denoting objects its number known exclusively in Provence, for which there is no correspond- ing term in the sister speech. Many plants have simple, familiar names, for which the FRfeDfeRIC 64 MISTRAL French must substitute a name that only approximate, or Words of every learned category is either and pedantic. exist to express usages that are exclusively Provengal. The study of the modern language confirms the results, as regards etymology, reached by Diez and Fauriel and others, who have busied themselves with the Old Provencal. The great mass of the words are traceable to Latin etyma, as in all Romance dialects a large portion of Germanic words are found. Greek and Arabic Basque words are comparatively numerous. and Celtic have contributed various elements, and, as in French, there is a long list of words the origin of which is undetermined. The language shares with the other southern Romance languages a fondness for diminutives, augmentatives, and pejoratives, and is far richer than French in terminations of these classes. Long suffixes abound, and the style becomes, in consequence, frequently high-sounding and exaggerated. One of the most evident sources of new words in the language of Mistral is in its suf- Most of these are common to the other Romance languages, and have merely undergone fixes. THE MODERN PROVENQAL LANGUAGE 65 the phonetic changes that obtain in this form of speech. In many instances, however, they differ in meaning and in application from their corre- sponding forms in the sister languages, and a vast number of words are found the formation of which is peculiar to the language under consideration. These suffixes contribute largely to give the language its external appearance ; and while a thorough and scientific study of them cannot be given here, enough will be presented to show some of the special developments of Mistral's language in this direction. -a. This suffix marks the infinitive of the first conjugation, and also the past participle. It answers to the French forms in -er and -e. As the first conjugation is a so-called " living " conjugation, it is the termination of many new verbs. -a, -ado. -ado is the termination of the feminine of the past participle. This often becomes an abstract feminine noun, answering to the French ter- mination -ee; armSe in Mistral's language is FRfeD:fcRIC 66 armado. MISTRAL Examples of forms peculiar to Pro- vengal are dulivo, an olive. duliva, to gather olives. dulivado, olive gathering, pi ado, footprint. pie, foot. -age (masc). This sufl&x is the equivalent of the French and is a suffix of frequent occurrence in forming new words. Oulivage is a synonym of A rather curious dulivado, mentioned above. word is the adverb arrage, meaning at random, -age, haphazard. It appears to represent a Latin adverb, erratice. Mourtau, mourtalo, mortal, gives the noun mourtalage, a massacre. -agno (fem.). An interesting example of the use of this suffix is seen in the word eigagno, dew, formed from aigo, water, as though there had been a Latin word aquanea. -aio (fem.). This ending corresponds to the French -aille. poulo, a hen. poulaio, a lot of hens, poultry. THE MODERN PROVENQAL LANGUAGE 57 (masc). -aire This represents the Latin -ator (one who). The corresponding feminine in Mistral's works has always the diminutive form -arello. toumba, to fall. cantaire, cantarello, singer. toumbaire,toumbarello, one panie, basket. who falls or one who fells. panieraire, basket maker. duliva, to gather olives. caligna, to court. dulivaire, dulivarello, olive calignaire, suitor. gatherer. paternostriaire, one c&nta,, to sing. forever praying. who is Like the corresponding French nouns in -eur, these nouns in -aire, as well as those in -dire, are also used as adjectives. -aire = -arium. The suffix sometimes represents the Latin A curious word is vejaire, meaning -arium. opinion, manner of seeing, as though there had been a Latin word videarium. It sometimes has the form jaire or chaire, through the loss of the first syllable. -an, -ano. This suffix is common in the Romance languages. Fihan, filial^ seems to be peculiar to the Provencal. FRfeDfeRIC 68 MISTRAL -anci (fern.). This is the form corresponding to the French Abundance is in Mistral's dialect ahoun- -ance. ddnci. -ant, -anto. This is the termination of the present participle and verbal adjective derived from verbs These words sometimes have a special meaning, as toumbant, declivity. in -a. -ard, -ardo. Gaiard is Proven9al for the French gaillard. -ari. This represents the Latin -arius. Abouticari is Provengal for apothecary. -as. This is an augmentative suffix of very fre- quent use. pore, hog. pourcas, great hog. serp, snake. serpatas, great serpent. cast^u, fort. castelas, /or<rcss. rouco, rock. roucas, great rock. THE MODERN PROVENQAL LANGUAGE 59 -asso. This is a pejorative suffix. vidasso, wretched life. vido, life. -astre. In French this suffix has the form -4tre. dulivastre (Fr. olivatre), olive in color. -at. Constat is in French cotS (side). The suffix is often diminutive. auc, a gander. aucat, gosling. passero, sparrow. passerat, small sparrow. -au, -alo. This -al. is the form of the widely used suffix Mistral uses paternau for paternal., and also the adjective formed upon paire, father^ peirenau, peirenalo, fatherly. bourg, city. bourgau, bourgalo, civil. -edo (fem.). pin, pine. pinedo, pine-grove. clapo, stone. claparedo, stony plain. dulivo, olive. dulivaredo, olive-orchard. FRfeD]fcRIC eo MISTRAL -eire, -erello. This suffix corresponds to the suffix mentioned above. It is -aire, appended to the stem of verbs not of the first conjugation. courre, to run. courrfeire, courerello, run' ner. legeire, legerello, reader. legi, to read. -eja. This is an exceedingly common verb-suffix, corresponding to the Italian -eggiare. toumbarfeu, kind of cart. toumbaraleja, to cart. f arandolo, farandole. farandouleja, to dance the farandole. poutoun, ^c poutouneja, to kiss. poumpoun, caress. poumpouneja, to caress. segnour, lord. segnoureja, to lord it over. mistral, wind of the Rhone mistraleja, to roar like the mistral. valley. poudro, powder. poudreja, tojire a gun. clar, bright. clareja, to brighten. -en (masc), -enco (fem.). This is a common adjective-suffix. souleu, sun. souleien, souleienco, sunny. mai, May. maien, maienco, relating to Madaleno, Magdalen. madalenen, May. madalenenco, like Magdalen. THE MODERN PROVENgAL LANGUAGE 61 -es (masc), -esso (fem.). This suffix corresponds to the French -ais, -aise. Liounes = lyonnais. -et (masc), -eto (fem.). This is perhaps the commonest of the diminutive suffixes. ome, man. oumenet, little man. fiho, daughter. fiheto, dear daughter. enfan, child. enfantounet, little child. vfent, wind. ventoulet, breeze. toiimba, to fall. toumbaraleto, little leaps. chato, girl. chatouueto, little girl. malaut, ill. malautounet, sickly. It will be observed that the double diminu- tive termination is the most frequent. Sometimes the -et is not diminutive. Ouliveto may mean a small olive or a field planted with olives. -eu (masc), -ello (fem.). This suffix is often diminutive. paorin, poor chap. paurineu, paurinello, poor pin, ^in«. pinateu, young pine. little fellow or girl. pinatello, forest of young pines. sauvage, wild. sauvageu, sauvagello, some- what wild. FR^DflRIC MISTRAL 62 Sometimes it is not. toumbareu, toumba, to fall. -ello, likely to fall canta, to sing. cantareu, -ello, songful. crese, to believe. cresereu, -ello, inclined to belief. -i. This is a verb-suffix, marking the infinitive of a " living " conjugation. bourgau, civil. abourgali, to civilize. -ie (fem.). Carestie, dearness, stands in contrast to the Italian carestia. priva, to train, to tame. privadie, sweet food given in training animals. -ie (masc), -iero (fem.). This is the equivalent of the French -ier. dulivie, olive tree. pinatie, bouchid, butcher. pinatiero, \| J a dwelling among pines. -ieu (masc), -ivo (fem.). This is the form corresponding to the French -if, -ive. ablati^u, ablative. vieu, vivo, lively. THE MODERN PROVENQAL LANGUAGE 63 -ige (m.). According to Mistral, it this represents the We incline to think rather that Latin -ities. corresponds to -age, being added chiefly to -age fits rather upon stems in a. words in e. gounflige, swelling. gounfle, swollen. Felibre. Felibrige. paure, poor. paurige, poverty. -iho (fern.). This suffix makes collective nouns. pastre, shepherd. pastriho, company of shep- herds. pauriho, the poor. paure, j)oor. -in (m.), -ino (fem.). This is usually diminutive or pejorative, paurin, poor wretch. -ioun (fem.). This corresponds to the French -ion. nacioun, nation. abdicacioun, abdication, erme, desert. asserma, to dry up. assermacioun, thirst, dryness. FRiiDfiRIC 64 -is MISTRAL (masc), -isso (fern.). Crida, to cry. cridadisso, cries of woe. chapla, to slay. chapladis, slaughter. coula, to flow. couladis or couladisso, flow- abareja, to throw pell-mell. abarejadis, confusion. toumba, to fall. toumbadis, ing. tottering -isso, (adj.). This suffix added to the past participle is stem -isoun (fem.). This suffix forms nouns from verbs in -i. abalauvi, to make dizzy, to abalauvisoun, vertigo, confound. -men (masc). This corresponds to the French -ment ; basti- men = batiment, ship. abouli, to abolish. aboulimen, abolition. toumba, to fall. toumbamen, fall. -men (adverb), urous, urouso, happy. urousamen, happily. It is to be noted here that the adverb has the vowel of the old feminine termination a, and not the modern o. THE MODERN PROVENQAL LANGUAGE 65 -ot (masc), -oto (fern.). A diminutive suffix. viloto, little town. vilo, toum. Sometimes the stem no longer exists separately. mignot, mignoto, darling. pichot, pichoto, little little hoy, girl. -oto (fern.), passaroto, ^(M«m^ to amfyro. passa, to />as9. -ou (masc). This is a noun-suffix of very frequent use. It seems to be for Latin -or and -orium. jougadou, player. jouga, to play. abla, to brag (cf. Fr. hdbler). abausi, to abuse, to exag^ abladou, braggart. abausidou, braggart. gerate. courre, to run. courredou, corridor. lava, to wash. lavadou, lavatory. espande, to expand. espandidou, expanse, pano- escourre, to flow out. escourredou, passage, hollow. toumba, to fall. toumbadou, water-fall, rama. abeura, to water. abeuradou, drinking-trough. passa, to sift. passadou, sieve. mounda, to winnow. mouudadou, sieve. FRfeD^RIC MISTRAL 66 -ouge. This is an adjective suffix, ivernouge, wintry. iver, winter. -oun (masc), -ouno (fern.). A diminutive suffix. enfan, child. enfantoun,enfantouno,^i«/e pauriho, the poor. paurihoun, poor wretch. child. -ounge (masc). A suffix forming nouns from adjectives. viM, old. vieiounge, old age. -our (fern.). This is like the above, vifeiour, old age. vifei, old. -ous, -ouso. This is the Latin -osus ; French -eux, -euse. It forms many new words in Mistral. urous (Fr, heureux), happy. aboundous, abundant. pouderous (It. and Sp. poderoso), powerful. pin, pine. pinous, covered with pines. escalabra, to climb. escalabrous, precipitous. THE MODERN PROVENQAL LANGUAGE 67 -ta (fern.). This the equivalent of the is French -te. Latin -tas, In Mistral's language it is usually preceded by a connecting vowel e. mouudaneta, worldliness. soucieta, society. paureta, poverty. -u (masc.)» -udo (fern.). This ending terminates the past participles of verbs whose infinitive ends in e. It also forms many new adjectives. astre, star. malastru, ill-starred. sabe, to know. saberu, learned. The feminine form often becomes a noun, escourre, to run out. escourregudo, excursion. -un (masc). This is a very common noun-suffix. clar, bright. clarun, brightness. rat, rat. ratun, lot of rats, smell of rats. paure, poor. paurun, poverty. dansa, to dance. dansun, love of dancing. plagne, to pity. plagnun, complaining. viei, old. vieiun, old age. FR6d6rIC mistral 68 -uro (fern.). toumba, to fall. toumbaduro, a fall. escourre, to flow away. escourreduro, what flows away. bagna, to wet. bagnaduro, dew. This partial survey of the subject of the suffixes in Mistral's dialect will suffice to show that it is possible to create words indefinitely. There is no academy to check abuse, no large, cultivated forms. the public to disapprove of new A fond- The Felibres have been free. ness for diminutives marks all the languages of southern Europe, and a love of long terminations generally distinguished Spanish latinity. The language of the Felibres is by no means free from the grandiloquence and pomposity that results from the employment of these high- sounding and long terminations. Toumbarelado, toumbarelaire^ are rather big in the majesty of their five syllables to denote a cart-load and its driver respectively. The abundance vocabulary is at any rate manifest. of this We have here not a poor dialect, but one that began with a large vocabulary and in possession of the power of indefinite development and recreation out of its own resources. It forms compounds THE MODERN PROVENQAL LANGUAGE 69 with greater readiness than French, and the learner is impressed by the unusual number of compound adverbs, some of very peculiar formation. Tourna-mai (again) is an example. Somewhat on the model of the French va-etvient is the word li mounto-davalo, the ups and downs. Un regardo-veni means a look-out. Noun-ren is nothingness. Ped-terrou8 (earthy foot) indicates a peasant. Onomatopoetic words, like zounzoun, vounvoun^ dinddnti^ are common. Very interesting as throwing light upon the Proven9al temperament are the numerous and constantly recurring interjections. in the This trait man of the Midi is one that Daudet has brought out humorously in the Tartarin books. It is often difficult in serious situations to take these explosive monosyllables seriously. In his study of Mistral's poetry, Gaston Paris calls attention to the fact that the Provencal vocabulary offers many words of low association, or at least that these words suggest what is low or trivial to the French reader ; he admits that the effect upon the Proven9al reader may not be, and is likely not to be, the same ; but even the latter must occasionally experience a feeling FR^DfeRIC MISTRAL 70 of surprise or slight shock to find such words For the English reader used in elevated style. it is even Many such expressions could worse. not be rendered literally at all. Mistral resents this criticism, and maintains that the words in question are employed in current usage with- out calling up the image of the low association. This statement, of course, must be accepted. It is true of all languages that words rise and fall in dignity, and their origin and association are momentarily or permanently forgotten. The undeniably great Provengal literature revival of the dialect. success of this justifies new completely the As Burns speaks from his soul only in the speech of his mother's fireside, so the Proven9al nature can expressed in the home-dialect. wrote for Provencals only. associates early only be fully Roumanille Mistral and his became more ambitious. His works have been invariably published with French translations, and more readers know them through the translations than through the But they are what they are because originals. they were conceived in the patois, and because their author was fired with a love of the lan- guage itself. THE MODERN PROVENgAL LANGUAGE 71 As to the future of this rich and beautiful idiom, nothing can be predicted. The Felibrige movement appears to have endowed southern France with a literary language rivalling the French; it appears to have given an impulse toward the unification of the dialects and sub- But the patoisants dialects of the langue cfoc. are numerous and powerful, and will not abdicate their right to continue to speak and write their local dialects in the face of the superiority of the Felibrige literature. Is it to be expected that Frenchmen in the south will hereafter know and use three languages and three literatures — the local dialect, the language of the Felibres, and the national language and literature ? One is inclined to think not. The two literamore than most men can become practical difficulties are very great ; tures are familiar with. However, this much is certain: a rich, harmonious language has been saved forever and crystallized in works of great beauty ; its revi- val has infused a fresh, intellectual activity into the people whose birthright it studied with delight by many born in sunny Provence ; is ; it has been who were not a very great contri- FK^D^RIC MISTRAL 72 bution is made through it to philological study. Enthusiasts have dreamed of its becoming an international language, on account of its inter- mediary position, that it is its simplicity, and the fact not the language of any nation. Enthusiasm has here run pretty high, as is apt to be the case in the south. In connection with the revival of all these dialects the opinion of two men, eminent in the science of education, is of the greatest interest. Eugene Lintilhac approves the view of a professor of Latin, member of the Institute, who had often noticed the superiority of the peasants of the frontier regions over those from the interior, and who said, "It is not surprising, do they not pass their lives translating ? " Michel Breal considers the patois a great help in the study of the official language, on the principle that a term of comparison study of a language. is necessary in the As between Provencal and French this comparison would be between words, rather than in syntax. Often the child's respect for his home would be increased if he sees the antiquity of the speech of his fireside if, as Breal puts it, he is shown that his dialect conforms frequently to the speech of Henri IV THE MODERN PROVENQAL LANGUAGE or St. Louis. 73 " If the province has authors like Jasmin, Roumanille, or Mistral, let the child read their books from time to time along with his French books; he proud of will feel his province, and will love France only the more. The clergy is well aware of this power of the knows how to turn it to native dialect, and account, and your culture is often without root and without depth, because you have not recognized the strength of these bonds that bind to a locality. The school must be fast to the soil and not merely seem to be standing upon it. There need be no fear of thereby shak- ing the authority of the official language necessity of the latter is ; the continually kept in sight by literature, journalism, the administration of government." The revival of this speech could not fail to interest lovers of literature. descendant, it is If not a lineal at least a descendant, of the language that centuries ago brought an era of beauty and light to Europe, that inspired Dante and Petrarch, and gave to modern literatures the poetic forms that still bear their Provengal The modern dialect is devoted to other uses now it is still a language of brightness names. ; 74 FR^D^RIC MISTRAL and sunshine, graceful and artistic, but instead of giving expression to the conventionalities of courtly love, or tending to soften the natures of fierce feudal barons, it now sings chiefly of the simple, genuine sentiments of the human heart, of the real beauties of nature, of the charm of wholesome, outdoor life, of healthy the love of home and toil and simple living, of country, and brings at least a message of hope and cheer at a time when greater literatures are burdened with a weight of discouragement and pessimism. CHAPTER IV THE VERSIFICATION OF THE F^LIBRES The versification of the Felibres follows in the main the rules observed by the French poets. As in all the Romance languages the verse consists of a given number of syllables, and the number of stressed syllables in the line not constant. The few differences to be noted between French verse and Provencal verse arise from three differences in the languages. The Provengal has no e mute^ and is therefore all the syllables theoretically counted are distinctly heard, and the masculine and the feminine rhymes are fully distinguished in pronunciation. The new language possesses a number of diphthongs, and the unaccented part of the diphthong, a m or an i, constitutes a consonant either before or after a vowel in another word, being really a w> or a y. This prevents hiatus, which is banished from Provencal verse as it is from French, and here again theory and 76 MISTRAL FR^DlfcRIC 76 practice are in accord, for the elision of the e mute where this e follows a vowel readmits hiatus into the French line, and no such phe- nomenon is known to the Provencal. Thirdly, word is strongly the stressed syllable of each marked, and verse exists as strongly and regularly accentual as English or German. in This is seen in the numerous poems written to be sung to an air already existing. The accents in these pieces fall with the rhythmic beat the English ear is accustomed to and which it so misses on first acquaintance with French verse. A second consequence of this stronger stress is that verse is written without rhyme ; the entire Poem of the Rhone is written in ten-syllable feminine verses unrhymed. " O terns di viei, d'antico bounoumio, Que lis oustau avien ges de sarraio E que li gent, k Coundrieu coume au nostre, Se gatihavon, au caleu, p^r rire " ! Mistral has (Canto I.) made use of all the varieties of One of the verse known to the French poets. poems in the Isclo d' Or fourteen-syllable verse ; dou (The Belvedere). stanzas ; — offers an example of it is called L'AmiraHere are the first two THE VERSIFICATION OF THE FELIBRES * 77 Au casteu de Tarascoun, i'a 'no reino, i'a 'no fado Au casteu de Tarascoun I'a 'no fado que s'escound. ** Aqn^n que ie durbira la presoun ounte es clavado Aqueu que ie durbira Belfeu elo Tamara." ^ We may note here instances of the special features of Provencal versification mentioned above. The i in French il y a, is i'a, the equivalent of the really a consonant. This i occurs again in the fourth of the lines quoted, so that there is no hiatus between que and iS. In like manner the u of heleu, in the last line, stands with the sound of the English w between this and elo. The e of ounte is elided. It will be observed that there is a caesura between the seventh and eighth syllables of the long line, and that the verse has a marked rhythmic beat, with decided trochaic movement, 1 — In the castle at Tarascon there is a queen, there is a fairy, In the castle of Tarascon There is a fairy in hiding. The one who shall open the prison wherein she is confined, The one who shall open for her, Perhaps she will love him. FR^D^RIC MISTRAL 78 In his use of French Alexandrine, or twelvesyllable verse, Mistral takes few liberties as to No ternary verses are found in Mireio^ caesura. that is, verses that fall into three equal parts. In general, it may be said that his Alexandrines, except in the play La Reino Jano^ represent the classical type of the French poets. To be noted, however, is the presence of feminine caesuras. These occur, not theoretically or intentionally, but as a consequence of pronunciation, and are an additional beauty in that they vary the The unstressed vowel movement of the lines. at the hemistich, theoretically elided, is pro- nounced because of the natural pause intervening between the two parts of the verse. " Per duliva tant d'aubre I — H6u, tout ac6 se fai !" (Mirfeio, Canto I.) In one of the divisions of Lou Tambour cfArcolo (The Drummer of Arcole), the poet uses tensyllable verse with the caesura after the sixth an exceedingly unusual syllable, caesura, imi- tated from the poem G-irard de Boussillon. " Ah lou pichot tambour devengufe flbri do en plen souleu, Davans touto I'arma I — \| F^r estelk soun front \| \| I d'un rai de glbri," etc. THE VERSIFICATION OF THE F:fcLIBRES 79 Elsewhere he uses this verse divided after the fourth syllable, and less frequently after the fifth. The stanza Mireio and used by Mistral throughout Calendau is his own invention. Here is the first stanza of the second canto of Mireio : — " Cantas, cantas, magnanarello, Que la culido es cantarello Galant soun li magnan e s'endormon di tres Lis amourie soun plen de fiho Que lou beu terns escarrabiho, Courae un vou de bldundis abiho Que raubon sa melico i roumanin ddu gres." This certainly is a stanza of great beauty, and eminently adapted to the language. Mistral is exceedingly skilful in the use of it, distributing pauses effectively, breaking the monotony of the repeated feminine verses with enjambements, and continuing the sense from one stanza to the next. This stanza, like the language, is pretty and would scarcely be a suitable vehicle for poetic expression requiring great liness. depth or state- Provencal verse in general cannot be said to possess majesty or the rich orchestral quality Brunetiere finds in Victor qualities are sweetness, Hugo. daintiness, Its rapidity, 80 FR:feD:feRIC MISTRAL grace, a merry, tripping flow, great smoothness, and very musical rhythm. Mireio contains one ballad and two lyrics in a measure differing from that of the rest of The ballad of the Bailiff Suffren has the swing and movement a sea ballad should The stanza is of six lines, of ten sylpossess. the poem. lables each, with the syllable, the caesura after " Lou Baile Sufrfen In the third \| fifth que sus mar coumando." canto occurs the famous song Magali, so popular in Provence. is the rhymes being a55, aha. The melody printed at the end of the volume. Mireio's prayer in the tenth canto is in five-syllable verse with rhymes ahhdb. The poems of the laclo d' Or offer over eighty varieties of strophe, a most remarkable number. This variety is produced by combining in different manners the verse lengths, and by changes in the succession of rhymes. Whatever inge- nuity Mistral has exercised in the creation of rhythms, the impression must not be created that inspiration has suffered through attention to mechanism, or that he is to be classed with the old Provencal versifiers or those who flourished in THE VERSIFICATION OF THE F^LIBRES 81 northern France just before the time of Marot. Artifice is always strictly subordinated, and the No violence poet seems to sing spontaneously. is ever done to the language in order to force it into artificial moulds, there is no punning in rhymes, there nothing that can be charged is against the poet as beneath the real dignity of his art. Let us look at some of the more striking of these verse forms. The second of Li Cansoun^ Lou Bastimen, offers the following form : — "Lou bastimen ven de Maiorco Emd d'arange un cargamen An courouna de vferdi torco : L'aubre-mestre ddu bastimen Urousamen Ven de Maiorco Lou bastimen." * This stanza reproduces in the sixth line the word of the first, and in the seventh the last word of the fourth. last An excellent example of accentual verse set to an already existing melody Bon Prouvenfau. 1 The air is : — is seen in Li The ship comes from Majorca with a cargo of oranges the mainmast of the ship has been crowned with green gar- lands : safely the ship arrives from Majorca. 9 FRfeD^RIC MISTRAL 82 " Si le roi m'avait donnd Paris, sa grand' vUle." We quote tlie first stanza — : " Bouf o, au siecle mounte sian Uno auro superbo Que vou faire rfen qu'un tian De tduti lis erbo Nautri, li bon Prouven<jau Aparan lou viei casau Ounte fan I'aleto Nosti dindouleto." ^ This poem scans itself with perfect regu- and the rhythm of the tune is evident who may never have heard the larity, to the reader actual music. The stanza of La Tourre de Barhentano is as follows : — " L'Evesque d'Avignoun, Mounsen Grimau, A fa basti 'no tourre k Barbentano Qu' enrabio vent de mar e tremountano E fai despoutenta I'Esprit ddu mau. Assegurado Sus lou roucas Forto e carrado Escounjiurado '^ There blows, in this age, a proud wind, which would make a mere hash of all herbs we, the good Provencals, defend the old home over which our swallows hover. : THE VERSIFICATION OF THE F^LIBRES 83 Porto au soulfeu soun front bouscas Mememen i fenestro, dins lou cas Que vouguesse lou Diable intra di vitro, A fa Mounsen Grimau grava sa mitro." * Here is a stanza of Lou Renegat : — " Jan de Gounf aroun, pres pfer de courskri, Dins li Janisskri Sfet an a servi Fau, enco di Turc, ave la coudeno Facho k la cadeno Emai au rouvi." * The stanza employed in La CadSno de MbustiS is remarkable in having only one masculine and one feminine rhyme in its seven lines : — " Presounie di Sarrasin, Engimbra coume un caraco, Em' un calot cremesin Que lou blanc soulfeu eidraco, En virant la pouso-raco, 1 The bishop of Avignon, Monseigneur Grimoard, hath built a tower at Barbentane, which excites the rage of the sea wind and the northern blast, and strips the Spirit of Evil of his power. Solid upon the rock, strong, square, freed of demons, it lifts its fierce brow sunward likewise upon the windows, in case the devil might wish to enter thereby, Monseigneur Grimoard has had his mitre carved. 2 John of Gonfaron, captured by corsairs in the Janissaries, served seven years. Among the Turks a man must use his skin to chains and rust. ; FRfeDfeRIC 84 MISTRAL Rico-raco, Blacasset pregavo ansin." * The " roumanso " of La Reino Jano offers a stanza containing only five rhymes in fourteen lines : — " Fieu de Maiano S'ere vengu ddu terns De Dono Jano, Quand ero k soun print^ms E soubeirano Coume feron autre-tfems, S^nso autro engano Que soun regard courous, Auri^u, d'elo amourous, Trouva, i^u benurous, Tant fino cansouneto Que la bello Janeto M'auri^ douna 'n mantra Pfer parfeisse i castfeu." * The rhythm of the noble Saume de la Penitenei is as follows : — " Segnour, k la fin ta coul^ro Largo si tron 1 Prisoner of the Saracens, accoutred like a gypsy, with a crimson turban, dried by the while sun, turning the creaking water-wheel, Blac prayed thus. * A son of Maillane, if I had come in the days of Queen Joanna when she was in her springtime and a sovereign such as they were in those days, with no other diplomacy than her THE VERSIFICATION OF THE F^LIBRES 85 Sus nosti front E dins la nine nosto gal^ro Pico d'a pro Contro U ro." ^ Another peculiar stanza is exhibited in Lou Prego-DiSu : — " Ero un tantost d'aquest estidu Que ni vihave ni dourmidu Fasieu miejour, tau que me plaise, Lou cabass6u Toucant lou s6u, A I'aise." ^ Perhaps the most remarkable of all in point of originality, not to say queerness, is Lou Blad de Luno. The rhyme in lin is repeated through- out seventeen stanzas, and of course no word is used twice. " La luno barbano Debano De lano. bright glance, in love with her, I should have found, lucky I, me a mantle to appear in the castles. 1 This poem will be found translated in full at the end of so fine a song that the fair Joanna would have given the book. * It was an afternoon of this summer, While I neither woke nor slept, I was taking my noonday rest, as is my pleasure, My head touching the ground at ease. FRfeD^RIC MISTRAL 86 S'entend peralin L'aigo que lalejo E batarelejo Darrid lou moulin. La luuo barbano Debano Delin."! The little poem, Auhencho^ is interesting as offering two rhymes in its nine lines. Mistral's sonnets offer some peculiarities. He has one composed of lines of six syllables, others of eight, besides those considered regular in French, consisting, namely, of twelve syl- The following sonnet addressed Roumania appears to be unique in form to lables. : — " Quand lou chaple a pres fin, que lou loup e la russi An rousiga lis os, lou soul^u flamejant Esvalis gaiamen lou brumage destriissi E lou prat bataid tourno leu verdejant. " Aprfes lou long trep^ di Turc emai di Riissi T'an visto ansin renaisse, o nacioun de Trajan, Coume I'astre lusfent, que sort ddu negre esliissi, Em^ lou nouvelun di chato de quinge an. 1 The ghostly moon is unwinding wool. Afar off is heard the gurgling water shaking the clapper behind the mill. The ghostly moon is unwinding flax. THE VERSIFICATION OF THE FilLIBRES 87 " E li ra90 latino A ta lengo argentino An couneigu I'ounour que dins toun sang V avi^ " E t'apelant germano, La Prouven90 roumano Te mando, o Roumanio, un rampau d'dulivi^."* It would be a hopeless task for an English poems translator to attempt versions of these that should forms. reproduce the original strophe A few such translations have been made into German, which possesses a much greater wealth of rhyme than English. Let us repeat that it must not be imputed to Mistral as a fault that he is too clever a versifier. His strophes are not the artificial complications of the Troubadours, and if these greatly varied 1 When the slaughter is over, when the wolf and the buz- zard have gnawed the bones, the flaming sun scatters merrily the hurtful vapors and the battlefield soon becomes green once more. After the long trampling of the Turks and Russians, thou, too, art seen thus reborn, O nation of Trajan, like the shining star coming forth from the dark eclipse, with the youth of a maiden of fifteen. And the Latin races, in thy silvery speech, have recognized the honor that lay in thy blood ; and calling thee sister, the Romance Provence sends thee, Roumania, an olive branch. MISTRAL FRiJDfeRIC 88 forms cost him effort to produce, his art is most More likely it is that marvellously concealed. the almost inexhaustible abundance of rhymes in the Provencal, and the ease of construction of merely syllabic verse, explain in great measure his fertility in the production of stanzas. Some others of the F^libres, even Aubanel, in our opinion, have produced verse that is very Verse may be made too ordinary in quality. and fluent rhymed language that merely expresses commonplace seneasily in this dialect, timent may readily be mistaken for poetry. The wealth of rhyme in the Provengal language appears to be greater than in any other form of Romance speech. As compared with Italian and Spanish, it may be noted that the Provencal has no proparoxytone words, and hence a whole class of words is brought into the two categories possible Though the number of diphthongs is Provengal. in different vowels and greater than in these two lan- guages, only three consonants are found as finals, w, r, « (I very rarely). great abundance of rhymes insistence upon the rich scarcely attainable in The consequent limited by an is rhyme French ; to an extent in fact, the THE VERSIFICATION OF THE FifeLIBRES merely sufficient unfortunate rhyme that so is very rare. many of 89 It is the feminine rhymes terminate in o. In the Poem of the Mhone, composed entirely in feminine verses, passages occur where nine successive lines end in this letter, and the verses in a vastly out- number all others. assonance is In this unrhymed poem, very carefully avoided. The play, Queen Joanna, is remarkable among the productions of Mistral as being the only work of any length he has produced that makes extensive use of the Alexandrine. In fact, the versification is precisely that of any modern French play written in verse and we may note here the liberties as to csesura and enjambements which are now usual in French verse. We remark elsewhere the lack of in; dependence in the dialect of Avignon, that its vocabulary alone gives it life. Not only has it no syntax of its own, but it really has been a difficulty of the poet in translating his own Alexandrines into French prose, not to produce verses ; nor has he always avoided them. Here, for instance, is a distich which not only becomes French when translated word for word, but also reproduces exactly metre and rhyme : — FR^DfeRlC MISTRAL 90 " En un mot tout me dis que lou cfeu predestino Un revieure de glori k la terro latino. " En un mot tout me dit que le ciel predestine Un renouveau de gloire k la terre latine." The effectiveness, the charm, and the beauty who understand and of this verse, for those feel the language, cannot be denied ; and this poetic literature did not if meet a want, it could not exist and grow as it does. The fact that the prose literature is so slight, so scanty, is highly significant. The poetry that goes straight to the heart, that speaks to the inner feeling, that calls forth composed in the a response, must be home speech. It is exceed- ingly unlikely that a prose literature of any importance will ever grow up in Provence. No great historians or dramatists, and few novelists, will ever write in this dialect. The people of Provence will acquire their knowl- edge and thelir general higher culture in French But they will doubtless enjoy that poetry best which sings to them of themselves literature. in the speech of their firesides. Mistral has endowed them with a verse language that has high artistic possibilities, some of which he THE VEKSIFICATION OF THE F^LIBRES has realized most completely. 91 The music of his verse is the music that expresses the nature of his people. It is the music of the gai savoir. Brightness, merriment, movement, quick and sudden emotion, — not often deep or sustained, — exuberance and enthusiasm, love of light and life, are free predominant ; and the verse, absolutely from strong and heavy combinations of consonants, ripples and glistens with its pretty terminations, full of color, full of vivacity, full of the sunny south. CHAPTER V MISTRAL'S DICTIONARY OF THE PROVENgAL LANGUAGE Au MiEJOUB Sant Jan, vfengue meissoun, abro si fi6 de joio Amount sus I'aigo-vers lou pastre pensatieu, En I'ounour ddu pais, enausso uno mount-joio £ marco li pasquie mounte a passa I'esti^u. — Emai i^u, en laurant e quichant moun ancboio, Per lou noum de Prouven90 ai fa 90 que poudi^u E, Dieu de moun pres-fa m'aguent douna la voio, Dins la rego, k geinoui, vuei r^nde grkci h Dieu. En terro, fin qu'au sistre, a cava moun araire E lou brounze rouman e Tor dLs emperaire Treluson au souleu dintre lou blad que sort. . . . O pople ddu Miejour, escouto moun arengo Se vos recounquista I'empferi de ta lengo. Per t'arnesca de n6u, pesco en aqu^u Tresor. "Saint John, at harvest time, kindles his bonfires; high up on the mountain slope the thoughtful shepherd places a pile of stones in 02 DICTIONARY OF PROVENgAL LANGUAGE 93 honor of the country, and marks the pastures where he has passed the summer. " I, too, tilling and living frugally, have done what I could for the fame of Provence; and God having permitted on me to complete my my knees in the furrow, I task, to-day, offer thanks to Him. " My plough has dug into the soil down to the rock ; and the of the emperors Roman bronze and the gold gleam in the sunlight among the growing wheat. "Oh, people of the South, heed my saying: you wish to win back the empire of your language, equip yourselves anew by drawing upon this Treasury." If Such is the sonnet, dated October 7, 1878, which Mistral has placed at the beginning of his vast dictionary of the dialects of southern France. The title of the work is Lou Tresor d6u Felihrige or Dictionnaire provengal-frangais. It is published in two large quarto volumes, This great work offering a total of 2361 pages. occupied the poet some ten years, and is the most complete and most important work of its kind that has been made. The statement that this work represents for the ProvenQal dialect U TRtDtmC MISTRAL what Littre's monumental dictionary is for the French, is not exaggerated. Nothing that Mistral has done entitles him in a greater degree to the gratitude of students of Romance philology, and the fact that the work has been done in so masterful a fashion by one who is not first of all a philologist excites our wonder and admiration. And let us not forget that it was above all else a labor of love, such as probably never was undertaken elsewhere, unless the work of Ivar Aasen in the Old Norse dialects be counted as such and there is some; thing that appeals strongly to the imagination in the thought of this poet's labor to render imperishable the language so dear to him. Years were spent in journeying about among all classes of people, questioning workmen and sailors, asking them the names they applied to the objects they use, recording their proverbial expressions, noting their peculiarities of pronunciation, listening to the songs of the peasants ; and then all was reduced to order and we have a work that is really monumental. The dictionary professes to contain all the words used in South France, with their mean- DICTIONARY OF PROVENQAL LANGUAGE ing in French, their proper and 95 figurative acceptations, augmentatives, diminutives, with Along with each examples and quotations. word we have all its various forms as they appear in the different dialects, its forms in the older dialects, the closely related forms in the other Romance languages, and its etymol- A special feature of the work in view ogy. of its destination is the placing of numerous synonyms along with each word. The dictionary almost contains a grammar, for the conjugation of regular and of irregular verbs in all the dialects is given, and each word is treated in its grammatical relations. terms of all arts and trades ; Technical popular terms in natural history, with their scientific equivalents ; all the geographical names of the region in all their forms ; family names proper historical names common in the south; explana- tions as to customs, manners, institutions, traditions, and beliefs ; biographical, bibliographical, and historical facts of importance ; and a com- plete collection of proverbs, riddles, and popular idioms — such are the contents of this prodigious work. If any weakness is to be found, it is, of course. FR^DfeRIC MISTRAL 06 in the etymological part. Even here we can but pay tribute to Mistral. If he can be accused, now and then, of suggesting an etymology that is impossible or unscientific, let it be gratefully conceded that his desire is to offer the etymologist all possible help by placing at his disposal all the material that can be found. The pains Mistral has taken to look up all possibly related words in Greek, Arabic, Basque, and English, to say nothing of the Old Provencal and Latin, would alone suffice to call forth the deepest gratitude on the part of all students of the subject. This dictionary makes order out of chaos, and although the language of the Felibres is justly said to be an artificial literary language, we have in this work along with the form adopted or created by the poet an orderly presentation of langiie d'oc as of the people. all the speech-forms of the they really exist in the mouths PART SECOND THE POETICAL WOKKS OF MISTRAL CHAPTER I THE FOUR LONGER POEMS I. MiBEio (Mireille) The publication of this poem in 1859 is an event of capital importance in the history of modern Provencal literature. Recognized im- mediately as a master-work, it fired the ambitions of the Felibres, enlarged the horizon of possibilities for the new speech, and earned for its author the admiration of critics in and out of France. Original in language and in con- ception, full of the charm of rustic life, taining a pathetic tale of love, a sweet interest, and glowing with con- human pictures of the strange and lovely landscapes of Provence, the poem charmed all readers, and will doubtless always rank as a work that belongs to general literature. Of no other work written in this dialect can the same be asserted. Mistral has not had an equal success since, and in spite of 99 FR^D^RIC MISTRAL 100 the merit of his other productions, his literary fame will certainly always be based upon this poem. Whatever be the destiny of this revival, the author of Mireio has probably already taken his place among the immortals of literature. He has incarnated in this poem all that is sweetest and best, all that is most typical in the life of his region. The tale is told, in general, with complete simplicity, sobriety, and concise- The poet's heart and soul are in his work from beginning to end, and it seems more genuinely inspired than any of the long poems ness. he has written subsequently. In the first canto the author says, — " Car cantan que per vautre, o pastre e gent di mas." For we sing for you alone, O shepherds and people of the farms, and when he wrote this verse, he was doubtless sincere. Later, however, he must have become conscious that a work of great artistic beauty was growing under his hand, and that it would find a truly appreciative public more probably among the cultivated classes than among the peasants of Provence. translation; Hence the French prose and hence, furthermore, a paradox in the position Mistral assumed. Since those THE FOUR LONGER POEMS 101 who really appreciate and admire his poetry are the cultivated classes who know French, and since the peasants who use the dialect cannot feel the artistic tion, or worth of his literary produc- even understand the elevated diction he is forced to employ, should he not, after all, have written in French? The idea of Rou- manille was simpler and less ambitious than that of Mistral; he aimed to give the classes about him a humble within literature their reach, that should give them moral lessons, and appeal to the best within them. Mistral, de- veloping into a poet of genius while striving to attain the same object, could not fail to change the object, and this contradiction be- comes apparent in Mireio, and constitutes a problem in any discussion of his literary work. The story of MirMo may be told in a few words. She is a beautiful young girl of fifteen, living at the mas of her father, Ramoun. falls in She love with a handsome, stalwart youth, Vincen, son of a poor basket-maker. difference in worldly wealth is But the too great, her father and mother violently oppose their union, and so, one night, the maiden, in despair, rushes FRfeD^RIC MISTRAL 102 away from home, across the great plain of the Crau, across the Rhone, across the island of Camargue, to the church of the three Maries. Vincen had told her to seek their aid in any time of trouble. Here she prays to the three saints to give Vincen to her, but the poor girl has been overcome by the terrible heat of the sun in crossing the treeless plains and is found by her parents and friends unconscious before the altar. Vincen comes also and joins his lamen- tations to theirs. The holy caskets are lowered from the chapel above, but no prayers avail to save the maiden's life. She expires, with words of hope upon her lips. This simple tale is told in twelve cantos; it aims to be an epic, and in its external form is such. It employs freely the merveilleux chri- condemned by Boileau, and in one canto. La Maseo (The Witch), the poet's desire to embody the superstitions of his ignorant landsmen has led him entirely astray. The opening stanza tien^ begins in true epic fashion : — " Cante uno chato de Prouvfen90 Dins lis amour de sa jouvfen^o." I sing a maiden of Provence In her girlhood's love. THE FOUR LONGER POEMS The invocation is addressed to Christ: 103 — Thou, Lord God of my native land, Who wast born among the shepherd-folk, Fire my words and give me breath. The epic character of the poem is sustained further than in its mere outward form; the The art of the manner of telling is truly epic. poet is throughout singularly objective, his narrative is a narrative of actions, his personages speak and move before us, without intervention on the part of the author to analyze their He is absent from his thoughts and motives. work even in the numerous descriptions. Everything is presented from the outside. From the outset the poem enjoyed great success, and the enthusiastic praise of Lamartine contributed greatly thereto. this. Mistral In gratitude for dedicated the work to Lamartine in one of his most happy inspirations, and these dedicatory lines appear in Lis Iselo cf Or and in all the subsequent editions of Mireio. Mistral had professed great admiration for the author of Joeelyn even before 1859, but as poets they stand in marked contrast. We may partly define Mistral's art in stating that it is utterly unlike that of Lamartine. Mistral's inspiration FR^D^RIC MISTRAL 104 is not that of a Romantic ; his art sense is de- rived directly from the study of the Greek and Roman classics. there is very In all that Mistral has written little sonal sorrows. that springs from his per- The great body of his poetry best of his work in is epic in character, and the the lyric form gives expression not to merely personal emotion, but to the feeling of the race to which he belongs. The action of the poem begins one day that Vinc^n and his father Meste Ambroi, the basket-makers, were wandering along the road in Their conversation makes search of work. them known, and depicts for us the old Mas des Micocoules^ the home of the prosperous father We learn of his wealth in lands, in olives, in almonds, and in bees. We watch the of Mirdio. farm-hands coming home at evening. When the basket-makers reach the gate, they find the daughter of the house, who, having just fed her silkworms, is now twisting a skein. The man and the youth ask to sleep for the night upon a haystack, and stop in friendly talk with Mireio. The poet describes Vincen, a dark, stalwart youth of sixteen, and tells of trade. his skill at his Mdste Ramoun invites them in to supper. THE FOUR LONGER POEMS Mireio runs to serve them. 105 In exquisite verse the poet depicts her grace and beauty. When all have eaten, at the request of the farm-hands, to which Mireio adds hers, Mdste Ambroi sings a stirring ballad about the naval victories of Suffren, and the gallant conduct of the ProvenQal sailors who whipped the British tars. " And the old basket-maker finished his naval song in time, for his voice was about to break in tears, but too soon, surely, for the farm-hands, for, without moving, with their heads intent and lips parted, long after the song had ceased^ they were listening still." And then the men go about their affairs and leave Vinc^n and Mirdio alone together. talk is full of charm. Their Vinc^n is eloquent, like a true southerner, and tells his experiences with flashing eye and animated gestures. Here we learn of the belief in the three Maries, who have their church in the Camargue. Here Vin- c^n narrates a foot-race in which he took part at Nimes, and Mireio listens in rapt attention. " It seems to me," said she to her mother, "that for a basket-maker's fully. child he talks wonder- O mother, it is a pleasure to sleep fr^dIjric mistral 106 now the night is too bright to in winter, but sleep, but let us listen awhile yet. I could pass my evenings and my life listening to him." The second canto opens with the exquisite stanza beginning, " Cantas, caiitas, magnanarello Que la culido es cantarello I and the poet evidently fell in love with music, for he repeats several times it, its with slight variations, during the canto. This second canto is a delight from beginning to end ; Mistral is here in his element ; he is at his very best. The girls sing merrily in the lovely sunshine as they gather the silkworms, Mireio among them. Vincen passes along, and the two engage in conversation. Mistral cannot be praised too highly for the sweetness, the naturalness, the animation of this scene. Mireio learns of Vincen's lonely winter evenings, of his sister, who is like Mireio but not so fair, and they forget to work. lost, only But they make good the time now and then their fingers meet as they put the silkworms into the bag. And then they find a nest of little birds, and the saying goes that when two find a nest at the top of a tree a year cannot pass but that Holy THE FOUR LONGER POEMS 107 Church unite them. So says Mireio but Vincen adds that this is only true if the young escape ; " Jesu moun before they are put into a cage. Dieu take care," cries the young girl, " catch them carefully, for this concerns us." So Vincen gets the young birds, and Mirdio puts them ! and and must be transferred to Vincen's cap and then the branch breaks, and the two fall together in close embrace upon the soft The poet breaks into song grass. " Fresh breezes, that stir the canopy of the woods, let your merry murmur soften into silence over the young couple! Wandering carefully into her bodice; but they dig scratch, ; : zephyrs, — breathe softly, give time to dream, give them time at least to dream of happiness! Thou that ripplest o'er slowly, slowly, little brook ! sound among the stones, thy bed, go Make not so much make not so much sound, for the two souls have gone off, in same beam of fire, like a swarming hive the — let them hover in the starry air But Mireio quickly releases herself; the young man is full of anxiety lest she be hurt, and curses the devilish tree "planted a Friday But she, with a trembling she cannot control, tells ! ! FRfeDfeRIC 108 MISTRAL of an inner torment that takes away hearing and sight, and keeps her heart beating. Vincen won- ders if it may not be fear of a scolding from her mother, or a sunstroke. Then Mir^io, in a sud- den outburst, like a Wagnerian heroine, confesses her love to the astonished boy, who remains dazed, and believes for a time that she is cruelly trifling with him. ately. She reassures him, passion- " Do not speak so," cries the boy, " from me to you there is a labyrinth; you are the queen of the Mas, all bow before you I, peasant of Valabregue, am nothing, Mireio, but a worker in the fields!" "Ah, what is it to me ; whether my beloved be a baron or a basket- Why, O Vincen, in your rags do you appear to me weaver, provided he is pleasing to me. so handsome?" And then the young man is as inspired, and in impassioned, well-nigh extravagant language tells of his love for Mireio. He is like a fig grew thin and miserable out of a rock near Vaucluse, and once a year the water comes and the tree quenches its And the thirst, and renews its life for a year. youth is the fig tree and Mireio the fountain. "And would to Heaven, would to Heaven, tree he once saw that THE FOUR LONGER POEMS that I, 109 poor boy, that I might once a year, as now, upon my knees, sun myself in the beams of thy countenance, and graze thy fingers with a trembling kiss." And then her mother calls. Mireio runs to the house, while he stands motionless as in a dream. No resume or even translation can give the beauty of this canto, its brightness, its music, its vivacity, the perfect harmony between words and sense, the graceful succession of the rhymes and the cadence of the stanzas. Elsewhere in the chapter on versification a reference is made to the mechanical difficulties of translation, but there are difficulties of a deeper order. The Felibres put forth great claims for the richness of their vocabulary, and they undoubtedly exaggerate. Yet, how shall we render into English or French the word embessouna when describing the of Mireio writes : — and Vincdn from the tree. fall Mistral " Toumbon, embessouna, sus lou souple margai." Bessoun (in French, hesson) means a twin, and the participle expresses the together like twins. idea, clasped (Mistral translates, " serres comme deux jumeaux.") An expression of FR^DfeRIC MISTRAL 110 this sort, of course, adds little to the prose lan- guage ; but this power, untrammelled by aca- word for the moment, is essential to the freshness of poetic demic traditions, of creating a style. What is to be praised above all in these two exquisite cantos is the pervading naturalness. The similes and metaphors, however bold and original, are always drawn speakers. from the life of the Meste Ambroi, declining at first to sing, says " lA mirau soun creha ! " (The mirrors are broken), referring to the membranes of the locust that make its song. under the hammer," " Their " Like a scythe heads leaning together like two marsh-flowers in bloom, blowing in the merry wind," " His words flowed abundantly like a sudden shower on an after- math in May," " When your eyes beam upon me, it seems to me I drink a draught of per- fumed wine," " My sister is burned like a branch of the date tree," " You are like the asphodel, and the tanned hand of Summer dares not caress your white brow," "Slender as a dragon-fly," are comparisons taken at random. Of Mireio the poet says, " The merry sun hath hatched her out," " Her glance is like dew, THE FOUR LONGER POEMS 111 her rounded bosom is a double peach not yet ripe." The background of the action is obtained by the simplest description, a cart casting the shadow of its great wheels, a bell now and then sounding afar off across the marshes, references to the owl adding its plaint to the song of the nightingale, to the crickets who stop to listen now and then, and the recurring verses about the " magnanarello " reminds us now and then, like a lovely leitmotiv, of the group of singing girls about the amorous pair. The next canto is called La Descoucounado (The Opening of the Cocoons), and it must be confessed that there is a slight falling off in interest. All that describes the country-folk is full of life of the sustained charm, but Mistral has not escaped the dangers that beset the modern poet who aims at the epic style. Here begins the recounting of the numerous superstitions of the ignorant peasants, and the wonders of Provence are interpolated at every turn. The maidens, while engaged in stripping the cocoons, make known a long list of popular beliefs, and then branch tion about love. off into a conversa- They are surprisingly well li2 FRfeDljRIC MISTRAL acquainted with the writings of Jean de Nostradamus, to whom the Felibres are indebted for a lot of erroneous ideas concerning the Troubadours and the Courts of Love. This literary conversation is not convincing, and we are pleased when Noro sings the pretty song of Magali, which, composed to be sung to an air well known in Provence, has become very popular. The idea is not new the young girl sings ; of successive forms she will assume, to avoid the attentions of her suitor, and he, ingeniously, finds the transformation necessary to overcome her. For instance, when she becomes a rose, he changes into a butterfly to kiss her. At last the maiden becomes convinced of the love of her pursuer, and is won. The fourth canto, Li Demandaire (The Suitors), recalls the Homeric style, and is among the finest of the poem. Alari, the shepherd, Veran, the keeper of horses, and Ourrias, who has herds of bulls in the Camargue, present themselves successively for the hand of Mireio. The " transhumance des troupeaux " scribed in verse full of vigorous the sheep are taken is de- movement up into the Alps for the summer, and then in the fall brought down to THE FOUR LONGER POEMS 113 the great plain of the Crau near the Delta of the Rhone. The whole description made is with bold, simple strokes of the brush, offering a vivid picture not to be forgotten. too, offers a marvellously Alari, carved wooden cup, adorned with pastoral scenes. Veran owns a hundred white mares, whose manes, thick and flowing like the grass the marshes, are of untouched by the shears, and float above their necks, as they bound fiercely along, like a fairy's They are never subdued, and often, after years of exile from the salt meadows of the scarf. Camargue, they throw off their rider, and gallop over twenty leagues of marshes to the land of their birth, to breathe the free salt air of the sea. Their element is the sea ; they have surely broken loose from the chariot of Neptune are they and when the sea white with foam ; still ; roars and darkens, when the ships break their cables, the stallions of the Camargue neigh with joy. And Ramoun welcomes Veran, and hopes that Mireio will wed him, and calls his daughter, who gently refuses. rias, has this The third suitor, OurThe account of no better fortune. man's giant strength, the narrative of his fr6dI:ric mistral 114 exploits in subduing the wild bulls, are quite The Homeric. story told of the scar he is bears, how one of the fiercest bulls that he had branded carried him along, threw him ahead on the ground, and then hurled him high into the air. The strong, fierce man presents his suit, describing the Camargue life women lead the in the but before he has her love, "his ; trident will bear flowers, the hills will melt away like wax, and the journey to Les Baux will be by sea." This canto and the next, recounting the fierce combat between Ourrias and Vincen, are really splendid narrative poetry. The style is marvellously compressed, and the story thrilling. The sullen anger of Ourrias, his insult that does not spare Mireio, the indig- nation of Vincen, that fires him with unwonted two men out alone mighty Pont du Gard, strength, the battle of the in the fields near the Vincen's victory in the treachery of Ourrias, trial of strength, who the sneaks back and enemy down with the trident. " With a mighty groan the hapless boy rolls at strikes his full length upon the grass, and the grass yields, bloody, and over his earthy limbs the ants of the fields already make their way." The THE FOUR LONGER POEMS rapidity, the compactness of impressed Gaston the 115 sentences, Paris as very remarkable. The assassin gallops away upon his mare, and seeks by night to cross the Rhone. A singu- larly felicitous use of the supernatural is made Ourrias is carried to the bottom of the by the goblins and spirits that come out and hover over it at night. There is a certain here. river terror in this termination, something that recalls parts of the Inferno. fears are the effect of his guilty conscience. Ourrias's superstitious The souls of the damned, their weird ceremonial, are but the outward rendering of the inward terror he feels. A less legitimate use of the supernatural made in the succeeding canto, called La Masco (The Witch). In fact, the canto is is poem. Vinfound unconscious and carried to the really a blemish in the beautiful cen is Mas des Micocoules, and various remedies tried. He comes to himself, but the wound is deemed too serious to be healed by natural means, and Mireio, at the suggestion of one of her maiden friends, takes Vincen to the abode of the witch who lives in the Fairies' Hole under the rocks of Les Baux. Besides the obvious 116 rR:feD]feRIC MISTRAL objection that the magic cure could not have been made, there is the physical impossibility of Vincen's having walked, in his dying condition, through the labyrinth of subterranean passages, amid the wild scenes of a sort of Wal- purgis night. The poet was doubtless led into all the by legends and superstitious lore of Provence. Possibly he was led astray also by his desire to create an epic poem, in which a visit to The entire the lower regions is a necessity. episode is impossible and uninteresting, and this is error his desire to preserve a blot in the beautiful idyll. Later on, this desire to insert the supernatural leads the poet to interrupt the action of his poem, while the three Maries relate to the unconscious Mireio at great length the story of their Jerusalem to Provence. lore, or as coming from Interesting as folk- an evidence of the credulity of the Provengals, this narrative of the three Maries is out of place in the poem. It does not help us out to suppose that Mireio dreams the narrative, for it is full of theology, history, traditions and she could not possibly have con- The poem of Mireio and all Mistral's suffer from this desire to work into his work ceived. THE FOUR LONGER POEMS 117 poetry all the history, real and legendary, of his region. The three Maries are Mary Magdalen, Mary, the mother of James and John, and Mary, the mother of James the Less. fixion they embark After the Cruci- with Saint Trophime, and successfully battling with the storms of the they land finally in Provence, and by a sea, series of miracles convert the people of Aries. This canto never would have converted Boileau from his disapproval of the " merveilleux Chretien." The poet finds his true inspiration again in the life of the Mas, in the home-bringing of the crops, in the gathering of the workers about the table of Meste Ramoun. This picture of patriarchal life is like a bit out of an ancient we have a feeling of the archaic, of the primitive, we are amid the first elements of human life, where none of the complications of the modern man find a place. Meste Am- literature ; broi, whom Vincen has finally persuaded with passionate entreaties to seek the hand of Mireio for him, comes upon this evening scene. interview of the two old men play ; is like a The Greek their wisdom and experience are uttered FRfeD^RIC MISTRAL 118 in stately, sententious language, proverb falls from their lips. and many a Ramoun has inflexible ideas as to parental authority : " father is A must be done. a father, his will The herd that leads the herdsman, sooner or later, is crunched in the jaws of the wolf. If a son resisted his father in our day, the father would have slain him perhaps Therefore the families were strong, united, sound, resisting Doubtthe storm like a line of plane trees less they had their quarrels, as we know, but ! ! when Christmas night, beneath its starry tent, brought together the head of the house and his descendants, before the blessed table, before the table where he presided, the old man, with his wrinkled hand, washed it all away with his " benediction ! But Mireio and not Meste Ambroi makes known to her father that it is her hand Vincen seeks, and the mother and father break out in Ramoun's anger leads him to speak offensively to Meste Ambroi, who nobly maintains his dignity amid his poverty, anger against the maid. and recounts his services to his country that have been so ill repaid. Ramoun is equally proud of his wealth, earned by the sweat of THE FOUR LONGER POEMS his brow, and sternly refuses. 119 The other leaves, and then the harvesters continue their merry-making, with singing and farandoles, about a great bonfire in honor of Saint John. " All the hills were aglow as if stars had rained mad wind carried up in the darkness, and the the incense of the hills and the red gleam of the fires toward the saint, hovering in the blue twilight." That night Mireio grieved and wept for Vincen, and, remembering what he had told her of the three Saint Maries, rises before the dawn and flees away. Her journey across the Crau and the island of Camargue is narrated with numerous details and descriptions they ; are never extraneous to the action, and are a constant source of beauty and interest. The strange, barren plain of the Crau, covered with the stones that once destroyed a race of Giants, as the legend has the maiden flies of the June sun. it, is across vividly described, as it in the ardent rays She stops to pray to a saint that he send her a draught of water, mediately she comes upon a well. and imHere she meets a little Arlesian boy who tells her "in his golden speech" of the glories of Aries. FRilDfeRIC 120 MISTRAL " But," says the poet, " O soft, dark city, the child forgot to tell fertile land of Aries, thy supreme wonder O Heaven gives pure beauty thy daughters, as to ; gives grapes to the it autumn, and perfumes to the mountains and The little fellow talks of many things and leads her to his home. From wings to the bird." here the fisherman ferries her over the broad Rhone, and we accompany her over the Ca- A mirage deceives margue, down to the sea. her for a time, she sees the town and church, but it soon vanishes in air, and the maiden hurries on in the fierce heat. Her prayer in the chapel is written in another verse form : — O Santi Mario Que poudes en flour Chanja nosti plour Clinas Ifeu I'auriho De-vers ma doulour I O Holy Maries, who can change our tears to blossoms, incline quickly an ear unto my grief Before the prayer is ended, there begins the vision of the three Maries, descending to her from Heaven. THE FOUR LONGER POEMS 121 Meste Ramoun discovers the flight of the unhappy maiden, and with all his family starts After the first outburst of grief, he in pursuit. sends out a messenger. "Let the mowers and the ploughmen leave Say to the har- the scythes and the ploughs vesters to throw ! down their sickles, bid the shepherds leave their flocks, bid them come to me!" The boy goes out into the fields, among the mowers and gleaners, and everywhere solemnly delivers his message in the selfsame words. He goes down to the Crau, among the dwarf oaks, and toilers summons the shepherds. All these gather about the head of the farm and his wife, who await them in gloomy silence. Meste Ramoun, without making clear what misfortune has overtaken him, entreats the men to tell him what they have seen. And the chief of the haymakers, father of seven sons, tells of an evil omen, how, for the first time in thirty years, at the beginning of his day's work, moan the Then a mower from Tarascon tells how he had cut himself. more. The as he began his work he parents had discovered a nest wherein the young birds had been done to FR^DfeRIC MISTRAL 128 death by a myriad of invading ants. " the tale of Again woe was a lance-thrust for the father and mother." A third had been taken as with epilepsy, a shudder had passed over him, and through his dishevelled hair as through the heads of thistles he had felt Death pass like A fourth had seen Mireio just before a wind. the dawn, and had heard her say, " Will none among the shepherds come with me to the Holy Maries ? " And then while the mother laments, preparations are made to follow the maiden to the shrines out yonder by the sea. This poem, then, depicts for us the rustic life of Provence in all its outward aspects. The pretty tale and the description of the life of the Mas and of the Provengal landscapes are inseparably woven together, forming an harmonious whole. It is not a tragedy, all the characters are too utterly lacking in depth. Vincen and Mireio are but a boy and a girl, children just awakening to life. The reader may be re- minded of Hermann and Dorothea, of Gabriel and Evangeline, but the creations of the German and the American poet are greatly superior in all that represents study of the human mind and heart. THE FOUR LONGER POEMS 123 poem and Mistral's have several Hermann seeks to marry points of likeness. Goethe's against his father's wish, and the objection is The case is merely inverted. Both poems imitate the Homeric style, Goethe's more palpably than Mistral's, since the German poet has adopted the Homeric verse. the poverty of Dorothea. He affects, also, certain recurring terms of expression, "Also sprach sie" and the like, and there a rather artificial seeking after sim- is plicity of expression. Goethe's poem is more interesting because of the greater solidity of the characters, and because of the more closely knitted plot. The curiosity of the reader is kept roused as in a well-constructed romance. Mistral's poem has, after all, scarcely any more two real local color; the rustic life of the poems similar, allowing for geographical is differences, and we carry away quite as real a picture of Hermann's home and the fields about it as of the Mas of Meste Ramoun. Mistral's idyll terminates tragically in that Mireio dies of sunstroke, leaving her lover to the tenor of the mourn, but German poem is more serious and moves us more deeply the background of war contributes to this, but the source of our ; FRto^RIC MISTRAL 124 emotion is in the deep seriousness of the characters themselves. Vincen and Mireio are charming in their and unreflecting. They are children, and lacking in well-defined naivete, they are unspoiled personality. They have no knowledge of any- thing beyond the customs and superstitions of the simple folk about them. Their religion, which is so continually before us, furnishing the very mainspring of the fatal denouement, is of the most superficial sort, if it can be called religion at all. Whether you are bitten by a dog, a wolf, or a snake, or lose your eyesight, or are in danger of losing your lover, you run to the shrine of some saint for help. ligious feeling really runs no deeper. The reIn his outburst of grief upon seeing Mireio prone upon the floor of the chapel, the unhappy boy asks what he has done to merit such a blow. " Has he lit his pipe in a church at the lamp? or dragged the crucifix among Jews ? " thistles, like the Of the deeper, nobler consolations of religion, of the problems of human destiny, of the relations of religious conviction to human conduct, there is no inkling. All the characters are equally on the surface. THE FOUR LONGER POEMS 125 They are types rather than individuals. They have in common the gift of eloquence. They have no thought-life, no meditation. They are eminently sociable, frequently loquacious. They make you think of Daudet's statement concerning the is not man of the south, "When he talking, he is not thinking." But they and have to an eminent degree the Vinc^n's stories of what he has knows and seen are told most beautifully, and the poet never forgets himself by making the boy utter thoughts he could not have conThe boy is merely a child of his race. ceived. In any rustic gathering in southern France you talk well, gift of narrative. may hear a man of the people speak dramatiand thrillingly, with resonant voice and with a marvellous power of mimicry, and the faces of the listeners reflect all the emotions of the speaker. The numerous scenes, therefore, wherein a group of listeners cally vivid gestures, follow with keenest interest a tale that is told, are eminently true to life. The supreme merit of Mirdio lies in this power of narration that its author possesses. It is all action from begin- ning to end, and even the digressions and episodes, which occasionally arrest the flow of the FR^D^RIC MISTRAL 126 narrative, are in themselves admirable pieces of Most critics have found fault with these episodes and the frequent insertion of narrative. legends. In defence of the author, it may be said, that he must have feared while writing Mireio that it might be his last and only opportunity to address his countrymen in their own dialect, and in his desire to bring them back to a love of the traditions of Provence, he yielded crowd his poem rather more than he would otherwise have done. to the temptation to Mireio, then, is a lovely poem, an idyll, a charming, vivid picture of life in the rural parts of the Rhone region. Local color is its It is singularly original. very essence. Its thought and action are strictly circumscribed within the boundaries of the Crau and the Camargue, and its originality consists in this limitation, in the fact that a poet of this century has written a work that comes within the definition of an epic, with all the primitive simplicity of Bibli- cal or Classic writers, without any agitation of the problems of modern life, without any new thought or feeling concerning love or death, or man's relation to the universe, using a dialect unknown at the time beyond the region de- THE FOUR LONGER POEMS scribed. 127 success could scarcely have been Its attained without the poet's masterly prose translation, and yet it is evident that the poem could not have been conceived and carried out in French verse. The freshness, the artlessness, the lack of modernity, would have suffered if the poet had bent his inspiration to the cial language. offi- Using a new idiom, wherein he practically had no predecessor, he was free to create expression as he went along, and was not compelled to cast his thought in existing moulds. The poem cannot place its author among the very great poets of the world, if only because of this limitation. It lacks the breadth and depth, the everlasting interest. But it is a work of great beauty, of wonderful purity, a sweet story, told in lovely, limpid language, and will cause many eyes to turn awhile from other lands to the sunny landscapes of southern France. II. Calendau. (Calbndal.) Mistral spent seven years in elaborating his second epic, as he did in writing his first. The poem had not a popular success, and the reason FRfiDfiRIC 128 is The most striking limita- not far to seek. tion of the poet of flesh MISTRAL is his failure to create beings and blood. Even in Mireio this lack of well-defined individuality in the characters begins to be apparent, but, in general, the action of the earlier poem is confined to the world of realities, whereas in Calendau the poet has given free play to a brilliant and vivid imagination, launching and incredible, forth into the heroic yet without abandoning world of real time and real places. the Allegory and symbolism are the web and woof of Calendau. The poem, again, is overburdened with minute historic details and descriptions, which are greatly magnified in the eye of his imagination. A poet, of course, must be pardoned for this want of a sense of proportion, but even a Provengal reader cannot be kept in constant illusion as to the greatness of little places that can scarcely be found upon the map, or dazzled by the magnificence of achievements that really have left little or no impress upon the history As we follow the poet's work in its chronological development, we find this trait of the world. growing more and more pronounced. He sees his beloved Provence, its past and present, and THE FOUR LONGER POEMS its future, too, in a embellishes magnifying mirror that all it reflects ing colors, and exalts sal proportions. the spell of 129 with splendid, glow- little figures to colos- The reader falls easily under this exuberant enthusiasm and charmed by the poetic power evinced. The wealth of words, the beauty of the imagery is with which, for example, the humble, well-nigh unknown little port of Cassis and its fishing industry are described, carry us along and hold us in momentary illusion. poet's We see them in the magic mirror for the time. To the traveller or the sober historian all these things appear very, very different. With the Felibres the success of the poem was much greater; it is a kind of patriotic hymn, a glorification of the past of Provence, and a song of hope for its future. Its allegory, its learned literary allusions, its delving into obscure historic events, preclude any hope of popular success. Like Mireio, the poem is divided into twelve cantos, and the form of stanza employed is the same. The heroic tone of the poem might be thought to have required verse of greater stateliness; the recurrence of the three feminine FR^DfeRIC MISTRAL 130 rhymes in the shorter verses often seems too Like Mireio, the poem has the outward marks of an epic. Unlike Mireio, it reminds us frequently of the Chansons de geste, and we see pretty. that the author has been living in the world of the Old Provengal poets. This is apparent not merely in the constant allusions, in the reproductions of episodes, but in the manner in which the narrative moves along. Lamartine would not have been reminded of the ancient Greek poets had Calendau preceded Mireio. The conception of courtly love, the guiding, elevating inspiration of Beatrice, leading Dante on to greater, higher, more spiritual things, are the sources of the chief ideas contained in Cal- endau. Vinc^n and Mireio remain throughout the simple youth and maiden they were, but Calendau, " the simple fisherman of Cassis," develops into a great hero, performing Herculean tasks, like a knight of the days of chivalry, and rises higher and higher until he wins "the empire of pure love " — his lady's hand. Very beautiful is the invocation addressed to the " soul of his country that radiates, manifest in language and in its through the greatness of its its history — that memories saves THE FOUR LONGER POEMS hope for him." 131 It is the spirit that inspired the sweet Troubadours, and set the voice of Mirabeau thundering like the mistral. The " For the poet proclaims his belief in his race. waves of the ages and their storms and horrors mingle the nations and wipe out frontiers in vain. Mother Earth, Nature, ever feeds her sons with the same milk, her hard breast will ever give the fine oil to the olive ; Spirit, ever springing into joyous, proud, life, and living spirit that neighest in the noise of the Rhone and in the wind thereof harmo- spirit of the ! nious woods, and of the sunny bays, pious soul of the fatherland, I call thee ! be incarnate in " my Provengal verse We are medias res. ! plunged in orthodox fashion in The young fisherman is seated upon the rocky heights above the sea before the He does not know who she is; he has performed almost superhuman exploits to win her but there is an beautiful woman he loves. ; obstacle to their union. She relates that she is the last of the family of the Princes des Baux, who had their castle and city hewn out of the solid rock in the strange look the plain of Aries. mountains that over- She tells the mar- FR^D^RIC MISTRAL 132 vellous history of the family, evoking a vision of the days of courtly love when the Trouba- dours sang at the feet of the fair princesses. A panorama of the life of those days of poetry and song moves before us. The princess even de- scribes and defines in poetic language the forms of verse in vogue in the ancient days, the Ten8on, the Pastoral, the Ballad, the SirventSs, the Romance, the CongS, the Auhade, the Solace of She relates her marriage with the Count Love. Severan, who fascinated her by some mysterious power. he is At the wedding-feast she learns that a mere bandit, leader of a band of rob- She fled away through the mountains and found the grotto bers that infests the country. where she now lives. The fishermen, seeing her appear and vanish among the cliffs, take her to be the fairy Esterello, who is a sort of Loreley. Calendau determines that either and seeks him out. His splendid physical appearance and bold, de- Severan or he shall die, fiant manner arouse in the bandit a desire to get Calendau to join his company, and the women of the band are charmed with him. to hear the story 'of his life, They ask and the great body of the poem consists of the narrative by THE FOUR LONGER POEMS Calendau of his exploits. 138 After the last one Calendau has risen to the loftiest conception of pure love through the guidance of Esterello, Dante inspired by Beatrice. Then the Count holds an orgy and tries to tempt the like virtue of the hero. Calendau, after witnessing the lascivious dances, challenges the Count to The latter knows now who he and that Esterello is none other than the mortal combat. is, bride who fled after the marriage-feast. Cal- endau is overpowered and imprisoned, and the Count and his men set off in search of EsteBut Calendau is freed by Fourtuneto, one of the women, and journeys by sea from Cannes to Cassis to defend the Princess. Here a great combat takes place with the Count, who fires the pine-woods and perishes miserably, uttering blasphemous imprecations. The Cassidians fight the fire, and Calendau and the rello. blond Princess are saved. "The applause of two thousand lutes them and acclaims them. Calendau, let us plant the queror of Esterello. souls ' sa- Calendau, May for the con- He glorifies, he brings to the light our little harbor of fishermen, let us make him Consul, Consul for life! ' So saying FR^DfiRIC MISTRAL 134 the multitude accompanies the generous, happy pair of lovers, and the sun that God rules, the great sun, rises, illumines, and procreates endlessly new enthusiasms, new lovers." The poem clearly symbolizes the Provencal renascence; Calendau typifies the modern Pro- vengal people, rising to an ideal life and great achievements through the memory of their traditions, and this ideal, this memory, are per- sonified in the person of the beautiful Princess. The time of the action is the eighteenth century, before the Revolution. erate choice of the poet symbolism in mind. This is a delib- who has a temporal " I shall thus combine in my picture the three aspects of Provence on the eve of the Revolution: in the background, the noble legends of the past ; in the foreground the social corruption of the evil days; and before us the better future, the future and the reparation personified in the son of the work- ing classes, guardians of the tradition of the country." As regards the execution, it is masterly, and cannot be ranked below Mir Ho. There is the same enthusiastic love of nature, the same astonishing resources of expression, the same THE FOUR LONGER POEMS novelty and originality. 135 In place of the rustic nature of Mireio, we have the wild grandeur of mountains and sea. There is the same, nay, even greater, eloquence of the speakers, the same musical verse. " Car, d'aquesto ouro, ounto es la raro Que di delice nous separo, Jouine, amourous que siam, libre coume d'aucfeu? Regardo : la Nature brulo A noste entour, e se barrulo Dins li bras de I'Estieu, e chulo Lou devourant alen de soun nove roussfeu. " Li serre clar e blu, 11 colo Palo de la calour e molo, Boulegon trefouli si mourre ... Ve la mar Courouso e lindo courao un vhhe, D6u grand souleu i rai bevfeire Enjusqu'au founs se laisso veire, Se laisso coutiga per lou Rose e lou Var." : " For now, where is the limit that separates us from joy, young, amorous as we are, free as birds ! Look: Nature burns around us and rolls in the arms of Summer, and drinks in the devouring breath of her ruddy spouse. The and soft with the heat, are thrilled and stir their rounding summits. Behold the sea, glistening and limclear, blue peaks, the hills, pale frI:dI3ric 136 mistral pid as glass; in the thirsty rays of the great sun, she allows herself to be seen clear to the bottom, to be caressed by the Rhone and the Var." These are the words of Calendau when, seeking his reward after his final exploit, he learns that he has never poet won the love of Esterello. goes in the strain, and the cially further The voluptuous mere music of the words, espe- "Ve la mar" is exquisite. beginning They are found in the first canto. This scene wherein the Princess refuses to wed Calendau is typical of the poet. The northern temperament is tirades, full not of impressed with ejaculations these long and apostrophes; they are apt to seem unnatural, insincere, and theatrical. Intense feeling is not so verbose in the north. In this particular Mistral is true to his race. We quote entire the words of Calen- dau after the refusal of Esterello, itself — full exclamation and apostrophizing " Then I have but won the thirst, the weari: ness of the midshipman, when he is about to reach the summit of the mainmast, and sees gleaming at the limit of the liquid plain naught but water, water eternally I Well, if thou wilt THE FOUR LONGER POEMS hear it, listen it! I 137 and let the heath resound with It is thou, false woman that thou art, it is thou that hast deceived me, luring me on to believe that at the summit of the peaks I should find the splendor of a sublime dawn, that after winter spring would come, that there is nothing so good as the food earned by labor. Thou hast deceived me, for in the wilderness I found naught but drought; and the wind of this world and its idle noise, the embarrassment of luxury, and the din of glory, and what is called the enjoyment of triumph, are not worth a little hour of love beneath a pine tree! See, from my hand the bridle escapes, my skull is bursting, and I am not sure now that the people in their fear are not right in dreading thee like a ghost, now that I feel, as my reward, thy burning poison streaming through my heart. Yes, thou art the fairy Esterello, and thou art unmasked at last, cruel creature of thy refusal I have ! In the known the viper. art Esterello, bitter foe to chill Thou man, haunting the wild places, crowned with nettles, defending who clear the land. Thou art Esterello, the fairy that sends a shud- the desert against those der through the foliage of the woods and the MISTRAL FR]fcD6RIC 138 hair of the terrified hermit; that fires with the desire of her perfumed embrace her suitors and in malevolence them drives to despair with infernal longings. "My head is bursting, and since from the heights of my supernatural love a thunderbolt thus hurls me down, since, nothing, nothing moment on, can give me henceforth, from this joy, since, cruel woman, when thou couldst throw me a rope, thou leavest me, in dismay, to drink the bitter current — let death come, black hiding-place, bottomless abyss! let me plunge " down head first And when Esterello, ! fearing he will slay himself, clasps him about the neck, they stand silently embraced, gling, rain "the tears, in tender min- from their eyes; despair, agitation, a spell of happiness, keep their lips idle, and from hell, at one bound, they rise to para- dise." Like the creations of Victor Hugo's poetry, those of Mistral speak the language of the author. They have his eloquence, his violent energy of figurative speech, his love of the wild, sunny landscapes about them; they thrill as he does, at the memories of the past ; they love, as THE FOUR LONGER POEMS 139 he does, enumerations of trees and plants; they have his fondness for action. The poem is filled with interesting episodes. One that is very striking in the narrative of Esterello we shall here reproduce. We are at the wedding feast of Count Severan and the Princess des Baux. The merry- making begins to be riotous, and the Count has made a speech in honor of his bride, promising to take her after the melting of the snows to his Alpine palaces, where the walls are of steel, the doors of silver, the locks of gold, and when the sun shines their crystal roofs glitter like flame. " Scarcely from his lips had fallen these wild words, when the door of the banquet hall opens, and we see the head of an old man, wearing a bonnet and a garment of rough cloth; we see the dust and sweat trickling down his tanned cheeks. The bridegroom, with glance, like the lightning flash a terrible of a fearful storm, turns suddenly pale, and seeks to stop him; but he, whom the glance cannot harm, calmly, impassively, like God when he clothes himself like a poor man, to confound sometimes some rich evil-doer, slowly advances toward the bridegroom, crosses his arms, and scans FRl:D:fcRIC 140 his countenance. MISTRAL And he says not a word to any one, and all are afraid; a weight of lead lies upon every heart, and from without there seems to blow in upon the lamps an icy wind. "Finally, a few of them, shaking off their oppression, ' If there come not soon a famine to wipe out this hideous tribe, we shall be eaten by beggars within four days! To the merry bridal pair, what hast thou to say, old scullion? And they continue to taunt him cruelly. The outraged peasant holds his peace. 'With his blear eyes, his white pate, whither comes he trudging? ill his limping leg, Pelican, bird of omen, go to thy hole and hide thy sorry face.' The stranger swallows their insults, and casts toward the bridegroom a beseeching glance. "But others cry: 'Come on, old man, come on! Come on, fear not the company, the laughing and joking of these pretty gentlemen. Hunt about the tables for the dainties and the carcasses. Hast thou a good jaw ? Here, catch of pork and toss off a glass of this piece wine!' " ' No,' at length comes an answer from the THE FOUR LONGER POEMS 141 old man, in a tone of deep sadness, ' gentlemen, do not beg, and have never desired what 'His son! others leave: I seek my son.' I What is he saying — — the son of this seller about the Baroness of of eelskins hovering Aiglun ? "And they look at each other in doubt, in Then they said: Show thy son, come on! If, to mock us, thou lie, wretch, burning scorn. I listened. 'Where is thy son? and beware. at the highest gargoyle of the towers of Aiglun, without mercy, we'll hang thee! " Well, since I am disowned, and relegated ' to the sweepings,' the old man begins, draped in his sayon^ and with a majesty that frightens us, ' you shall hear the crow sing ! ' Then the Count, turning the color of the wall, cold as a bench of stone, said, 'Varlets, here, cast this dismal phantom ! ' out Two tears of fire, that pierced the ground, and that I still see shining, streamed down the countenance of the poor old man, ah! so bitter, that we all became white as shrouds. " ' Like Death, I come where I am forgotten, without summons. I am wrong ! ' broke out the unhappy man, 'but I wished to see my FRi:D:fcRIC 142 MISTRAL Come on, cast out this dis- daughter-in-law. mal phantom, who is, however, thy father, O splendid bridegroom ! "I uttered a cry; all the guests rose from their chairs. ' But the relentless old man went on My lords, to tear from the evil fruit its whole covering, I have but two words to say. Be seated, for I still see on the table dishes not yet eaten.' "Standing like palings, silent, anxious, the guests remained with hearts scarce beating. trembled, my eyes in mist. I We were like the dead of the churchyard about some funeral feast, full of terror and mystery. The Count grinned sardonically. " ' Thou shalt run in vain, wretch,' said the venerable father, 'the vengeance of God will To-day thou makest me bow my head but thy bride, if she have some honor, will presently flee from thee as from the surely reach thee ! ; pest, for God ' thou shalt some day hang, accursed of I rush to the arms of ! ' Stop, stop ; ' my father-in-law. but he, leaning down to my ear, Without knowing the vine or measuring the furrows, thou hast bought the wine, mad girl Go, thou didst not weep all thy tears in said : ! ' THE FOUR LONGER POEMS thy swaddling clothes ! 143 Knowest thou whom thou hast ? a robber-chief ! ' And the scene continues, weirdly dramatic, some old romantic tale of feudal days. Such scenes of gloom and terror are not frequent in Mistral. This one is probably the best of its kind he has attempted. On his way to seek Count Severan in his fastness, Calendau " enters, awestruck, into the like stupendous valley, deep, frowning, cold, saturnine, and fierce ; the daylight darts into this enclosure an instant upon the viper and the lizard, then, ishes. behind the jagged peaks, it van- The Esteron rolls below. Now, Calen- dau feels a shudder in his soul, and winds his horn. The call resounds in the depths of the gorges. It seems as though he calls to his aid the spirits of the place. And he thinks of the paladin dying at Roncevaux." For the sake of greater completeness, we summarize briefly the exploits of the hero. As has been stated, they compose the great body of the poem, and are narrated by him to the Count and his company of thieves and women. The narrative begins with the account of the little port of Cassis, his native place; and one of FR^DifeRIC 144 MISTRAL the stanzas is a setting for the surprising prov- erb: " Tau qu'a vist Paris, Se noun a vist Cassis, Pou dire : N'ai ren vist! He who has seen Paris, and has not seen Cassis, may say, " I have seen nothing." No less than forty stanzas are taken up with the wonders of Cassis, and more than half of those are devoted to sidians catch. naming the fish the Cas- It is to be feared that other than Provengal readers and students of natural history will fail to share the enthusiasm of the poet here. Calendau's father used to read out of an ancient book ; and the hero recounts the history of Provence, going back to the times of the Ligurians, telling us of the coming of the Greeks, who brought the art of sculpture for the future Puget. We hear of the founding of Marseilles, the days of Diana and Apollo, followed by the coming of the Romans. victory of Caius Marius is The celebrated, the con- the introduction of Christianity. We learn of We come down Raymond quest of Julius Caesar deplored. to Toulouse. the glorious days of of THE FOUR LONGER POEMS 145 " And enraptured to be free, young, robust, happy in the joy of living, in those days a whole people was seen at the feet of Beauty and singing blame or praises a hundred Troubadours flourished; and from its cradle, amid vicissitudes, Europe smiled upon our merry singing." " O flowers, ye came too soon bloom, the sword cut Bright sun of the Nation in ! down thy blossoming south, thou shonest too powerfully, and the thunder-storms gathered. Dethroned, made barefoot, and gagged, the Proven9al language, proud, however, as before, went off to live among the shepherds and the sailors." "Language of love, if there are fools and by Saint Cyr, thou shalt have the men of the land upon thy side, and as long bastards, ah! as the fierce mistral shall roar in the rocks, sensitive to an insult offered thee, we shall defend thee with red cannon-balls, for thou art the fatherland, and thou art freedom This love of the language all the work expressed it " ! itself pervades of our poet, but rarely has he more violently, than here. energetically, not to say FRiJD^RIC MISTRAL 146 Calendau reaches the point where he catches a glimpse of the Princess. first He tells of the legends concerning the fairy Esterello, and of the Fada (Les Enfees). This last is a name given to idiots or to the insane, who are sup- posed to have come under her spell. " E degun auso Se trufa d'eli, car an quicon de sacra " I And none dares mock them, for they have in them something sacred. The fisherman makes many attempts to find her again, and at last succeeds. She haughtily dismisses his suit. "Vai, noun sies proun famous, ni proun fort, ni proun fin." Go, thou art not famous enough, nor strong enough, nor fine enough. He realizes her great superiority, and, after a time of deep discouragement, rouses himself and sets about to deserve and win her by deeds of daring, by making a great name for himself. His first idea is to seek wealth, so he builds a great boat and captures twelve hun- dred tunny fish. The fishing scenes are de- picted with all the glow of fancy and brilliant THE FOUR LONGER POEMS word-painting for which Mistral able. 147 so remark- is Calendau is now rich, and brings jewels to his lady. She haughtily refuses them, and the fisherman throws them away. « — Eh ! bfen, i^ fau, d'abord, ingrato, Que toun cor dur ansin me trato E que de mi present noun t'enchau mai qu' ac6, E li bandisse Vagon au Diable ! Pataflbu ! — dins lou precepice." . . . " Well," said I to her, " since, ungrateful woman, thy hard heart treats me thus, and thou carest no more about my presents than that, let them go to the devil " and I hurled them, patajibu, into the precipice. I . Here the tone is reader finds serious ; . . not one that an English the sending the jewels to the Devil, in the presence of the beautiful lady, and the interjection, seem trivial. Evidently they are not so, for the Princess is mollified at once. " He was not very astute, he who made thee believe that the love of a proud soul can be won with a few trinkets ! Ah, where are the hand- some Troubadours, masters of love ? " She tells the love-stories of Geoffroy Rudel, of Ganbert de Puy-Abot, of Foulquet of Marseilles, of Guillaume.de Balaiin, of Guillaume 148 FRfeDfeRIC MISTRAL de la Tour, and her words fall upon Calendau's heart like a flame. He catches a glimpse of an existence of constant ecstasy. His second exploit is a tournament on the water, where the combatants stand on boats, and are rowed violently against one another, each striking his lance against the wooden breastplate of his adversary. His victory wins him the hatred of the Cassidians, for his enemy accuses him of cornering the fish. Esterello consoles him with more stories from the for Chansons de geste and the songs of the Troubadours. In the seventh canto is described in magnificent language Calendau's exploit on the Mont Ventoux. This is a remarkable mountain, visi- ble all over the southern portion of the Rhone valley, standing in solitary grandeur, like a great pyramid dominating the plain. mit is exceedingly pears to be the difiicult of access. first Its sum- It ap- mountain that literature records as having been ascended for pleasure. This ascent is the subject of one of Petrarch's letters. During nine days Calendau felled the larches that grew upon the flanks of the mighty moun- THE FOUR LONGER POEMS tain, and 149 hurled the forest piecemeal into the At the Rocher du Cire he is torrent below. stung by myriads of bees, during frightfully his attempt to obtain as a trophy for his lady a quantity of honey from this well-nigh inaccessible place. The kind of criticism that is appropriate for realistic literature is here quite must be said, however, that the episode is far from convincing. Calendau out of place. It compares his sufferings to those of a soul in hell, condemned to the cauldron of oil. Yet he makes a safe escape, and we never hear of the physical consequences of his terrible punish- ment. The canto, in its vivid language, its movement, its life, one of the most astonishing is that has come from the pen of offers beautiful its author. It examples of his inspiration in depicting the lovely aspects of nature. He finds words of liquid sweetness to describe the music of the morning breezes breathing through the mass of trees : — " La Ventoureso matiniero, En trespirant dins la sourniero Dis aubre, fernissie coume un pur cantadis, Ounte di colo e di vallado, FR^D^RIC MISTRAL 160 Tduti li voues en assemblado, Mandavon sa boiifaroulado. Li mk\e tranquilas, li mfele mescladis," etc. The morning breeze of the Mont Ventoux, breathing symphony of into the mass of trees, quivered like a pure song wherein all the voices of hill and dale sent their breathings. In the last line the word tranquilas is meant to convey the idea "in tranquil grandeur." This rutliless destruction of the forest brings down upon Calendau the anger of his lady has dishonored the noble mountain. ; he "Sacri- legious generation, ye have the harvest of the plains, the chestnut and the olives of the hillsides, but the beetling belong to brows of the mountains God !" and the lady continues an eloquent defence of the trees, "the beloved sons, the inseparable nurslings, the joy, the colossal ! glory of the universal nurse " and pictures the vengeance Nature wreaks when she is wronged. Calendau is humbled and departs. His next exploit is the settling of the feud between two orders of Masons. marvellous bravery in facing He displays the fighting crowds, and they choose him to be umpire. He delivers a noble speech in favor of peace, full THE FOUR LONGER POEMS allusions of to architectural the 151 glories of Provence, that grew up when " faith and union lent their torch." He tells the story of the building of the bridge of Avignon. " Noah himself with his ark could have passed beneath He touches their emotions each of its arches." with his appeal for peace, and they depart reconciled. And now Esterello begins to love him. She bids him strive for the noblest things, to love country and humanity, to become a knight, an apostle ; and after Calendau has performed the famous brigand Marco- feat of capturing the Mau, after he has been crowned in the feasts at Aix, and resisted victorious the wiles of the women that surround the Count Severan, and saved his lady in the fearful combat on the firesurrounded rock, he wins her. III. Nebto In spite of its utter unreality Nerto is a charming tale, written in a sprightly vein, with here and there a serious touch, reminding the reader fre- quently of Ariosto. The Devil, the Saints, and the Angels figure in it prominently ; but the Devil is not a very terrible personage in Provence, FR^D^RIC MISTRAL 152 and the Angels are entirely lacking in Miltonic The scene of the story is laid in the time of Benedict XIII, who was elected Pope at Avignon in 1394. The story offers grandeur. a lively picture of the papal court, remind- ing the reader forcibly mule. It is filled the description tale of the Pope's of found in Daudet's famous throughout with legends relating to the Devil, and with superstitious beliefs of the Middle Age. It is not always easy to determine when the poet is serious in his statement of religious belief, occasionally he appears to be so, and then a line or so shows us that he has a legend in mind. of the poem he says : — In the prologue " Crfeire, coundus h la vitbri. Douta, vaqui 1' endourmitbri E la pouisoun dins lou barrieu E la lachuslo dins lou rieu." To believe leads to victory. Doubt is the narcotic, and the poison in the barrel, and the euphorbia in the stream. " E, quand lou pople a perdu fe, L'infer abrivo si boufet." And when the people have lost faith, Hell sets its bellows blowing. THE FOUR LONGER POEMS Then later we read A wager Thousands : " What is this between Christ and the of years 163 world ? Demon. ago he challenged God, and when the great game began, they played with great loose rocks from the hills, at quoits, and if any one is unwilling to believe this, let him go to Mount Leberon and see the stone thrown by Satan." So we see that the theology was merely a means of leading up to a local legend. The story is briefly as follows all Mistral's heroines, is : Nerto, like exceedingly young, Her father, the Baron away everything he owned in this world, when she was a very little child, thirteen years of age. Pons, had gambled and while walking along a lonely road one night he met the Devil, who took advantage of his despair to tempt him with the sight of heaps of money. The wretched father sold his daughter's soul to the Evil One. Now on his death-bed he tells his child the fearful tale ; one — means of salvation lies open for her she must go to the Pope. Benedict XIII is besieged in the great palace at Avignon, but the Baron knows of a secret passage from his castle leading under the river Durance to one of the FRtD]fcRIC 154 MISTRAL He bids Nerto towers of the papal residence. go to seek deliverance from the bond, and to make known to the Pope the means of escape. Nerto reaches the palace at the moment when all is in great commotion, for the enemy have suc- ceeded in setting it on fire. She is first seen by the Pope's nephew Don Rodrigue, an exceedingly wicked young man, a sort of brawling Don Juan, who seems to have been guilty of numerous assassinations. He immediately begins to talk love to the maiden, as the means of saving her from the Devil, " the path of love is full of But Nerto has flowers and leads to Paradise. been taught that the road to Heaven is full of stones and thorns, and her innocence saves her from the passionate outburst of the licentious youth. And Nerto is taken to the Pope, whom she finds sadly enthroned in all his splendor, and brings him the news of a means of escape. The last Pope of Avignon bearing the sacred elements, pourtant soun Dieu,, follows the maiden through the underground passage, and escapes with all his followers. At Chateau- Renard he sets up his court with the King of Forcalquier, Naples, and Jerusalem and Donna lolanthe his Queen. Nerto asks the Pope to THE FOUR LONGER POEMS save her soul, but he is powerless. acle can save a soul sold to Satan. 155 Only a mirShe must enter a convent, and pray to the Saints continually. The Court is about to move to Aries, she shall enter the On the way, convent there. Don Rodrigue makes love to her assiduously, but the young girl's heart seems untroubled. At Aries we witness a great combat of animals, in which the lion of Aries, along with four bulls, is turned loose in the arena. lion kills all but one of the bulls. beast, enraged, gores the lion. The The fourth The royal among the spectators and makes for the King's throne. Nerto and the Queen are crouching in terror before him, when Don Rodbrute rushes rigue slays the animal, saving Nerto's life. Nay, he saves more than her life, for had she died then she would have been a prey to the flames of Hell. Nerto becomes a nun, but Don Rodrigue, with a band of ribald followers, succeeds in carrying her off with all the other nuns. They by the King's soldiers into the cemetery of the Aliscamps. Nerto wanders away during the battle and is lost among the tombs. At dawn the next day she strays far out to a are all driven frI;d6ric mistral 166 forest, where she finds a hermit. The old man welcomes her, and believes he can save her soul. The Angel Gabriel visits him frequently, and But the Angel disapproves, condemns the pride of the anchorite, and soars away to the stars without a word of hope or consolation, and so in great anxiety the pious man bids her go back to the convent, and he will speak to him. prays Saint Gabriel, Saint Consortia, Saint Tullia, Saint Gent, Saint Verdeme, Saint Julien, Saint Trophime, Saint Formin, and Saint Ste- phen to accompany her. Don Rodrigue is living in a palace built for him in one night by the Devil, wherein are seven halls, each sins. devoted to one of the seven mortal Hither Nerto wanders; here Rodrigue finds her, and begins his passionate love-making But Nerto remains true to her vows, although the germ of love has been in her heart afresh. since the day Rodrigue saved her from the lion. On learning that she is in the Devil's castle, she is filled with terror, believing the fatal day The has arrived. She confesses her love. maiden cries: "Woe is me, Nerto loves you, but if Hell should swallow us up, would there be any love for the damned? Rodrigue, no, THE FOUR LONGER POEMS 157 you would but break the if, with one happy wingstroke, you could soar up to the summits where lives last forever, where hearts vanish united in the bosom of God, I should be delivered, it seems to me, in the same upward there tie is none. If that binds you, impulse; for, in heaven or in the abyss, I am inseparable from you." Rodrigue replies sadly, that his past is too dreadful, that only the ocean could wipe it out. burst of repentance is " Rodrigue, one worth a long penance. Courage, come, only one look toward Heaven The Devil appears. this, his finest ! He swells with pride in triumph; black souls he has in plenty, but since the beginning of his reign over the lower regions he has never captured an immaculate victim like this soul. Rodrigue inverts his sword, and at the sign of the cross, a terrific hurricane sweeps away the palace, Don Rodrigue, and the Devil, and nothing is but a nun of stone who is still visible in left the midst of a field on the site of the chateau. In an Epilogue we learn from the Archangel who visits the hermit that the knight and the maiden were both saved. It is difficult to characterize the curious com- FRifeD^RIC 158 MISTRAL bination of levity and seriousness that runs through this reality tale. anywhere ; There there is is no illusion of no agony of soul in Baron Pon's confession ; Nerto's terror when she learns that she is the property of the Devil is from impressive, because she says too far much, with expressions that are too pretty, perhaps because the rippling octosyllabic verse, in Provengal at least, cannot be serious ; it is hardly worth while to mention the objection that if the Devil can be worsted at any time merely by inverting a sword, especially when the sword is that of an assassin and a rake, whose repentance is scarcely touched upon and is by no means disinterested, it is clear that the Demon has wasted his time at a very foolish game a religious mind might feel a deeper ; sort of reverence for the Archangels than is evinced here. poem it Yet it cannot be said that the parodies things sacred and sublime, and appears to be utterly without philosophical intention. Mistral really has to a surprising degree the naivete of writers of former centuries, and as regards the tale itself and its gen- eral treatment it could almost have been written by a contemporary of the events it relates. THE FOUR LONGER POEMS IV. 159 Lou PouBMo d6u Bose The Poem of the RJione^ the third of the poems in twelve cantos that Mistral has written, appeared in 1897. It completes the symmetry of his life work the former epics extolled the life of the fields, the mountains, and the sea, the ; the beautiful river that brings last glorifies life to his native soil. More than either of the other long poems, it is an act of affection Rhone of the poem is the Rhone of his early childhood, before the steamfor the past, for the packets churned its waters, or the railroads poured up their smoke along its banks. Al- though the poet has interwoven in it a tale of merest fancy, it is essentially realistic, differing notably in this respect from Calendau. This realism descends to the merest details, and the poetic quality of the work suffers considerably in many passages. The poet does not shrink from minute enumeration of cargoes, or technical description of boats, or word-for-word reproduction of the idle talk of boatwomen, or the apparently inexhaustible profanity of the boatmen. The life on the river is vividly por- trayed, and we put down the book with a sense fr6d6ric mistral 160 of really having made the journey from Lyons to Beaucaire with the fleet of seven boats of Master Apian. On opening the volume the reader is struck of first all with the novel versification. It is blank verse, the line being precisely that of Dante's Divina Commedia. there no rhyme, but assonance fully avoided. The effect Not only is of this is very care- unbroken succession of feminine verses is slightly monot- onous, though the poet shifts his pauses skilfully. The rhythm of the lines is marked, the effect upon the ear being quite like that of English The iambic pentameters hypercatalectic. absence of rhyme is the more noteworthy in that rhyme offers little difficulty in Provengal. Doubtless the poet was pleased to show an additional claim to superiority for his speech over the French as a vehicle for poetic thought ; for while on the one hand the rules of rhyme and hiatus give the poet writing in Provencal less when writing in French, on the other hand this poem proves that splendid blank verse may be written in the new trouble than language. The plan of the poem is briefly as follows: THE FOUR LONGER POEMS it 161 describes the departure of a fleet of boats from Lyons, accompanies them down the river to Beaucaire, describes the fair and the return up the river, the boats being hauled by eighty horses ; narrates the collision with a steamboat coming down the stream, which drags the animals into the water, setting the boats adrift in the current, destroying them and their cargo, and typifying as it were the ruin of the old The river itself is detraffic on the Rhone. scribed, its dangerous shoals, its beautiful towns and castles. We learn how the life on board the boats were manoeuvred banks, its ; and the ideas of the men are set before us minutely. Legends and stories concerning the and the river places along the shores abound, and into this general background is of course woven the tale of a Prince of Orange and a ; little maiden called the Anglore, two curiously half-real, half-unreal Mistral seems to love to create. comes on board the fleet, of the beings that The Prince intending to see Orange and Provence some day he is to be King of Holland, but has already sickened of court ceremonies and intrigues. ; " Uno foulid d'amour s'es mes en t^to." FRifeD^RIC 162 MISTRAL This dreamy, imaginative, blond Prince is in search of a Naiade and the mysterious "swanflower," wherein the fair nymph hidden. is This flower he wears as an emblem. When the boatmen see as the jieur de it, they recognize Rh6ne that the Anglore is it so fond The men get Jean Roche, one of their number, to tell the Prince who this mysterious Anglore is, and we learn that she is a little, laughing maiden, who wanders barefoot of culling. on the sand, so charming that any of the sailors, were she to make a sign, would spring into the water to go and print a kiss upon her little Not only is the Prince in search of a foot. nymph and a flower, not only does he wish to behold Orange, he wishes also to learn the lan- guage in which the Countess of Die sang lays of love with Raimbaud of Orange. He is full of thoughts of the olden days, he feels regret for the lost conquests. " But why should he feel regret, if he may recover the sunny land of his forefathers by drinking it in with eager What need is there of gleaming swords to seize what the eye shows us?" He cares eyes little I for royalty. " Strongholds crumble away, as may be seen THE FOUR LONGER POEMS 163 everything falls to ruin and But on thy summits, unchanging Nature, forever the thyme shall bloom, and the shepherds and shepherdesses frolic on the grass on all these hills is ; renewed. at the return of spring." The Prince apostrophizes the " empire of the sun," bordering like a silver hem the dazzling Rhone, the "poetic empire of Provence, that with its name alone doth charm the world," and he calls to mind the empire of the Bosonides, the memory of which survives in the speech of the boatmen; they call the east shore "em- pire," the west shore "kingdom." The journey is full of episodes. The owner of the fleet. Apian, is a sententious individual. He is devoted to his river life, full of religious fervor, continually crossing himself or praying to Saint Nicholas, the patron saint of sailors. This faith, falls into however, is not entire. If a man the water, the fellows call to him, " Recommend thyself to Saint Nicholas, but swim for dear life." it, As the English expression has " Trust to God, but keep your powder dry. Master Apian always says the Lord's Prayer when he puts off from shore, and solemnly utters the words, " In the name of God aloud FRtDfeRIC MISTRAL 164 and the Holy Virgin, Rhone " His piety, however, does not prevent him from in! the to terrupting his prayer to swear at the men most vigorously. Says he, "Let whoever would learn to pray, follow the water," but his argu- ments and experiences rather teach the vanity He is full of superstitious tales. He has views of life. of prayer. " Life is a journey like that of the bark. It The wise man, when has its bad, its good days. the waves smile, ought to know how to behave But man is He who rows gets He who is his pay at the end of the month. in the breakers he must go slow. born for toil, for navigation. afraid of blistering his hands takes a dive into the abyss of poverty." Napoleon in flight women who cried down He tells a story of the Rhone, of the out at him, reviling him, bidding him give back their sons, shaking their and crying out, " Into the Rhone with him." Once when he was changing horses at an inn, a woman, bleeding a fowl at the door, If I had exclaimed "Ha, the cursed monster fists : ! him here, I'd plant my knife into his throat like that " The emperor, unknown to her, draws "What did he do to you? " said he. "I near. ! THE FOUE LONGER POEMS 165 had two sons," replied the bereaved mother wrathfuUy, "two handsome boys, tall as towers. He killed them for me in his battles." "Their names will not perish in the stars," said Napoleon sadly. " Why could I not fall like them ? — for they died for their country on the field of glory." — "But who are you?" — "I am the emperor." — " Ah ! " The good woman fell upon her knees dismayed, kissed his hands, begged his forgiveness, and all in tears Here the story is interrupted. Wholly charming and altogether original is the tale of the little maiden whom the boatmen name L'Anglore, and whom Jean Roche loves. The men have named her so for fun. They knew her well, having seen her from earliest — childhood, half naked, paddling in the water along the shore, sunning herself like the little lizard they call anglore. Now she had grown, and eked out a poor living by seeking for gold in the sands brought down by the Ardeche. The little maid believed in the story of the Drac, a sort of merman, that lived in the Rhone, and had power to fascinate the women who ventured into the water. There was once a very widespread superstition concerning this Protean FR:feD:feRIC Id6 creature ; MISTRAL and the women washing in the river often had a figure of the Drac, in the form of a lizard, carved upon the piece of wood with which they beat the linen, as a sort of talisman against The mother of the Anglore had his seduction. told her of his wiles ; and one story impressed — the story of the young woman her above all who, fascinated by the Drac, lost her footing in down into At the end of seven years she re- the water and was carried whirling the depths. turned and told her tale. She had been seized by the Drac, and for seven years he kept her to nurse his little Drac. The Anglore was never afraid while seeking the specks of gold in the sunlight. night it was different. But at A gem of poetry is the scene in the sixth canto, full of witchery and charm, wherein the imagination of the little maid, wandering out along the water in the mysterious moonlight, causes her to fancy she sees the Drac in the form of a fair youth smiling upon her, offering her a wild flower, uttering sweet, mysterious words of love that die away She often came again to meet and she noticed that if ever she crossed in the water. him ; herself on entering the water, as she had always THE FOUR LONGER POEMS done when a little 167 the Drac would not girl, These three or four pages mark the appear. genuine poet and the master of language. The mysterious night, oppressively warm, the moonlight shining on the little white figure, the deep silence, broken only by the faint murmur of the river and the distant singing of a nightingale, the gleam of the glowworms, compose a scene of fantastic beauty. The slightest sounds startle her, whether it be a fish leaping at the surface of the water to seize a fly, the gurgling of a little eddy, or the shrill cry of a bat. There is a certain voluptuous beauty in the very sound of the words that describe the kissed by the moonbeams : — little nymph, " alusentido Pfer li rai de la luno que beisavon Soun fin coutet, sa jouino car ambrenco, Si bras poupin, sis esquino rabloto E si pousseto armouniouso e fermo Que s'amagavon coume dos tourtourd Dins I'esparpai de sa cabeladuro." The last three lines fall like a caress upon the ear. Mistral often attains a perfect melody of words with the harmonious succession of varied vowel sounds and the well-marked cadence of his verse. FRIJD^RIC mistral 168 When Apian's fleet comes down the river and passes the spot where the little maid seeks for gold, the men see her and invite her on board. She will go down to Beaucaire to sell her findings. Jean Roche offers himself in marriage, but she will have none of him; she loves the vision seen beneath the waves. Anglore spies the blond-haired turns pale and nearly swoons. ! When the Prince, she "'Tis he, 'tis and she stands fascinated. William, charmed with the little maid, says to her, "I recognize thee, O Rhone flower, blooming on the water flower of good omen that I saw in a dream." The little maid calls he " she cries, — him Drac, identifies the flower in his hand, and The boatmen lives on in this hallucination. consider that she has lost her reason, and say she must have drunk of the fountain of Tourne. The little maid hears them, and bids them speak low, for their fate is written at the fountain of Tourne and like a Sibyl, raising her bare arm, she describes the mysterious carvings on the rock, and the explanation given by a witch she knew. ; These carvings, according to Mistral's note, were dedicated to the god Mithra. The meaning given by the witch is that the day the THE FOUR LONGER POEMS 169 Drac shall leave the river Rhone forever, that day the boatmen shall perish. The men do not laugh, for they have already heard of the great boats that can make their way against the cur- Apian breaks out into furious imprecations against the men who would ruin the thousands that depend for their living upon the river. One is struck by this introduction of a question of political economy into a poem. During the journey to Avignon the Prince falls more and more in love with the little rent without horses. Anglore, whom no sort of evidence can shake out of her belief that the Prince is the Drac, for the Drac can assume any form at pleasure. Her delusion is so complete, so naive, that the prince, romantic by nature, is entirely under the spell. There come on board three Venetian women, who possess the secret of a treasure, twelve golden statues of the Apostles buried at Avi- The Prince leaves the boat to help them and the little maid suffers inBut he comes tensely the pangs of jealousy. gnon. find the place, back to her, and takes her all about the great That night, however, he fair at Beaucaire. wanders out alone, and while calling to mind MISTRAL FRifeDljRIC 170 the story of Aucassin and Nicolette, he is sand- The Anglore believes bagged, but not killed. he has left his human body on the ground so as to visit his caverns beneath the Rhone. William seems unhurt, and at the last dinner before they start to go up the river again, surrounded by the crew, he makes them a truly Felibrean speech: " Do you know, friends, to whom I feel like consecrating our last meal in Beaucaire ? To the patriots of the Rhodanian shores, to the dauntless men who, in olden days, maintained themselves in the strong castle that stands before our eyes, to the dwellers along the river* banks who defended so valiantly their customs, their free trade, and their great free Rhone. If the sons of those forefathers who fell bravely in the strife, to-day have forgotten their glory, much the worse for the sons well, so ! But you, my mates, you who have preserved the call. Empire ! and who, like the brave men you are, and defend the Rhone in its very will soon go life, fighting your last battle with me, a stranger, but enraptured and intoxicated with the light of your Rhone, come, raise your glasses to the " cause of the vanquished ! The love scenes between the Prince and the THE FOUR LONGER POEMS 171 Anglore continue during the journey up the river. Her devotion to him is complete ; she knows not whither she goes, if to perish, then In a moment of enthusiasm let it be with him. William makes a passionate declaration. "Trust me, Anglore, since I have freely chosen thee, since thou hast brought me thy deep faith in the beautiful wonders of the fable, since thou art she who, without thought, yields to her love, as wax melts in the sun, since thou livest free of all our bonds and shams, since in thy blood, in thy pure bosom, lies the renewal of the old sap, I, on my faith as a Prince, I swear to thee that none but me, O my Rhone flower, shall have the happiness to pluck thee as a flower of love and as a wife ! One day the But this promise is never kept. boats meet the steamer coming down the river. Apian, pale and silent, watches the magic bark whose wheels beat like great paws, and, raising great waves, come down steadily upon him. The captain cries, " One side ! " but, obsti- nate and angry. Apian tries to force the steamer to give way. The result is disastrous. The steamer catches in the towing cables and drags the horses into the water. The boats drift FR^DilRIC MISTRAL 172 back and are hurled against a bridge. Will- iam and the Anglore are thrown into the river and are lost. All the others escape with their Jean Roche is not sure but that he lives. was the Drac after all, who, foreseeing the shipwreck, had thus followed the boats, to carry the Anglore at last down into the depths of the Maitre Apian accepts his ruin philoriver. sophically. Addressing his men, he says " Ah, : my seven boats ! my splendid draught horses All gone, all ruined ! ness ! ! It is the end of the busi- Poor fellow-boatmen, you may well say, 'good-by to a pleasant life.' To-day the great Rhone has died, as far as we are concerned." The idea of the poem is, then, to tell of the To-day the river flows old life on the Rhone. almost as in the days when its shores were untrod by men. Rarely is any sort of boat seen upon its swift and dangerous current. Mistral portrays the life he knew, and he has done it with great power and vividness. The fanciful tale of the Prince and the Anglore, suggested by the beliefs and superstitions of the humble was introduced, doubtless, as a necessary The little maid Anglore, half mad in her illusion, is none the less a very sympa- folk, love story. THE FOUR LONGER POEMS 173 This thetic creation, and surely quite original. tale, however, running through the poem like a thread, is not the poem, nor does it fill proportionately a large place therein. The poem is, as its title proclaims, the Poem of the Rhone, a poem of sincere regret for the good old days when the muscular sons of Condrieu ruled the stream, the days of jollity, of the curious boat- ing tournaments of which one is described in Calendau, when the children used to watch the boats go by with a Condrillot at the helm, and the Rhone was swarming like a mighty beehive. The poet notes in sorrow that all is dead. river flows on, broad and silent, and The no vestige of all its past activity remains, but here and there a trace of the cables that used to rub along the stones. As we said at the outset, what is most strikis its realism. The poet revels in enumerating the good things the men ing about this poem had to eat at the feast of Saint Nicholas he describes with a wealth of vocabulary and a flood ; of technical terms quite bewildering every sort of boat, and all its parts with their uses ; he re- produces the talk of the boatmen, leaving unvarnished their ignorance and superstition, their FR^D^RIC MISTRAL 174 roughness and brutality ; he describes their ap- pearance, their long, hair and large earrings ; he explains the manner of guiding the boats down the swirling, treacherous waters, amid the dangers of shoals and hidden rocks ; he describes all the cargoes, not finding it beneath the dignity of an epic poem to tell us of the kegs of foamy beer that is destined for the thirsty throats of the drinkers at Beaucaire ; as the boats pass Condrieu, he reproduces the gossip of the boat- men's wives ; he does not omit the explanations Apian addressed to the Prince concerning fogs and currents he is often humorous, telling us of the heavy merchants who promenade their paunches whereon the watch-charms rattle against their snug little money carried in a belt of ; he describes the passengers, tells us their various trades and destinations, is even cynical ; tells of the bourgeois, who, once away from their wives, grow suddenly lavish with like pigs let loose in whole roadway ; their money, and the street, take up the he does not shrink from letting us know that the men chew a cud of tobacco while they talk; goods ; he mentions the price of he puts into the mouth of Jean Roche's mother a great many practical and material THE FOUR LONGER POEMS 175 considerations as to the matter of taking a wife, and a very wise and practical old lady she is he ; treats as " joyeuset^s " the conversation of the Venetian women who inform the Prince that in their city the noblewoman; once married, may have quite a number of lovers without exciting any comment, the husband being rather relieved than otherwise; he allows his boatmen to swear and call one another vile names, and a howling, brawling lot they frequently become and when at last we get to the fair at Beaucaire, there are pages of minute enumerations that can scarcely be called Homeric. In short, a very large part of the book is prose, animated, vigorous, often exaggerated, but prose. other long poems it is Like his singularly objective. Rarely does the author interrupt his narrative or description to give an opinion, to speak in his own name, or to analyze the situation he has created. Like the other poems, too, it is sprinkled with tales and legends of some of them charming. all sorts, Superstitions abound. Mistral shares the fondness of the Avignonnais for the number seven. Apian has seven boats, the Drac keeps his victim seven woman of Condrieu has seven sons. years, the FR^DfeRIC MISTRAL 176 The poem offers the same beauties as the others, an astonishing power of description first of Mistral all. is always masterly, always poetic in depicting the landscape and the life that moves thereon, and especially in evoking the life of the past. He revives for us the princesses and queens, the knights and trouba- and they move before us, a fascinating, pageant. The perfume of flowers, the sunlight on the water, the great birds flying dours, glittering in the air, the silent drifting of the boats in the broad valley, the reflection of the tall poplars in the water, the old ruins that crown the hilltops — all these things are exquisitely woven into the verse, and more than a mere word-painting they create a mood in the reader in unison with the mood of the person of whom he is reading. In touching truly deep and serious things Mistral is often superficial, and passes them off with a commonplace. is An instance in this poem the episode of the convicts on their way to the galleys at Toulon. No terrible indignation, no heartfelt pity, is expressed. Apian silences one of his crew who attempts to mock at the unhappy wretches. " They are miserable enough THE FOUR LONGER POEMS 177 without an insult! and do not seem to recognize them, for, branded on the shoulder, they Let this be an example to you They are going to eat beans at Toulon, seek the shade. all. poor fellows! All sorts of men are there, churchmen, rascals, nobles, notaries, even some who are innocent " ! And the poet concludes, "Thus the world, thus the agitation, the stir of life, good, evil, pleasure, pain, pass along swiftly, confusedly, between day and night, on the river of time, rolling along and fleeing." The enthusiasm of the poet leads him into exaggeration whenever he comes to a wonder of Provence. Things are relative in this world, and the same words carry different meanings. Avignon is scarcely a colossal pile of towers, and would not remind many of Venice, even at sunset, and we must make a discount when we hear that the boats are engulfed in the fierce (sic) arch of the colossal bridge of stone that Benezet, the shepherd, erected seven hundred years ago. A monlent later he refers daintily and accurately to the chapel of Saint Nicholas "riding on the bridge, slender and pretty." The epithets sound larger, too, in Provengal; FR:fcDl:RIC 178 the view of Avignon MISTRAL is " espetaclouso," the walls of the castle are "gigantesco." Especially admirable in expression is its sober, energetic the account of the Remonte, in the eleventh canto, wherein we see the eighty horses, grouped in fours, tug slowly up the river. "The long file on the rough-paved path, dragging the weighty train of boats, in spite of the impetuous waters, trudges steadily along. And beneath the lofty branches of the great white poplars, in the stillness of the Rhone valley, in the splendor of the rising sun, walk- ing beside the straining horses that drive a mist from their nostrils, the first driver says the prayer." With each succeeding poem the vocabulary of Mistral seems to grow, along with the bold- ness of expression. All his poems he has himself translated into French, and these translations are remarkable in more than one respect. That of the Poem of the Rhone is especially full of rare French words, and it cannot be imputed to the leader of the Provengal poets that he is not past master of the French vocabOften his French expression is as ulary. THE FOUR LONGER POEMS strange as the original. 179 Not many French writers would express themselves as he does in the following: — " Et il tressaille de jumeler le nonchaloir de sa jeunesse au renouveau de la belle ingenue." In this translation, preceding, there is also, more than in the occasionally an affectation of archaism, which rather adds to than detracts effect of his prose, and the number of lines in the prose translation that are from the poetic really ten-syllable verses is quite remarkable. On one page (page 183 of the third edition, Lemerre) more than half the lines are verses. Is the Poem of the Rhone a great poem ? Whether it is or not, it accomplishes admirably the purpose of its author, to fix in beautiful That much was inevitable; the nature of verse the former life of the Rhone. of it is prosaic the subject rendered it so. ties, It is full of beau- and the poet who wrote Mireio and comshown pleted it before his thirtieth year, has that in the last decade of his threescore years and ten he could produce a work as full of fire, energy, life, and enthusiasm as in the stirring days when the Felibrige was young. poem there occurs a passage In this put into the FRflDfeRIC 180 MISTRAL mouth of the Prince, which gives a view of life that we suspect is the poet's own. He here calls the Prince a young sage, and as we look back over Mistral's life, and review its aims, and the conditions in which he has striven, we incline to think that here, in a few words, he has condensed his thought. " For what is life but a dream, a distant appearance, an illusion gliding on the water, which, fleeing ever before our eyes, dazzles us and lures us on Ah, how good it is to sail on ceaselessly toward one's desire, even though it is but a dream! like a mirror flashing, entices I The time will come, it is near, perhaps, when men will have everything within their reach, when they will possess everything, when they will know and have proved everything; and, regretting the old mirages, who knows but what they will not grow weary of living " ! CHAPTER II LIS ISCLO D'OB The lover of poetry will probably find more to admire and cherish in this volume than in any other that has come from the pen of its author, excepting, possibly, the best passages of Mireio. It is the collection of his short poems that appeared from time to time in different Provencal publications, the earliest dat- ing as far back as 1848, the latest written in They are a very complete expression of among their numThe poet's lyre has ber gems of purest poesy. not many strings, and the strains of sadness, of 1888. his poetic ideas, and contain pensive melancholy, are almost absent. Mistral has once, and very successfully, tried the theme of Lamartine's Lac^ of Musset's Souvenir^ of Hugo's Tristesse c?' Olympio; but his poem is not an elegy, it has not the intensity, the passion, the deep undertone of any of the three great Romanticists. La Fin d6u MeissouniS is a 181 MISTRAL FRf:Dl:RIC 182 beautiful, pathetic, and touching tale, that easily brings a tear, and Lou Saume de la Penitenci is without doubt one of the noblest poems inspired any Frenchman by the disaster But these poems, though among the in the heart of of 1870. best according to the feeling for poetry of a reader from northern lands, are not characteristic of the volume in general. The dominant strain is energy, a clarion-call of life and light, an appeal to his fellow-countrymen to be strong and independent; the sun of Provence, the language of Provence, the ideals of Provence, the memories of Provence, these are his themes. His poetry is not personal, but social. Of his own joys and sorrows scarce a word, unless we say what is doubtless the truth, that his joys and sorrows, his regrets and hopes, are identical with those of his native land, and that he has blended his being completely with the about him. The volume contains a great number of pieces written for special occasions, life for the gatherings of the Felibres, for their Many of them are addressed to persons in France and out, who have been in weddings.. various ways connected with the Felibrige. Of these the greeting to Lamartine is especially LIS ISCLO D'OR 183 felicitous in expression, and the following stanza from it forms the dedication of Mir Ho: " Te counsacre Mireio : — eo moun cor e moun amo, Es la flour de mis an Es un rasin de Crau qu' em^ touto sa ramo Te porge un paisan." The entire poem, literally translated, is as follows If I : — have the good fortune to see my bark early upon the waves, Without fear of winter, Blessings upon thee, O divine Lamartine, Who hast taken the helm If my prow bears a bouquet of blooming laurel. It is thou hast made it for me If iny sail swelleth, it is the breath of thy glory That bloweth it. Therefore, like a pilot who of a fair church Clirabeth the hill And upon the altar of the saint that hath saved him at sea Hangeth a miniature ship. 'tis my heart and my soul, my years; I consecrate Mireio to thee ; 'Tis the flower of 'Tis a cluster of grapes from the Crau that with all its leaves A peasant offers thee. FRifeD^RIC 184 MISTRAL Grenerous as a king, when thou broughtest me fame In the midst of Paris, Thou knowest that, in thy home, the day thou saidst to me, " Tu Marcellus eris 1 Like the pomegranate in the ripening sunbeam, My heart opened. And, unable to find more tender speech. Broke out in tears. It is interesting to notice that the earliest poem of our author, La Bello d^Avoust, is a tale of the supernatural, a poem of mystery it is an ; order of poetic inspiration rather rare in his work, and this first poem is quite as good as anything of its kind to be found in Mireio or Nerto. It refrain : — has the form of a song with the Ye little nightingales, ye grasshoppers, be still! Hear the song of the beauty of August Margai of Val-Mairane, intoxicated with love, goes down into the plain two hours before the day. Descending the hill, she is wild. " In vain," she says, " I seek him, I have missed him. Ah, my heart trembles." The poem is full of imagery, delicate and pretty. Margai is so lovely that in the clouds LIS ISCLO D'OR 185 the moon, enshrouded, says to the cloud very softly, " Cloud, beautiful cloud, pass away, my face would let fall a ray on Margai, th}^ shadow hinders me." her, And the bird offers to console and the glow-worm guide her to her lover. offers his light to Margai comes and goes until she meets her lover in the shadow of the trees. She tells of her weeping, of the moon, the birdling, and the glow-worm. thy brow is dark, art thou ill ? to " But Shall I return my father's house ? " " If my face is sad, on my faith, it is because a black moth hovering about hath alarmed me." And Margai says, " Thy voice, once so sweet, to-day seems a trembling sound beneath the earth; I shudder at it." " If my voice is so hoarse, it is because while waiting for thee I lay upon my back in the grass." " I was dying with longing, but now it is For the day of our elopement, be" loved, thou wearest mourning with fear. ! " If my cloak be sombre and black, so is the night, and yet the night also glimmers." When the star of the shepherds began to FR6d6rIC mistral 186 pale, and when the king of stars was about to appear, suddenly off they went, upon a black And the horse flew on the stony road, horse. and the ground shook beneath the lovers, and 'tis said fantastic witches danced about them until day, laughing loudly. Then the white moon wrapped herself again, the birdling on the branch flew off in fright, even the glow-worm, poor little thing, put out his lamp, and quickly crept away under the And it is said that at the wedding of grass. poor Margai there was little feasting, little laughing, and the betrothal and the dancing took place in a spot where fire was seen through the crevices. "Vale of Val-Mairane, road never again o'er or plain Baux, did ye see Her mother prays and weeps, and Margai. will hill to the not have enough of speaking of her lovely shepherdess." This weird, legendary tale was composed in 1848. The next effort of the poet is one of his masterpieces, wherein his inspiration is truest and most poetical. La Fin d6u MeusouniS (The Reaper's Death) is a noble, genuinely pathetic tale, told in beautifully varied verse, LIS ISCLO D'OR full of the love of field 187 work, and aglow with sympathy for the toilers. The figure of the old man, stricken down suddenly by an accidental blow from the scythe of a young man mowing behind him, as he lies dying on the rough ground, urging the gleaners to go on and not mind him, praying to Saint John, patron of the harvesters, gotten. one not to be for- is The description of the mowing, the long line of fierce — — the toilers with their scythes, the sun making their blood boil, the sheaves falling by hundreds, the ruddy grain waving in the breath of the mistral, the old chief lead- ing the band, "the strong affection that urged the men on to cut down the harvest," — all is vividly pictured, and foretells the future poet of Mireio. The words of the old man are full of his energy and faith : " The wheat, swollen and ripe, is scattering in the summer wind do not leave to the birds and ants, O binders, the wheat that comes from God " " What good is your weeping? better sing with the young ; ! fellows, for I, before task. you all, have finished my Perhaps, in the land where I shall be presently, it will be hard for me, when evening comes, to hear no more, stretched out upon the FR^D^RIC MISTRAL 188 grass, as I used to, the strong, clear singing of the youth rising up amid the trees ; but it ap- pears, friends, that it was my star, or perhaps One above, seeing the ripe the Master, the Come, come, good-by, I grain, gathers it in. am going gently. Then, children, when you carry off the sheaves upon the cart, take away your chief on the load of wheat." And he begs Saint John to remember his olive trees, his family, who will sup at Christmas-tide without him. " If sometimes I have murmured, forgive me The sickle, meeting a stone, cries out, I O master Saint John, the friend of God, patron of the reapers, father of the poor, up there in Paradise, remember me." And after the old man's death " the reapers, silent, sickle in hand, go on with the work in haste, for the hot mistral was shaking the ears." Among these earlier poems are found some told, homely tales, with a pointed Such are La Plueio (The Rain), La Rascladuro de Petrin (The Scraping from the cleverly moral. Kneading-trough). They are really excellent, and teach the lesson that the tillers of the soil have a holy calling, of which they may be LIS ISCLO D'OR proud, and that 189 God sends them health and The second of happiness, peace and liberty. the poems just mentioned is a particularly amusing story of choosing a wife according to the care she takes of her kneading-trough, the idea being derived from an old fablieau. are one or There two others purely humorous and capitally told. After 1860, however, the poet abandoned these homely, simple tales, that doubtless realized Roumanille's ideas of one aspect of the literary revival he was seeking to bring about. The poems are not arranged chronologically, but are classified as Songs, Romances, Sirventes, Reveries, Plaints, Sonnets, Nuptial Songs, etc. The Cansoun (Songs) are sung at every reunion of the Felibrige. melodies well known in They are set to Provence, and are and vigorous indeed. The Germans who write about Provence are fond of making known the fact that the air of the famous Hymn to the Sun is a melody written by Kuecken. There is Lou Bastimen (The Ship), spirited as full of dash lad. and go as any English sea bal- La Coutigo (The Tickling) is a dialogue La between a mother and her love-sick son. FRfeD^RIC MISTRAL 190 Coupo (The Cup) is the song of the Felibres jpar excellence; it was composed for the recep- tion of a silver cup, sent to the Felibres by the The coupo felibrenco is now a feature The song expresses the of enthusiasm of the Felibres for their cause. The Catalans. all their banquets. refrain is, " Holy cup, overflowing, pour out in plenty the enthusiasms and the energy of the strong." The most significant lines are : — Of a proud, free people We are perhaps the end; And, if the Felibres fall, Our nation will fall. Of a race that germs anew Perhaps we are the first growth Of our land we are perhaps The pillars and the chiefs. Pour out for us hope And dreams of youth. The memory of the past And faith in the coming year. The ideas and sentiments, then, that are expressed in the shorter poems of Mistral, written since the publication of Mireio^ have been, in the main, the ancient glories and liberties of Provence, a clinging to national traditions, to LIS ISCLO D'OR local traditions, of ancestors, a 191 and to the religion and ideas profound dislike of certain modern ideas of progress, hatred of the levelling influence of Paris, love of the Provengal speech, belief in the Latin race, in the Roman Catholic Church, unshaken faith in the future, love of the ideal and hatred of what is servile and sordid, an ardent love of Nature, an intense love of life and movement. These things are reflected in every variety of word and figure. He is not the poet of the romantic type, selfcentred, filling his verse with the echoes of his own loves and joys and woes, nor is his poetry as large as humanity; Provence, France, the Latin race, are the limits beyond which it has no message or interest. Possibly no poet ever wrote as many lines to Such lines abound in each volume he has produced. laud the language he was using. " Se la lengo di moussu Toumbo en gargavaio Se tant d'escrivan coussu Pescon de ravaio, Nkutri, li bon Prouven9au Vers li serre li plus aut Enauren la lengo De nosti valengo." FRl:DfeRIC 192 If the sweepings, MISTRAL language of the messieurs falls among the if so many comfortably well-off writers small fry, fish for the highest we, the good summits, raise Proven9als, toward language the of our valleys. The Sirventes addressed to the Catalan poets begins : — " Fraire de Catalougno, escoutas Nous an di Que fasias peralin revieure e resplendi I Un di rampau de nosto lengo." Brothers from Catalonia, listen! We have heard that ye cause one of the branches of our language to revive and flourish yonder. In the same poem, the poet sings of the Troubadours, whom none have since surpassed, who in the face of the clergy raised the language of the common people, sang in the very ears of the kings, sang with love, and sang freely, the coming of a new world and contempt for ancient fears, and later on he says : — "From the Alps to the Pyrenees, hand in hand, poets, let us then raise up the old Romance speech ! It is the sign of the family, the sacra- ment that binds the sons to the forefathers, man to the soil ! It is the thread that holds the nest in the branches. Fearless guardians of LIS ISCLO D'OR our beautiful speech, let 193 us keep it and free pure, and bright as silver, for a whole people drinks at this spring ; for when, with faces on the ground, a people falls into slavery, if it holds its language, it holds the key that delivers it from the chains." The final stanza of the poem, written in honor of Jasmin in 1870, is as follows : "For our dead and our — fathers, and our sacred rights as a people and as poets, that yesterday were trampled beneath the feet of the usurper, and, outraged, cried out, now live Now, between the two seas the language of Oc triumphs. O Jasmin, thou again in glory ! hast avenged us " ! In the Rock of Sisyphus the poet says, " Formerly we kept the language that Nature upon our lips." In the Poem to the Latin Race we read "Thy mother tongue, the great stream that herself put : — spreads abroad in seven branches, pouring out love and light like an echo from Paradise, thy golden speech, O Romance daughter of the King-People, is the human as long reason." lips song that will • as speech live shall on have FR^D^RIC MISTRAL 194 Elsewhere we find : — " Oh, maintain thy historic speech. It is the proof that always thou carriest on high and free, thy coat of arms. In the language, a Each year mystery, an old treasure is found. the nightingale puts on new plumage, but keeps its song." One entire poem, Espouscado^ is a bitterly indignant protest against those who would suppress the dialect, against the regents and the rectors whom " we must pay with our pennies them scoff at the language that binds us to our fathers and our soil " And the poet cries out, "No, no, we'll keep our rebellious We'll speak it langue d'oc, grumble who will. to hear ! in the stables, at harvest-time, among the silk- worms, among etc. lovers, brotherhood. among neighbors, etc., be the language of joy and of It shall We'll joke and laugh with — and as for the army, it; we'll take it to the barracks to keep off homesickness." And his anger rising, he exclaims : — " O the fools, the fools, who wean their chil- dren from it to stuff them with self-sufficiency, fatuity, and hunger in the throng ! ! Let them get drowned But thou, O my Provence, be LIS ISCLO D'OR 195 not disturbed about the sons that disown thee and repudiate thy speech. They are dead, they are still-born children that survive, fed on bad milk." And he concludes : — "But, eldest born of Nature, you, the sun- browned boys, who speak with the maidens in the ancient tongue, fear not you shall remain -, the masters ! Like the walnuts of the plain, gnarled, stout, cahn, motionless, exploited and ill-treated as you may be, O peasants (as they call you), you will remain masters of the land!" This was written in 1888. The quotations might be multiplied ; these suffice, however, to show the intense love of the poet for " the language of the soil," the energy with which he has constantly struggled for its maintenance. He is far from looking upon the multiplication of dialects as an evil, points to the literary glory of Greece amid her many forms of speech, and does not even seek to impose his own language upon the rest of southern France. He sym- pathizes with every attempt, wherever made, the world over, to raise up a patois into a lan- guage. Statesmen will probably think other- mistral rRi2Di:Ric 196 wise, and there are nations which would at once take an immense stride forward if they could attain one language erature. to be and a purely national lit- The modern world does not appear marching in accordance with Mistral's view. The poems inspired by the love of the ancient ideals and literature of Provence are very beautiful. They have in general a fascinating swing and rhythm, and are filled with charming One of the best is L'Amiradou (The imagery. Belvedere), the story of a fairy imprisoned in the castle at Tarascon, love the one who "who will doubtless shall free her." knights attempt the rescue and there comes along a little fail. Three Then Troubadour, and sings so sweetly of the prowess of his forefathers, of the splendor of the Latin race, that the guard are charmed and the bolts fly back. And the fairy goes up to the top of the tower with the little mute with love, Troubadour, and they stand and look out over all the beautiful landscape, and the old monuments of Provence with their lessons. This is the king- dom of the fairy, and she bestows it upon him. " For he who knows how to read in this radiant LIS ISCLO D'OR 197 book, must grow above all others, and all that his eye beholds, without paying any tithe, is his in abundance." The lilt of this little romance^ with its pretty repetitions, is delightful, and the symbolism is, of course, perfectly obvious. There is the touching story of the Troubadour Catalan, slain by robbers in the Bois de Boulogne, where the Pre de Catalan there is now is ; the tale that accounts for the great chain that hangs across the gorge at Moustiers, a chain over six hundred feet long, bearing a star in the centre. A knight, being prisoner among the Saracens, vows to hang the chain before the chapel of the Virgin, if ever he re- turns home. " A ti ped, vierge Mario, Ma cadeno penjarai, Se jamai Tourne mai A Mousti^, dins ma patrio " I There is the tale of the Princess Clemence, daughter of a king of Provence. Her father was deformed, and the heir-presumptive to the French crown sought her in marriage. In order that the prince might be sure she had FR^DfeRIC MISTRAL 198 inherited none of the father's deformity, she was called upon to show herself in the garb of Lady Godiva before his ambassadors. This rather delicate subject is handled with consummate art. The idea of federalism is found expressed with sufficient clearness in various parts of these poems of the Golden Isles, and the patriotism of the poet, his love of France, is perfectly evi- dent, in spite of all that has been said to the contrary. In the poem addressed to the Cat- numerous allusions to the dissensions and rebellions of bygone days, we read "Now, however, it is clear; now, however, alans, after : — we know that in the divine order all is for the best ; the Proven9als, a unanimous part of great France, frankly, loyally flame, are ; the Cat- alans, with good-will, are part of magnanimous Spain. For the brook must flow to the sea, and the stone must fall on the heap ; the wheat is best protected from the treacherous cold wind when planted close ; and the little boats, if they are to navigate safely, when the waves are black and the air dark, must sail together. it is ' For good to be many, it is a fine thing to say, We are children of France " I ' LIS ISCLO D'OR 199 But in days of peace let each province de- velop its own life in its own way. "And France and Spain, when they see their warming themselves together in the children sunbeams of the fatherland, singing matins out of the same book, will say, ' The children have sense enough, let them laugh and play together, now they are old enough to be free.' "And we shall see, I promise you, the ancient freedom come down, O happiness, upon the smallest city, and love together; tyrant and alone bind the races ever the black talon of if is seen, all t!ie the races will bound up to drive out the bird of prey!" Of all the poems of Mistral expressing this order of ideas, the one entitled The Countess made the greatest stir. It appeared in 1866, and called forth much angry discussion and imputation of treason from the enemies of the new movement. The Countess is an allegorical representation of Provence ; the fair descend- ant of imperial ancestors is imprisoned in a con- vent by her half-sister France. Formerly she possessed a hundred fortified towns, twenty seaports ; she had olives, fruit, and grain in abundance; a great river watered her fields; FRfeD^RIC MISTRAL 200 a great wind vivified the land, and the proud noblewoman could live without her neighbor, and she sang so sweetly that all loved her, poets and suitors thronged about her. Now, in the convent where she is cloistered all are dressed alike, all obey the rule of the same bell, all joy is gone. The half-sister has broken her tambourines and taken away her vineyards, and gives out that her sister is dead. Then the poet breaks into an appeal to the strong to break into the great convent, to hang the abbess, and say to the Countess, " Appear again, O splendor ! Long life to joy! " Away with grief, away Each stanza is followed by the refrain "Ah! se me sabien entendre : — I Ah se me voulien seguil " Ah if they could understand me Ah if they would follow me I ! Mistral disdained to reply to the storm of accusations and publication I 1 ! incriminations raised by the of this poem. Lou Saume de la Penitenoiy that appeared in 1870, set at rest all doubts concerning his deep and sincere patriotism. Uhe Psalm of Penitence is possibly the finest LIS ISCLO D'OR of the short poems. 201 It is certainly surpassed by no other in intensity of feeling, in genuine inspiration, in nobility and beauty of expression. It is a hymn of sorrow over the woes of France, a prayer of humility and resignation after the disaster of 1870. The reader must accept the idea, of course, that the defeat of the was a visitation French of Providence in punishment for sin. " Segnour, h la fin ta coul^ro Largo si tron Sus nosti front E dins la nine nosto galero Pico d'a pro Contro li ro." Lord, at last thy wrath hurls its thunderbolts upon our foreheads And in the night our vessel strikes its prow against the rocks. France was punished for irreligion, for closing the temples, for abandoning the sacraments and commandments, for losing faith in all except selfish interest and so-called progress, for contempt of the Bible and pride in science. The poet makes confession : — " Segnour, sian tis enfant proudigue Mai nkutri sian Ti vifei crestian FRfeDfeRIC 202 MISTRAL Que ta Justigo nous castigue, Mai au trepas Nous laisses pas " I Lord, we are thy prodigal sons; but we are thy Christians of old: Let thy justice chastise us, but give us not over unto death 1 Then the poet prays in the name of all the brave men who gave up their lives in battle, in name of all the mothers who will never again see their sons, in the name of the poor, the strong, the dead, in the name of all the the defeats and tears and sorrow, the slaughter and the fires, the affronts endured, that God disarm his justice, and he concludes : — " Segnour, voulen deveni d'ome En liberty. Pos nous bouta I Sian Gau-Rouraan e gentilome, E marchan dre Dins noste endrd. " Segnour, ddu mau sian pas I'encauso. Mando ei<;abas Un rai de pas I Segnour, ajudo nosto Causo, E reviduren E t'amaren." LIS ISCLO D'OR Lord, we desire to become men ; 203 thou canst set us free ! We are Gallo-Romans and of noble race, and we walk upright in our land. Lord, we are not the cause of the evil. Send down upon us a ray of peace Lord, aid our Cause, and we shall live again and love thee. ! The poem called The Stone of Sisyphus completes sufficiently the evidence necessary to ex- culpate Mistral of the charge of antipatriotism and makes Provence was clear his thought. once a nation, she consented years ago to lose Now her identity in the union with France. it is proposed to heap up all the old traditions, the Gai Savoir, the glory of the Troubadours, the them on a pyre. Well, France is a great people and But some would go further, Vive la nation. some would suppress the nation " Down with old language, the old customs, and burn : the frontiers, national glories are an abomination ! Wipe out the past, man is God Vive Our patrimony we repudiate. ! rhumanitS f" What are Joan of Arc, Saint Louis, and Turenne? Then ^^ All that is old rubbish. the people JEmperaire, siegues cry with maudi, nous as vendu" and hurl Victor maudi, down the Hugo, maudi! Vendome column, burn Paris, slaughter the priests, and FR^D^RIC MISTRAL 204 then, worn out, commence again, like Sisyphus, to push the rock of progress. So much for the conservatism of Mistral. We shall conclude this story of the shorter poems with some that are not polemical or essentially Provencal; three or four are especially noteworthy. The Drummer of Arcole, Lou Prego-Bieu, Rescontre (Meeting), might properly find a place in any anthology of general poetry, and an ode on the death of Lamartine is sincere and beautiful. Such poems must be read in the original. The first one, The Drummer of Arcole^ is the story of a drummer boy who saved the day at Arcole by beating the charge; but after the wars are over, he is forgotten, and remains a drummer as before, becomes old and regrets his life given up to the service of his country. But one day, passing along the streets of Paris, he chances to look up at the Pantheon, and there in the huge pediment he reads the words, ^^ Aux grands hommes la patrie reconnaissante.^'' " ' Drummer, raise thy head ! ' calls out a The one up there, hast thou seen passer-by him?' Toward the temple that stood superb Just the old man raised his bewildered eyes. ! ' LIS ISCLO D'OR 206 then the joyous sun shook his golden locks above enchanted Paris . . . " When the soldier saw the dome of the Pan- theon rising toward heaven, and with his drum hanging at his side, beating the charge, as if it were real, Arcole, he recognized himself, the boy of away up there, right at the side of the great Napoleon, intoxicated with his for- mer fury, seeing himself, so high, in full relief, above the years, the clouds, the storms, in glory, azure, sunshine, he felt a gentle swell- ing in his heart, and fell dead upon the pave- ment." Lou Prego-DiSu is a sweet poem embodying Prego-dieu is the name of a a popular belief. little insect, so called from the peculiar arrange- ment of its legs and antennae that makes it appear to be in an attitude of prayer. Mistral's poetic ideas have been largely suggested to him by popular beliefs and the stories he heard at his fireside when a boy. This poem is one of the best of the kind he has produced, and, being eminently characteristic, will find juster treat- ment in a literal translation than in a commentary. The first half was written during the time he was at work upon Mireio, in 1856, the FR^DfeRIC MISTRAL 206 second in 1874. We quote the first stanza in the original, for the sake of showing its rhythm. " Ero un tantost d'aquel estieu Que ni vihave ni dourmi^u Fasieu miejour, tau que me plaise, Lou cahess6u Toucant lou s6u A I'aise." was one afternoon this summer, while I was neither awake nor asleep. I was taking a It noon siesta, as is my pleasure, my head at ease upon the ground. And greenish among the stubble, upon a spear of blond barley, with a double row of seeds, I saw a prego-dieu. " Beautiful insect," said I, " I have heard that, as a reward for thy ceaseless praying, God hath given thee the gift of divination. " Tell me now, good friend, if she I love hath slept well ; tell what she is thinking at this hour, and what she is doing ; tell me if she is laughing or weeping." The insect, that was kneeling, stirred upon the tube of the tiny, leaning ear, and unfolded and waved his little wings. LIS ISCLO D'OR 207 And his speech, softer than the softest breath of a zephyr wafted in a wood, sweet and mysterious, reached my ear. " I see a maiden," said he, " in the cool shade beneath a cherry tree; the waving branches touch her; the boughs hang thick with cherries. " The cherries are fully ripe, fragrant, solid, red, and, amid the smooth leaves, make one hungry, and, hanging, tempt one. " But the cherry tree offers in vain the sweetness and the pleasing color of its bright, firm fruit, red as coral. " She sighs, trying to see if she can jump high enough to pluck them. Would that my lover might come He would climb up, and throw them down into my apron." So I say to the reapers " Reapers, leave behind you a little corner uncut, where, during the summer, the prego-dieu may have shelter." ! : II This autumn, going down a sunken road, I wandered off across the fields, lost in earthly thoughts. And, once more, amid the stubble, I saw, FR:feD6RIC 208 MISTRAL clinging to a tiny ear of grain, folded up in his double wing, the prego-dieu. " Beautiful insect," said I then, " I have heard that, as a reward for thy ceaseless praying, God hath given thee the gift of divination. " And that if some child, lost amid the harvest fields, asks of thee his way, thou, little creature, showest him the way through the wheat. " In the pleasures and pains of this world, I see that I, poor child, am astray; for, as he grows, man feels his wickedness. " In the grain and in the chaff, in fear and in pride, in budding hope, alas for me, I see my ruin. " I love space, and I am in chains ; among thorns I walk barefoot ; Love is God, and Love sins ; every enthusiasm after action is disap- pointed. " What we accomplished is wiped out ; brute instinct is satisfied, and the ideal is not reached we must be born amid tears, and be stung among the flowers. " Evil is hideous, and it smiles upon me ; the flesh is fair, and it rots ; the water is bitter, and I would drink ; I am languishing, I want to die and yet to live. " I am falling faint and weary ; O pr^go-dieu, LIS ISCLO D'OR 209 cause some slight hope of something true to upon me show me the way." I saw that the insect stretched forth its slender arm toward Heaven mysterious, mute, earnest, it was praying. shine And ; straightway Such reference to religious doubt is elsewhere absent from Mistral's work. His faith is strong, and the energy of his life-work has its source largely, not only in this religious faith, but in his firm belief in himself, in his race, and in the mission he has felt called upon Reflected obviously in the above to undertake. poem is the growth of the poet in experience and in thought. Lastly, among the poems of his Iselo d' Or^ we wish to call attention to one that, in its theme, recalls Le Lac^ La Tristesse d' Olympio^ and Le Souvenir. The poet comes upon the scene of his first love, and apostrophizes the natural objects about him. All four poets intone the strain, " Ye rocks and trees, guard the memory of our love." " O coumbo d'Uriage Bos fresqueirous, Ounte aven fa lou viage Dis amourous, P FR:feDfeRIC 215 MISTRAL O van qu'aven noumado Noste univers, Se perdes ta ramado Gardo mi vers." O vale of Uriage, cool wood, where we made our lovers' journey O vale that we called ; our world, if thou lose thy verdure, keep my verses. Ye flowers of the high meadows that no man knoweth, watered by Alpine snows, ye are less pure and fresh in the month of April than the little mouth that smiles for me. Ye thunders and stern voices of the peaks, murmurings of wild woods, torrents from the mountains, there is a voice that dominates you aU, the clear, beautiful voice of my love. Alas ! vale of Uriage, we may never return to thy leafy nooks. air, and I, She, a star, vanisheth in folding my tent, go forth into the wilderness. Apart from the intrinsic worth of the thought or sentiment, there is found in Mistral the essential gift of the poet, the power of expression — of clothing in words that fully embody the meaning, and seem to sing, in spontaneous musical flow, the inner inspiration. He is superior LIS ISCLO D'OR 211 to tlie other poets of the F^librige, not only in the energy, the vitality of his personality, and in the fertility of his ideas, but also in this Even if he creates his vocabulary as he goes along, somewhat after great gift of language. the fashion of Ronsard and the Pleiade, he does this in strict accordance with the genius of his him, untrammelled by dialect, fortunately for traditions, and, what is significant, acceptably. He poets proclaim No one who is the master. he does it His fellow- and acclaim his supremacy. penetrated to any degree has into the genius of the Romance languages can fail to agree that in this of one of its forms. point exists a master CHAPTER III THE TRAGEDY, LA RfeiNO JANO The peculiar qualities and limitations of Mistral are possibly nowhere better evidenced than in this play. Full of charming passages, fre- quently eloquent, here and there very poetic, it is scarcely dramatic, and certainly not a trag- edy either of the French or the Shakespearian type. The most striking lines, the most elo- quent tirades, arise less from the exigences of the drama than from the constant desire of the poet to give expression to his love of Provence. The attention of the reader is diverted at every turn from the adventures of the per- sons in the play to the glories and the beauties of the lovely land in which our poet was born. The matter of a play is certainly contained in the subject, but the energy of the author has not been spent upon the invention of strong situations, upon the clash 212 of wills, upon the THE TRAGEDY, LA RfeiNO JANO 213 psychology of his characters, upon the interplay of passions, but rather upon strengthening in the hearts of his Provencal hearers the love of the good Queen Joanna, whose life has some of the romance of that of Mary, Queen of Scots, and upon letting them hear from her lips and from the lips of her courtiers the praises of Provence. Mistral enumerates treating the life eight dramatic works They are a Magnon of his heroine. tragedy in five acts and a verse by (Paris, 1656), called Jeanne P^, reine de Naples; a tragedy in five acts and in verse by Laharpe, produced in 1781, entitled, Jeanne de Naples; an opera-comique in three acts, the book by De Leuven and Brunswick, the music by Monpon and Bordese, produced in 1840 an Italian tragedy, La Regina Giovanna, by the Marquis ; of Casanova, written about 1840 ; an Italian opera, the libretto by Ghislanzoni, who is known as the librettist of Aida^ the music by Petrella (Milan, 1875) ; a play in verse by Brunetti, called G-iovanna I di Napoli (Naples, 1881) ; a Hungarian play by Rakosi, Johanna es JSndre, and lastly the trilogy of Walter Savage Landor, Andi-ea of Hungary^ Criovanna of Naples, and FRlfeDfeRIC 214 MISTRAL Fra Rupert (London, 1853). is dated May, 1890. Mistral's play It may be said concerning the work of der, which is a Lan- poem in dramatic form rather than a play, that it offers scarcely any points of resemblance with Mistral's beyond the few essential facts in the lives of Andrea and Joanna. Both poets take for granted the innocence of It is worth noting that Provence the Queen. is but once referred to in the entire work of the English poet. The introduction that precedes Mistral's play life of the Queen quotes the account of the from the Dictionnaire of Moreri (Lyons, 1681), which we here translate. " Giovanna, first of the name. Queen of Jerusalem, Naples, and Sicily, Duchess of Apulia and Calabria, Countess of Provence, etc., was a daughter of Charles of Sicily, Duke of Calabria, who died in 1328, before his father Robert, and of Marie of Valois, his second wife. She was only nineteen years of age when she assumed the government of her dominions after her grandfather's death in 1343. She had already been married by him to his nephew, Andrea of Hungary. This was not a happy marriage ; for THE TRAGEDY, LA r4:IN0 JANO 215 the inclinations of both were extremely contrary, and the prince was controlled by a Fran- ciscan monk named Robert, and the princess by a washerwoman called Filippa Catenese. These indiscreet advisers brought matters to extremes, so that Andrea was strangled in 1345. The disinterested historians state ingenuously that Joanna was not guilty of this crime, although the others accuse her of it. She married again, on the 2d of August, 1346. Her second hus- band was Louis of Tarento, her cousin and she was obliged to leave Naples to avoid the armed attack of Louis, King of Hungary, who committed acts of extreme violence in this ; state. Joanna, however, quieted all these things by her prudence, and after losing this second husband, on the 25th of March, 1362, she married not long afterward a third, James of Aragon, Prince of Majorca, who, however, tarried not long with her. So seeing herself a widow for the third time, she made a fourth match in 1376 with Otto of Brunswick, of the House of Saxony and as she had no children, ; she adopted a relative, Charles of Duras. . . . This ungrateful prince revolted against Queen Joanna, his benefactress. . . . He captured MISTRAL FRlfeDfeRIC 216 Naples, and laid siege to the Castello Nuovo, Queen was. She surrendered. Charles of Duras had her taken to Muro, in the Basilicata, and had her put to death seven or eight months afterward. She was then in her fifty-eighth year. Some authors say where the . . . that he caused her to be smothered, others that she was strangled ; but the more probable view is that she was beheaded, May. in 1382, on the 5th of It is said that a Provencal astrologer, doubtless a certain Anselme who lived at that time, and who is very famous in the history of Provence, being questioned as to the future hus- band of the young princess, replied, 'Maritabitur cum ALIO.' This word is composed of the initials of the names of her four husbands, Andrea, Louis, James, and Otto. This princess, furthermore, was exceedingly clever, fond of the sciences and of men of learning, of whom she had a great many at her court, liberal and and not lacking, in She it is that sold Avignon to the Boccaccio, Balde, and other scholars beautiful, prudent, wise, piety. popes. of her time speak of her with praise." In offering an explanation of the great popularity enjoyed by Joanna of Naples among the THE TRAGEDY, LA RfeiNO JANO 217 people of Provence, the poet does not hesitate to acknowledge that along with her beauty, her personal charm, her brilliant arrival on the gorgeous galley at the court of Clement VI, came, whither she eloquent and proud, to exculpate herself, her long reign and its vicissitudes, her generous efforts to reform abuses, must be counted also the grewsome procession of her four husbands; and this popularity, he The centuries. among such historic figures as says, is still alive, after five poet places her King Arthur, Count Raymond of Toulouse, the good King Rene, Anne of Brittany, Roland, the Cid, to which the popular mind has attached heroic legends, race traditions, and mysterious monuments. The people of Provence still look back upon Caius the Marius, days of Ossian, their independence when she reigned, a sort of good fairy, as the good old times of Queen Joanna. bridges, churches, life among this and ruins, towers name. Countless castles, monuments, testify to her people. Roads and aqueducts, bear her enthusiastic Proverbs exist wherein it is preserved. " For us," says Mistral, " the fair Joanna what Mary Stuart is for the Scotch, is — a mirage retrospective of MISTRAL FRfeDfeRIC 218 love, a regret of youth, of nationality, of poetry passed away. And anal- ogies are not lacking in the lives of the royal, tragic enchantresses." two Petrarch, speak- ing of her and her young husband surrounded by Hungarians, refers to them as two lambs among wolves. In a letter dated from Vancluse, August, 1346, he deplores the death of the King, but makes no allusion to the complicity of the Queen. Boccaccio proclaims her the special pride of Italy, so gracious, she seemed rather and kindly, that companion than the gentle, the queen of her subjects. Our author cites likewise some of her accusers, and considers most of the current sayings against her as apocryphal. Some of these will not bear quotation in English. Mistral evi- dently wishes to believe her innocent, and he makes out a pretty good case. He approves the remark of Scipione Ammirato, that she contracted four successive marriages through a desire to have direct heirs. Another notices that had she been dissolute," she would have preferred the liberty of remaining a widow. The poet cites Pope Innocent VI, who gave THE TRAGEDY, LA RfeiNO JANO 219 her the golden rose, and sets great store upon the expression of Saint Catherine of Siena, who calls her "Venerabile madre in Gesu Cristo," and he concludes by saying, "We prefer to concur in the judgment of the good Giannone (1676-1748), which so well agrees with our traditions." The first act opens with a picture that might The Queen tempt a painter of Italian scenes. and her gay court are seated on the lawn of the palace garden at Naples, overlooking the bay and islands. At the very outset we hear of the Gai Savoir, and the Queen utters the es- sentially Provengal sentiment that "the chief glory the world should strive for is light, for joy and love are the children of the sun, and art and literature the great torches." She calls upon Anfan of Sisteron to speak to her of her Provence, " the land of God, of song and youth, the finest jewel in her crown," and Anfan, in long and eloquent tirades, tells of Toulouse and Nice and the Isles of Gold, reviews the settling of the Greeks, the domination of the Romans, and the sojourn of the Saracens; Aix and Aries, les Baux, Toulon, are glorified again; we hear of the old liberties of these FRjfeD^RIC 220 towns where men MISTRAL and shout, and sleep, sing, of the magnificence of the papal court at Avi- gnon. " Enfin, en Avignoun, i'a lou papo ! grandeur Poud^, magnificenci, e poumpo e resplendour, Que mestrejon la terro e fan, senso messorgo, Boufa I'alen de Di^u i ribo de la Sorgo." Lastly, in Avignon, there's the Pope 1 greatness, power, magnificence, pomp, and splendor, dominating the earth, and without exaggeration, causing the breath of God to blow upon the banks of the Sorgue. We learn that the brilliancy and animation of the court at of Avignon outshine the glories Rome, and in language that fairly glitters with its high-sounding, highly colored words. We hear of Petrarch and Laura, and the associations of Vaucluse. At this juncture the Prince arrives, and is struck by the resemblance of the scene to a court of love; he wonders if they are not discussing the question whether love is not drowned in the nuptial holy water font, or whether the lady inspires the lover as much with her presence as when absent. And the Queen defends her mode of life and temperament she cannot brook the cold and gloomy ; THE TRAGEDY, LA R^INO JANO 221 ways of the north. Were we to apply the methods of Voltaire's strictures of Corneille to this play, it might be interesting to see how many vers de comidie could be found in these scenes of dispute between the prince consort and his light-hearted wife. " A I'avans Go ahead ! ! zdu ! en ffesto arrouinas lou Tresor " ! that's right, ruin the treasury with your feasts! and to his objections to so many flattering courtiers, the Queen replies : — " Voules que moan palais devengue un mounastie ? " Do you want my palace to become a monastery ? Joanna replies nobly and eloquently to the threats of her husband to assume mastery over her by violent means, and, in spite of the anachronism (the poet makes her use and seemingly invent the term Renascence')^ her defence and science of her time is forceful and enthusiastic, and carries the reader along. That this sort of eloquence is dramatic, appears, of the arts however, rather doubtful. The next scene interests us more directly in The Prince, left alone the characters before us. with his confidant, Fra Rupert, gives expression to his passionate love for the Queen, and pours FRfeDfeRIC 222 MISTRAL forth the bitterness of his soul to see it unre- The fierce Hungarian monk denounces, quited. rather justly, it appears to us, the license and levity of the Italian court, and incites Andrea to an appeal to the Pope, "a potentate that has no army, whose dominion extends from pole to pole, who binds and unbinds at his will, up- holds, makes, or unmakes thrones as an almighty master." But Andrea fears the Queen would never pardon him. " E se noun ai en plen Ion m^u de si caresso, L'empferi universau m'es un gourg d'amaresso " ! And if I have not fully the honey of her caresses The empire of the world is to me a gulf of bitterness. Finally the monk and La Catanaise stand This woman is the Queen's alone before us. nurse, who loves her with a fierce sort of pas- sion, and it is she who commits the crime that causes the play to be called a tragedy. This final scene brings out a flood of the most violent vituperation from this veritable virago, some of it exceedingly low in tone. The friar leaves with the threat to have a red-hot nail run through her hellish tongue, and La Catanaise, THE TRAGEDY, LA RfeiNO JANO 223 standing alone, gives vent to her fury in threats of murder. The next act reveals the Hall of Honor in Andrea in anger the Castel-Nuovo at Naples. proclaims himself king, and in the presence of the Queen and the Italian courtiers gives away one after another all the offices and honors of the realm to his Hungarian followers. A conflict with drawn swords is about to ensue, when the Queen rushes between the would-be combatants, reminding them of the decree of the Pope but Andrea in fury accuses the Queen of conduct worthy a shameless adventuress, and cites the ; reports that orgies. liken her to Semiramis in her The Prince of Taranto throws down his glove to the by a threat enraged Andrea, who replies to bring him to the executioner. The Prince of Taranto answers that the executioner may be the supreme law for a king, " Mai per un qu'a I'ounour dins lou pi^s e dins I'amo, Uno escorno, cousin, se purgo eme la lamo." But for one who has honor in his breast and his soul, An insult, cousin, is purged with the sword. Andrea turns to his knights, and leaving the room with them points to the flag bearing the frI:d6ric mistral 224 block and axe as emblems. The partisans of Joanna remain full of indignation. La Catanaise addresses them. The Sicilians, she says, waste no time in words, but have a speedier method of punishing a wrong, and she reminds them of the massacre at Palermo. The Prince of Taranto discountenances the proposed crime, for the Queen's fair name would suffer. the fierce woman points to the flag. But " Do you see that axe hanging from a thread ? You are me act alone." And the all cowards I Let Prince nobly replies, " Philippine, battles are men of our renown, men of my stamp, do not crouch down in the fought in the sunlight ; dark shadow of a plot." again shows the flag. falling upon the block ? And the Catanaise " Do you see the axe " Joanna enters to offer the Prince her thanks for his chivalrous defence of her fair name, and dismisses the other courtiers. brief scene is really The ensuing between the Queen and the Prince very eloquent and very beautiful. The Queen recalls the fact that she was married at nine to Andrea, then only a child too ; she has never known love. The and poorest of the shepherdesses on the mountains of Calabria THE TRAGEDY, LA RfelNO JANO 225 may quench her thirst at the spring, but she, the Queen of the Sun, if to pass away the time, or to have the appearance of happiness, she loves to listen to the echo of song, to behold the joy and brilliancy of a noble her fete, And the Prince very smile becomes criminal. reminds her that she is the Provengal queen, and that in the great times of that people, if the consort were king, love was a god, and he recalls the names of all the ladies made famous by the Troubadours. Thereupon the Queen in an outburst of enthusiasm truly Felibrean invokes the God of Love, the God that slew Dido, and speaks in the spirit of the days of courtly love, " O thou God of Love, hearken unto me. If my fatal beauty is destined sooner or later to bring about my death, let this flame within me be, at least, the pyre that shall kindle the song of the poet ! Let my beauty be the luminous star exalting men's hearts to lofty visions The chivalrous Prince is ! and dismissed, Joanna is alone with her thoughts. The little page Dragonet sings outside a plaintive song with the refrain : — " Que regret Jamai digues toun secret." Q What regret I Never tell thy secret. FR^D^RIC MISTRAL 226 La Catanaise endeavors to excite the fears of the Queen, insinuating that the Pope may give the crown to Andrea. Joanna has no fear. "We shall have but to appear before the country with this splendor of irresistible grace, and like the smoke borne away by the breeze, suddenly my enemies shall disappear." We may ask whether such self-praise comes gracefully from the Queen herself, whether she might not be less conscious of her own charm. La Catanaise is again alone on the scene, threat"The bow is drawn, the hen setting." ening. This last comparison, the reader will remark, would be simply impossible as the termination of an act in a serious English play. This last scene, too, is wofully weak and purposeless. The conversation of three courtiers at the beginning of Act III apprises us of the fact that the Pope has succeeded in bringing about a reconciliation between the royal pair, and that they are both to be crowned, and as a matter of precaution, the nurse Philippine, and the monk Fra Rupert are to be sent upon their several ways. The scene is next filled by the conspirators. La Catanaise directing the details of the plots. It is made clear that the Queen is THE TRAGEDY, LA RkmO JANO 227 utterly ignorant of these proceedings, which are after all useless ; for we fail to see what valid motive these plotters have to urge them on to their contemptible deed. A brilliant banquet Anfan of Sisteron sings scene ensues, wherein a song of seven stanzas about the fairy M^lusine, and seven times Dragonet sings the refrain, "Sian de la rago di lesert" race of the lizards). (We are of the And there are enthusias- tic tirades in praise of the Queen and of Pro- But Andrea spills salt upon the table, which evil augury seems to be vence, and all is merry. taken seriously. This little episode is foolish, and unworthy of a tragedy. We are on the verge of an assassination. Either the gloomy forebodings and the terror of the event should be impressed upon us, or the exaggerated gayety and high spirits of the revellers should by contrast make the coming event seem more terri- ble; but the spilling of salt is utterly trivial. After the feast La Catanaise and her daughter proceed to their devilish work, in the room now lighted only by the pale rays of the moon, while the voice of the screech-owl is heard outside. The trap is set for the King ; he is strangled just out of sight with the silken noose. The FR6d6rIC mistral 228 Queen is roused by her nurse. The palace is in an uproar, and the act terminates with a passionate demand for vengeance and justice on the part of Fra Rupert. And now the Fourth Act. Here Mistral is in his element ; here his love of rocky landscapes, and golden islands, of song and of azure seas festivity, finds gotten, the full play. The tragedy is for- dramatic action completely inter- — never mind. We accompany the Queen on her splendid galley all the way from She leaves amid the Naples to Marseilles. rupted, acclamations of the Neapolitans, recounts the splendors of the beautiful bay, and promises to return "like the star of night coming out of the mist, laurel in hand, on the white wings of her The boat starts, the rowers sing their plaintive rhythmic songs, the Queen Provengal galley." is enraptured by the beauty of the fleeing shores, the white sail glistens in the glorious blue above. She is lulled by the motion of the boat and the waving of the hangings of purple and gold. Midway on her journey she receives a visit from the Infante of Majorca, James of Aragon, who seems to be wandering over that part of the sea; then the astrologer Anselme THE TRAGEDY, LA RtlINO JANO 229 predicts her marriage with Alio and her death. She shall be visited with the sins of her ancestors ; the blood spilled by Charles of Anjou cries The Queen passes through a moment of gloom. She dispels it, exclaiming " Be it so, strike where thou wilt, O fate, I am for vengeance. a queen ; I shall fight, if need be, until death, to uphold If my cause and my womanly honor. my wild planet is destined to sink in a sea of blood and tears, the glittering trace I shall leave on the earth will show at least that I was worthy to be thy great Provence queen, O brilliant " ! She descends into the ship, and the rowers resume their song. Later we arrive at Nice, where the Queen is received by an exultant throng. She forgets the awful predictions and is utterly filled with delight. She will visit all the cities where she is loved, her ambition is to see her flag greeted all along the Mediterranean with shouts of joy and love. to be a Proven9ale. She feels herself " Come, people, here I am; breathe me in, drink me in ! It is sweet to me to be yours, and sweet to please you; and you may gaze in love and admiration upon me, for I am your queen ! FRfeD^RIC MISTRAL 230 We pass the Isles The journey is resumed. of Gold, and the raptures are renewed. At Marseilles the Queen is received by the Consuls, and swears solemnly to respect all the rights, customs, and privileges of the land, and the Consul exacts as the last oath that she swear to see that the noble speech of Aries shall be main- tained and spoken in the land of Provence. The act closes with the sentiment, "May Provence triumph in every way The last act brings us to the great hall of ! the papal palace at Avignon, where the Pope is to pronounce judgment upon the Queen. Fra Rupert, disguised as a pilgrim, harangues the throng, and two Hungarian knights are beaten in duel by Galeas of Mantua. its alternate cries of Zou ! Zou ! is Dau difficult to ! This duel, with Dau ! Te ! Te ! take seriously and The Queen enters in The Hungarian conversation with Petrarch. reminds us of Tartarin. knights utter bitter accusations against the Queen, who gives them in place of iron chains the golden chains about her neck, whereupon the knights gallantly declare their hearts are won forever. The doors open at the back and we see the papal court. Bertrand des Baux THE TRAGEDY, LA RillNO JANO 231 a hideous account of the torture and gives death of those who had a hand in the death of Andrea. The Queen makes a long speech, ex- pressing her deep grief at the calumnies and slander that beset her. The court and people resolve themselves into a kind of opera chorus, expressing their various sentiments in song. The Queen next reviews her life with Andrea, — and concludes " And it seemed to me noble and worthy of : a queen to melt with a glance the cold of the frost, to make the almond tree blossom with a smile, to be amiable to and lead all, affable, generous, my people with a thread of wool Yes, all the thought of my mad youth was to be loved and to reign by the power of love. Who could have foretold that, afterward, on the day of the great disaster, all this should be made a reproach against me! that I should be accused, at the age of twenty, of instigating crime an awful ! And she breaks down weeping. The page, the people, the pilgrim, and the astrologer again sing in a sort of operatic ensemble their various emotions. The Pope absolves the Queen, the pilgrim denounces the verdict furiously, and FRfeDfeRIC 232 is put to death by MISTRAL Gal^as of Mantua. So ends the play. La Reino Jano is a pageant rather than a It is full of song and sunshine, glow tragedy. and glitter. The characters all talk in the exaggerated and exuberant style of Mistral, who is not dramatist enough to create independent being, living before us. The central person- no sense a tragic character. The fanatical Fra Rupert and the low, vile-tongued age is in The psy- Catanaise are not tragic characters. chology throughout is decidedly upon the surface. The author in his introduction warns us that to judge this play we must place ourselves at the point of view of the Provencals, in whom many an expression or allusion that leaves the ordinary reader or spectator untouched, will possibly awaken, as he hopes, some particular emotion. This is true of all his literature ; the Provencal language, the traditions, the memories of Provence, are the web and woof of it all. It is interesting to note the impression made by the language upon a Frenchman and a critic He says in of the rank of Jules Lemaitre. concluding his review of this play : — THE TRAGEDY, LA RfeiNO JANO 233 "The language is too gay, it has too much sing-song, it is too harmonious. It does not possess the rough gravity of the Spanish, and has too few of the i's and sonority of the Italian. it is I e's that soften the may venture to say too expressive, too full of onomatopoeia. Imagine a language, in which to say, "He bursts out laughing," one must use the word e'escacalasso ! There are too many ow's and omw's and too much ts and dz in the pronunciation. So that the Provencal language, in spite of everything, keeps a certain patois vulgarity. It forces the poet, so to say, to perpetual song- making. It must be very difficult, in that language, to have an individual style, still more difficult to express abstract ideas. But it is a merry language." The play has never yet been performed, and until a trial is made, one is inclined to think it would not be effective, except as a spectacle. It is curious that the Troubadours produced no dramatic literature whatever, and that the same lack is found in the modern revival. Aubanel's Lou Pan d6u Pecat (The Bread of Sin), written in 1863, and performed in 1878 at Montpellier, seems to have been successful, 234 FRfeDfeRIC MISTRAL and was played at Paris at the Theatre Libre in 1888, in the verse-translation made by Paul Arene. Aubanel wrote two other plays, Lou Pastre^ which is lost, and Lou Raubatdn, a work that must be considered unfinished. Two plays, therefore, constitute the entire dramatic duction in the new language. pro- PART THIRD CONCLUSIONS CONCLUSIONS It would be idle to endeavor to determine whether Mistral is to be classed as a great poet, or whether the Felibres have produced a great is defined when the made that Mistral is or is not a literature, and nothing statement is great poet. His genius may be said to be lim- ited geographically, for nated all if from it were elimi- that pertains directly to Provence, the remainder would be almost nothing. The only human nature known to the poet is the human nature of Provence, and while it is perfectly true that a human being in Provence could be typical of human nature in general, and arouse interest in all men through his humanity common to all, the fact is, that Mistral has not sought to express what is of universal inter- est, but has invariably chosen to present human life in its Provengal aspects and from one of view only. point A second limitation is found in 287 FR^D^RIC MISTRAL 238 the unvarying exteriority of his method of pre- senting human nature. Never does he probe deeply into the souls of his Provengals. Very vividly indeed does he reproduce their words and gestures ; but of the deeper under-currents, the inner conflicts, the agonies of doubt and indecision, the bitterness of disappointments, the lofty aspirations toward a higher inner life or a closer communion with the universe, the moral problems that shake a human soul, not a syllable. Nor is he a poet who pours out his own soul into verse. External nature is for him, again, nature as fields The rocks and trees, the and the streams, do not awaken in him a stir of emotions because of their power to com- seen in Provence. pel a mood in any responsive poetic soul, but they excite him primarily as the rocks and trees, the fields and streams of his native region. is no mere word-painter. He Rarely do his descrip- tions appear to exist for their own sake. They furnish a necessary, fitting, and delightful back- ground to the action of his poems. They are too often indications of what a Provengal ought to consider admirable or wonderful, they are sometimes spoiled by the poet's excessive par- CONCLUSIONS tiality for his own little land. 239 His work is ever the work of a man with a mission. There is no profound treatment of the theme Each of the long poems and his play of love. have a love story as the centre of interest, but the lovers are usually children, and their love utterly without complications. There is every- where a lovely purity, a delightful simplicity, a straightforward naturalness that is very charming, but in this theme as in the others. Mistral is incapable of tragic depths and heights. So it is as regards the religious side of man's The poet's work is filled with allunature. sions to religion ; there are countless legends concerning saints and hermits, descriptions of churches and the papal palace, there is the detailed history of the conversion of Provence to Christianity, but the deepest religious spirit Only twice in all his work do we come upon a profounder religious sense, in the second half of Lou Prego-BiSu and in Lou Saume de la Penitenei. There is no doubt is not his. that Mistral is a believer, but religious feeling has not a large place in his work ; there are no other meditations upon death and destiny. And this dme du Midi, spirit of Provence, the FRfiDfeRIC 240 MISTRAL genius of his race that he has striven to express, How shall it be defined or formu- what is it? lated? Alphonse Daudet, who knew loved it, whose it, and Parisian life and world-wide success did not destroy in him the love of his native Provence, who loved the very food of the Midi above all others, and jumped up in joy when a southern intonation struck his ear, and who was continually beset with longings to return to the beloved region, has well defined it. He was the friend of Mistral and followed the poet's efforts and achievements with deep and affectionate interest. It is not difficult to see that the satire in the " Tartarin " series is not Daudet approved of the Felibrige movement, though what he himunkind, nor is it untrue. self wrote in Provengal is insignificant. He believed that the national literature could be best vivified by those who most loved their homes, that the best originality could thus be He has said :^ attained. — " The imagination of the southerners differs from that of the northerners in that it does not mingle the different elements and forms in literature, and remains lucid in its outbreaks. 1 See Bevue de Paris, 15 avril, 1898. In CONCLUSIONS 241 our most complex natures you never encounter the entanglement of directions, relations, and figures that characterizes a Carlyle, a Browning, or a Poe. For this reason the man of the north always finds fault with the man of the south for his lack of depth and darkness. "If we consider the most violent of human passions, love, we see that the southerner makes it the great affair of his life, but does not allow himself to become disorganized. He likes the talk that goes with it, its lightness, its change. He hates the slavery of it. It furnishes a pre- text for serenades, fine speeches, light scoffing, caresses. He finds it difficult to comprehend the joining together of love and death, which lies in the northern nature, and casts a shade of melancholy upon these brief delights." Daudet notes the ease with which the southerner is carried away and duped by the mirage of his own fancy, his semi-sincerity in excite- ment and enthusiasm. He admired the natural He found a justi- eloquence of his Provengals. fication for their exaggerations. " Is it right to accuse a man of lying, who is intoxicated with his own eloquence, who, with- out evil intent, or love of deceit, or any instinct 242 FRfeDifeRIC MISTRAL of scheming or false trading, seeks to embellish his own life, and other people's, with stories he knows to be illusions, but which he wishes were Is Don Quixote a liar? Are all the true? poets deceivers who aim to free us from realities, to among Each go soaring off into space? After all, no deception. there southerners, is one, within himself, restores things to their proper proportions." Daudet had Mistral's love of the sunshine. He needed it to inspire him. He believed it explained the southern nature. Concerning the absence of metaphysics in the race he says : — "These reasonings may culminate in a state of mind such as we see extolled in Buddhism, a colorless state, joyless and painless, across which the fleeting splendors of thought pass like stars. Well, the man of the south cares naught for that sort of paradise. tion is The vein of real sensa- freely, perpetually open, open to life. The side that pertains to abstraction, to logic, is lost in mist." We have referred to the power of storytelling among sponsiveness as the Provengals and their re- listeners. Daudet mentions CONCLUSIONS 243 the contrast to be observed between an audi- ence of southerners and the stolid, self-contained attitude of a crowd in the north. The evil side of the southern temperament, accompany these traits, are plainly stated by the great novelist. Enthe faults that thusiasm turns to hypocrisy, love of at what any cost ; or brag ; the glitters, to a passion for luxury sociability, the desire to please, become weakness and fulsome flattery. orator beats his breast, his voice is The hoarse, choked with emotion, his tears flow conveniently, he appeals to patriotism and the noblest sentiments. There is a legend, according to Daudet, which says that when Mirabeau cried out, "We will not leave unless driven out at the point of the bayonet," a voice off at one side corrected the utterance, murmuring sarcastically, tracks ! "And if the bayonets come, we make " The southerner, when he converses, is roused to animation readily. His eye flashes, his words are uttered with strong intonations, the im- pressiveness of a quiet, earnest, self-contained manner is unknown to him. Daudet is a novelist and a humorist. Mis- FRfeD^RIC MISTRAL 244 tral is a poet ; hence, although aim at a he professes to expression of the "soul of his full Provence," there are many aspects of the Provengal nature that he has not touched upon. He has omitted all the traits that lend themselves to satirical treatment, and, although he is many ways a remarkable realist, he has in very little dramatic power, and seems to lack the gift of searching analysis character. of individual It is hardly fair to reckon it as a shortcoming in the poet and apostle of Provence that he presents only what is most beauti- The novelist offers us a faithful and vivid image of the men of his own day. The poet glorifies the past, clings to ful in the life about him. tradition, and exhorts his countrymen to return to it. Essentially and above all else a conservative. Mistral has the gravest doubts about so-called modern progress. Undoubtedly honest in de- siring the well-being of his fellow Proven9als, he believes that this can be preserved or attained only by a following of tradition. There must be no breaking with the past. Daudet, late in adhered to this doctrine. His son quotes life, him as saying : — CONCLUSIONS 246 "I am followiDg, with gladness, the results of the impulse Mistral has given. Return to tradition ! that is our salvation in the present going to pieces. I have always came to me felt this in- clearly only a few stinctively. It years ago. It is a bad thing to become wholly loosened from the church soil, to forget the village Curiously enough spire. poetry at- taches only to objects that have come down to us, that have had long use. What is called progress^ a vague and very doubtful term, rouses The higher parts vibrate the better for what has moved the lower parts of our intelligence. and inspired a long series of imaginative minds, inheriting each from a predecessor, strength- ened by the sight of the same landscapes, by the same perfumes, by the touch of the same Very ancient im- furniture, polished by wear. pressions sink into the depth of that obscure memory which we may call the race-memory^ out of which is woven the mass of individual memories." Mistral is can best see truly the poet of the Midi. One how superior he is as an artist in words by comparing him with the foremost of his fellow-poets. He is a master of language. FR^DjfcRIC 240 MISTRAL He has the eloquence, the enthusiasm, the optimism of his race. His poetic earnestness saves his tendency to exaggerate. its superiority, is His style, in all a southern style, full of in- terjections, full of long, sonorous words. His thought, his expressions, are ever lucid. His art is almost wholly objective. His work has extraordinary unity, and therefore does not es- cape the monotony that was unavoidable when the poet voluntarily limited himself to a single purpose in life, and to treatment of the themes thereunto pertaining. Believers in material progress, those who look for great changes in political and social conditions, will turn from indifference. His contentment Mistral with with present things, and his love of the past, are likely to irritate them. Those who seek in a poet consolation in the personal trials of life, new message concerning human destiny, a new note in the everlasting themes that the a great poets have sung, will be disappointed. A word must be said of him as a writer of French. In the earlier years he felt the weight of the Academy. He did not feel that French would allow full freedom. He was scrupulous and timid. He soon shook off this timidity CONCLUSIONS 247 and became a really remarkable wielder of the French tongue. His trafislations of his own works have doubtless reached a far wider public than the works themselves, characterized and are certainly by great boldness, clearness, and an astonishingly large vocabulary. His earlier work clearly inspired is love of Greek literature, and those in Latin literature by his qualities wherein the Greek genius shines through, possibly also by some mysteri- ous affinity with the Greek spirit resulting from climate or atavism. him. This never entirely left When later he writes of Provence in the Middle Age, of the days of the Troubadours, his manner does not change ; his work offers no analogies here with the French Romantic school. No poet, it would seem, was ever so in love with his own language ; no artist ever so loved the mere material he was using. Mistral loves the words he uses, he loves their sound, he loves to hear them from the lips of those about him; he loves the intonations and the cadences of his verse ; his love is for the speech itself aside from any meaning it conveys. A beautiful in- strument it is indeed. Possibly nothing is more 248 FRifeD^RIC MISTRAL peculiarly striking about him than this extreme enthusiasm for his golden speech, his lengo d'or. To him must be conceded the merit of originality, great originality. In seeking the source many of his conceptions, one is led to the conclusion, and his own testimony bears it out, that they are the creations of his own fancy. If there is much prosaic realism in the Poem of of the Hhone, the Prince and the Anglore are purely the children of Mistral's almost naive imagination, and Calendau and Esterello are attached to the real world of history by the slenderest bonds. When we seek for resem- blances between his conceptions and those of other poets, we can undoubtedly find them. Mireille now and then reminds of Daphnis and Chloe, of Hermann and Dorothea, of Evangeline, but the differences are far more in evidence than the resemblances. Esterello is in an atti- tude toward Calendau not without analogy to that of Beatrice toward Dante, but it would be impossible to find at any point the slightest imitation of Dante. Some readers have been reminded of Faust in reading Nerto, but beyond the scheme of the Devil to secure a woman's CONCLUSIONS 249 soul, there is little similarity. Nothing could be more utterly without philosophy than Nerto. Mistral has drawn his inspirations from within himself ; he has not worked over the poems and legends of former poets, or sought much of his subject-matter in the productions ages. of former He has not suffered from the deep re- flection, the pondering, and the doubt that destroy originality. If Mistral had written his poems in French, he would certainly have stood apart from the would have been impossible to attach him to any of the socalled " schools " of poetry that have followed general line of French poets. It one another during this century in France. He is as unlike the Romantics as he is unlike the Par- M. Brunetiere would find no difficulty in applying to his work the general epithet nassians. French main current, for of "social" that so well characterizes literature considered in its Mistral always sings to his fellow-men to move them, to persuade them, to stir their hearts. Almost all of his poems in the lyrical form show him as the spokesman of his fellows or He is as the leader urging them to action. therefore not of the school of " Art for Art's FR:feDfeRIC 250 MISTRAL sake," but his art is consecrated to the cause he represents. His thought is ever pure and high ; his les- sons are lessons of love, of noble aims, of energy and enthusiasm. He is full of love for the best in the past, love of his native soil, love of his native landscapes, love of the love of his country. men about him, He is a poet of the "Gai Saber," joyous and healthy, he has never felt a trace of the bitterness, the disenchantment, the gloom and the pain of a Byron or a Leopardi. He is eminently representative of the race he seeks to glorify in its own eyes and in the world's, himself a type of that race at its very best, with all its exuberance and energy, with its need of outward manifestation, life and movement. to An important place must be assigned him among those who have bodied their forth poetic conceptions in the various eupho- nious forms of speech descended from the ancient speech of Rome. In Provence, and far beyond its borders, he is known and loved. His activity has not ceased. His voice is still heard, clear, strong, hopeful, inspiring. Mireille is sung in the ruined Roman theatre at Aries, museums are founded CONCLUSIONS 261 to preserve Provengal art and antiquities, the Felibrean feasts continue with unabated enthuis a successful life; he has revived a language, created a literature, siasm. Mistral's life inspired a people. So potent is art to-day in the old land of the Troubadours. All the charm and beauty of that sunny land, all that is enchanting in its past, all the best, in the ideal sense, that may be hoped for in its future, is expressed in his musical, limpid, lovely verse. Such a poet and such a leader of men is rare in Such complete oneness the annals of literature. of purpose men. and of achievement is rare among APPENDIX We offer here a literal prose translation of the Psalm of Penitence. THE PSALM OF PENITENCE I Lord, at last thy wrath hurleth its thunderbolts upon our foreheads, and in the night our vessel strikes its prow against the rocks. Lord, thou cuttest us down with the sword of the barbarian like fine wheat, and not one of the cravens that we shielded comes to our defence. Lord, thou twistest us like a willow wand, thou breakest down to-day all our pride is ,• there none to envy us, who but yesterday were so proud. Lord, our land goeth to ruin in war and strife; and if thou withhold thy mercy, great and small will devour one another. Lord, thou art terrible, thou strikest us upon the back; in awful turmoil thou breakest our power, compelling us to confess past evil. 268 FRfeD^RIC MISTRAL 264 n Lord, we had strayed away from the austerity of the old laws and ways. Virtues, domestic customs, we had destroyed and demolished. Lord, giving an evil example, and denying thee like the heathen, we had one day closed up thy temples and mocked thy Holy Christ. Lord, leaving behind us thy sacraments and commandments, we had brutally lost belief in all but self-interest and progress ! Lord, in the waste heavens we have clouded thy light with our smoke, and to-day the sons mock the nakedness and purity of their fathers. Lord, we have blown upon thy Bible with the breath of false knowledge ; and holding ourselves up like the poplar trees, we wretched beings have declared ourselves gods. Lord, we have left the furrow, we have trampled all respect under foot ; and with the heavy wine that intoxicates us we defile the innocent. Ill Lord, we are thy prodigal children, but we are thy Christians of old ; let thy justice chastise us, but give us not over unto death. APPENDIX 255 Lord, in the name of so many brave men, who went forth fearless, valiant, docile, grave, and then fell in battle Lord, in the name of so many mothers, who are about to pray to God for their sons, and who next year, alas and the year thereafter, shall see them no more Lord, in the name of so many women who have at their bosoms a little child, and who, poor creatures, moisten the earth and the sheets ! of their beds with tears Lord, in the name of the poor, in the name of the strong, in the die for their name of the dead who shall country, their duty, and their faith Lord, for so many defeats, so many tears and woes, for so many towns ravaged, for so much brave, holy blood many adversities, for so much mourning throughout our France, for so many Lord, for so insults upon our heads IV Lord, disarm thy justice. Cast down thine eye upon us, and heed the cries of the bruised and wounded FR6d6rIC mistral 258 Lord, if the rebellious cities, through their luxury and folly, have overturned the scale-pan of thy balance, resisting and denying thee Lord, before the breath of the Alps, that praiseth God winter and summer, all the trees of the fields, obedient, bow together Lord, France and Provence have sinned only through forgetf ulness offences, for ; do thou forgive us our we repent of the evil of former days. Lord, we desire to become men, thou canst set us free. We are Gallo-Romans, and of noble race, and we walk upright in our land. Lord, we are not the cause of the evil, send down upon us a ray of peace. Lord, help our cause, and we shall live again and love thee. THE PRESENT CAPOULIE OF THE FELIBRIGE. M. Pierre Devoluy, of the town of Die, was elected at Aries, in April, 1901. was presided over by Mistral. The Consistory BIBLIOGRAPHY The following list contains the most important works that have been published concerning Mistral and the F^librige. all Numerous articles have appeared in nearly Of the languages of Europe in various magazines. these only such are mentioned as seem worthy of special notice. WOKKS CONCERNING THE FfiLlBRIGE IN GENERAL America Janvier, Thomas A., Numerous articles in the Century Magazine, New York, 1893, and following years. An Embassy to Provence. New York, 1893. Preston, Harriett, Mistral's Calendau. The Atlantic Monthly, New York, 1874. AuhaneVs Mibugrano entreduberto. Monthly, New York, 1874. The Atlantic England Craig, Duncan, Miejour or Provencal Legend, Life, Language, and Literature. London. The Handbook of the Modern Provencal Language. Crombie, J. W., The Poets and Peoples of Foreign Lands: Frederic Mistral. Elliot, London, 1890. Hartog, Cecil, Poets of Provence. London Contemporary Review, 1894. 269 frI:d6ric mistral 260 France BoissiK, FiRMiN, Le Midi litteraire contemporain. Doular doure, Toulouse, 1887. De Bouchaud, Roumanille et le Felibrige. Mougin, Lyons, 1896. Brun, C, L' Evolution felibreenne. Paquet, Lyons, 1896. DoNN ADIEU, F., Les Precurseurs des Fe litres. Quantin, Paris, 1888. Hennion, C, Les Fleurs felibresques. Paris, 1893. JouRDANNE, G., Histoire du Felibrige. Roumanille, Avignon, 1897. LiNTiLHAC, E., Les Felibres a travers leur monde et leur Lemerre, Paris, 1895. poesie. Precis de la litterature franfaise. Paris, 1890. Legre, L., Le Poke Theodore Aubanel. Paris, 1894. Margox, a. de, Les Precurseurs des Felibres. Beziers, 1891. Marieton, Paul, La Terre provenfale. Lemerre, Paris, 1894. Article Felibrige in the Grande Encyclopedic. Article Mistral in the Grande Encyclopedie. Michel, S., La Petite Patrie. Roumanille, Avignon, Essai sur I'histoire litteraire 1894. NouLET, B., midi de lier, la France, au des patois XVIII* siecle. du Montpel- 1877. Paris, Gaston, Penseurs et poetes. Calmann-L^vy, Paris, 1896. Restori, Histoire de temps les pellier, plus 1895. la litterature provent^ale recule's jusqu'a depuis nos jours. les Mont- (Translated from the Italian.) BIBLIOGRAPHY 261 Roque-Ferrier, a., Melanges de critique litteraire et de philologie. Montpellier, 1892. Saint-Rene-Taillandier, v., Etudes litteraires. Plon et Cie, Paris, 1881. Tavernier, E., La Renaissance provenfale et Roumanille. Gervais, Paris, 1884. Le mouvement litteraire provengal et Lis Isclo d''Or de Frederic Mistral. Aix, 1876. De Terris, J., Roumanille et la litterature provenfale. Blond, Paris, 1894. De Vinac, M., Les Fe'libres. Richaud, Gap, 1882. Germany BOhmer, E., Die provenzalische Dichtung der Gegenwart. HeUbronn, 1870. KOSCHWITZ, E., Ueher die provenzalischen Feliber und ihre Vorganger. Berlin, 1894. Grammaire historique de la langue des Fe'libres. Greifswald and Paris, 1894. A study of Bertuch's translation of Nerto in the Litteraturhlatt fur germanische und romanische Philologie. 1892. A study of Proven9al phonetics with a translation of the Cant dau Souleu. Sonderahdruck aus der Sprache und Littera- ZeitscTirift fiir franzosische tur. Berlin, 1893. Schneider, B., Bernerkungen zur liiterarischen Beioegung auf neuprovenzalischem Sprachgebiete. Berlin, 1887. Welter, N., Frederi Mistral, der Dichter der Provence. Marburg, 1899. i 1 The present work was completed in manuscript before the reception of Welter's book. FRfeDfiRIC 262 MISTRAL Italy LiCBR, Maria, / Felibri, in the Roma letteraria. June, 1893. Portal, E., Appunti Sulla poesia provenzale. letterari: Pedone, Palermo, 1890. La Letteratura provenzale moderna. Reber, Palermo, 1893. Scritti vari di letteratura classica provenzale moderna. Reber, Palermo, 1895. Restori, a., Letteratura provenzale. Hoepli, Milan, 1892. ZuccARO, L., Un avvenimento letteraria; Mistral tragico, in the Scena illustrata. Florence, 1891. // Felibrigio, rinascimento delle lettere provenzali, Con- Novara, 1892. cordia. Spain TuBiNO, Historia del renacimiento literario contemporaneo en Calaluna, Baleares y Valencia. Madrid, 1881. MISTRAL'S WORKS Mirfeio. 1859. Calendau. Avignon, 1867. Lis Isclo d'Or. Nerto. Paris, Lemerre, 1887. 1876. Hachette, Paris, 1884. Lou Tresor ddu Felibrige. Aix, 1886. La Reino Jano. Lemerre, Paris, 1890. Lou Pouemo ddu Rose. Lemerre, Paris, 1897. TRANSLATIONS OF MISTRAL'S WORKS H. Grant, An English Version of F. Mistral's Mireio/rom London. the Original Provencal. BIBLIOGRAPHY 263 Harriett Preston, Mistral's Mireio. A Provencal Poem Translated. Roberts Bros., Boston, 1872. Second edition, 1891. A. Bertuch, Der Trommler von Arcole. tung, Dresden, 1890. Nerto. Triibner, Strassburg, 1890. Mireio. Triibner, Strassburg, 1892. Espouscado. Deutsche Dich- Zeitschrift f iir f ranzosische Sprache und Litteratur, XV^, p. 267. Hennion, Mireille. Traduction en vers fran9ais. E. RiGAUD, Mireille. Metrical translation into French, with the original form of stanza. Jaroslav Vrchlichky. Translation of several poems of Mistral into Bohemian, under the title, Mistralovych, in the Review, Kvety. Z bdsni Prague, 1886. Contains seven Hostem u Basniku. Prague, 1891. poems by Aubanel and thirteen by Mistral. DoM SiGiSMOND Bouska, Le Tambour d' Arcole, in the Review, Lumir. Prague, 1893. Cantos IV and V of Mireio, in the Review, Vla^U Prague, 1894. Pelay Boiz, Mireio, in Catalan. RocA Y RoCA, Calendau. Lo Gay Saber, Barcelona, 1868. C. Barallat y Falguera, Mireya, poema provenzal de Frederico Mistral puesto en prosa espanola. Maria Licer, L' Angela (Canto VI of Nerto). Italian. Iride, Casal, 1889. A. Naum, Traduceri. Jassy, 1891. (Translation into Rumanian of Canto IV of Mireio, The Song of Magali, and The Drummer of Arcole.) T. Caxnizzaro, La Venere d'Arli, in Vita Intima. 1891. Milan, INDEX Countess, the, 199. Aasen, Ivan, 94. Alexandrine verse, 78, 89. Cup, 31, 32, 190. Alpilles, 11. Amiradou, 76, 196. Dante, 40, 73, 130, 133, 160, Arfene, Paul, 21, 234. 248. Ariosto, 20, 151. Darmesteter, 41. Armana prouven^au, 17, 28. Aubanel, Theodore, 15, 17, Daudet, 21, 36, 88, 233. 9, 21, 69, 152, 240 seq. Dictionary of the Proven gal Aucassin and Nicolette, 170. language, 20, 92. Drac, 165 seq. Balagenr, Victor, 31, 32. Drummer of Arcole, 78, 204. Bello d'Avoust, 184. Berluc-F^russis, 33. Espouscado, 194. Boileau, 102. Evangeline, 122. Bonaparte- Wyse, 31, 33. Bornier, Henri de, 33. Faust, 248. Br^al, Michel, 34, 72. F^libre, 5, 27. Brunet, Jean, 16. F^librige, 24 seq. Brunetifere, 79, 249. F^librige de Paris, 16, 20, 33. Byron, 250. F^librige, foundation of, 15. Calendau, 18, 79, 127. Fin dou Meissouni6, 186. Floral games, 20, 32, 35. Font-S^gugne, 17. Four6s, Auguste, 37. F^librige organized, 19, 34. Capouli^, 19, 36, 36. Catalans, 31. Cigale. Soci^t^ de la, 20, 33. 266 FRiiDiiRIC 266 MISTRAL Mary, Queen of Scots, 213, Garcin, Eupfene, 16. Gi^ra, Paul, 15. 217. Mas, 11. Mathieu, Anselme, 13, 15, 21, Goethe, 123. Gounod, 18. Gras, F^lix, 36, 37, 38. 26. Gr6vy, 20. Meissoun, 14. Homer, 13, 123. Hugo, Victor, 79, Meyer, Paul, 33. Mila y Fontanals, 34. Mirabeau, 131, 243. 138, 181, 203. Mirfeio, 12, 17, 28, 79, 99. Mistral's marriage, 19. Mistral's Memoirs, 21. Isclo d'Or, 19, 181. Mont-Ventoux, 148. Janvier, Mrs. Thomas A., 38. Museum of Aries, 21. Musset, 181. Jasmin, 6, 14, 29, 43, 73, 193. Jeanroy, 27. Jourdanne, 24, 37. Napoleon, 164. Nerto, 20, 161. Noulet, 43. Koschwitz, 49. Paris, Gaston, 34, 69, 115. Lamartine, 17, 29, 103, 130, 181, 182, 183, 204. Landor, Walter Savage, 213, 214. Latin race, 30, 191, 193. Legouvfi, 20. Lemaitre, Jules, 232. Leopardi, 260. Lintilhac, Eugfene, 72. Littr^, 94. Longfellow, 6. Petrarch, 18, 19, 33, 34, 36, 73, 148, 220. Poem of the Rhone, 21, 76, 89, 169. Political separatism, 15. Prfego-Di6u, 84, 204, 206 seq., 239. Provencal language, 43, 191 seq. Psalm of Penitence, 84, 182, 200 seg., 239, 263. Maillane, 10, 12. Marot, 81. Queens of the F^Ubrige, 36. INDEX 267 R6ino Jano, 21, 89, 212. Tartarin, 69, 230, 240. Rock of Sisyphus, 193, 203. Tavan, Alphonse, 15. Ronsard, 211. Roumanille, 7, 9, 14, 15, 17, Translation, 87, 89, 178, 247. 21, 26, 30, 36, 70. Tresor d6u Felibrige, 20, 92. Troubadours, 40, 44, 87, 112, Saboly, 6. 132, 147, 226, 261. Sainte-Beuve, 6. Saint-R^my, 7, 10. Simon de Montfort, 37. Versification, 76. Songs, 189. Virgil, 13. Sonnets of Mistral, 86. Voltaire, 221. Villemain, 29. qasz^ University of California SOUTHERN REGIONAL LIBRARY FACILITY Return this material to the library from which it was borrowed. UC SOUTHERN REGIONAL LIBRARY FACILITY A 000 679 070 3
https://openalex.org/W2797009699	https://zenodo.org/records/2534646/files/RRP_article_25528.pdf	English	null	Correction of morphofunctional disturbances arising when modelling Preeclampsia with resveratrol and nicorandil	Research results in pharmacology	2,018	cc-by	9,491	Research Results in Pharmacology 4(1): 59–71 UDC: 618.3-06-08:577.112.385.2 DOI 10.3897/rrpharmacology.4.25528 Research Results in Pharmacology 4(1): 59–71 UDC: 618.3-06-08:577.112.385.2 DOI 10.3897/rrpharmacology.4.25528 Research Article Abstract Introduction: Preeclampsia is one of the most serious diseases of the second half of pregnancy and is surely amongst the top three causes of maternal mortality. Therefore, the creation of new drugs for preventing and correcting pre eclampsia is an urgent task. Methods: In the experiment, an ADMA-like L-NAME-induced model of preeclampsia was reproduced. To assess the emerging morphofunctional disorders, the following parameters were used: blood pressure, endothelial dysfunction coef ficient, microcirculation in the placenta, proteinuria, fluid content in the large omentum, concentration of terminal metab olites in the blood plasma, morphological state of the placenta and kidneys and morphometric parameters of the foetus. Results and Discussion: Injection of L-NAME into the animals from the 14th to the 20th day of pregnancy causes dis orders: an increase in systolic and diastolic blood pressure by 1.4 and 1.5 times, an increase in proteinuria by 3.3 times and an increase in the fluid content in a large omentum from 45.82 ± 1.82% to 54.73 ± 1.96%, which correspond to disorders due to preeclampsia in pregnant women. There was also a disturbance of endothelial function, as evidenced by an increase in the coefficient of endothelial dysfunction (CED) by 2.9 times. The use of resveratrol leads to a pro nounced correction in the changes that occur: a decrease in systolic and diastolic arterial pressure by 1.2 and 1.3 times, a decrease in proteinuria by a factor of 1.9 and a decrease in the fluid content in the large omentum to 50.00 ± 1.25%. The use of nicorandil leads to a pronounced correction in the resulting changes: a decrease in the diastolic blood pressure by 1.14 times, a decrease in proteinuria by a factor of 1.7 and a decrease in the fluid content in the large omentum to 50.57 ± 2.08%. CED decreased 1.7 times. When combining their use with amlodipine, the positive effects increased: systolic and diastolic blood pressure decreased 1.13 and 1.24 times and 1.14 and 1.23 times, respectively, proteinuria decreased 2.7 and 2.3 times, the fluid content in the large omentum was reduced to 44.54 ± 1.80% and 46.73 ± 1.30%. CED decreased 1.7 and 2.3 times. The administration of glibenclamide together with resveratrol and nicorandil removes a significant part of their positive effects. Conclusion: Resveratrol and nicorandil have a significant positive effect in the correction of morphofunctional disor ders in animals with ADMA-like preeclampsia. Correction of morphofunctional disturbances arising when modelling Preeclampsia with resveratrol and nicorandil Elena G. Stupakova1, Galina A. Lazareva1, Vladimir V. Gureev1 1 Kursk State Medical University, 3 K. Marx St., Kursk 305041 Russia Corresponding author: Elena G. Stupakova (miss.stupackova@yandex.ru) Academic editor: Mikhail Korokin ♦ Received 17 January 2018 ♦ Accepted 19 March 2018 ♦ Published 28 March 2018 Citation: Stupakova EG, Lazareva GA, Gureev VV (2018) Correction of morphofunctional disturbances arising when modelling Preeclampsia with resveratrol and nicorandil. Research Results in Pharmacology 4(1): 59–71. https://doi. org/10.3897/rrpharmacology.4.25528 Abstract Activation of K+ ATP channels plays a significant role in the realisation of their positive effects. Methods The experiment was performed on 250 white Wistar fema le rats weighing 250–300 g. To form the groups of preg nant animals with predetermined periods, the females (3 animals), which had been kept separately, were caged with males (2 animals) for 24 hours. Then the animals were se parated and, 10 days later, in the condition of etheric sleep, determined pregnancy by palpation. In our experiments, pregnancy occurred in 30–40%. An ADMA-like agent, a non-selective NO-synthase blocker, N-nitro-L-arginine methyl ester (L-NAME), was administered intraperito neally at a dose of 25 mg/kg/day for seven days (14-20 days of gestation). According to literature, this moderately prolonged average dose regimen of L-NAME administra tion results in the blockade of NO-synthase in the endot helium of the placental vessels, which leads to destructive changes in placental tissues, arterial hypertension and pro teinuria (Gureev et al. 2014, Gureev 2012, Tsukimori et al. 2008). On the 21st day of pregnancy, under anaesthesia (chloral hydrate 300 mg/kg), a catheter was inserted into the left carotid artery to record haemodynamic parameters and bolus administration of pharmacological agents was performed in the right femoral vein. The haemodynamic parameters: systolic blood pressure (SBP), diastolic arteri al pressure (DBP) and heart rate (HR) were measured con tinuously by means of a sensor and a hardware complex for invasive measurement of haemodynamic parameters With regard to the pathogenesis of preeclampsia, although a large number of uncertainties remain, its main conditions are determined and all the newly discovered information complements pathological events. It is generally accepted that the obligatory component of the pathogenesis of pree clampsia is endothelial dysfunction (Bloshhinskaja 2003, Gureev 2012, Scioscia et al. 2015, Sánchez-Aranguren et al. 2014). Endothelium, or internal cellular lining of vessels, provides selective permeability between intravascular and interstitial space, which is ensured by its specific structure. A histological study of the placenta in females with preeclampsia revealed incomplete invasion of the cytotrop hoblast into the spiral arteries of the mother. The level of maturity of the spiral arteries themselves in cases of pree clampsia does not reach the level of normal pregnancy (Pe reira et al. 2015, Verlohren et al. 2010, Ducray et al. 2011). This leads to ischemia of the trophoblast and an increase in the permeability of the foetoplacental barrier (Ducray et al. 2011, van Oppenraaij et al. 2011, Wang et al. 2015). Keywords Keywords Keywords resveratrol, nicorandil, preeclampsia, pharmacological preconditioning, K+ ATP channels, rats, ADMA akova EG et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permi se, distribution, and reproduction in any medium, provided the original author and source are credited. Stupakova EG et al.: Correction of morphofunctional disturbances arising when modelling... 60 and oxidative stress (Sidorova 2003, Bloshhinskaja 2003, Sánchez-Aranguren et al. 2014, Bloshchinskaja 2003). The consequence of the described events is a decrease in the formation of nitric oxide, which has not only a vasodi lating effect, but also suppresses apoptosis in the placenta and plays a significant role in the realisation of the effects of many growth factors. Thus, the vicious circle closes: angiogenesis abnormality provokes placenta ischemia and placental ischemia aggravates angiogenesis abnormality. Introduction Preeclampsia is a multisystem, highly variable and preg nancy-specific condition that occurs after the 20th week of gestation and is determined by the presence of arterial hy pertension and proteinuria. For many years, preeclampsia has been one of the frequent and threatening complicati ons of pregnancy and childbirth, leading to disability of mothers and their children (Gureev et al. 2014, Kolgush kina 2000, Reznikova 2013, Shuvalov et al. 2014, Save lyeva et al. 2011, Federal State Statistics Service). The described spectrum of pathogenetic mechanisms of pathogenesis, which is far from being complete, shows that this disease is associated with the emergence of se veral vicious circles, the integral components of which are endothelial dysfunction and ischemia of the placenta. According to the Ministry of Healthcare of the Russi an Federation, over the past decade, hypertensive com plications in pregnancy have been ranked third in the list of causes of maternal mortality and, in 2014, accounted for 15.7% of the structure of maternal losses, in 2015 – 10.2% and by the end of 2016, this figure was 11.7% (Shuvalov et al. 2014, Methodical letter 2016). Earlier in our laboratory, the endoteleoprotective properties of resveratrol and nicorandil (Danilenko 2013, Kochkarov 2009, Kochkarov et al. 2006, Kochkarov 2008) were studied which could activate K+ATP channels (Tanaka et al. 2010, Novaković et al. 2015); therefore we considered them to be the most promising for our studies. According to the statistics, the frequency of pree clampsia in pregnant women has increased in recent years and ranges from 7% to 20% (Kolgushkina 2000, Reznik ova 2013, Shuvalov et al. 2014, Federal State Statistics Service, Methodical letter 2016). Objective Perinatal morbidity is 56% and perinatal mortality is 3-4 times higher than that of the population, reaching 12% (Savelyeva et al., 2011). To experimentally substantiate the prospects of using drugs which activate K+ ATP channels: resveratrol and ni corandil in correcting morphofunctional disorders of ex perimental preeclampsia. The etiological aspects of preeclampsia development still provoke heated discussions in the scientific commu nity. There are several theories of preeclampsia develop ment: the immunity theory, the theory of disadaptation, the theory of placental ischemia, the hormonal theory, the theory of toxic effects, the hereditary theory, etc. (Shu valov et al. 2014, Savelyeva et al. 2011, Sidorova 2003). Methods Determination of the amount of protein in daily urine was carried out using the pyrogallol red method on a spectrophotometer PE-5400B. 8. Modelling ADMA-like preeclampsia + Nicorandil (2x10 mg/kg/day orally) + Glibenclamide (50 mg/kg). 9. Modelling ADMA-like preeclampsia + Resveratrol (2 mg/kg/day orally) + Glibenclamide (50 mg/kg). 9. Modelling ADMA-like preeclampsia + Resveratrol (2 mg/kg/day orally) + Glibenclamide (50 mg/kg). To study the liquid content in the large omentum, it was weighed, followed by drying at 37°C for 24 hours and re-weighing (Reznikova 2013, Ivanova et al. 2012).i For the morphological confirmation of the development of the simulated pathological processes and for the com plex evaluation of the efficacy of the preparations, a his tological examination (in all series of the experiment) of kidneys and placenta was carried out. The material is fixed in 10% formalin, followed by pouring into paraffin. Sec tions of the kidneys were taken perpendicular to the main axis of the organ through the pelvis. Histological sections of the placenta were made in a strictly vertical direction through the middle of the placental disc with the capture of all layers of the placenta and the walls of the uterine horn. Microslide studies, photorecording and morphometry were performed on a Leica DM4000B microscope with a video registration and imaging system. For all morphological stu dies, staining with haematoxylin and eosin was used. Methods The response to ischemia is the formation of a large number of vasoactive humoral factors; besides, foetal an tigens entering the bloodstream of the mother are likely to cause the accumulation of methylated analogues of L-argi nine (ADMA, MMA) in the plasma. The latter causes dys function of the endothelium, secondary ischemic lesions Research Results in Pharmacology 4(1): 59–71 61 of Biopac (USA) and by the computer programme Asq Knowledge 3.8.1 (Gureev et al. 2014, Gureev 2012). ranking was assessed using the Mann-Whitney analysis of nonparametric data (Glanz 1999, Sydorenko 2003). All the calculations were performed using the Microsoft Ex cel 7.0 statistical software package. The degree of endothelial dysfunction in the experi mental animals was assessed by the ratio of indices of endothelium-dependent vasodilation and endothelium-in dependent vasodilation with subsequent calculation of the coefficient of endothelial dysfunction (CED) (Korokin et al. 2011, Pokrovsky et al. 2006). According to the objective, all the animals were distri buted amongst the following groups: 1. Control group (animals with oral administration of NaCl in equivalent doses from 14th to 20th days of pregnancy). The level of NO metabolites (the total concentration of nitrates and nitrites, NOx) was determined by the colori metric method for the development of colour in the diazo tisation reaction by sulphonamide nitrite, which is part of the reagent (Metelskaya et al. 2004, Oganov et al. 2004). 2. Modelling ADMA-like preeclampsia (animals with in traperitoneal administration of L-NAME at a dose of 25 mg/kg once daily from 14th to 20th days of gestation). 3. Modelling ADMA-like preeclampsia + Amlodipine (0.5 mg/kg/day orally). Measurement of microcirculation in the placenta was carried out with the help of Biopacsystems equipment: MP100 polygraph with LDF100C laser doppler flow metry module (LDF100C) and invasive needle probe TSD144 [1, 25], which was mounted directly on to the placental disc projection. LDF was recorded and proces sed using the AcqKnowledge version 3.8.1. The microcir culation values were expressed in perfusion units (PEU). 4. Modelling ADMA-like preeclampsia + Nicorandil (2x10 mg/kg/day orally). 5. Modelling ADMA-like preeclampsia + Resveratrol (2 mg/kg/day orally). 6. Modelling ADMA-like preeclampsia + Nicorandil (2x10 mg/kg/day orally) + Amlodipine (0.5 mg/kg/ day orally). 7. Modelling ADMA-like preeclampsia + Resveratrol (2 mg/kg/day orally) + Amlodipine (0.5 mg/kg/day orally). Collection of urine was carried out within 12 hours with the use of special metabolic cells. Results and discussion: Effect of resveratrol and nicorandil on morphofunctional disorders in ADMA-like preeclampsia. The administration of resveratrol (2 mg/kg/day) to ani mals with experimental preeclampsia resulted in a statis tically significant (p <0.05) decrease in systolic and dia stolic blood pressure from 181.3 ± 4.58 and 144.7 ± 4.09 mm Hg to 154.6 ± 7.53 and 110.90 ± 8.71 mm Hg, respec tively, but the level of the intact animals was not reached (133.8 ± 2.45 and 95.36 ± 2.82 mm Hg, respectively). The administration of nicorandil (2x10 mg/kg/day) resulted in a statistically significant (p <0.05) decrease in only di astolic blood pressure, blood pressure reaching 174.1 ± 6.14 and 126.9 ± 6.31 mm Hg, respectively. For subsequent statistical processing, the degree of morphological changes was ranked. The foetuses were re moved from the uterine cavity and weighed; their growth (craniocaudal size) was measured, followed by calcula ting the height-weight coefficient (Mironov 2012). The ratio of endothelium-dependent vasorelaxation and endothelium-independent vasorelaxation, expressed by CED, was not influenced by resveratrol. Nicorandil statistically significantly reduced CED from 3.36 ± 0.23 to 1.96 ± 0.13 (p <0.05), but not to the level of the intact animals (1.17 ± 0.10). For all the data, descriptive statistics were applied: the data were checked for the normality of the distribution. The type of distribution was determined by the Shapi ro-Wilk criterion. In the case of a normal distribution, the mean value (M) and the standard error of the mean (m) were calculated. The intergroup differences were analy sed by means of parametric (Student’s t-test) or nonpa rametric (Mann-Whitney test) methods, depending on the type of distribution. The statistical significance of the differences between the morphological changes after their In the animals with ADMA-like preeclampsia, when res veratrol and nicorandil were administered, an increase in the microcirculation in the placenta was observed from 213.7 ± 14.97 PUn to 452.4 ± 27.16 PUn and 377.9 ± 18.8 PUn, res pectively (p<0 , 05), which did not differ statistically from the level of the intact animals (409.9 ± 30.57 PUn). Stupakova EG et al.: Correction of morphofunctional disturbances arising when modelling... 62 In the animals with ADMA-like preeclampsia, a statis tically significant (p<0.05) decrease in the concentration of final metabolites of NO in blood plasma was observed in comparison with the intact animals from 2.22 ± 0.08 μmol/dl to 1.27 ± 0.04 μmol/dl. Results and discussion: Effect of resveratrol and nicorandil on morphofunctional disorders in ADMA-like preeclampsia. Using resveratrol and ni corandil in the animals with ADMA-like preeclampsia, their statistically significant increase was observed to 1.78 ± 0.05μmol/dl and 1.80 ± 0.07 μmol/dl, respectively. tration of resveratrol and nicorandil resulted in a decrease in the level of swelling in the tissues of the large omen tum to a level statistically indistinguishable in relation to the intact pregnant animals – 50.00 ± 1.25% and 50.57 ± 2.08%, respectively. A morphological examination of the renal parenchyma in the animals with ADMA-like preeclampsia revealed: anaemia and spasm of renal vessels, thickening of their walls, thickening of the basement membrane of the glo meruli capillaries in the form of a wire loop (Fig. 1(B)). No noticeable reaction of mesangium was observed. Thus, the average number of cells per glomerulus in the group with experimental preeclampsia was 39.60 ± 1.88 (in the normal condition 40.60 ± 2.39). The administration of an ADMA-like agent to the pregnant animals from the 14th to 20th days of gestation resulted in an increase in proteinuria from 0.69 ± 0.70 g/l to 2.30 ± 0.14 g/l (p<0.05). The administration of res veratrol and nicorandil to the animals with experimental preeclampsia resulted in a statistically significant decre ase in the urinary protein concentration to 1.22 ± 0.11 g/l and 1.39 ± 0.10 g/l, respectively (p<0.05).l In the placenta of the animals with ADMA-like pree clampsia, massive deposition of fibrin and significant changes in ischemic genesis were observed (Fig. 2(B)). A study of the fluid content in the large omentum of the animals with ADMA-like preeclampsia revealed its incre ase in comparison with the intact pregnant animals from 45.82 ± 1.82% to 54.73 ± 1.96% (p<0.05). The adminis The integral index of analysis of the morphological study in animals with ADMA-like preeclampsia was 5-6 points (p <0.05). B Figure 1. Histological picture of the kidney in the modelling of ADMA-like preeclampsia. Note: (A) – the glomerule of an intact animal. (B) the glomerule of an animal with ADMA-like preeclampsia. ×200. A B A B A Figure 1. Histological picture of the kidney in the modelling of ADMA-like preeclampsia. Note: (A) – the glomerule of an intact animal. (B) the glomerule of an animal with ADMA-like preeclampsia. ×200. B Figure 2. Histological picture of the placenta in the modelling of ADMA-like preeclampsia. Note: (A) type of intact placenta. (B) ADMA-like preeclampsia. Results and discussion: Effect of resveratrol and nicorandil on morphofunctional disorders in ADMA-like preeclampsia. Vacuolar degeneration of giant cell trophoblast; dystrophic changes in the decidual layer, foci of necrosis at the border of the giant cell trophoblast and decidual tissue. ×200. A B A B A Figure 2. Histological picture of the placenta in the modelling of ADMA-like preeclampsia. Note: (A) type of intact placenta. (B) ADMA-like preeclampsia. Vacuolar degeneration of giant cell trophoblast; dystrophic changes in the decidual layer, foci of necrosis at the border of the giant cell trophoblast and decidual tissue. ×200. 63 Research Results in Pharmacology 4(1): 59–71 Effect of resveratrol and nicorandil when combined with amlodipine on morphofunctional disorders in ADMA-like preeclampsia The administration of resveratrol and nicorandil to the animals with ADMA-like preeclampsia led to significant positive morphological changes in the kidneys and placen ta. In the kidneys, less significant changes were recorded (Fig. 3(A)). There was no noticeable mesangium reaction (42.20 ± 1.91 cells when administering resveratrol and 39.40 ± 1.53 cells when administering nicorandil). In the placenta, moderately significant fibrous deposits, less sig nificant dystrophic changes in the giant cell trophoblast and decidual layer and no necrosis foci were observed. The integral index of the analysis of the morphological study in the animals with the correction of ADMA-like preeclampsia by resveratrol and nicorandil was 3 points (p <0.05). The administration of nicorandil in combination with am lodipine to the animals with experimental ADMA-like preeclampsia resulted in a statistically significant (p <0.05) decrease in systolic and diastolic blood pressure (Table 2). It should be noted that in monotherapy, statistically neither amlodipine nor nicorandil significantly reduced systolic pressure. Therefore, a decrease in systolic pressure with their combined use can be regarded as a potentiation of their action. Addition of amlodipine to resveratrol did not lead to an increase in its hypotensive effect. The administration of resveratrol and nicorandil in com bination with amlodipine to the animals with ADMA-like preeclampsia led to a more significant decrease in CED, compared to the groups of animals where these pharmaco logical agents were used as monotherapy (Table 2). The study of the features of foetal development in the animals with ADMA-like preeclampsia revealed a mod erately significant foetal hypotrophy, which manifested itself in a decrease in mass and length (growth). It should be noted that the mass of the foetuses was most affect ed. With the administration of resveratrol, only the mass of the foetus decreased. Results and discussion: Effect of resveratrol and nicorandil on morphofunctional disorders in ADMA-like preeclampsia. With the administration of nico randil, neither the weight nor height indices differed from those of intact foetuses (Table 1). The administration of resveratrol and nicorandil in combination with amlodipine to the animals with AD MA-like preeclampsia led to an increase in the microcir culation in the placenta (p <0.05) (Fig. 4(A)).i In the biochemical study of the final metabolite con centration of NO in the blood plasma, their statistically significant increase (p <0.05) was recorded (Fig. 4(B)). The most significant effect was observed when amlodipi ne was combined with nicorandil. In the animals of this Thus, the results of the series of experiments indicate a pronounced protective activity of resveratrol and nico randil in the correction of morphofunctional disorders occurring in the animals when modelling ADMA-like preeclampsia. A B Figure 3. Morphological picture of kidneys and placenta in animals with correction of ADMA-like preeclampsia by resveratrol. Note: (A) kidney; (B) placenta. ×200. B A B A Figure 3. Morphological picture of kidneys and placenta in animals with correction of ADMA-like preeclampsia by resveratrol. Note: (A) kidney; (B) placenta. ×200. of Resveratrol and Nicorandil on the Height-weight Indices of Foetal Development in ADMA-like Preeclampsia (M Table 1. Effect of Resveratrol and Nicorandil on the Height-weight Indices of Foetal Development in ADMA-like Preeclampsia (M ± m; n = 10). Group Index Pregnant intact Pregnant + L-NAME Pregnant + L-NAME + Resveratrol Pregnant + L-NAME + Nicorandil 1.68±0.09y 1.38±0.07* 1.45±0.06* 1.53±0.06 Foetus weight, g 23.79±0.42y 21.82±0.34* 22.59±0.61* 22.91±0.61 Foetus height, mm 14.01±0.28y 15.81±0.43* 15.58±0.35* 14.97±0.43 Notes: * - p<0.05 in comparison with intact pregnant females; y - р < 0.05 in comparison with L-NAME group. Table 1. Effect of Resveratrol and Nicorandil on the Height-weight Indices of Foetal Development in ADMA-like Pree ± m; n = 10). Stupakova EG et al.: Correction of morphofunctional disturbances arising when modelling... 64 group, the microcirculation rate in the placenta was at the level of intact animals. omentum to a level statistically distinct when compared with “untreated” animals (Figure 4(D)). The administration of resveratrol and nicorandil in combination with amlodipine to the animals with ex perimental preeclampsia led to a statistically signifi cant decrease in urinary protein concentrations (p<0.05) (Fig. 4(C)). The combined use of these drugs in the animals with ADMA-like preeclampsia led to significant positive mor phological changes in the kidneys and placenta. In the kidneys, there was a moderate vasospasm. Results and discussion: Effect of resveratrol and nicorandil on morphofunctional disorders in ADMA-like preeclampsia. No symptoms of the wire loop were observed (Fig. 5). There was no noticeable mesangium reaction (38.10 ± 2.23 cells and 40.50 ± 2.33 cells, respectively) (in the normal condition 40.60 ± 2.39 cells). The combined administration of amlodipine with resveratrol or nicorandil to the animals with ADMA-like preeclampsia resulted in reduced swelling of the large Table 2. Results of Correction of ADMA-like Preeclampsia with Resveratrol and Nicorandil in Combination with Amlodipine (M ± m; N = 10). Group SBP mm Hg DBP mm Hg CED relative units Intact 133.8±2.45y 95.36±2.82 y 1.17±0.10 y L-NAME 181.3±4.58* 144.7±4.09* 3.36±0.23* L-NAME + Amlodipine 167.0±7.67* 122.9±7.49y 2.24±0.19y L-NAME +Resveratrol 154.6±7.53 y 110.90±8.71y 3.36±0.41* L-NAME +Nichorondil 174.1±6.14* 126.9±6.31y 1.96±0.13y L-NAME +Resveratrol+Amlodipine 159.9±4.36y 116.5±4.41y 2.02±0.19y L-NAME +Nicorondil+Amlodipine 158.5±3.84y 117.2±3.70y 1.49±0.15y Notes: SBP, DBP - systolic and diastolic blood pressure (mmHg); CED - coefficient of endothelial dysfunction (r.u.); * - p <0.05 in comparison with the group of intact animals; y - р<0.05 in comparison with the L-NAME group. orrection of ADMA-like Preeclampsia with Resveratrol and Nicorandil in Combination with Amlodipine (M orrection of ADMA-like Preeclampsia with Resveratrol and Nicorandil in Combination with Amlodipine (M Table 2. Results of Correction of ADMA-like Preeclampsia with Resveratrol and Nicorandil in Combination with ± m; N = 10). Notes: SBP, DBP - systolic and diastolic blood pressure (mmHg); CED - coefficient of endothelial dysfunction (r.u.); * - p <0.05 in comparison with the group of intact animals; y - р<0.05 in comparison with the L-NAME group. Figure 4. Effect of resveratrol and nicorandil in combination with amlodipine on microcirculation in the placenta, plasma NOx con tent, proteinuria and swelling of the large omentum with ADMA-like preeclampsia. Notes: * - р<0.05 in comparison with the group of intact animals; y - р<0.05 in comparison with the L-NAME group. D C A B A B B A D C C D Figure 4. Effect of resveratrol and nicorandil in combination with amlodipine on microcirculation in the placenta, plasma NOx con tent, proteinuria and swelling of the large omentum with ADMA-like preeclampsia. Notes: * - р<0.05 in comparison with the group of intact animals; y - р<0.05 in comparison with the L-NAME group. 65 Research Results in Pharmacology 4(1): 59–71 In the placenta, weakly expressed fibrous deposits were observed; dystrophic changes of the giant cell trophoblast and decidual layer were not significantly expressed; there were no necrosis foci (Fig. 5). Results and discussion: Effect of resveratrol and nicorandil on morphofunctional disorders in ADMA-like preeclampsia. The integral index of the ana lysis of the morphological study in the animals with the cor rection of ADMA-like preeclampsia by resveratrol and ni corandil in combination with amlodipine is given in Table 3. when combined with amlodipine to correct morphofunc tional disorders occurring in the animals when modelling ADMA-like preeclampsia. It should be noted that the combinations used have a more significant correction of morphofunctional disorders in comparison with the drugs used in monotherapy. A study of the features of foetal development in the animals with ADMA-like preeclampsia revealed no foetal hypotrophy when administering resveratrol and nicoran dil in combination with amlodipine (Table 4). Role of K+ ATP channels in the positive effects of res veratrol and nicorandil in ADMA-like preeclampsia. In a biochemical study of the concentration of terminal metabolites of NO in the blood plasma, a statistically sig nificant decrease in glibenclamide when using resveratrol and nicorandil to correct morphofunctional disorders in the animals with ADMA-like preeclampsia was revealed (Fig. 6(B)). Nevertheless, this indicator was at a level sta tistically different from that in the group of the “untrea ted” animals (p <0.05). Thus, the results of the series of experiments conduc ted indicate a significant decrease in the positive effects of resveratrol and nicorandil by glibenclamide. Since glibenclamide is a blocker of K+ ATP channels, the series of experiments makes it possible to assert the significant role of K+ ATP in revealing protective effects of resver atrol and nicorandil when correcting morphofunctional disorders in ADMA-like preeclampsia. Incomplete can cellation of their positive effects is due to the fact that Adding glibenclamide to resveratrol and nicorandil caused an increase in proteinuria compared to the groups of the animals in which these pharmacological agents were used in the monotherapy (Fig. 6(C)) (р<0.05). Table 5. Results of Correction of ADMA-like Preeclampsia with Resveratrol and Nicorandil Affected by Glibenclamide (M ± m; n = 10). Group SBP mm Hg DBP mm Hg CED relative units Intact 133.8±2.45y 95.36±2.82 y 1.17±0.10 y L-NAME 181.3±4.58* 144.7±4.09* 3.36±0.23* L-NAME + Resveratrol 154.6±7.53y* 110.90±8.71y 3.36±0.41* L-NAME + Nicorandil 174.1±6.14* 126.9±6.31y 1.96±0.13y L-NAME + Resveratrol + Glibenclamide 158.1±5.16y 120.8±3.16y 2.95±0.22* L-NAME + Nicorandil + Glibenclamide 186.0±7.46* 134.5±8.92* 2.55±0.0.17y* Notes: SBP, DBP – systolic and diastolic blood pressure (mmHg); CED – coefficient of endothelial dysfunction (r.u.); * - p<0.05 in comparison with the group of intact animals; y - р<0.05 in comparison with the L-NAME group. Table 6. Effect of Resveratrol and Nicorandil Affected by Glibenclamide on the Comprehensive Assessment of Pathomorphological Changes in Kidneys and Placenta in ADMA-like Preeclampsia (M ± m; n = 10). Series of experiments Comprehensive assessment in points Pregnant intact 0–1y Pregnant + L-NAME 5–6* Pregnant + L-NAME + Resveratrol 3y Pregnant + L-NAME + Nicorandil 3y Pregnant + L-NAME + Resveratrol + Glibenclamide 4y Pregnant + L-NAME + Nicorandil + Glibenclamide 4–5y Notes: * - p<0.05 in comparison with pregnant intact females; y- р<0.05 in comparison with L-NAME group. Table 5. Results of Correction of ADMA-like Preeclampsia with Resveratrol and Nicorandil Affected by Glibenclamide (M ± m; n = 10). Role of K+ ATP channels in the positive effects of res veratrol and nicorandil in ADMA-like preeclampsia. The administration of resveratrol and nicorandil in com bination with an inhibitor of K+ ATP channels to the ani mals with experimental ADMA-like preeclampsia led to Thus, the results of a series of experiments indicate a significant protective activity of resveratrol and nicorandil A B Figure 5. Morphological picture of kidneys and placenta in animals with correction of ADMA-like preeclampsia by nicorandil in combination with amlodipine. Note: (A) kidney; (B) placenta. ×200. B A B A Figure 5. Morphological picture of kidneys and placenta in animals with correction of ADMA-like preeclampsia by nicorandil in combination with amlodipine. Note: (A) kidney; (B) placenta. ×200. Table 3. Effect of Resveratrol and Nicorandil in Combination with Amlodipine on Comprehensive Assessment of Pathomorpholog ical Changes in Kidneys and Placenta in ADMA-like Preeclampsia (M ± m; n = 10). le 3. Effect of Resveratrol and Nicorandil in Combination with Amlodipine on Comprehensive Assessment of Pathomo Changes in Kidneys and Placenta in ADMA-like Preeclampsia (M ± m; n = 10). Series of experiments Comprehensive assessment in points Pregnant intact 0–1y Pregnant + L-NAME 5–6* Pregnant + L-NAME + Amlodipine 4y Pregnant + L-NAME + Resveratrol 3y Pregnant + L-NAME + Nicorandil 3y Pregnant + L-NAME + Resveratrol+ Amlodipine 2y Pregnant + L-NAME + Nicorandil+ Amlodipine 1-2y Notes: * - p<0.05 in comparison with intact pregnant females; y- р<0.05 in comparison with L-NAME group. Table 4. Effect of Resveratrol and Nicorandil in Combination with Amlodipine on Height-weight Indicators of Foetal Development in ADMA-like Preeclampsia (M ± m; n = 10). of Resveratrol and Nicorandil in Combination with Amlodipine on Height-weight Indicators of Foetal Development Preeclampsia (M ± m; n = 10). f p g g p in ADMA-like Preeclampsia (M ± m; n = 10). Group Indicator Foetus weight, g Foetus height, mm Height / weight, mm/g Pregnant intact 1.68±0.09y 23.79±0.42y 14.01±0.28y Pregnant + L-NAME 1.38±0.07* 21.82±0.34* 15.81±0.43* Pregnant + L-NAME + Amlodipine 1.40±0.09* 22.09±0.40* 15.78±0.51* Pregnant + L-NAME + Resveratrol 1.45±0.06* 22.59±0.61 15.58±0.35* Pregnant + L-NAME + Nicorandil 1.53±0.06 22.91±0.61 14.97±0.43 Pregnant + L-NAME + Resveratrol+ Amlodipine 1.60±0.07y 23.19±0.53 14.49±0.48 Pregnant + L-NAME + Nicorandil+ Amlodipine 1.59±0.05y 22.92±0.53 14.42±0.53 Notes: * - p<0.05 in comparison with intact pregnant females; y - р<0.05 in comparison with L-NAME group. Stupakova EG et al.: Correction of morphofunctional disturbances arising when modelling... Role of K+ ATP channels in the positive effects of res veratrol and nicorandil in ADMA-like preeclampsia. 66 The administration of resveratrol and nicorandil in combination with glibenclamide led to an increased swelling of the tissues of the large omentum to a level statistically indistinguishable when compared to that of the “untreated” animals (Fig. 6(D)). a decrease in the intensity of their positive effects. Thus, an increase in systolic and diastolic arterial pressure was observed in comparison with the groups of the animals in which resveratrol and nicorandil were used in monothera py (Table 5). It should be noted that the diastolic pressure in the group of the animals with combined administration of nicorandil and glibenclamide did not statistically differ from that in the group of the “untreated” animals. The administration of resveratrol and nicorandil in combination with glibenclamide in the animals with AD MA-like preeclampsia aggravated the morphological pat tern in the kidneys and placenta. In the kidneys, there was a pronounced spasm of the vessels. There were symptoms of a wire loop. There was no noticeable mesangium reac tion (40.20 ± 1.68 cells and 38.60 ± 1.63 cells, respective ly) (in the normal condition 40.60 ± 2.39 cells).i The administration of nicorandil in combination with glibenclamide to the animals with ADMA-like pree clampsia led to an increase in CED, compared to the groups of the animals where nicorandil was used as a monotherapy (Table 5), but this coefficient continued to differ statistically significantly from that in the group of the “untreated” animals (p <0.05). In the placenta, there were observed fibrous deposits; the dystrophic changes of the giant cell trophoblast and the decidual layer were not significantly expressed; there were isolated foci of necrosis. The integral index of the analysis of the morphological study in the animals with correction of ADMA-like preeclampsia by resveratrol and nicorandil affected by glibenclamide is given in Table 6. Adding glibenclamide to nicorandil and resveratrol when correcting ADMA-like preeclampsia led to redu ced microcirculation in the placenta to a level statistically distinct from that in the animals with correction by these drugs in the monotherapy (Fig. 6(A)) (p <0.05), while ad ding resveratrol almost completely removed its positive effect on microcirculation. Investigation of peculiarities of foetal development in the animals with ADMA-like preeclampsia revealed foe tal hypotrophy when administering resveratrol and nico randil in combination with glibenclamide (Table 7). The mass of the foetus was most affected. Role of K+ ATP channels in the positive effects of res veratrol and nicorandil in ADMA-like preeclampsia. Group SBP mm Hg DBP mm Hg CED relative units Intact 133.8±2.45y 95.36±2.82 y 1.17±0.10 y L-NAME 181.3±4.58* 144.7±4.09* 3.36±0.23* L-NAME + Resveratrol 154.6±7.53y* 110.90±8.71y 3.36±0.41* L-NAME + Nicorandil 174.1±6.14* 126.9±6.31y 1.96±0.13y L-NAME + Resveratrol + Glibenclamide 158.1±5.16y 120.8±3.16y 2.95±0.22* L-NAME + Nicorandil + Glibenclamide 186.0±7.46* 134.5±8.92* 2.55±0.0.17y* Notes: SBP, DBP – systolic and diastolic blood pressure (mmHg); CED – coefficient of endothelial dysfunction (r.u.); * - p<0.05 in comparison with the group of intact animals; y - р<0.05 in comparison with the L-NAME group. tion of ADMA-like Preeclampsia with Resveratrol and Nicorandil Affected by Glibenclamide (M ± m; n = 10). – systolic and diastolic blood pressure (mmHg); CED – coefficient of endothelial dysfunction (r.u.); * - p<0.05 in the group of intact animals; y - р<0.05 in comparison with the L-NAME group. Resveratrol and Nicorandil Affected by Glibenclamide on the Comprehensive Assessment of Pathomorphological eys and Placenta in ADMA-like Preeclampsia (M ± m; n = 10). Changes in Kidneys and Placenta in ADMA-like Preeclampsia (M ± m; n = 10). Series of experiments Comprehensive assessment in points Pregnant intact 0–1y Pregnant + L-NAME 5–6* Pregnant + L-NAME + Resveratrol 3y Pregnant + L-NAME + Nicorandil 3y Pregnant + L-NAME + Resveratrol + Glibenclamide 4y Pregnant + L-NAME + Nicorandil + Glibenclamide 4–5y Notes: * - p<0.05 in comparison with pregnant intact females; y- р<0.05 in comparison with L-NAME group. 67 Research Results in Pharmacology 4(1): 59–71 concentration of terminal metabolites of NO in the blood plasma, increased proteinuria, an increased fluid content in the large omentum, disorders in the placenta of ische mic origin and foetal hypotrophy. The described symptom complex of morphofunctional changes quite accurately represents the clinical picture of syndromes arising during preeclampsia. Thus, ADMA-like preeclampsia fully mat ches the model chosen for our experiment. resveratrol and nicorandil have a pronounced biological activity in their implementation in which K+ ATP channels do not participate. Conclusion 2014), plays an important role, since relaxation of the myometrium helps improve microcirculation in it.i Against the background of the significant complex protective effect of resveratrol on the development of morphofunctional disturbances arising when modelling ADMA-like preeclampsia, the lack of its positive effect on the coefficient of endothelial dysfunction was somewhat unexpected for the authors, especially as in the model of ADMA-like pathology in males, there was a significant de crease in CED (Kochkarov 2009). We believe that this fact can be explained by an increased gestogenic background in pregnant females, with which resveratrol, being a phy toestrogen, enters into some antagonistic relationships. Thus, administration of drugs with different mechanis ms of action to animals with ADMA-like preeclampsia leads to more significant protective effects compared to those in the groups in which these drugs were used in the monotherapy. It becomes clear that the use of new phar macological agents for treating preeclampsia is most ap propriate with the drugs within the standard therapy. The available information on the ability of resveratrol and nicorandil to activate K+ ATP channels logically pre determined the positive effects of their use in correcting morphofunctional disorders in ADMA-like preeclampsia. It is clear that the improvement of microcirculation due to a reduced tone of the myointrium and the preconditioning effect indirectly improves the endothelial function by re ducing placental ischemia. However, the drugs under stu dy have a whole spectrum of biological activities that can also bring about positive effects. Therefore, a further series of our experiments were aimed at elucidating the role of K+ ATP channels in the mechanism of protective effects of resveratrol and nicorandil in ADMA-like preeclampsia. The administration of nicorondil led to a statistically significant decrease in diastolic pressure and CED, an in crease in the microcirculation in the placenta, a decrease in the protein content in the urine and swelling of the large omentum and an increase in the NO-synthesising functi on of the endothelium. The morphological examination of the kidneys and placenta revealed moderately pronounced pathological changes of a functional nature.f The pronounced protective effects of nicorandil are linked to its ability to activate K+ ATP channels and to be a donor of nitric oxide (Tanaka et al. 2010, Gayet et al. 2011, Holzmann 1983). Activation of K+ ATP channels leads to hyperpolarisation of the membrane, reduction of Ca2+ current and relaxation of small arterial vessels. Conclusion The administration of a nonselective inhibitor of NOS - L-NAME resulted in an increased systolic and diastolic pressure, a disrupted relationship between endothelium-de pendent and endothelium-independent vasodilating reacti ons, a reduced microcirculation in the placenta, a decreased The administration of resveratrol led to a statistically significant decrease in systolic and diastolic arterial pres sure, an increase in microcirculation in the placenta, a de crease in the protein content in the urine, swelling of the Figure 6. Effect of resveratrol and nicorandil in combination with glibenclamide on microcirculation in the placenta, proteinuria, plasma NOx concentration and swelling of the large omentum with ADMA-like preeclampsia. Notes: * - р<0.05 in comparison with he group of intact animals; y- р<0.05 in comparison with L-NAME group. A D C B A B B A C D D C Figure 6. Effect of resveratrol and nicorandil in combination with glibenclamide on microcirculation in the placenta, proteinuria, plasma NOx concentration and swelling of the large omentum with ADMA-like preeclampsia. Notes: * - р<0.05 in comparison with the group of intact animals; y- р<0.05 in comparison with L-NAME group. ect of Resveratrol and Nicorandil in Combination with Glibenclamide on the Height-weight Foetal Development Ind DMA-like Preeclampsia (M ± m; n = 10). Table 7. Effect of Resveratrol and Nicorandil in Combination with Glibenclamide on the Height-weight Foetal Development Indi cators in ADMA-like Preeclampsia (M ± m; n = 10). Group Indicator Foetus weight, g Foetus height, mm Height / weight, mm/g Pregnant intact 1.68±0.09y 23.79±0.42y 14.01±0.28y Pregnant +L-NAME 1.38±0.07* 21.82±0.34* 15.81±0.43* Pregnant + L-NAME + Resveratrol 1.45±0.06* 22.59±0.61* 15.58±0.35* Pregnant +L-NAME + Nicorandil 1.53±0.06* 22.91±0.61* 14.97±0.43* Pregnant + L-NAME + Resveratrol + Glibenclamide 1.44±0.09* 22.05±0.67* 15.31±0.54* Pregnant + L-NAME + Nicorandil + Glibenclamide 1.47±0.07* 21.88±0.48* 14.88±0.61* Notes: * - p<0.05 in comparison with pregnant intact females; y- р<0.05 in comparison with L-NAME group. Stupakova EG et al.: Correction of morphofunctional disturbances arising when modelling... 68 large omentum and an increase in the NO-synthesising function of the endothelium. The morphological exami nation of the kidneys and placenta revealed moderately pronounced pathological changes of a functional nature. A certain role in the positive effects of nicorandil can be played by its having an anti-inflammatory activity (Li et al. 2015, Serizawa et al. 2014, Chao et al. 2016). Conclusion In addition, the ability of nicorandil to relax isolated strips of myomentry of the pregnant uterus, which is realised through K+ ATP channels, can improve microcirculation in the placenta (Matsui et al. 2015, Hong et al. 2016). large omentum and an increase in the NO-synthesising function of the endothelium. The morphological exami nation of the kidneys and placenta revealed moderately pronounced pathological changes of a functional nature. One of the mechanisms for preventing the develop ment of morphofunctional disorders in ADMA-like pree clampsia by resveratol is its pronounced endothelial-pro tective properties (Kochkarov 2009). Another positive aspect is an antisense activity in resveratrol (Camont et al. 2010, Camont et al. 2012). By reducing the number of free radicals, it thereby increases the bioavailability of the NO formed. An equally important endotheletoprotec tive mechanism is an ability of resveratrol to increase the activity of NO-synthase, promoting greater formation of NO and a subsequent increase in the level of cGMP that leads to vasodilation (Leikert et al. 2002, Wallerath et al. 2002). Under the conditions of oxidative stress, resver atrol restores the activity of DDAH, which is an enzyme metabolising ADMA (Frombaum et al. 2011). This is of great importance, since an increase in ADMA inhibits eNOS and contributes to an impaired endothelial function (Bivalacqua et al. 2001, White et al. 2006). Thus, the use of drugs possessing preconditioning properties for correcting morphofunctional disorders in ADMA-like preeclampsia leads to pronounced positive effects, which indicates the correctness of the chosen ap proach. The administration of the tested pharmacological agents in combination with a prolonged form of the an tagonist of the Ca2+-channels of the dihydropyridine se ries of amlodipine led to a more significant correction of morphofunctional disorders in ADMA-like preeclampsia. This can be explained by the fact that amlodipine has its own endothelium-protective activity, which is fulfilled through the mechanisms different from those in the tested drugs. There are published data on the improvement of endothelium-dependent vasodilation due to an increased level of NO (Parimala et al. 2006) and an increased level of endothelial NO synthase (Parimala et al. 2006, Koro kin et al. 2011, Xu et al. 2016). Besides, Ca2+ antagonists have an angioprotective effect due to a decrease in Ca2+ current through L-channels.f The ability of resveratrol to induce a dose-dependent relaxation in isolated strips of the pregnant uterus myo metrium, which is realised through K+ ATP channels (Rose et al. References Belousov YB (2012) The use of nicorandil in cardiovascular diseas es and the optimization of its designation. The Archives of Internal Medicine 1(3): 46–51. [In Russian] Gureev VV (2012) Endothelial dysfunction is the central link in the pathogenesis of gestosis. Research Bulletins of Belgorod State Uni versity. Medicine. Pharmacy 17/1(4(123)): 5–12. [In Russian] Gureev VV (2012) The role of iNOS in correcting endothelial dys function in ADMA-like gestosis by short episodes of ischemia-reper fusion in an experiment. Fundamental Research 8(2): 298–301. [In Russian] Gureev VV (2012) The role of iNOS in correcting endothelial dys function in ADMA-like gestosis by short episodes of ischemia-reper fusion in an experiment. Fundamental Research 8(2): 298–301. [In Russian] Bivalacqua TJ, Hellstrom WJ, Kadowitz PJ, Champion HC (2001) Increased expression of arginase II in human diabetic corpus cav ernosum: in diabetic-associated erectile dysfunction. Biochemi cal and Biophysical Research Communications 283(4): 923–927. https://doi.org/10.1006/bbrc.2001.4874 [Full text] [PubMed] Gureev VV, Pokrovsky MV, Korokin MV (2014) ADMA – eNOS – deterministic ways of pharmacological correction of preeclampsia. Belgorod Publishing House of Belgorod State National Research University, Belgorod, 260 pp. [In Russian] Bloshchinskaja IA (2003) The role of the basic vasoactive factors of the vascular endothelium in the development of gestosis. Russian Bulletin of Obstetrician-Gynecologist 4: 7–10. [In Russian] Bloshhinskaja IA (2003) Functional state of the vascular endothe lium and microcirculatory disturbances in pregnancy complicated by gestosis, and the impact of normobaric hypoxic therapy on them [dissertation]. Far Eastern State Medical University, Khabarovsk. Holzmann S (1983) Cyclic GMP as possible mediator of coronary arterial relaxation by nicorandil (SG-75). Journal of Cardiovascular Pharmacology 5(3): 364–370. https://doi.org/10.1097/00005344- 198305000-00004 [PubMed] Camont L, Collin F, Couturier M, Thérond P, Jore D, Gardès-Al bert M, Bonnefont-Rousselot D (2012) Radical-induced oxida tion of trans-resveratrol. Biochimie 94(3): 741–747. https://doi. org/10.1016/j.biochi.2011.11.005 [Full text] [PubMed] Hong SH, Kyeong KS, Kim CH, Kim YC, Choi W, Yoo RY, Kim HS, Park YJ, Ji IW, Jeong EH, Kim HS, Xu WX, Lee SJ (2016) Regulation of myometrial contraction by ATP-sensitive potassium (KATP) channel via activation of SUR2B and Kir 6.2 in mouse. The Journal of Veterinary Medical Science 78(7): 1153–1159. https://doi. References org/10.1292/jvms.15-0700 [Full text] [PubMed] Camont L, Collin F, Marchetti C, Jore D, Gardes-Albert M, Bon nefont-Rousselot D (2010) Liquid chromatographic/electrospray ionization mass spectrometric identification of the oxidation end-products of trans-resveratrol in aqueous solutions. Rapid Com munications in Mass Spectrometry 24(5): 634–642. https://doi. org/10.1002/rcm.4427 [PubMed] Ivanova LB, Karamysheva VI, Perfilova VN, Tjurenkov IN (2012) The effect of GABA-derivatives on endothelial function in rats with experimental preeclampsia. Problems of reproduction 1: 28–30. [In Russian] Chao HH, Hong HJ, Cheng TH, Shih NL, Loh SH, Liu JC, Chen JJ, Sung LC (2016) Nicorandil Inhibits Cyclic Strain-Induced In terleukin-8 Expression in Human Umbilical Vein Endothelial Cells. Pharmacology 98(1–2): 42–50. https://doi.org/10.1159/000445075 [Full text] [PubMed] Kochkarov VI (2008) Comparative research of endothelio- and car dioprotective effects of resveratrol and its combinations with enal april and losartan potassium in hypoestrogen-induced endothelial dysfunction. Kuban Scientific Medical Bulletin 5: 82–85. [In Rus sian] Danilenko LM (2013) Towards the implementation of distant isch emic preconditioning and pharmacological preconditioning by nicorandil in L-NAME induced deficiency of nitric oxide. Modern Problems of Science and Education 5: 20–21. [In Russian] Kochkarov VI (2009) Resveratrol and its combinations with the main antihypertensive drugs in pharmacological correction of endo thelial dysfunction [dissertation]. Kursk State Medical University, Smolensk. [In Russian] Ducray JF, Naicker T, Moodley J (2011) Pilot study of compara tive placental morphometry in pre-eclamptic and normotensive pregnancies suggests possible maladaptations of the fetal compo nent of the placenta. European Journal of Obstetrics & Gynecology and Reproductive Biology 156(1): 29–34. https://doi.org/10.1016/j. ejogrb.2010.12.038 [Full text] [PubMed] Kochkarov VI, Pokrovsky MV, Korneev MM, Pokrovskaya TG, Gladchenko MP, Artyushkova EB, Metelskaya VA, Tumanova NG, Faitelson AV, Dudka VT, Klyavs YP, Zelenkova TI, Gudyrev OS (2006) Endothelioprotective effects of resveratrol and its combina tions with enalapril and losartan in the experimental modeling of nitric oxide deficiency. Kuban Scientific Medical Bulletin 9(90): 150–152. [In Russian] Federal State Statistics Service (2018) Federal State Statistics Ser vice. http://www.gks.ru/wps/wcm/connect/rosstat_main/rosstat/ru/ statistics/population/healthcare/#/ [Electronic resource] Kolgushkina TN (2000) Gestosis: (etiopathogenesis, clinical picture, diagnosis, treatment): method. Recommendations. Publishing House of Minsk State Medical Institute, Minsk. [In Russian] Frombaum M, Therond P, Djelidi R, Beaudeux JL, Bonnefont-Rous selot D, Borderie D (2011) Piceatannol is more effective than resver atrol in restoring endothelial cell dimethylarginine dimethylamino hydrolase expression and activity after high-glucose oxidative stress. Conclusion Vasodilati on is also facilitated by the formation of NO by activating guanylate cyclase (Tanaka et al. 2010, Wei et al. 2016). In addition, the positive effects of nicorandil are enhanced by its having a preconditioning effect (Belousov 2012), endo thelium-protective properties (Malysheva et al. 2011) and its ability to reduce apoptosis (Staroseltseva 2012). To determine the role of K+ ATP channels in revealing the positive effects of the tested pharmacological agents, we used a blocker of these channels – glibenclamide. The administration of glibenclamide in combination with resveratrol and nicorandil significantly reduced the effectiveness of these drugs, but not completely. Thus, the results of the series of experiments make it possible to conclude that K+ ATP channels play a significant role in revealing the positive effects of resveratrol and nicorandil in correcting morphofunctional disorders in ADMA-like preeclampsia, but do not completely determine them. Research Results in Pharmacology 4(1): 59–71 69 References https://doi.org/10.1016/j.jri.2015.01.009 [Full text] [PubMed] Matsui TC, Coura GM, Melo IS, Batista CR, Augusto PS, Godin AM, Araújo DP, César IC, Ribeiro LS, Souza DG, Klein A, de Fáti ma Â, Machado RR, Coelho MM (2015) Nicorandil inhibits neu trophil recruitment in carrageenan-induced experimental pleurisy in mice. European Journal of Pharmacology 769: 306–312. https://doi. org/10.1016/j.ejphar.2015.11.034 [Full text] [PubMed] Serizawa K, Yogo K, Tashiro Y, Aizawa K, Ishizuka N (2014) GATA-4 transcription factor regulates cardiac COX-2 expression in duced by nicorandil in left ventricle of rats. Pharmacology 93(3–4): 129–136. https://doi.org/10.1159/000360008 [Full text] [PubMed] Shuvalov MP, Frolova OG, Ratushnyak SS (2014) Preeclampsia and eclampsia as a cause of maternal mortality. Obstetrics and Gynecol ogy 1: 81–87. [In Russian] Metelskaya VA, Gumanova NG, Litinskaya OA (2004) Potentialities of laboratory diagnosis of the NO-producing function of the vascular endothelium. Clinical Laboratory Diagnostics 9: 86. [In Russian] Methodical letter (2016) Methodical letter of the Ministry of Health care of The Russian Federation of 14.12.2016 No. 15-4/10/2-7955 “Results of confidential audit of maternal mortality in the Russian Federation in 2015”. [In Russian] [Electronic resource] Sidorova IS (2003) Gestosis. Medicine, Moscow, 416 pp. [In Russian] Staroseltseva OA (2012) The use of pharmacological precondition ing with nicorandil for the correction of endothelial dysfunction in the model of L-NAME-induced nitric oxide deficiency. Kursk State Medical University. [In Russian] Mironov AN (2012) Manual for conducting preclinical studies of me dicinal products. Part 1. Grif and K, Moscow, 944 pp. [In Russian] Sydorenko EV (2003) Methods of mathematical processing in psychol ogy. Rech Publishing House, Saint-Petersburg, 350 pp. [In Russian] Novaković R, Radunović N, Marković-Lipkovski J, Ćirović S, Be leslin-Čokić B, Ilić B, Ivković B, Heinle H, Živanović V, Gojkov ić-Bukarica LJ (2015) Effects of the polyphenol resveratrol on con tractility of human term pregnant myometrium. Mol Hum Reprod 21(6): 545–551. https://doi.org/10.1093/molehr/gav011 [Full text] [PubMed] Tanaka K, Kato K, Takano T, Katagiri T, Asanoi H, Nejima J, Na kashima M, Kamijo T, Sakanashi M (2010) Acute effects of intrave nous nicorandil on hemodynamics in patients hospitalized with acute decompensated heart failure. Journal of Cardiology 56(3): 291–299. References Free Radical Research 45(3): 293–302. https://doi.org/10.3109/1071 5762.2010.527337 [Full text] [PubMed] Korokin MV (2011) Ways of pharmacological correction of patho genetic disorders of nitric oxide metabolism when modeling endo thelial dysfunction. Kursk State Medical University, Belgorod State National Research University, Moscow. [In Russian] Korokin MV, Pokrovsky MV, Novikov OO, Gureev VV, Denisyuk TA, Korokina LV, Polyanskaya OS, Ragulina VA, Pokrovskaya TG, Danilenko LM, Belous AS (2011) Effect of L-arginine, vitamin B6 and folic acid on parameters of endothelial dysfunction and micro circulation in the placenta in modeling of L-NAME-induced NO deficiency. Bulletin of Experimental Biology and Medicine 152(7): 77–79. https://doi.org/10.1007/s10517-011-1456-z [In Russian] Gayet JL, Paganelli F, Cohen-Solal A (2011) Update on the medi cal treatment of stable angina. Archives of Cardiovascular Diseases 104(10): 536–544. https://doi.org/10.1016/j.acvd.2011.08.001 [Full text] [PubMed] Glanz С (1999) Biomedical statistics (translation from English). Praktika Publishing House, Мoscow, 459 pp. [In Russian] Stupakova EG et al.: Correction of morphofunctional disturbances arising when modelling... 70 Rose KM, Parmar MS, Cavanaugh JE (2014) Dietary supplementa tion with resveratrol protects against striatal dopaminergic deficits produced by in utero LPS exposure. Brain Res 1573: 37–43. https:// doi.org/10.1016/j.brainres.2014.05.028 [Full text] [PubMed] Leikert JF, Räthel TR, Wohlfart P, Cheynier V, Vollmar AM, Dirsch VM (2002) Red wine polyphenols enhance endothelial nitric oxide synthase expression and subsequent nitric oxide release from endo thelial cells. Circulation 106(13): 1614–1617. [Full text] [PubMed] Li W, Wu N, Shu W, Jia D, Jia P (2015) Pharmacological precon ditioning and postconditioning with nicorandil attenuates ischemia/ reperfusion-induced myocardial necrosis and apoptosis in hypercho lesterolemic rats. Experimental and Therapeutic Medicine 10(6): 2197–2205. https://doi.org/10.3892/etm.2015.2782 [Full text] [PubMed] Sánchez-Aranguren LC, Prada CE, Riaño-Medina CE, Lopez M (2014) Endothelial dysfunction and preeclampsia: role of oxida tive stress. Frontiers in Physiology 5: 372. https://doi.org/10.3389/ fphys.2014.00372 [Full text] [PubMed] Savelyeva GM, Shalina RI, Panina OB, Kurtzer MA (2011) Obstetrics. GEOTAR-Media Publishing Group, Moscow, 656 pp. [In Russian] Malysheva AM, Martsevich SY, Ginzburg ML (2011) Cardioprotec tive drug Nicorandil in coronary heart disease. Therapeutic Archive 83(9): 14–19. [In Russian] Scioscia M, Karumanchi SA, Goldman-Wohl D, Robillard PY (2015) Endothelial dysfunction and metabolic syndrome in pre eclampsia: an alternative viewpoint.Journal of Reproductive Immu nology 108: 42–47. References https://doi.org/10.1016/j.jjcc.2010.06.009 [Full text] [PubMed] Tanaka K, Kato K, Takano T, Katagiri T, Asanoi H, Nejima J, Na kashima M, Kamijo T, Sakanashi M (2010) Acute effects of intrave nous nicorandil on hemodynamics in patients hospitalized with acute decompensated heart failure. Journal of Cardiology 56(3): 291–299. [Full text] [PubMed] Oganov RG, Metelskaya VA, Akhmedzhanov NM, Litinskaya OA, Vasyutina EI, Gumanova NG (2004) Characteristics of biochemical markers of vascular endothelium function: development of a mod el system using cell cultures Questions of Biological, Medical and Pharmaceutical Chemistry 2: 34–39. [In Russian] Parimala K, Peedicayil J, Peedicayil A, Nelson N, Ernest K (2006) The effect of nicorandil congener on isolated human myometrium. European Journal of Obstetrics & Gynecology and Reproductive Bi ology 126(2): 176–9. Https://doi.org/10.1016/j.ejogrb.2005.08.014 [Full text] [PubMed] Tsukimori K, Komatsu H, Fukushima K, Kaku T, Nakano H, Wake N (2008) Inhibition of nitric oxide synthetase at mid-gestation in rats is associated with increases in arterial pressure, serum tumor necrosis factor-alpha, and placental apoptosis. American Journal of Hypertension 21(4): 477–481. https://doi.org/10.1038/ajh.2007.80 [Full text] [PubMed] Pereira RD, De Long NE, Wang RC, Yazdi FT, Holloway AC , Raha S (2015) Angiogenesis in the placenta: the role of reactive oxygen species signaling. BioMed Research International 2015: 814543. https://doi.org/10.1155/2015/814543 [Full text] [PubMed] van Oppenraaij RH, Bergen NE, Duvekot JJ, de Krijger RR, Hop Ir WC, Steegers EA, Exalto N (2011) Placental vascularization in early onset small for gestational age and preeclampsia. Reproductive Sci ences 18(6): 586–593. https://doi.org/10.1177/1933719110396231 [Full text] [PubMed] Pokrovsky MV, Kochkarov VI, Pokrovskaya TG, Gladchenko MP, Artyushkova EB, Pashin EN, Brusnik MV, Chulukova TN, Kljavs JP, Korneev MM, Zelenkova TI, Malyhin VA, Belous AS, Zaloznyh JI, Majakov AI (2006) Methodical approaches for the quantitative estimation of development endothelial dysfunction at L-NAME-the induced model of deficiency of nitric oxide in experiment. Kuban Scientific Medical Bulletin 10(91): 72–77. [In Russian] Verlohren S, Geusens N, Morton J, Verhaegen I, Hering L, Herse F, Dudenhausen JW, Muller DN, Luft FC, Cartwright JE, Davidge ST, Pijnenborg R, Dechend R (2010) Inhibition of trophoblast-induced spiral artery remodeling reduces placental perfusion in rat pregnan cy. Hypertension 56(2): 304–310. https://doi.org/10.1161/HYPER TENSIONAHA.110.153163 [Full text] [PubMed] Reznikova LB (2013) Endothelioprotective activity of GABA de rivatives with experimental gestosis [dissertation]. Volgograd State Medical University, Volgograd. References [In Russian] Wallerath T, Deckert G, Ternes T, Anderson H, Li H, Witte K, Förstermann U (2002) Resveratrol, a polyphenolic phytoalexin Research Results in Pharmacology 4(1): 59–71 71 cardiacmyocytes. Pflügers Archiv 468(4): 693–703. https://doi. org/10.1007/s00424-015-1763-8 [Full text] [PubMed] present in red wine, enhances expression and activity of endo thelial nitric oxide synthase. Circulation 106(13): 1652–1658. https://doi.org/10.1161/01.CIR.0000029925.18593.5C [Full text] [PubMed] present in red wine, enhances expression and activity of endo thelial nitric oxide synthase. Circulation 106(13): 1652–1658. https://doi.org/10.1161/01.CIR.0000029925.18593.5C [Full text] [PubMed] White AR, Ryoo S, Li D, Champion HC, Steppan J, Wang D, Nyhan D, Shoukas AA, Hare JM, Berkowitz DE (2006) Knock down of arginase I restores NO signaling in the vasculature of old rats. Hypertension 47(2): 245–251. https://doi.org/10.1161/01. HYP.0000198543.34502.d7 [Full text] [PubMed] Wang QJ, Song BF, Zhang YH, Ma YY, Shao QQ, Liu J, Qu X (2015) Expression of RGC32 in human normal and preeclamptic placen tas and its role in trophoblast cell invasion and migration. Placen ta 36(4): 350–356. https://doi.org/10.1016/j.placenta.2014.12.012 [Full text] [PubMed] Xu R, Cai A, Zheng D, Qiu R, Li L, Zhou Y, Feng Y, Mai W (2016) Amlodipine suppresses Ang-II-induced endothelium dysfunction by diminishing ROCK1expression. Clinical and Experimental Hyper tension 38(2): 166–72. https://doi.org/10.3109/10641963.2015.1081 212 [Full text] [PubMed] Xu R, Cai A, Zheng D, Qiu R, Li L, Zhou Y, Feng Y, Mai W (2016) Amlodipine suppresses Ang-II-induced endothelium dysfunction by diminishing ROCK1expression. Clinical and Experimental Hyper tension 38(2): 166–72. https://doi.org/10.3109/10641963.2015.1081 212 [Full text] [PubMed] Wei J, Watanabe Y, Takeuchi K, Yamashita K, Tashiro M, Kita S, Iwamoto T, Watanabe H, Kimura J (2016) Nicorandil stimulates a Na⁺/Ca²⁺ exchanger by activating guanylate cyclase in guinea pig Contributors Elena G. Stupakova, Assistant to the Department of Obstetrics and Gynaecology, Faculty of Postgraduate Edu cation, Kursk State Medical University of the Ministry of Healthcare of the Russian Federation. e-mail: miss.stu packova@yandex.ru. The author had a leading role in planning and performing the experiment, analysing the data and literature and writing the article. Elena G. Stupakova, Assistant to the Department of Obstetrics and Gynaecology, Faculty of Postgraduate Edu cation, Kursk State Medical University of the Ministry of Healthcare of the Russian Federation. e-mail: miss.stu packova@yandex.ru. The author had a leading role in planning and performing the experiment, analysing the data and literature and writing the article. Galina A. Lazareva, Doctor of Medical Sciences, Professor, Head of the Department of Obstetrics and Gynae cology, Faculty of Postgraduate Education, Kursk State Medical University of the Ministry of Healthcare of the Russian Federation. e-mail: akush.fpo@gmail.com. The author took part in planning experiments, analysed the literature and participated in interpreting the data. Vladimir V. Gureev, Doctor of Medical Sciences, Associate Professor of the Department of Pharmacology and Clinical Pharmacology. e-mail: gureev@bsu.edu.ru. The author participated in planning the experiments, analysed the literature and participated in interpreting the data. Vladimir V. Gureev, Doctor of Medical Sciences, Associate Professor of the Department of Pharmacology and Clinical Pharmacology. e-mail: gureev@bsu.edu.ru. The author participated in planning the experiments, analysed the literature and participated in interpreting the data.
https://openalex.org/W2165616869	https://inria.hal.science/inria-00568922/document	English	null	Network Non-neutrality Debate: An Economic Analysis	Lecture notes in computer science	2,011	cc-by	8,693	To cite this version: Eitan Altman, Arnaud Legout, Yuedong Xu. Network Non-Neutrality Debate: An Economic Analy- sis. 10th IFIP Networking Conference (NETWORKING), IFIP Technical Committee on Communi- cation Systems (TC 6), May 2011, Valencia, Spain. pp.68-81, 10.1007/978-3-642-20798-3_6. inria- 00568922v2 Network Non-Neutrality Debate: An Economic Analysis Eitan Altman, Arnaud Legout, Yuedong Xu To cite this version: Eitan Altman, Arnaud Legout, Yuedong Xu. Network Non-Neutrality Debate: An Economic Analy- sis. 10th IFIP Networking Conference (NETWORKING), IFIP Technical Committee on Communi- cation Systems (TC 6), May 2011, Valencia, Spain. pp.68-81, 10.1007/978-3-642-20798-3_6. inria- 00568922v2 Network Non-Neutrality Debate: An Economic Analysis Eitan Altman, Arnaud Legout, Yuedong Xu Distributed under a Creative Commons Attribution 4.0 International License HAL Id: inria-00568922 https://inria.hal.science/inria-00568922v2 Submitted on 19 Mar 2011 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 ⋆⋆Corresponding author: Prof. Eitan Altman. The work was supported by the INRIA ARC Meneur on Network Neutrality. Network Non-neutrality Debate: An Economic Analysis ⋆ Eitan Altman ⋆⋆, Arnaud Legout, and Yuedong Xu Eitan Altman ⋆⋆, Arnaud Legout, and Yuedong Xu INRIA Sophia Antipolis, 2004 Route des Lucioles, France {eitan.altman,arnaud.legout}@inria.fr,yuedong.xu@gmail.com INRIA Sophia Antipolis, 2004 Route des Lucioles, France {eitan.altman,arnaud.legout}@inria.fr,yuedong.xu@gmail.com Abstract. This paper studies economic utilities and quality of service (QoS) in a two-sided non-neutral market where Internet service providers (ISPs) charge content providers (CPs) for the content delivery. We pro- pose new models that involve a CP, an ISP, end users and advertisers. The CP may have either a subscription revenue model (charging end users) or an advertisement revenue model (charging advertisers). We formulate the interactions between the ISP and the CP as a noncoop- erative game for the former and an optimization problem for the latter. Abstract. This paper studies economic utilities and quality of service (QoS) in a two-sided non-neutral market where Internet service providers (ISPs) charge content providers (CPs) for the content delivery. We pro- pose new models that involve a CP, an ISP, end users and advertisers. The CP may have either a subscription revenue model (charging end users) or an advertisement revenue model (charging advertisers). We formulate the interactions between the ISP and the CP as a noncoop- erative game for the former and an optimization problem for the latter. Our analysis shows that the revenue model of the CP plays a signiﬁcant role in a non-neutral Internet. With the subscription model, both the ISP and the CP receive better (or worse) utilities as well as QoS in the presence of the side payment at the same time. With the advertisement model, the side payment impedes the CP from investing on its contents. g Our analysis shows that the revenue model of the CP plays a signiﬁcant role in a non-neutral Internet. With the subscription model, both the ISP and the CP receive better (or worse) utilities as well as QoS in the presence of the side payment at the same time. With the advertisement model, the side payment impedes the CP from investing on its contents. Keywords: Network Non-neutrality, Side Payment, Nash Equilibrium, Bargaining ⋆This version has made some changes to rectify small typos and misinterpretations in the published one. Keywords: Network Non-neutrality, Side Payment, Nash Equilibrium, Bargaining 1 Introduction Network neutrality, one of the foundations of Internet, is commonly admitted that ISPs must not discriminate traﬃc in order to favor speciﬁc content providers [1]. However, the principle of network neutrality has been challenged recently. The main reason is that new broadband applications cause huge amount of traﬃc without generating direct revenues for ISPs. Hence, ISPs want to get additional revenues from CPs that are not directly connected to them. For instance, a residential ISP might want to charge Youtube in order to give a premium quality of service to Youtube traﬃc. This kind of monetary ﬂows, which violates the principle of network neutrality, are called two-sided payment. We use the term side payment to name the money charged by ISPs from CPs exclusively. On the one hand, the opponents of network neutrality argue that it does not give any incentive for ISPs to invest in the infrastructure. This incentive issue is even more severe in two cases: the one of tier-one ISPs that support a high load, but do not get any revenue from CPs; and the one of 3G wireless networks that need to invest a huge amount of money to purchase spectrum. On the other hand, advocates of network neutrality claim that violating it using side payment will lead to unbalanced revenues among ISPs and CPs, thus a market instability. Recent work addressed the problem of network neutrality from various per- spectives [2,3,4,5,6,7,8,9]. Among these work, [2,3,4,13] are the closest to our Network Non-neutrality Debate: An Economic Analysis 2 work. Musacchio et al. [2] compare one-sided and two-sided pricing of ISPs. However, they only investigate an example where the joint investments of CPs and ISPs bring revenue from advertisers to CPs. In [3], the authors show how side payment is harmful for all the parties involved such as ISP and CP. Altman et al. in [4] present an interesting bargaining framework to decide how much the ISP should charge the CP. However, their models might give a biased conclusion by overlooking the end users’ sensitivity towards the prices of the CP and the ISP. Hermalin et.al in [13] study the impact of QoS diﬀerentiation (removing product-line constraint) in a two sided Internet market. The monopoly ISP is able charge a continuum of CPs based on the qualities of connections chosen by them. The CPs diﬀer in the attractiveness of their content. 1 Introduction The authors show that the product-line restriction has an ambiguous overall eﬀect, but is usually harmful. In this work, they diﬀerentiate the sources of CPs’ income for the ﬁrst time. Diﬀerent from [13], we investigate the QoS provision of the ISP to the CP instead of the QoS diﬀerentiation among a continuum of CPs. By using sim- ple two-sided market models, we answer the question when the side payment is beneﬁcial. In this paper, we unravel the conﬂicts of the side payment in a more general context. We consider a simpliﬁed market composed of one ISP, one CP, some advertisers, and a large number of end users. The ISP charges end-users based on their usage and sets their QoS level according to the price paid. The CP can either have a subscription based or an advertisement based revenue model. For the subscription based revenue model, the CP gets its revenue from the subscription paid by end-users. End-users adapt their demand of contents based on the price paid to the ISP and the CP. For the advertisement based revenue model, the CP gets its revenue from advertisers. End users adapt the demand according to the price paid to the ISP and the CP’s investment on its contents. Our work diﬀers from related work [2,3,4,13] by: i) incorporating the QoS provided by the ISP, ii) studying diﬀerent revenue models of the CP, and iii) in- troducing the relative price sensitivity of end users in the subscription model. Es- pecially, in the subscription model, the relative price sensitivity decides whether the side payment is beneﬁcial (or harmful) to the ISP and the CP. Our ﬁnding contradicts the previous work (e.g. [3]) that argues that the side payment is harmful for all parties involved. In the advertisement model, the ability of the CP’s investment to attract the traﬃc of end users plays a key role. It deter- mines whether the side payment is proﬁtable for the ISP and the CP. Our main contributions are the following. – We present new features in the mathematical modeling that include the QoS, the relative price sensitivity of end-users, and the CP’s revenue models. – We analytically show that the side payment from the CP to the ISP is beneﬁcial to the ISP and the CP in terms of proﬁts under certain conditions. 2.1 Revenue Models We consider a simpliﬁed networking market with four economic entities, namely the advertisers, the CP, the ISP and end users. All the end users can access the contents of the CP only through the network infrastructure provided by the ISP. The ISP collects subscription fees from end users. It sets two market parameters (ps, q) where ps is the non-negative price of per-unit of demand, and q is the QoS measure (e.g. delay, loss or block probability). End users can decide whether to connect to the ISP or not, or how much traﬃc they will request, depending on the bandwidth price and the QoS. The CP usually has two revenue models, the user subscription and the advertisement from clicks of users. These two models, though sometimes coexisting with each other, are studied separately in this work for clarity. The CP and the ISP interact with each other in a way that depends on the CP’s revenue models. In the subscription based model, the CP competes with the ISP by charging users a price pc per-unit of content within a ﬁnite time. End users respond to ps, pc and q by setting their demands elastically. Though pc has a diﬀerent unit as ps, it can be mapped from the price per content into the price per bps (i.e. dividing the price of a content by its size in a ﬁnite time). The price pc not only can stand for a ﬁnancial disutility, but also can represent the combination of this disutility together with a cost per quality. Thus a higher price may be associated with some better quality (this quality would stand for parameters diﬀerent from the parameter q which we will introduce later). Without loss of generality, ps and pc can be positive or 0. For the advertisement based model, instead of charging users directly, the CP attracts users’ clicks on online advertisements. The more traﬃc demands the end users generate, the higher the CP’s revenue. To better understand network neutrality and non-neutrality, we describe the monetary ﬂows among diﬀerent components. The arrows in Figure 1 represent the recipients of money. A “neutral network” does not allow an ISP to charge a CP for which it is not a direct provider for sending information to this ISP’s users. On the contrary, monetary ﬂow from a CP to an ISP appears when “network neutrality” is violated. 2 Basic Model In this section, we ﬁrst introduce the revenue models of the ISP and the CP. Then, we formulate a game problem and an optimization problem for the selﬁsh ISP and the CP. Finally, we describe the bargaining games in a two-sided market. 1 Introduction – We utilize bargaining games based on [4] to investigate how the side payment is determined. – We utilize bargaining games based on [4] to investigate how the side payment is determined. The rest of this work is organized as follows. In section 2, we model the economic behaviors of ISP, CP, advertisers and end users. Section 3 and 4 study the impact of the side payment and its bargaining outcomes. Section 5 presents numerical study to validate our claims. Section 6 concludes this paper. Network Non-neutrality Debate: An Economic Analysis Network Non-neutrality Debate: An Economic Analysis 3 Network Non-neutrality Debate: An Economic Analysis Network Non-neutrality Debate: An Economic Analysis We present market demand functions for the subscription and the advertise- ment based revenue models. Subscription model Let us deﬁne the average demand of all users by D that has D(ps, pc, q) = max{0, D0 −α(ps + ρpc) + βq}, (1) (1) where D0, α, β and ρ are all positive constants. The parameter D0 reﬂects the total potential demand of users. The parameters α and β denote the responsive- ness of demand to the price and the QoS level of the ISP. The physical meaning of (1) can be interpreted in this way. When the prices of the ISP and the CP in- crease (resp. decrease), the demand decreases (resp. increases). If the QoS of the ISP is improved, the demand from users increases correspondingly. The param- eter ρ represents the relative sensitivity of pc to ps. We deliberately set diﬀerent sensitivities of end users to the prices of the ISP and the CP because pc and ps refers to diﬀerent type of disutilities. If ρ = 1, the prices of the ISP and the CP are regarded as having the same eﬀect on D. When ρ > 1, users are more sensitive to the change of pc than ps. The positive prices ps and pc can not be arbitrarily high. They must guarantee a nonnegative demand D. We denote SCP to be the pricing strategy of the CP that has SCP = {pc : pc ≥0}. The utility (or revenue equivalently) of the CP is expressed as Ucp = (pc −pt)D(ps, pc, q). (2) Ucp = (pc −pt)D(ps, pc, q). (2) Note that the variable D(ps, pc, q) is interchangeable with D all the time. Next, we present the utility of the ISP with QoS consideration. We assume that the pricing strategy of the ISP is deﬁned by SISP = {(ps, q) : ps ≥0; 0 < q ≤qmax}. To sustain a certain QoS level of users, the ISP has to pay the costs for operating the backbone, the last-mile access, and the upgrade of the network, etc. Let u(D, q) be the amount of bandwidth consumed by users that depends on the demand D and the QoS level q. We assume that u(D, q) is a positive, convex and strictly increasing function in the 2-tuple (D, q). 1 The QoS metric can be the functions of packet loss rate or expected delay etc. 2.1 Revenue Models The ISP may charge the CP an additional amount of money that we denote by f(D) = ptD where pt is the price of per-unit of demand. We denote by δ ∈[0, 1] the tax rate of this transferred revenue imposed by the regulator or the government. Fig. 1. Money ﬂow of a non-neutral network. Fig. 1. Money ﬂow of a non-neutral network. 4 Network Non-neutrality Debate: An Economic Analysis This is reasonable because a larger demand or higher QoS usually requires a larger bandwidth of the ISP. We now present a natural QoS metric as the expected delay 1. The expected delay is computed by the Kleinrock function that corresponds to the delay of M/M/1 queue with FIFO discipline or M/G/1 queue under processor sharing [10]. Similar to [10], instead of using the actual delay, we consider the reciprocal of its square root, q = 1 √Delay = p u(D, q) −D. Thus, the cost C(D, q) can be expressed as C(D, q) = pru(D, q) = prD +prq2, where pr is the price of per-unit of bandwidth invested by the ISP. Therefore, the cost of the ISP is denoted by C(D, q) = pru(D, q). The utility of the ISP is deﬁned as the diﬀerence between revenue and cost: expressed as C(D, q) = pru(D, q) = prD +prq2, where pr is the price of per-unit of bandwidth invested by the ISP. Therefore, the cost of the ISP is denoted by C(D, q) = pru(D, q). The utility of the ISP is deﬁned as the diﬀerence between revenue and cost: Uisp = (ps −pr)D(ps, pc, q) + (1 −δ)ptD(ps, pc, q) −prq2. (3) (3) Advertisement model Nowadays, a small proportion of CPs like Rapidshare and IPTV providers get their income from end users. Most of other CPs provide contents for free, but collect revenues from advertisers. The demand from users is transformed into attentions such as clicks or browsing of online advertisements. To attract more eyeballs, a CP needs to invest money on its contents, incurring a cost c. The investment improves the potential aggregate demand D0 in return. Let D0(c) be a concave and strictly increasing function of the investment cost c. 1 The QoS metric can be the functions of packet loss rate or expected delay etc. 5 Network Non-neutrality Debate: An Economic Analysis With abuse of notations we denote the strategy of the CP by SCP = {c : c > 0}. Hence, the demand to the CP and the ISP is written as D = D0(c) −αps + βq. (4) (4) The utility of the ISP is the same as that in (3). Next, we describe the economic interaction between advertisers and the CP. Lemma 1. The optimal demand Da is a strictly decreasing function of pa if the pdf x(v) is nonzero in (0, v). The proofs of all lemmas and theorems can be found in [12]. From (6), we can observe that the optimal price pa∗is obtained at D = MB · (1 −X(pa∗))/pa∗. Here, we denote a function y(·) such that pa∗= y(D). According to the demand curve of attentions, y(·) is a decreasing function of D. The utility of the CP is a function of the demand D and the cost c, i.e. Ucp = y(D) · D −ptD −c. Network Non-neutrality Debate: An Economic Analysis There are M advertisers interested in the CP, each of which has a ﬁxed budget B in a given time interval (e.g., daily, weekly or monthly). An advertiser also has a valuation v to declare its maximum willingness to pay for each attention. The valuation v is a random variable in the range [0, v]. Suppose that v is characterized by probability den- sity function (PDF) x(v) and cumulative distribution function (CDF) X(v). We assume that the valuations of all advertisers are i.i.d. Let pa be the price of per attention charged by the CP. We denote by Da(pa) the demand of attentions from advertisers to the CP. Therefore, Da can be expressed as [11] Da = MB · Prob(v ≥pa)/pa = MB · (1 −X(pa))/pa. (5) (5) When the CP increases pa, the advertisers will reduce their purchase of atten- tions. It is also easy to see that the revenue of advertising, pa·Da, decreases with regard to pa either. However, the attentions that the CP can provide is upper bounded by the demand of users through the ISP. Then, we can rewrite Da as that in [11] by Da = min{D, MB · (1 −X(pa))/pa} Da = min{D, MB · (1 −X(pa))/pa}. (6) (6) Correspondingly, subtracting investment from revenue, we obtain the utility of the CP b Correspondingly, subtracting investment from revenue, we obtain the utility of the CP by (7) Ucp = (pa −pt)Da −c. (7) (7) Lemma 1. The optimal demand Da is a strictly decreasing function of pa if the pdf x(v) is nonzero in (0, v). Lemma 1. The optimal demand Da is a strictly decreasing function of pa if the pdf x(v) is nonzero in (0, v). 2.3 Bargaining Game The side payment serves as a ﬁxed parameter in the above two problems. A subsequent and important problem is how large the side payment should be. When the ISP decides the side payment unilaterally, it might set a very high pt in order to obtain the best utility. However, this leads to a paradox when the ISP sets ps and pt at the meantime. With the subscription based model, if the ISP plays a strategy (pt, ps, q) and the CP plays pc, the noncooperative game leads to a zero demand and hence a zero income. This can be easily veriﬁed by taking the derivative of Uisp over pt (also see [3]). In other words, the ISP cannot set pt and ps simultaneously in the price competition. Similarly, with the advertisement based model, the ISP meets with the same paradox. There are two possible ways, the Stackelberg game and the bargaining game, to address this problem. Their basic principle is to let the ISP to choose pt and ps asynchronously. In this work, we consider the bargaining game in a market where the ISP and the CP usually have certain marketing powers. Our analysis in this work is close to the one presented in [4], but comes up with quite diﬀerent observations. p [ ], p q We here analyze the bargaining games of the side payments that are played at diﬀerent time sequences. The ﬁrst one, namely pre-bargaining, models the situation that the bargaining takes place before the problems (G1) or (G2). The second one, deﬁned as post-bargaining, models the occurrence of bargaining after the problems (G1) or (G2). Let γ ∈[0, 1] be the bargaining power of the ISP over the CP. They negotiate the transfer price pt determined by pt∗= arg maxpt (Uisp)γ(Ucp)1−γ. Since the utilities can only be positive, the optimal pt maximizes a virtual utility U (12) pt∗= arg max pt U = arg max pt (1 −γ) log Ucp + γ log Uisp. (12) We use (12) to ﬁnd pt as a function of the strategies of the ISP and the CP. We use (12) to ﬁnd pt as a function of the strategies of the ISP and the CP. 3 Price Competition of the Subscription Model In this section, we ﬁrst investigate how the relative price sensitivity inﬂuences the price competition between the ISP and the CP. We then study the choice of the side payment under the framework of bargaining games. 2.2 Problem Formulation With the subscription model, the strategy proﬁle of the ISP is to set the 2- tuple (ps, q) and that of the CP is to set pc. This is actually a game problem in which the ISP and the CP compete by setting their prices. Since the ISP’s QoS is tunable, we call this game “QoS Agile Price competition”. With the advertisement model, the strategy of the ISP is still the price paid by end users, while that of the CP is to determine the investment level c. The ISP and the CP maximize their own utilities selﬁshly, but do not compete with each other. We name this maximization as “Strategic Pricing and Investment”. Deﬁnition 1. QoS Agile Price Competition In the subscription model, the CP charges users based on their traﬃc demands. If the Nash equilibrium NE(1) = {ps∗, pc∗, q∗} exists, it can be expressed as (G1) Uisp(ps∗, pc∗, q∗) = max {ps,q}∈SISP Uisp(ps, pc∗, q), (8) Ucp(ps∗, pc∗, q∗) = max pc∈SCP Ucp(ps∗, pc, q∗). (9) (9) Network Non-neutrality Debate: An Economic Analysis 6 Deﬁnition 2. Strategic Pricing and Investment In the advertisement model, the ISP sets (ps, q) and the CP sets c to optimize their individual utilities. If there exists an equilibrium {ps∗, q∗, c∗}, it can be solved by Deﬁnition 2. Strategic Pricing and Investment In the advertisement model, the ISP sets (ps, q) and the CP sets c to optimize their individual utilities. If there exists an equilibrium {ps∗, q∗, c∗}, it can be solved by (G2) Uisp(ps∗, q∗, c∗) = max {ps,q}∈SISP Uisp(ps, q, c∗), (10) Ucp(ps∗, q∗, c∗) = max c∈SCPUcp(ps∗, q∗, c). (11) (11) 3.1 Properties of Price Competition This subsection investigates the impact of the side payment on Nash Equilibrium of the noncooperative game G1. Before eliciting the main result, we show some basic properties of the subscription based revenue model. Lemma 2. The utility of the CP, Ucp(ps, pc, q), in (2) is a ﬁnite, strictly concave function with regard to (w.r.t.) pc. Similarly, we draw the following conclusion. Network Non-neutrality Debate: An Economic Analysis 7 Lemma 3. The utility of the ISP, Uisp(ps, pc, q), in (3) is a ﬁnite, strictly con- cave function w.r.t. the 2-tuple (ps, q) if the market parameters satisfy 4αpr > β2. For the QoS Agile Price Competition, we summarize our main results as below. Lemma 4. When the ISP and the CP set their strategies selﬁshly, – the Nash equilibrium (ps∗, pc∗, q∗) is unique; ( ) – the QoS level q∗at the NE is inﬂuenced by the side payment in the ways: • improved QoS with ρ + δ < 1; • degraded QoS with ρ + δ > 1; • unaﬀected QoS with ρ + δ = 1 if (ps∗, pc∗, q∗) satisfy ps∗> 0, pc∗> 0, 0 < q∗< qmax and 4αpr > β2. if (ps∗, pc∗, q∗) satisfy ps∗> 0, pc∗> 0, 0 < q∗< qmax and 4αpr > β2. if (ps∗, pc∗, q∗) satisfy ps∗> 0, pc∗> 0, 0 < q∗< qmax and 4αpr > β2 When the NE (ps∗, pc∗, q∗) is not at the boundary, we can yield the following expressions by solving the best response equations. q∗= β(D0 −αpr + αpt(1 −ρ −δ)) 6αpr −β2 , (13) pc∗= 2pr(D0 −αpr + αpt(1 −ρ −δ)) ρ(6αpr −β2) + pt, (14) ps∗= 2pr(D0 −αpr + αpt(1 −ρ −δ)) 6αpr −β2 + pr −(1 −δ)pt, (15) D∗= 2prα(D0 −αpr + αpt(1 −ρ −δ)) 6αpr −β2 , (16) (13) (15) (16) For the case that the NE is at the boundary, interested users can ﬁnd it in the technical report [12]. Lemma 4 means that that the QoS provision of the ISP is inﬂuenced by the side payment. We interpret the results by considering ρ and δ separately. When users are indiﬀerent to the price set by the ISP and that by the CP (i.e., ρ = 1), a positive tax rate δ leads to the degradation of q in the presence of the side payment. 3.1 Properties of Price Competition Next, we let δ be 0 and investigate the impact of ρ. If users are more sensitive to the price of the ISP (i.e. ρ < 1), the side payment is an incentive of the ISP to improve its QoS. Otherwise, charging side payment leads to an even poorer QoS of the ISP. Therefore, if users are more sensitive to the CP’s price, a good strategy of the ISP is to share its revenue with the CP so that the latter sets a lower subscription fee. 3.2 Bargaining of the Side Payment To highlight the bargaining of the side payment, we make the following two simpliﬁcations: i) the tax ratio δ is 0, and ii) pt can be positive, zero or negative. We let δ = 0 because it turns out to have the similar eﬀect as ρ. We have shown that a negative pt might beneﬁt both the ISP and the CP in some situations. Hence, we do not require pt to be positive in the bargaining game. When q reaches qmax, the QoS is reﬂected as a ﬁxed parameter in the demand model. To avoid considering the boundary cases of q, we also remove the constraint q ≤qmax. t Pre-bargaining: In the pre-bargaining, pt is chosen based on the NE of the ISP and the CP. The equations (13)∼(15) yield the expression of U U = 4 log D0 −αpr + αpt(1 −ρ) + constant . (17) (17) 8 Network Non-neutrality Debate: An Economic Analysis The utility U is increasing or decreasing in pt depending on the sign of (1 −ρ). If ρ < 1, a positive pt improves not only the QoS level of the ISP, but also the utilities of the ISP and the CP. As pt increases, ps decreases and pc increases consequently until ps hits 0. Hence, in the pre-bargaining, ps∗= 0. The prices pt∗and pc∗are computed by pt∗and pc∗are computed by pt∗= pr(4αpr + 2D0 −β2) 4αpr + 2ραpr −β2 . (18) pc∗= 2pr(D0 −αpr + αpt∗(1 −ρ)) ρ(6αpr −β2) + pt∗. (19) (18) (19) On the contrary, when ρ > 1, a negative pt beneﬁts both of them. Then, pt∗is a negative value such that pc∗is 0. When ρ = 1, the QoS and the utilities are unaﬀected by any pt. Among all these cases, the selection of pt is uninﬂuenced by the bargaining power γ. y g g p γ Post-bargaining: For the post-bargaining, the ISP and the CP compete for the subscription of users ﬁrst, knowing that they will bargain over pt afterwards [4]. In brief, we ﬁnd pt as a function of ps, pc and q ﬁrst. Then, the ISP and the CP compete with each other by setting the prices. To solve the maximization in (12), we let dU dpt be 0 and obtain pt = γpc −(1 −γ)(ps −pr) + (1 −γ)prq2/D. 3.2 Bargaining of the Side Payment (20) 0) to Uisp, we rewrite the ISP’s utility by (20) Uisp = γ (ps + pc −pr)(D0 −α(ps + ρpc) + βq) −prq2 . (21) (21) The utility of the CP is proportional to that of the ISP, i.e. Uisp γ = Ucp 1−γ . After knowing pt, we compute the derivatives dUisp dps , dUisp dq and dUcp dpc by The utility of the CP is proportional to that of the ISP, i.e. Uisp γ = Ucp 1−γ . After knowing pt, we compute the derivatives dUisp dps , dUisp dq and dUcp dpc by dUisp dps = γ(D −α(ps + pc −pr)), (22) dUisp dq = γ(β(ps + pc −pr) −2prq), (23) dUcp dpc = (1 −γ)(D −αρ(ps + pc −pr)). (24) (22) (23) (24) The best responses of Uisp and Ucp will not happen at the same time unless ρ = 1 or ps + pc −pr = 0. The condition ps + pc −pr = 0 does not hold because it leads to a zero demand D and zero utilities. When ρ is not 1, only one of (22) and (24) is 0. Here, we consider the case ρ > 1. The utility Ucp reaches its maximum upon D = αρ(ps + pc −pr), while Uisp is still strictly increasing w.r.t. ps. Thus, the ISP increases ps until the demand D is 0, which contradicts the condition of a nonzero D. If D = α(ps + pc −pr), dUcp dpc is negative and dUisp dps is 0. Then, the CP decreases pc until 0 and the ISP sets ps to achieve its best utility accordingly. By letting (24) be 0, we can ﬁnd (ps, q) at the Nash equilibrium q∗= β(D0 −αpr) 4αpr −β2 and ps∗= 2pr(D0 −αpr) 4αpr −β2 + pr. The price of the side payment, pt∗, is thus computed by The price of the side payment, pt∗, is thus computed by pt = −(1 −γ)D0 −αpr 2α . (25) (25) When ρ = 1, ps∗and pc∗can be arbitrary values in their feasible region that satisfy ps∗+ pc∗= pr + 2pr(D0−αpr) 4prα−β2 . Similar result has been shown in [4]. The analysis of ρ < 1 is omitted here since it can be conducted in the same way. Network Non-neutrality Debate: An Economic Analysis 9 9 4 Price, QoS and Investment settings of the Advertisement Model 4 Price, QoS and Investment settings of the Advertisement Model This subsection analyzes how the side payment inﬂuences the optimal strategies of ISP and CP with the advertisement model. The bargaining games are adopted to determine the amount of the side payment. Compared with subscription based model, the advertisement based model exhibits quite diﬀerent behaviors. 4.1 Properties of the Advertisement Mode In general, the subscription model is limited to ﬁle storage CDNs, newspaper corporations, or big content owners such as movie producers. Most of CPs are not able to provide enough unique contents so that they do not charge users, but make money from online advertisements. In this subsection, we present the general properties of the advertisement model and a couple of case studies. Lemma 5. For any feasible investment c of the CP, there exists a best strategy of the ISP, (ps, q). When c increases, the price and the QoS (i.e. ps and q) become larger. Lemma 5. For any feasible investment c of the CP, there exists a best strategy of the ISP, (ps, q). When c increases, the price and the QoS (i.e. ps and q) become larger. In G2, the CP and the ISP do not compete with each other. On one hand, the ISP sets the two-tuple (ps, q) with the observation of c. On the other hand, the CP adjusts c based on (ps, q). The investment of the CP brings more demand of end users, which increases the revenues of not only the ISP, but also the CP. Hence, diﬀerent from G1, the problem G2 is not a game. Instead of studying the NE, we look into the optimal strategies of the ISP and the CP in G2. Theorem 1. There exists a unique best strategy, namely (ps∗, q∗, c∗), with the advertisement model if the revenue of the CP, D · y(D), is a concave function w.r.t. D ≥0. Lemma 6. The side payment from the CP to the ISP leads to a decreased in- vestment on the contents when the best strategy (ps∗, q∗, c∗) has ps∗> 0, q∗> 0 and c∗> 0. 4.2 Case Study In this subsection, we aim to ﬁnd the best strategies of the ISP and the CP when the valuation of advertisers follows a uniform distribution or a normal distribution. Due to the page limit, the case of normal distribution is put in the technical report [12]. [ ] Recall that the potential aggregate demand of users, D0(c), is strictly increas- ing and concave w.r.t c. When the CP invests money on contents, D0 becomes larger, while its growth rate shrinks. Here, we assume a log function of D0(c), D0(c) = D0 0 + K log(1 + c), (26) (26) where the constant K denotes the ability that the CP’s investment brings the demand. The nonnegative constant D0 0 denotes the potential aggregate demand of end users when c is zero (the CP only provides free or basic contents). The utility of the ISP remains unchanged. Uniform Distribution: Suppose v follows a uniform distribution in the range [0, v]. Then, the CDF X(pa) is expressed as pa v . The optimal price pa is obtained Network Non-neutrality Debate: An Economic Analysis 10 when D = MB pa · (1 −pa v ) in the range [0, v] (see subsection 2.1). Alternatively, there has pa = MBv MB + Dv . (27) (27) The above expressions yield the utility of the CP by Ucp = MBvD MB + Dv −c −ptD. (28) (28) Deriving Ucp over c, we obtain Deriving Ucp over c, we obtain dUcp dc = ( (MB)2v (MB + Dv)2 −pt) · K 1 + c −1. (29) (29) We let (29) be 0 and get c = K( (MB)2¯v (MB + D¯v)2 −pt) −1. (30) (30) The rule of the ISP to decide (ps, q) is the same as that in the subscription model, except that the aggregate demand is not a constant, but a function of c, c = exp( D 2prα(4prα −β2) −D0 0 + αpr −(1 −δ)ptα K ) −1. (31) (31) Note that (30) is strictly decreasing and (31) is strictly increasing w.r.t. D. They constitute a ﬁxed-point equation for the 2-tuple (D∗, c∗). In the beginning, we assume that the optimal strategies are not on the boundary. When D approaches inﬁnity, (30) is negative while (31) is positive. When D is zero, if (30) is larger than (31), there exists a unique ﬁxed-point solution. 4.2 Case Study In this ﬁxed point, the ISP and the CP cannot beneﬁt from changing their strategy unilaterally. We can solve c∗and D∗numerically using a binary search. If (30) is smaller than (31) when D is 0, the best strategy of the CP is exactly c = 0. The physical interpretation is that the increased revenue from advertisers cannot compensate the investment on the contents. Once D∗and c∗are derived, we can solve ps∗and q∗subsequently. In this ﬁxed-point equation, pt greatly inﬂuences the optimal investment c∗. When pt grows, the right sides of (30) and (31) decrease at the mean time. The crossing point of two curves, (30) and (31), shifts toward the direction of smaller c as shown in Lemma 6. Intuitively, the contents of the CP become less when the ISP charges a positive pt. The boundary case of the optimal strategies as well as the bargaining games over pt are analyzed in [12]. 5 Evaluation Advertisement Model: CP’s in- vestment at the equilibrium Advertisement Model: In the advertisement model, we consider the de mand function D = K log(1 + c) −10ps + 0.5q. The coeﬃcient K reﬂects th eﬃciency of the CP’s investment to attract end users. The valuation of eac click/browsing follows uniform distribution in the range [0, 10]. The total bud get of advertisers is set to 1000. We conduct two sets of experiments. The ﬁrs one is to evaluate the impact of the side payment on the best strategies of th ISP and the CP. The second one is to ﬁnd the optimal pt in the pre-bargainin game. In ﬁgure 5, the CP’s investment is a decreasing function of pt. When pt i large enough, c reduces to 0. Figure 6 illustrates the utility of the CP when p 0 2 4 6 280 300 320 340 360 380 400 The price of side payment pt Utility Utilities of ISP and CP (ρ = 1.5) 0 2 4 6 8 400 450 500 550 600 The price of side payment pt Utility Utilities of ISP and CP (ρ = 0.5) ISP Utility CP Utility ISP Utility CP Utility Fig. 2. Subscription Model: Utilities of the ISP and the CP 0 2 4 6 8 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 The price of side payment pt QoS Level QoS level provided by ISP ISP QoS ρ = 1.5 ISP QoS ρ = 0.5 Fig. 3. Subscription Model: The QoS level provided by the ISP −15 −10 −5 0 5 10 15 20 Best pt charged by ISP Pre−bargaining of the price of side payment pt when α=5 pt when α=10 pt when α=15 20 40 60 80 100 120 The CP’s Investment The CP’s investment at the equilibrium Investment at the Equilibrium: K = 10 Investment at the Equilibrium: K = 20 Investment at the Equilibrium: K = 30 0 2 4 6 280 300 320 340 360 380 400 The price of side payment pt Utility Utilities of ISP and CP (ρ = 1.5) 0 2 4 6 8 400 450 500 550 600 The price of side payment pt Utility Utilities of ISP and CP (ρ = 0.5) ISP Utility CP Utility ISP Utility CP Utility Fig. 2. Subscription Model: Utilities of the ISP and the CP Fig. 3. 5 Evaluation 0 2 4 6 8 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 The price of side payment pt QoS Level QoS level provided by ISP ISP QoS ρ = 1.5 ISP QoS ρ = 0.5 Fig. 3. Subscription Model: The QoS level provided by the ISP 0 2 4 6 280 300 320 340 360 380 400 The price of side payment pt Utility Utilities of ISP and CP (ρ = 1.5) 0 2 4 6 8 400 450 500 550 600 The price of side payment pt Utility Utilities of ISP and CP (ρ = 0.5) ISP Utility CP Utility ISP Utility CP Utility Fig. 2. Subscription Model: Utilities of the ISP and the CP 0 2 4 6 8 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 The price of side payment pt QoS Level QoS level provided by ISP ISP QoS ρ = 1.5 ISP QoS ρ = 0.5 Fig. 3. Subscription Model: The QoS level provided by the ISP 0 2 4 6 280 300 320 340 360 380 400 The price of side payment pt Utility Utilities of ISP and CP (ρ = 1.5) 0 2 4 6 8 400 450 500 550 600 The price of side payment pt Utility Utilities of ISP and CP (ρ = 0.5) ISP Utility CP Utility ISP Utility CP Utility Fig. 2. Subscription Model: Utilities of the ISP and the CP 0 2 4 6 8 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 The price of side payment pt QoS Level QoS level provided by ISP ISP QoS ρ = 1.5 ISP QoS ρ = 0.5 Fig. 3. Subscription Model: The QoS level provided by the ISP 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 −20 −15 −10 −5 0 5 10 15 20 ρ: the end users’ sensitivity of prices Best pt charged by ISP Pre−bargaining of the price of side payment pt when α=5 pt when α=10 pt when α=15 Fig. 4. Subscription Model: Pre- bargaining of pt 0 1 2 3 4 5 6 7 0 20 40 60 80 100 120 The price of side payment pt The CP’s Investment The CP’s investment at the equilibrium Investment at the Equilibrium: K = 10 Investment at the Equilibrium: K = 20 Investment at the Equilibrium: K = 30 Fig. 5. 5 Evaluation We present some numerical results to reveal how the QoS, prices of the ISP and the CP, as well as their utilities evolve when the price of the side payment changes. The impact of bargaining power on the side payment is also illustrated. More numerical examples are demonstrated in the technical report [12]. Subscription Model: We consider a networking market where the demand function is given by D = 200 −10(ps + ρpc) + 0.5q. The operational cost of per-unit of bandwidth is set to pr = 1. Two situations, ρ = 0.5 and ρ = 1.5, are evaluated. The tax rate δ is set to 0 for simplicity. According to the above analysis, the side payment is beneﬁcial (resp. harmful) to the ISP and the CP if Network Non-neutrality Debate: An Economic Analysis 11 ρ is less (resp. greater) than 1. In ﬁgure 2, pt has diﬀerent impacts on utilities of the ISP and the CP. When ρ > 1, end users are more sensitive to the change of pc than ps. A positive pt leads to the increase of pc, causing a tremendous decrease of demand. Hence, both the ISP and the CP lose revenues w.r.t. a positive pt. Figure 3 further shows that a positive pt yields a better QoS if ρ < 1 and a worse QoS if ρ > 1. Next, the ISP and the CP bargain with each other to determine pt. We relax the choice of pt so that it can be negative. In the pre-bargaining game, pt is independent of the bargaining power γ. The optimal pt is obtained when ps∗ decreases to 0 for ρ > 1 and pc∗decreases to 0 for ρ < 1. We evaluate pt by changing ρ and α in ﬁgure 4. When ρ increases from 0.2 to 2, pt decreases until it becomes negative. A negative pt means that the ISP needs to transfer revenue to the CP instead. When ρ = 1, pt can be an arbitrary value as long as the sum of ps∗and pc∗is the ﬁxed value computed in section 3.2. Figure 4 also shows that a larger α results in a smaller absolute value of pt. 5 Evaluation Subscription Model: The QoS level provided by the ISP Fig. 2. Subscription Model: Utilities of the ISP and the CP 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 −20 −15 −10 −5 0 5 10 15 20 ρ: the end users’ sensitivity of prices Best pt charged by ISP Pre−bargaining of the price of side payment pt when α=5 pt when α=10 pt when α=15 Fig. 4. Subscription Model: Pre- bargaining of pt 0 1 2 3 4 5 6 7 0 20 40 60 80 100 120 The price of side payment pt The CP’s Investment The CP’s investment at the equilibrium Investment at the Equilibrium: K = 10 Investment at the Equilibrium: K = 20 Investment at the Equilibrium: K = 30 Fig. 5. Advertisement Model: CP’s in- vestment at the equilibrium Advertisement Model: In the advertisement model, we consider the de- mand function D = K log(1 + c) −10ps + 0.5q. The coeﬃcient K reﬂects the eﬃciency of the CP’s investment to attract end users. The valuation of each click/browsing follows uniform distribution in the range [0, 10]. The total bud- get of advertisers is set to 1000. We conduct two sets of experiments. The ﬁrst one is to evaluate the impact of the side payment on the best strategies of the ISP and the CP. The second one is to ﬁnd the optimal pt in the pre-bargaining game. In ﬁgure 5, the CP’s investment is a decreasing function of pt. When pt is large enough, c reduces to 0. 5 Evaluation Figure 6 illustrates the utility of the CP when pt 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 −20 −15 −10 −5 0 5 10 15 20 ρ: the end users’ sensitivity of prices Best pt charged by ISP Pre−bargaining of the price of side payment pt when α=5 pt when α=10 pt when α=15 0 1 2 3 4 5 6 7 0 20 40 60 80 100 120 The price of side payment pt The CP’s Investment The CP’s investment at the equilibrium Investment at the Equilibrium: K = 10 Investment at the Equilibrium: K = 20 Investment at the Equilibrium: K = 30 4 0.6 0.8 1 1.2 1.4 1.6 ρ: the end users’ sensitivity of prices 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 ρ: the end users’ sensitivity of prices 2 3 4 5 The price of side payment pt 2 3 4 5 The price of side payment pt Fig. 4. Subscription Model: Pre- Fig. 5. Advertisement Model: CP’s in- bargaining of pt vestment at the equilibrium Advertisement Model: In the advertisement model, we consider the de- mand function D = K log(1 + c) −10ps + 0.5q. The coeﬃcient K reﬂects the eﬃciency of the CP’s investment to attract end users. The valuation of each click/browsing follows uniform distribution in the range [0, 10]. The total bud- get of advertisers is set to 1000. We conduct two sets of experiments. The ﬁrst one is to evaluate the impact of the side payment on the best strategies of the ISP and the CP. The second one is to ﬁnd the optimal pt in the pre-bargaining game. In ﬁgure 5, the CP’s investment is a decreasing function of pt. When pt is large enough, c reduces to 0. Figure 6 illustrates the utility of the CP when pt Network Non-neutrality Debate: An Economic Analysis 12 0 1 2 3 4 5 6 7 0 50 100 150 200 250 300 The price of side payment pt The utility of the CP The CP utility at the equilibrium The CP revenue : K = 10 The CP revenue : K = 20 The CP revenue : K = 30 Fig. 6. 6 Conclusion and Future Work In this paper, we ﬁrst answer under what situations the side payment charged by the ISP is beneﬁcial for the ISP (or the CP). Then, we study how the price of the side payment is determined. Our models take account of three important features, the relative price sensitivity, the CP’s revenue models, and the QoS provided by the ISP. With the subscription model, the relative price sensitivity determines whether the ISP should charge the side payment from the CP or not. With the advertisement model, the charge of the side payment depends on the ability of the CP’s investment to attract the demand. 5 Evaluation Advertisement Model: The ISP’s utility at the equilibrium 0 1 2 3 4 5 6 7 0 50 100 150 200 250 300 350 400 450 The price of side payment pt The utility of the ISP The ISP utility at the equilibrium The CP revenue : K = 10 The CP revenue : K = 20 The CP revenue : K = 30 Fig. 7. Advertisement Model: The ISP’s utility at the equilibrium 0 1 2 3 4 5 6 7 0 50 100 150 200 250 300 The price of side payment pt The utility of the CP The CP utility at the equilibrium The CP revenue : K = 10 The CP revenue : K = 20 The CP revenue : K = 30 Fig. 6. Advertisement Model: The ISP’s utility at the equilibrium 0 1 2 3 4 5 6 7 0 50 100 150 200 250 300 350 400 450 The price of side payment pt The utility of the ISP The ISP utility at the equilibrium The CP revenue : K = 10 The CP revenue : K = 20 The CP revenue : K = 30 Fig. 7. Advertisement Model: The ISP’s utility at the equilibrium Fig. 6. Advertisement Model: The ISP’s utility at the equilibrium Fig. 7. Advertisement Model: The ISP’s utility at the equilibrium and K change. The CP’s utility increases ﬁrst and then decreases with K = 10 when pt increases. For the cases K = 20 and 30, the increase of pt usually leads to the decrease of revenues. In ﬁgure 7, the utility of the ISP with K = 10 and 20 increases ﬁrst and then decreases when pt grows. These curves present im- portant insights on the interaction between the CP and the ISP. If the contents invested by the CP can bring a large demand, the side payment is not good for both the ISP and the CP. On the contrary, when the eﬃciency K is small, a smaller investment of the CP might not signiﬁcantly reduce the demand from users. The ISP is possible to be better by extracting revenues from the CP with the low eﬃciency K. 4. E. Altman, M.K. Hanawal and R. Sundaresan, ”Nonneutral network and the role of bargaining power in side payments”, NetCoop, Ghent, Belgium, Nov. 2010. 6. V. Claudia Saavedra “Bargaining power and the net neutrality debate”, working paper, 2010. Available at http://sites.google.com/site/claudiasaavedra/ 5. N. Economides and J. Tag, “Net neutrality on the internet: A two-sided market anal- ysis”, working paper. Available at http://ideas.repec.org/p/net/wpaper/0714.html ysis”, working paper. Available at http://ideas.repec.org/p/net/wpaper/0714.html 6. V. Claudia Saavedra “Bargaining power and the net neutrality debate”, working paper, 2010. Available at http://sites.google.com/site/claudiasaavedra/ References 1. R. Hahn and S. Wallsten, “The economics of net neutrality,” The Berkeley Economic Press Economists’ Voice, Vol.3, No.6, 2006, pp:1-7. 2. J. Musacchio, G. Schwartz and J. Walrand, “A two-sided market analysis of provider investment incentives with an application to the net-neutrality issue”, Review of Network Economics, 8(1), 2009. ( ) 3. E. Altman, P. Bernhard, S. Caron, G. Kesidis, J. Rojas-Mora and S.L. Wong. ”A Study of Non-Neutral Networks with Usage-based Prices”, 3rd ETM Workshop of ITC conference, 2010. 4. E. Altman, M.K. Hanawal and R. Sundaresan, ”Nonneutral network and the role of bargaining power in side payments”, NetCoop, Ghent, Belgium, Nov. 2010. 5. N. Economides and J. Tag, “Net neutrality on the internet: A two-sided market anal- ysis”, working paper. Available at http://ideas.repec.org/p/net/wpaper/0714.html 6. V. Claudia Saavedra “Bargaining power and the net neutrality debate”, working paper, 2010. Available at http://sites.google.com/site/claudiasaavedra/ Network Non-neutrality Debate: An Economic Analysis 13 7. J.P. Choi and B.C. Kim, “Net Neutrality and Investment Incentives”, working pa- per, 2008 http://ideas.repec.org/p/ces/ceswps/ 2390.html 8. T.B. Ma, D.M. Chiu, J.C.S. Lui et.al, “On Cooperative Settlement Between Con- tent, Transit and Eyeball Internet Service Providers”, to appear in IEEE/ACM Trans. on Networking. 9. P. Bangera and S. Gorinsky, “Impact of Preﬁx Hijacking on Payments of Providers”, Proc. of COMSNETS 2011, Jan.,2011. f 10. R. El-Azouzi, E. Altman and L. Wynter, “Telecommunications Network Equilib- rium with Price and Quality-of-Service Characteristics”, in Proc. of ITC, 2003. R. El-Azouzi, E. Altman and L. Wynter, “Telecom 11. J. Liu, D.M. Chiu, “Mathematical Modeling of Competition in Sponsored Search Market”, in ACM Workshop on NetEcon’10, Vancouver, 2010. 12. E. Altman, A. Legout and Y.D. Xu, “Network Non-neutrality De- bate: An Economic Analysis”, in Technical Report, 2010. Available at http://arxiv.org/abs/1012.5862 13. B.E. Hermalin and M.L. Katz, “The Economics of Product-Line Restrictions With an Application to the Network Neutrality Debate”, Information Economics and Policy, Vol.19, pp. 215-248, 2007.
https://openalex.org/W4280616444	https://www.frontiersin.org/articles/10.3389/fncel.2022.884788/pdf	English	null	Focused Ultrasound Promotes the Delivery of Gastrodin and Enhances the Protective Effect on Dopaminergic Neurons in a Mouse Model of Parkinson’s Disease	Frontiers in cellular neuroscience	2,022	cc-by	10,404	ORIGINAL RESEARCH published: 17 May 2022 doi: 10.3389/fncel.2022.884788 Keywords: focused ultrasound, blood–brain barrier, gastrodin, Parkinson’s disease, drug delivery Reviewed by: Zengbing Lu, The Chinese University of Hong Kong, Hong Kong SAR, China Sheng Zhang, Zhejiang Provincial People’s Hospital, China Reviewed by: Zengbing Lu, The Chinese University of Hong Kong, Hong Kong SAR, China Sheng Zhang, Zhejiang Provincial People’s Hospital, China Correspondence: Moxian Chen chenmoxian@kmmu.edu.cn Lijuan Ao 13508710081@qq.com †These authors have contributed equally to this work Specialty section: This article was submitted to Cellular Neuropathology, a section of the journal Frontiers in Cellular Neuroscience Received: 27 February 2022 Accepted: 19 April 2022 Published: 17 May 2022 1 School of Rehabilitation, Kunming Medical University, Kunming, China, 2 Yunnan Cancer Center, Department of Radiology, Yunnan Cancer Hospital, The Third Afﬁliated Hospital of Kunming Medical University, Kunming, China, 3 Department of Physical Medicine and Rehabilitation, National Taiwan University Hospital, National Taiwan University College of Medicine, Taipei City, Taiwan Edited by: Feng Zhang, Third Hospital of Hebei Medical University, China Edited by: Feng Zhang, Third Hospital of Hebei Medical University, China Parkinson’s disease (PD) is the second most common chronic neurodegenerative disease globally; however, it lacks effective treatment at present. Focused ultrasound (FUS) combined with microbubbles could increase the efﬁcacy of drug delivery to speciﬁc brain regions and is becoming a promising technology for the treatment of central nervous system diseases. In this study, we explored the therapeutic potential of FUS-mediated blood–brain barrier (BBB) opening of the left striatum to deliver gastrodin (GAS) in a subacute PD mouse model induced by 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP). The concentration of GAS in the left hemisphere was detected by ultra-high performance liquid chromatography electrospray Q-Orbitrap mass spectrometry (UHPLC/ESI Q-Orbitrap) and the distribution of tyrosine hydroxylase (TH) neurons was detected by immunohistochemical staining. The expression of TH, Dopamine transporter (DAT), cleaved-caspase-3, B-cell lymphoma 2 (Bcl-2), brain- derived neurotrophic factor (BDNF), postsynaptic density protein 95 (PSD-95), and synaptophysin (SYN) protein were detected by western blotting. Analysis showed that the concentration of GAS in the left hemisphere of PD mice increased by approximately 1.8-fold after the BBB was opened. FUS-mediated GAS delivery provided optimal neuroprotective effects and was superior to the GAS or FUS control group. In addition, FUS enhanced GAS delivery signiﬁcantly increased the expression of Bcl-2, BDNF, PSD-95, and SYN protein in the left striatum (P < 0.05) and reduced the levels of cleaved-caspase-3 remarkably (P = 0.001). In conclusion, the enhanced delivery by FUS effectively strengthened the protective effect of GAS on dopaminergic neurons which may be related to the reinforcement of the anti-apoptotic activity and the expression of synaptic-related proteins in the striatum. Data suggests that FUS-enhanced GAS delivery may represent a new strategy for PD treatment. Study Design In order to test the safety of the BBB opening induced by FUS, healthy C57BL/6J mice were randomly divided into Sham group and FUS output voltage groups of 100, 150, and 200 mV. The opening of the BBB was visualized by Evans blue (EB, Millipore Sigma, Burlington, MA, United States) that exudated from the blood stream, and the safety was veriﬁed by hematoxylin- eosin (H&E) as well as Nissl staining. By considering the EB- stained area of the brain section and the results of pathological staining, the optimal FUS parameters to open the BBB were determined. Next, we investigated the eﬃcacy of FUS-induced BBB opening to delivery GAS in PD mice. After adaptation for 2 weeks, 48 male C57BL/6J mice were randomly divided into ﬁve subgroups: a Sham group (n = 8), an MPTP group (n = 8), an MPTP + GAS group (n = 12), an MPTP + FUS group (n = 8) and an MPTP + FUS + GAS group (n = 12). To establish a subacute model of PD on C57BL/6J mice, MPTP (Sigma- Aldrich, St. Louis, MO, United States) was dissolved in saline and subcutaneously injected 30 mg/kg for 5 consecutive days. The Sham group was injected with an equal volume of saline instead. Next, the MPTP + GAS group and the MPTP + FUS + GAS group were intraperitoneally injected with GAS (100 mg/kg/d) for 19 consecutive days; the other groups were injected with saline. FUS sonication was carried out once every 3 days with a total of 6 times. The Sham group, the MPTP group, and the MPTP + GAS group received placebo FUS sonications. Mice in the MPTP + FUS + GAS group were intraperitoneally injected with GAS immediately after the opening of the BBB to facilitate its delivery into the brain. We employed UHPLC/ESI Q-Orbitrap to assess the concentration of GAS in the left hemisphere. Behavioral tests were performed on the 1st, 7th, 13th, and 19th days after MPTP injection to evaluate the motor function of mice. Gastrodin, the main active component of Gastrodia elata, exerts neuroprotective eﬀects in various neurological diseases. Many studies have conﬁrmed the eﬃcacy of GAS in treating PD (Yan et al., 2019; He et al., 2021). The mechanism of action may involve the anti-oxidation eﬀect (Wang et al., 2014), anti-inﬂammation eﬀect (Li et al., 2012) and the inhibition of apoptosis (Chen et al., 2017) by GAS. INTRODUCTION (including the striatum), thus enhancing the protective eﬀect of GAS on dopaminergic neurons (Figure 1A). After conﬁrming that FUS can safely and eﬀectively open the BBB of the left striatum, we established a subacute PD mouse model by injecting 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). ultra-high performance liquid chromatography electrospray Q-Orbitrap mass spectrometry (UHPLC/ESI Q-Orbitrap) was then used to detect the concentration of GAS in the left hemisphere of the PD mice. Finally, the therapeutic eﬀect of GAS delivered by FUS on PD mice was evaluated by immunohistochemical staining and western blotting. Parkinson’s disease (PD) is characterized with resting tremor, bradykinesia, rigidity, and postural balance disorders, which is accompanied by non-motor symptoms such as anxiety and depression, and seriously damages patients’ quality of life (Armstrong and Okun, 2020). In 2016, approximately 6.1 million people were diagnosed with PD worldwide, which was about 2.4-fold higher than the number in 1990 (GBD, 2018). Drugs such as carbidopa-levodopa and dopamine agonists can alleviate the dyskinesia caused by early PD; however, their eﬃcacy ﬂuctuates with long-term use, leading to adverse reactions such as dyskinesia and on-oﬀphenomena, thus making it diﬃcult to control the patient’s condition (Armstrong and Okun, 2020). Citation: Wang Y, Luo K, Li J, Liao Y, Liao C, Chen W-S, Chen M and Ao L (2022) Focused Ultrasound Promotes the Delivery of Gastrodin and Enhances the Protective Effect on Dopaminergic Neurons in a Mouse Model of Parkinson’s Disease. Front. Cell. Neurosci. 16:884788. doi: 10.3389/fncel.2022.884788 May 2022 \| Volume 16 \| Article 884788 1 Frontiers in Cellular Neuroscience \| www.frontiersin.org Noninvasive Drug Delivery Improves PD Wang et al. Animals The protocol of this experiment was approved by the Animal Ethics Committee of Kunming Medical University (KMMU2019076). All the experimental procedures followed the guidelines for the care of laboratory animals. All male C57BL/6J mice (8 weeks old, 20–22 g) were purchased from the Experimental Animal Center of Kunming Medical University and housed at 25◦C ± 2◦C with a ﬁxed 12 h light/dark cycle. All mice had free access to food and water. MATERIALS AND METHODS p g The blood–brain barrier (BBB) blocks the entry of certain therapeutic drugs into the brain and represents a key obstacle in terms of treating PD. Under physiological conditions, 98% of drugs with a molecular weight of fewer than 400 Daltons and almost 100% of drugs with a molecular weight of more than 500 Daltons cannot pass through the tight junctions of BBB (Pardridge, 2005). Focused ultrasound (FUS) is a new method that could open the BBB and is highly penetrative, non-invasive with good localization and reversibility. In animal and clinical trials, researchers have demonstrated that the combination of FUS and microbubbles can safely as well as reversibly induce the opening of the BBB under speciﬁc parameters (Rezai et al., 2020; Wu et al., 2020; Pouliopoulos et al., 2021; Yang et al., 2021). Recently, our team found that the delivery of gastrodin (GAS) via FUS-induced BBB opening could improve memory impairment and neuropathology in a mouse model of Alzheimer’s disease (Luo et al., 2022). Moreover, studies have shown that the FUS-mediated delivery of neurotrophic factors and genes can eﬀectively inhibit the rapid progression of neurodegeneration in a mouse model of PD and improve neurological function (Mead et al., 2017; Ji et al., 2019). Frontiers in Cellular Neuroscience \| www.frontiersin.org Study Design (C,D) Acoustic pressure distribution of the lateral and axial direction The body weight of the mice was monitored every 3 days once. The mice were sacriﬁced on the 20th day for relevant biochemical tests (Figure 1B). United Kingdom). The lateral and axial sound pressure distributions are shown in Figures 1C,D, which indicates that the ultrasound transducer was well focused. The mice were anesthetized with isoﬂurane in the animal chamber and then the heads were ﬁxed with prone position. The anesthesia was maintained continuously by inhaled isoﬂurane with a concentration of 1% through an anesthesia machine (RWD R500, Shenzhen, Guangdong, China). Next, we shaved the fur on the top of the mouse’s head oﬀand removed any remaining fur with hair removal cream. The ultrasonic transducer was placed in an adapter designed for directional FUS delivery and ﬁxed to the left striatum region (0.5 mm front Bregma, 2 mm left). Then, we ﬁlled the gap between the mouse scalp and the ultrasound adapter with ultrasound couplant. SonoVue (Bracco Imaging BV, Study Design However, only a tiny amount of GAS can enter and disperse throughout the brain in terms of intravenous or oral administration (Lin et al., 2008). Kumar et al. (2013) found that increasing the dose of orally administered GAS could enhance the therapeutic eﬀect of GAS in PD. In another study, Haddadi et al. (2018) attempted to inject GAS into the substantia nigra of rats directly by microinjection; although the concentration of drug increased in speciﬁc brain region, the invasive nature of the method was challenging from a clinical point of view. Therefore, it is necessary to identify a non-invasive method to increase the concentration of GAS in speciﬁc brain regions without increasing the drug dose as this may represent an eﬀective method to improve the treatment eﬃciency of PD. We hypothesize that FUS mediated BBB opening can safely and eﬀectively increase the concentration of GAS in the brain May 2022 \| Volume 16 \| Article 884788 Frontiers in Cellular Neuroscience \| www.frontiersin.org 2 Noninvasive Drug Delivery Improves PD Wang et al. FIGURE 1 \| (A) Hypothesis of the delivery of gastrodin (GAS) by focused ultrasound (FUS)-induced blood–brain barrier (BBB) opening. FUS induced BBB opening increased the delivery of GAS to speciﬁc brain regions in Parkinson’s disease (PD) mice, thereby enhanced neuroprotective effect on dopaminergic neurons. (B) Details relating to experimental timing including behavioral testing and FUS operation. MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; UHPLC/ESI Q-Orbitrap, ultra-high performance liquid chromatography electrospray Q-Orbitrap mass spectrometry. (C,D) Acoustic pressure distribution of the lateral and axial direction. ypothesis of the delivery of gastrodin (GAS) by focused ultrasound (FUS)-induced blood–brain barrier (BBB) opening. FUS induced BBB opening very of GAS to speciﬁc brain regions in Parkinson’s disease (PD) mice, thereby enhanced neuroprotective effect on dopaminergic neurons. g to experimental timing including behavioral testing and FUS operation. MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; UHPLC/ESI high performance liquid chromatography electrospray Q-Orbitrap mass spectrometry. (C,D) Acoustic pressure distribution of the lateral and axial FIGURE 1 \| (A) Hypothesis of the delivery of gastrodin (GAS) by focused ultrasound (FUS)-induced blood–brain barrier (BBB) opening. FUS induced BBB opening increased the delivery of GAS to speciﬁc brain regions in Parkinson’s disease (PD) mice, thereby enhanced neuroprotective effect on dopaminergic neurons. (B) Details relating to experimental timing including behavioral testing and FUS operation. MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; UHPLC/ESI Q-Orbitrap, ultra-high performance liquid chromatography electrospray Q-Orbitrap mass spectrometry. Blood–Brain Barrier Opening Induced by Focused Ultrasound The FUS sonication system consisted of a waveform generator (RIGOL DG4202, Suzhou, China), a power ampliﬁer (Mini- circuits LZY-22+, New York, United States), and a focused ultrasonic transducer (fundamental frequency: 1 MHz, focal length: 4 cm, diameter: 15 mm). Two-dimensional sound ﬁeld distributions were measured using a calibrated needle hydrophone (2010, Precision Acoustics, Dorchester, May 2022 \| Volume 16 \| Article 884788 Frontiers in Cellular Neuroscience \| www.frontiersin.org 3 Noninvasive Drug Delivery Improves PD Wang et al. 180◦, and recorded the time that mice fell oﬀthe grid. The upper limit was up to 90 s. Milan, Italy) microbubbles (1.25 µl/g) were injected into the tail vein. Ten seconds later, the mice were sonicated with FUS. The pulse repetition frequency of FUS was 1 Hz, with a pulse width of 10 ms, and an exposure time of 60 s, output voltage of 100, 150, and 200 mV were used. Detection of Gastrodin Concentration by UHPLC/ESI Q-Orbitrap p We examined GAS concentration in the left hemisphere of mouse after the ﬁrst time of BBB opening by FUS. Mice in the MPTP + GAS and MPTP + FUS + GAS groups (n = 4 for each group) were randomly selected and sacriﬁced 30 min after the injection of GAS. The tissue of the left hemisphere was separated and placed in a cryotube at −80◦C. A standard sample of GAS (G299059-5 g, 62499-27-8, ≥98%, Aladdin, Shanghai, China) was weighed and mixed with pure methanol to prepare a 5.0 mg/ml reserve solution which was then diluted into 1000, 500, 200, 100, and 50 ng/mL standard solutions for analysis and to create a GAS standard curve. The tissue samples were weighed, mixed with methanol, grinded and then centrifuged; the supernatant was taken for testing subsequently. Levels of GAS in the left hemisphere were then quantiﬁed with a UltiMate 3000 RS chromatograph (Thermo Fisher Scientiﬁc, Waltham, MA, United States) and Q-Exactive high-resolution mass spectrometer (Thermo Fisher Scientiﬁc, Waltham, MA, United States). We used a positive electrospray ionization source (ESI); the electrospray voltage was 3.2 kV, the capillary temperature was 300◦C, high purity argon was used as the impact gas, the sheath gas was 40 Arb, and the auxiliary gas was 15 Arb. The system was equipped with a Waters T3 150 × 2.1 mm, 3- µm column with an automatic injection volume of 5 µl and a ﬂow rate of 0.30 ml/min. Chromatogram acquisition and the integration of analytes were processed by Xcalibur 4.1 software (Thermo Fisher Scientiﬁc, Waltham, MA, United States). Linear regression was performed with a weighting factor of 1/X. Immunohistochemistry Staining y g In order to evaluate the eﬀects of MPTP administration and diﬀerent treatments on dopaminergic neurons in the nigrostriatal pathway, we performed tyrosine hydroxylase (TH) immunohistochemical staining to the striatum and substantia nigra brain tissue of each group. Paraﬃn sections of brain tissue (4-µm-thick) were prepared from each mouse. Following antigen repair and sealing, the sections were incubated with TH antibody (Cat. No. ab137869, dilution 1:500, Abcam, Cambridge, United Kingdom) at 4◦C. A biotinylated goat anti-rabbit secondary antibody (Service GB23303, dilution 1:200, Wuhan, China) was added the next day and TH staining was observed under a microscope after 50 min incubation at room temperature. Western Blotting Fresh left striatum and substantia nigra were collected and stored at −80◦C for use. The total proteins were extracted by adding radioimmunoprecipitation assay (RIPA) buﬀer and phenylmethanesulfonyl ﬂuoride (PMSF) protease inhibitor. A bicinchoninic acid (BCA) protein concentration determination kit (Beyotime, China) was used to measure the protein concentration of each sample. Supernatants were dissolved in sample buﬀer at a protein concentration of 20 µg and Disruption and Safety Twenty-four male C57BL/6J mice were randomly divided into a Sham group (n = 6) and the FUS group. The FUS group was further divided into 100, 150, and 200 mV groups (n = 6 for each group) to determine the optimal output voltage for FUS-induced BBB opening when other parameters were ﬁxed. Immediately after FUS sonication, 2% of EB dye (6.25 µl/g) was injected through the tail vein. We injected microbubbles and EB dye in the Sham group with placebo FUS sonication. The mice were euthanized after 4 h of EB circulation and then the brain was ﬁxed in paraformaldehyde solution for 24 h (n = 3 for each group). Next, we prepared coronal brain slices which covered the center of the ultrasound-sonicated region. The BBB opened area was visualized by EB penetration. Brain tissues from mice in the Sham and FUS group were collected at 4 h after FUS sonication (n = 3 for each group) to test the safety of FUS-induced BBB opening. After dehydration and ﬁxation, tissues were embedded in paraﬃn and then cut into coronal sections with the thickness of 4 µm. Paraﬃn sections of the center of sonicated brain region were dewaxed in xylene and anhydrous ethanol and then stained with H&E (Service G1003, Wuhan, Hubei, China). For Nissl staining, the paraﬃn sections were dewaxed and stained with toluidine blue, diﬀerentiated with glacial acetic acid, cleared with xylene, and then sealed with neutral gum. After the optical parameters were determined on normal C57BL/6J mice, we carried out BBB opening of left striatum by FUS for six times with the optimal parameters on PD mice and reassessed the safety by H&E and Nissl staining. Images of the left cortex and striatum in the brain sections were acquired using a dissecting microscope (Olympus, Japan). Assessment of Motor Function Pole Test As previously described, we performed the pole test to assess motor function of mice. A 50-cm–long pole was wrapped with gauze to prevent mice from slipping and a wooden ball was placed on the top (Zhang et al., 2017). The bottom was covered with bedding to protect the mice from fall injury. Mice were trained three times in advance to ensure that all mice could bow their heads down when placed on the ball. Each mouse was placed on the wooden ball and the total time that mouse taken to climb from the top of the pole to the bottom was recorded. Paw Grip Endurance Test The Paw grip endurance test can evaluate comprehensive motor function, such as muscle strength, muscle tone, and ataxia (Hutter-Saunders et al., 2012). We chose a 45 cm × 30 cm rectangular stainless steel screen, the built-in mesh is 1 cm2, and the screen is 25 cm high from the pad. During the test, we placed the mice horizontally on the grid and then quickly ﬂipped the grid May 2022 \| Volume 16 \| Article 884788 Frontiers in Cellular Neuroscience \| www.frontiersin.org Noninvasive Drug Delivery Improves PD Wang et al. separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Separated proteins were then transferred to a polyvinylidene ﬂuoride (PVDF) membrane (Millipore Sigma, Burlington, MA, United States). After blocking with 5% skimmed milk for 2 h, the PVDF membrane was incubated with primary antibodies and placed overnight in a refrigerator at 4◦C. The primary antibodies included TH (Cat. No. ab137869, dilution 1:5000, Abcam, Cambridge, United Kingdom), Dopamine transporter (DAT, Cat. No. 22524- 1-AP, dilution 1:2000, Proteintech, Rosemont, IL, United States), B-cell lymphoma 2 (Bcl-2, Cat. No. 26593-1-AP, dilution 1:2000, Proteintech, Rosemont, IL, United States), caspase-3 (Cat. No. 19677-1-AP, dilution 1:2000, Proteintech, Rosemont, IL, United States), postsynaptic density protein 95 (PSD-95, Cat. No. 20665-1-Ig, dilution 1:2000, Proteintech, Rosemont, IL, United States), synaptophysin (SYN, Cat. No. 60191-1-Ig, dilution 1:2000, Proteintech, Rosemont, IL, United States), brain- derived neurotrophic factor (BDNF, Cat. No. 66292-1-Ig, dilution 1:2000, Proteintech, Rosemont, IL, United States), GAPDH (Cat. No. 60004-1-Ig, dilution 1:2000, Proteintech, Rosemont, IL, United States), β-Actin (Cat. No. sc-47778, dilution 1:2000, Santa Cruz Biotechnology, Dallas, TX, United States), and α-Tubulin (cat. No. 66031-1-Ig, dilution 1:2000, Proteintech, Rosemont, IL, United States). Following incubation with primary antibodies, the membranes were incubated with anti-rabbit/anti- mouse immunoglobulin G (IgG) enzyme-linked antibody labeled with secondary anti-horseradish peroxidase (HRR-) for 90 min at room temperature. Finally, after washing with TBST, the membranes were developed on a gel developer and the gray values of diﬀerent protein bands were quantiﬁed. was shallow and limited to the cortex. There was prominent EB inﬁltration in the left striatum of the 150 and 200 mV groups, and the EB inﬁltration area was larger in the 200 mV group. However, H&E staining showed that the sonicated cortex and striatum in the 200 mV group had obvious erythrocyte exudation. In contrast, there was no bleeding in other groups, the cells were evenly arranged, and the nucleoli were normal (Figure 2B). Statistical Analysis All data are expressed as mean ± standard error of the mean (SEM). We used SPSS version 25.0 (IBM, Armonk, NY, United States) for statistical analysis. The independent sample t-test was used to compare the mean diﬀerence between the two groups. One-way ANOVA was used to compare diﬀerences in means among the ﬁve subgroups. When analysis of variance showed signiﬁcant diﬀerences, pairwise comparisons between means were performed with the least signiﬁcant diﬀerence (LSD) test for post hoc analysis. Statistical graphs were generated using GraphPad Prism software version 8.0 (GraphPad Software, Inc., San Diego, CA, United States). Diﬀerences were considered to be signiﬁcant when P < 0.05 (bilateral). Movement Defect Were Induced by 1-Methyl-4-Phenyl-1,2,3,6- Tetrahydropyridine but Recovered 7 Days Later The climbing time of the pole test in each group was statistically longer than that of the Sham group (P < 0.05), while the grid grasping time in the paw grip endurance test was signiﬁcantly shorter when compared to the Sham group on day 1 (P < 0.05). These data suggest that MPTP induced dyskinesia and the PD model was established successfully. The climbing time of the pole test as well as the grid grasping time in the paw grip endurance test between all groups at day 7, 13, and 19 lacked statistical signiﬁcance (P > 0.05), indicating an auto recovery of movement defect (Supplementary Figures 1A,B). Paw Grip Endurance Test For Nissl staining, the neurons of mice in 100 mV, 150 mV, and Sham group were in good shape and obvious abnormality was absent (Figure 2C). Therefore, 150 mV was selected as the optimal output voltage. After six times’ opening of BBB in PD mice, we again performed histological analysis. Likewise, H&E staining showed no hemorrhage in the ultrasound-sonicated brain area (Figure 2D). The distribution and size of Nissl bodies in the MPTP group and MPTP + FUS group were homogeneous (Figure 2E). The body weight changes of the mice during the experiment are shown in Figure 2F. During the treatment period, there was no statistical diﬀerence of body weight between the MPTP + FUS, MPTP + FUS + GAS and the MPTP groups (P > 0.05). These data show that it is safe and feasible to use FUS to repeatedly induce the opening of the BBB. Focused Ultrasound-Induced Blood–Brain Barrier Opening Increased the Uptake of Gastrodin in the Left Hemisphere of Parkinson’s Disease Mice The concentration of GAS in the left hemisphere was 0.3679 ng/mg in the MPTP + GAS group, After FUS-induced BBB opening, the concentration of GAS increased signiﬁcantly (P = 0.03), reaching to 0.6619 ng/mg, approximately 1.8-fold higher than the MPTP + GAS group (Figure 3). Efﬁcacy and Safety of Focused Ultrasound-Induced Blood–Brain Barrier Opening First, we investigated the eﬃcacy of FUS-induced BBB opening by evaluating the EB penetration. As shown in Figure 2A, all FUS groups were stained by EB, indicating that FUS increased the permeability of the BBB and facilitated EB entry into the brain. However, in the 100 mV group, the penetration depth To investigate the eﬀect of FUS + GAS treatment on dopaminergic neurons in the nigrostriatal pathway of PD mice, we used immunohistochemistry assay to stain TH, and the results are shown in Figures 4A–C. Compared with the MPTP group, May 2022 \| Volume 16 \| Article 884788 Frontiers in Cellular Neuroscience \| www.frontiersin.org 5 Noninvasive Drug Delivery Improves PD Wang et al. FIGURE 2 \| Efﬁcacy and safety of focused ultrasound (FUS)-induced blood–brain barrier (BBB) opening. (A) Penetration of Evans blue in the brains of mice in different groups 4 h after FUS-induced BBB opening, n = 3. (B) H&E staining results of sonicated cerebral cortex and striatum in different groups 4 h after FUS-induced BBB opening, n = 3, scale bar = 50 µm. (C) Nissl staining results of sonicated cerebral cortex and striatum in different groups 4 h after FUS-induced BBB opening, n = 3, scale bar = 50 µm. (D) H&E staining results of sonicated cerebral cortex and striatum in Parkinson’s disease (PD) mice after a total of six times’ BBB opening induced by FUS, n = 3. Scale bar = 50 µm. (E) Nissl staining results of sonicated cerebral cortex and striatum in PD mice after a total of six times’ BBB opening induced by FUS n = 3 Scale bar = 50 µm (F) The body weight change curve of mice in each group FIGURE 2 \| Efﬁcacy and safety of focused ultrasound (FUS)-induced blood–brain barrier (BBB) opening. (A) Penetration of Evans blue in the brains of mice in different groups 4 h after FUS-induced BBB opening, n = 3. (B) H&E staining results of sonicated cerebral cortex and striatum in different groups 4 h after FUS-induced BBB opening, n = 3, scale bar = 50 µm. (C) Nissl staining results of sonicated cerebral cortex and striatum in different groups 4 h after FUS-induced BBB opening, n = 3, scale bar = 50 µm. (D) H&E staining results of sonicated cerebral cortex and striatum in Parkinson’s disease (PD) mice after a total of six times’ BBB opening induced by FUS, n = 3. Efﬁcacy and Safety of Focused Ultrasound-Induced Blood–Brain Barrier Opening Scale bar = 50 µm. (E) Nissl staining results of sonicated cerebral cortex and striatum in PD mice after a total of six times’ BBB opening induced by FUS, n = 3. Scale bar = 50 µm. (F) The body weight change curve of mice in each group. May 2022 \| Volume 16 \| Article 884788 Frontiers in Cellular Neuroscience \| www.frontiersin.org Frontiers in Cellular Neuroscience \| www.frontiersin.org 6 Wang et al. Noninvasive Drug Delivery Improves PD FIGURE 3 \| Changes of GAS concentration in the mice’s left hemisphere of the MPTP + GAS group and the MPTP + FUS + GAS group. Compared to the MPTP + GAS group, P < 0.05; T-test, n = 4, mean ± SEM. FIGURE 4 \| (A) Comparison of the immunohistochemical staining of tyrosine hydroxylase (TH)-positive nerve ﬁbers in the left striatum and substantia nigra of mice in each group, n = 3. Scale bars = 50 µm for images in stratum; 200 µm for images in substantia nigra. (B) Changes of TH-positive nerve ﬁbers in the left striatum of mice in each group. (C) Number changes of TH-positive cells in the left substantia nigra of mice in each group. (D,E) Representative images of the protein levels of TH and Dopamine transporter (DAT) in the left striatum and substantia nigra of mice in each group. (Continued) FIGURE 3 \| Changes of GAS concentration in the mice’s left hemisphere of the MPTP + GAS group and the MPTP + FUS + GAS group. Compared to the MPTP + GAS group, P < 0.05; T-test, n = 4, mean ± SEM. the density of TH- positive ﬁbers and the number of TH-positive cells in the nigrostriatal pathway of the MPTP + FUS + GAS group recovered signiﬁcantly (P = 0.016). Further qualitative analysis of the protein expression levels of TH and DAT in the left striatum and substantia nigra showed that the protein expression levels of TH and DAT in the MPTP group were lower than the levels of the Sham group (P < 0.05). When dealt with FUS + GAS treatment, the protein levels of TH as well as DAT in the left striatum and substantia nigra increased and were signiﬁcantly higher than those of the MPTP group (P < 0.05), while there was no signiﬁcance (P > 0.05) between the Sham group and the FUS + GAS group (Figures 4D–I). Efﬁcacy and Safety of Focused Ultrasound-Induced Blood–Brain Barrier Opening Compared with the MPTP group, the number of TH neurons in the left nigrostriatal pathway and the protein expression levels of TH and DAT were increased with varying degrees after FUS or GAS treatment, although there was no statistical signiﬁcance (P > 0.05). Collectively, these results suggest that FUS-induced BBB opening allows the entry of GAS, thus promoting dopamine synthesis and eﬀectively ameliorated MPTP-induced dopaminergic neuron death. The Focused Ultrasound Mediated Gastrodin Delivery Enhanced Anti-apoptotic Effects FIGURE 4 \| (A) Comparison of the immunohistochemical staining of tyrosine hydroxylase (TH)-positive nerve ﬁbers in the left striatum and substantia nigra of mice in each group, n = 3. Scale bars = 50 µm for images in stratum; 200 µm for images in substantia nigra. (B) Changes of TH-positive nerve ﬁbers in the left striatum of mice in each group. (C) Number changes of TH-positive cells in the left substantia nigra of mice in each group. (D,E) Representative images of the protein levels of TH and Dopamine transporter (DAT) in the left striatum and substantia nigra of mice in each group. (Continued) We found that the level of cleaved-caspase-3 protein in the left striatum after MPTP injection was signiﬁcantly higher than that of the Sham group (P = 0.014) and the level of Bcl-2 signiﬁcantly decreased (P < 0.001), suggesting that the administration of MPTP led to remarkable apoptosis. When dealt with FUS + GAS treatment, the expression level of cleaved-caspase-3 in the left striatum were signiﬁcantly lower than that of the MPTP group May 2022 \| Volume 16 \| Article 884788 Frontiers in Cellular Neuroscience \| www.frontiersin.org 7 Noninvasive Drug Delivery Improves PD Wang et al. administration (Su et al., 2020), but these methods have obvious limitations. Hypertonic solution and electroacupuncture stimulation open the BBB extensively rather than locally, resulting in the high risk of pathogens and toxic substances to enter the brain. Electroporation and intracranial injection are invasive treatments and face big challenge to repeated operations. Enzymes quickly degrade drugs administered intranasally, and the clearing function of nasal cilia reduces the time that drug get contact with the nasal epithelial cells, which decreases drug absorption. Under appropriate parameters, FUS combined with microbubbles can open the BBB non-invasively, locally, and reversibly thus enhances the delivery of antibodies (Jordão et al., 2010), neurotrophic factors (Lin et al., 2016; Karakatsani et al., 2019), nanoparticles (Ohta et al., 2020) and macromolecular drugs (Park et al., 2017; Wei et al., 2021) to speciﬁc brain regions. Due to the drug’s short half-life, it is necessary to open the BBB to deliver the drug repeatedly. Several previous studies reported that when the BBB was continuously opened 8 times in non-human primates at a frequency of every 15 days once, there were no pathological changes in the EEG and somatosensory evoked potentials (Horodyckid et al., 2017). The Focused Ultrasound Mediated Gastrodin Delivery Enhanced Anti-apoptotic Effects Another research group opened the BBB every 2 days once in rodents and found that repeated BBB opening by FUS at low sound pressure with appropriate microbubbles dose did not cause tissue damage or behavior change. Nevertheless, there were slight and transient behavioral changes when the pressure was signiﬁcantly higher than required or with excessive microbubbles dose (Tsai et al., 2018). We opened the BBB every 3 days once for a total of 6 times in this experiment. During the experiment, there was no signiﬁcant diﬀerence in mice’s body weight between the FUS group and the MPTP group. At the same time, the pathological sections of the striatum and cortex in the sonicated site showed no abnormality, indicating that the ultrasonic parameters used in this experiment are safe and feasible, exerting great potential to repeated delivery of drugs by FUS FIGURE 4 \| (F–I) The left striatum and substantia nigra of mice in each group and changes in the protein expression of TH and DAT. Compared to MPTP group, P < 0.05; P < 0.01; P < 0.001, no signiﬁcance (ns), P > 0.05. One-way ANOVA with LSD test; n = 5, mean ± SEM. FIGURE 4 \| (F–I) The left striatum and substantia nigra of mice in each group and changes in the protein expression of TH and DAT. Compared to MPTP group, P < 0.05; P < 0.01; P < 0.001, no signiﬁcance (ns), P > 0.05. One-way ANOVA with LSD test; n = 5, mean ± SEM. (P = 0.001). Furthermore, the expression levels of Bcl-2 were signiﬁcantly up-regulated by FUS + GAS treatment compared with the MPTP group (P = 0.001); the anti-apoptotic eﬀect of FUS + GAS was stronger than GAS treatment alone (P < 0.05) (Figures 5A,B). Focused Ultrasound Mediated Gastrodin Delivery Upregulated the Expressions of Brain-Derived Neurotrophic Factor, Synaptophysin, and Postsynaptic Density Protein 95 Next, we performed western blotting to investigate the eﬀect of FUS + GAS treatment on the expressions of BDNF, SYN, and PSD-95 in the left striatum of PD mice (Figure 6). We found that the protein expression levels of BDNF, SYN, and PSD-95 decreased after the injection of MPTP (P < 0.05) while increased after FUS or GAS treatment. The protein levels of BDNF (P < 0.001), SYN (P < 0.001) and PSD-95 (P = 0.003) in the FUS + GAS group were also signiﬁcantly higher than those of the MPTP group (Figures 6A–C). The BDNF level was higher in FUS + GAS group in comparison of FUS and GAS group (P < 0.01). For the expression levels of SYN and PSD-95, the mean values of the FUS + GAS group were higher than that of FUS as well as GAS group, but there was no statistical diﬀerence (P > 0.05). These results suggest that the delivery of GAS by FUS increased the expression of BDNF, SYN, and PSD-95 in the left striatum of MPTP mice. Frontiers in Cellular Neuroscience \| www.frontiersin.org DISCUSSION et al., 2018), and intranasal May 2022 \| Volume 16 \| Article 884788 Frontiers in Cellular Neuroscience \| www.frontiersin.org 8 Noninvasive Drug Delivery Improves PD Wang et al. FIGURE 5 \| (A) Changes in the expression of cleaved-caspase-3 in the left striatum of mice in each group. (B) Changes in the expression of B-cell lymphoma 2 (Bcl-2) protein in the left striatum of mice in each group. Compared to MPTP group, P < 0.05; P < 0.01; P < 0.001, no signiﬁcance (ns), P > 0.05. One-way ANOVA with LSD test; n = 5; mean ± SEM. FIGURE 5 \| (A) Changes in the expression of cleaved caspase 3 in the left striatum of mice in each group (B) Changes in the expression of B cell lymphoma 2 FIGURE 5 \| (A) Changes in the expression of cleaved-caspase-3 in the left striatum of mice in each group. (B) Changes in the expression of B-cell lymphoma 2 (Bcl-2) protein in the left striatum of mice in each group. Compared to MPTP group, P < 0.05; P < 0.01; P < 0.001, no signiﬁcance (ns), P > 0.05. One-way ANOVA with LSD test; n = 5; mean ± SEM. GAS has a short half-life and is poorly permeable to BBB, which limits its therapeutic eﬀect (Liu Y. et al., 2018). Opening the BBB is a good way to increase the GAS concentration in the brain and to prolong the half-life. It was shown that during the 24 h of BBB opening, GAS in CSF maintained at a high concentration (Kung et al., 2021). In this study, we used FUS to non-invasively as well as eﬀectively enhance the transport of GAS into the left hemisphere, which increased the concentration of GAS in the hemisphere of mice by approximately 1.8 times, eﬀectively improved the therapeutic eﬃcacy of GAS. Compared with the single intraperitoneal injection therapy, FUS induced BBB opening and transferred greater amount of GAS into the brain parenchyma and should reduce the drug consumption by plasma proteins and enzymes. Wang et al. (2009) found that GAS was concentrated in the cortex and cerebellum when administration group. Therefore, increasing the release of drugs in the nigrostriatal pathway may be an eﬀective way to improve drug eﬃcacy for the treatment of PD. GAS has a short half-life and is poorly permeable to BBB, which limits its therapeutic eﬀect (Liu Y. et al., 2018). DISCUSSION g y It was hypothesized that the degeneration of substantia nigra neurons in PD originate from the distal axon (Cheng et al., 2010). It is reported that approximately 30% of the dopamine neurons in the substantia nigra can be damaged while in the striatum it can be as severe as 50–70% in PD (Ma et al., 1997; Cheng et al., 2010). On this basis, the striatum may be an ideal target for PD treatment. MPTP induces PD-related symptoms by damaging dopaminergic neurons and decreasing the density of axons as well as dendrites in the nigrostriatal pathway. Therefore, we selected the MPTP-induced subacute PD model to simulate the symptoms of PD and took striatum as the treatment target. We found that FUS-induced opening of the BBB followed by intraperitoneal injection of GAS signiﬁcantly increased the number of TH- positive nerve ﬁbers and the expression of TH as well as DAT in the left striatum of PD mice. The concentration of GAS in the left hemisphere increased after FUS-induced BBB opening; the increased concentration enables more GAS to act directly on the damaged dopaminergic neurons. In the study of Ji et al. (2019) FUS was used to open the BBB of the striatum and substantia nigra. The curative eﬀect of the FUS + BDNF group was stronger than that of the FUS group and the BDNF intranasal In this study, we demonstrated that (1) FUS combined with microbubbles can repeatedly, eﬀectively and non-invasively open the BBB in the striatum of rodents without causing tissue damage. This method also increased the concentration of GAS in the left hemisphere by approximately 1.8-fold; (2) When delivered by FUS, GAS eﬀectively increased the number of dopaminergic neurons in the nigrostriatal pathway; (3) When delivered by FUS, GAS eﬀectively enhanced the anti-apoptotic ability in the striatum and promoted the expression of BDNF and synaptic-related proteins. This study innovatively identiﬁed that FUS-induced BBB opening can promote the intraperitoneally injected GAS into the brain and signiﬁcantly enhances the neuroprotective eﬀect of GAS on dopaminergic neurons in the nigrostriatal pathway. There are several methods facilitating the delivery of drugs into the brain including intra-arterial infusion of hypertonic solution (Kiviniemi et al., 2017), electroacupuncture stimulation (Zhang et al., 2020), electroporation (Lorenzo et al., 2019), intracranial injection (Liu Y. X. Frontiers in Cellular Neuroscience \| www.frontiersin.org DISCUSSION Opening the BBB is a good way to increase the GAS concentration in the brain and to prolong the half-life. It was shown that during the 24 h of BBB opening, GAS in CSF maintained at a high concentration (Kung et al., 2021). In this study, we used FUS to non-invasively as well as eﬀectively enhance the transport of GAS into the left hemisphere, which increased the concentration of GAS in the hemisphere of mice by approximately 1.8 times, eﬀectively improved the therapeutic eﬃcacy of GAS. Compared with the single intraperitoneal injection therapy, FUS induced BBB opening and transferred greater amount of GAS into the brain parenchyma and should reduce the drug consumption by plasma proteins and enzymes. Wang et al. (2009) found that GAS was concentrated in the cortex and cerebellum when Many studies have found that the therapeutic eﬃcacy of GAS is closely related to the therapeutic dose. In terms of AD treatment, 200 mg/kg of GAS was shown to restore the learning and memory ability of AD mice and reduced the deposition of Aβ plaques in the brain; the eﬃcacy of this dose was much higher than that of 100 and 50 mg/kg (Zhou et al., 2016). In another study, Doo et al. (2014) compared the therapeutic eﬀects of GAS at doses of 200, 400, and 800 mg/kg in rats with PD. The improvement of motor function of the 800 mg/kg group was greater than other groups; furthermore, the 800 mg/kg group showed a therapeutic eﬀect earlier than the low-dose group. Although increasing the dose can improve the drug’s eﬃcacy, May 2022 \| Volume 16 \| Article 884788 9 Noninvasive Drug Delivery Improves PD Wang et al. E 6 \| (A–C) Changes in the protein expression of brain-derived neurotrophic factor (BDNF), postsynaptic density protein 95 (PSD-95), and synaptophysin n the left striatum of mice in each group. Compared to MPTP group, P < 0.05 compared to MPTP group; P < 0.01; P < 0.001, no signiﬁcance (ns), 05. One-way ANOVA with LSD test; n = 5, mean ± SEM. FIGURE 6 \| (A–C) Changes in the protein expression of brain-derived neurotrophic factor (BDNF), postsynaptic density protein 95 (PSD-95), and synaptophysin (SYN) in the left striatum of mice in each group. Compared to MPTP group, P < 0.05 compared to MPTP group; P < 0.01; P < 0.001, no signiﬁcance (ns), P > 0.05. DISCUSSION One-way ANOVA with LSD test; n = 5, mean ± SEM. FIGURE 6 \| (A–C) Changes in the protein expression of brain-derived neurotrophic factor (BDNF), postsynaptic density protein 95 (PSD-95), and synaptophysin (SYN) in the left striatum of mice in each group. Compared to MPTP group, P < 0.05 compared to MPTP group; P < 0.01; *P < 0.001, no signiﬁcance (ns), P > 0.05. One-way ANOVA with LSD test; n = 5, mean ± SEM. Apoptosis is one of the main causes of dopaminergic neuron death in PD mice. The inhibition of apoptosis has been considered as a potential therapeutic strategy for PD. In the present study, we found that MPTP increased the expression of cleaved-caspase-3 protein in the striatum and decreased the expression of Bcl-2 protein, that is MPTP promoted apoptosis. However, the apoptosis was reversed by FUS + GAS treatment. Therefore, we speculate that the protective eﬀect of FUS + GAS entered the brain, and only 4.2% of the drugs could enter the striatum. We used FUS to open the BBB in the striatal region so that the drug can be concentrated in a speciﬁc brain region to give play to its eﬀect. Therefore, non-invasive and local BBB opening by FUS may be an eﬀective way to enhance GAS delivery and to reduce the injection dose or frequency of administration to a certain extent and exerts broad prospects for clinical application. May 2022 \| Volume 16 \| Article 884788 Frontiers in Cellular Neuroscience \| www.frontiersin.org 10 Noninvasive Drug Delivery Improves PD Wang et al. on dopaminergic neurons is partly due to the enhanced anti- apoptotic eﬀect in the striatum. A previous study also found that GAS increased the expression of Bcl-2 and inhibited MPTP-induced caspase-3 activation and protected dopaminergic neurons, in which the anti-apoptotic eﬀect increased as the dosage of GAS increased (Kumar et al., 2013). In the present study, the anti-apoptotic eﬀect of the FUS + GAS group was signiﬁcantly higher than the GAS group; this may also be related to the increase of GAS concentration after the opening of the BBB. were damaged, the striatum itself has a special compensatory mechanism, that is, the remaining dopaminergic neurons may release more dopamine and result in a signiﬁcant increase in the proportion of dopamine metabolites in the striatum (Luchtman et al., 2009; Zhang et al., 2017), thus improve the dyskinesia. What’s more, Rousselet et al. DISCUSSION (2003) suggested that dopamine in the prefrontal cortex may be transferred to the nigrostriatal system to play a compensatory role. The recovery of activity in MPTP mice may also be related to the increase of norepinephrine content. One study found a signiﬁcant norepinephrine increase in the striatum of MPTP- induced subacute PD mice, which may lead to overactivity (Rousselet et al., 2003). The reduction of dopamine in the striatum of PD may be related to ﬁber degeneration or loss and the synaptic reduction in the nigrostriatal pathway, while the eﬀective transport of dopamine is closely related to the integrity of the nigrostriatal pathway (Villalba and Smith, 2018). After injury to the nervous system, the levels of SYN reﬂect the degree of synaptic remodeling; furthermore, the accumulation of PSD- 95 in synapses can promote synaptic maturation as well as excitatory synapse enhancement (Kim et al., 2007). It has been found that MPTP reduces the density of dendritic spines in the striatum of mice and increases the expression of SYN while PSD-95 can restore the density of dendritic spines and relieve the symptoms of PD, at least to some extent (Toy et al., 2014). In addition, the increased expression of SYN and GAP43 can promote axonal regeneration and synaptic remodeling, thereby repairs the damaged dopamine transport pathway and increases dopamine release (Wang et al., 2008). BDNF can promote the maturation and genesis of synapses, and the increased expression of BDNF is essential to the increase of synaptic activity (Lipsky and Marini, 2007; Sen et al., 2016). In the present study, we found that FUS + GAS treatment could increase the expression of BDNF signiﬁcantly and improve the reduced PSD-95 and SYN level in the PD model induced by MPTP. That is, FUS + GAS may increase the number of terminal synaptic vesicles and the striatal synaptic density of dopaminergic neurons by increasing the expression of BDNF, which favors the enhancement of synaptic transmission, and promotes the release of dopamine. In addition, BDNF is also closely related to the growth and development of dopaminergic neurons (Palasz et al., 2020); therefore, an increase of BDNF may also improve the neuroprotective eﬀect of FUS + GAS. There are some limitations in the present study. First of all, the GAS concentration in the hemisphere was detected quantitatively by UHPLC/ESI Q-Orbitrap. ETHICS STATEMENT The animal study was reviewed and approved by the Animal Ethics Committee of Kunming Medical University. CONCLUSION In this study, FUS was employed to induce the BBB opening of striatal repetitively and safely in a mouse model of PD, which signiﬁcantly increased the concentration of GAS in the sonicated hemisphere and eﬀectively promoted the protective eﬀect of GAS on dopaminergic neurons, representing an exciting option for the treatment of PD. The enhanced delivery of GAS by FUS induced BBB opening may provide a promising alternative for the treatment of chronic neurodegenerative diseases. In this study, FUS was employed to induce the BBB opening of striatal repetitively and safely in a mouse model of PD, which signiﬁcantly increased the concentration of GAS in the sonicated hemisphere and eﬀectively promoted the protective eﬀect of GAS on dopaminergic neurons, representing an exciting option for the treatment of PD. The enhanced delivery of GAS by FUS induced BBB opening may provide a promising alternative for the treatment of chronic neurodegenerative diseases. DATA AVAILABILITY STATEMENT The pole and paw grip endurance tests are the most used behavioral methods to test MPTP-induced dopamine damage in the substantia nigra and striatum. Previous studies described dyskinesia of subacute PD mice induced by the injection of MPTP (Koppula et al., 2021; Qi et al., 2021). However, some studies have found that dyskinesia is not always apparent in this model, it may even present hyperactivity (Rousselet et al., 2003; Zhang et al., 2017). In the present study, the immunohistochemical staining of mice at the 20 days showed the dopaminergic neurons in the nigrostriatal pathway were severely damaged; however, the impairment of motor function in the experimental mice was not consistent with the pathological manifestations. This may be related to the compensatory ability of the dopaminergic system. It has been found that when the dopamine neurons in the nigra striatum The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. DISCUSSION However, the distribution and metabolism of GAS in the brain are not clear, which demands further research. Secondly, the dose of GAS (100 mg/kg) that was intraperitoneal injected in this study originates from the past experimental reports in rodents (60–800 mg/kg) (Doo et al., 2014; Wang et al., 2014; Liu Y. et al., 2018). In the view that opening of BBB will increase GAS concentration in the hemisphere of mice, it is necessary to explore the optimal dose of GAS under the appearance of FUS-induced BBB opening in terms of treating PD. Furthermore, the eﬀective maintaining time of GAS in the brain which is delivered by FUS-induced BBB opening need to be further clariﬁed to determine the best frequency of BBB opening induced by FUS. Frontiers in Cellular Neuroscience \| www.frontiersin.org REFERENCES doi: 10.3969/j.issn.1673-5374.2012.05.001 Haddadi, R., Poursina, M., Zeraati, F., and Nadi, F. (2018). Gastrodin microinjection suppresses 6-OHDA-induced motor impairments in parkinsonian rats: insights into oxidative balance and microglial activation in SNc. Inﬂammopharmacology 26, 1305–1316. doi: 10.1007/s10787-018-0470-4 g j Lin, C. Y., Hsieh, H. Y., Chen, C. M., Wu, S. R., Tsai, C. H., Huang, C. Y., et al. (2016). Non-invasive, neuron-speciﬁc gene therapy by focused ultrasound- induced blood-brain barrier opening in Parkinson’s disease mouse model. J. Control Rel. 235, 72–81. doi: 10.1016/j.jconrel.2016.05.052 He, J., Li, X., Yang, S., Li, Y., Lin, X., Xiu, M., et al. (2021). Gastrodin extends the lifespan and protects against neurodegeneration in the Drosophila PINK1 model of Parkinson’s disease. Food Funct. 12, 7816–7824. doi: 10.1039/ d1fo00847a Lin, L. C., Chen, Y. F., Lee, W. C., Wu, Y. T., and Tsai, T. H. (2008). Pharmacokinetics of gastrodin and its metabolite p-hydroxybenzyl alcohol in rat blood, brain and bile by microdialysis coupled to LC-MS/MS. J. Pharm. Biomed. Anal. 48, 909–917. doi: 10.1016/j.jpba.2008.07.013 Horodyckid, C., Canney, M., Vignot, A., Boisgard, R., Drier, A., Huberfeld, G., et al. (2017). Safe long-term repeated disruption of the blood-brain barrier using an implantable ultrasound device: a multiparametric study in a primate model. J. Neurosurg. 126, 1351–1361. doi: 10.3171/2016.3.JNS151635 Lipsky, R. H., and Marini, A. M. (2007). Brain-derived neurotrophic factor in neuronal survival and behavior-related plasticity. Ann. N. Y. Acad. Sci. 1122, 130–143. doi: 10.1196/annals.1403.009 Hutter-Saunders, J. A., Gendelman, H. E., and Mosley, R. L. (2012). Murine motor and behavior functional evaluations for acute 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP) intoxication. J. Neuroimmune Pharmacol. 7, 279– 288. doi: 10.1007/s11481-011-9269-4 Liu, Y., Gao, J., Peng, M., Meng, H., Ma, H., Cai, P., et al. (2018). A Review on Central Nervous System Eﬀects of Gastrodin. Front. Pharmacol. 9:24. doi: 10.3389/fphar.2018.00024 Ji, R., Smith, M., Niimi, Y., Karakatsani, M. E., Murillo, M. F., Jackson-Lewis, V., et al. (2019). Focused ultrasound enhanced intranasal delivery of brain derived neurotrophic factor produces neurorestorative eﬀects in a Parkinson’s disease mouse model. Sci. Rep. 9:19402. doi: 10.1038/s41598-019-55294-5 p Liu, Y. X., Liu, W. J., Zhang, H. R., and Zhang, Z. W. (2018). Delivery of bevacizumab by intracranial injection: assessment in glioma model. Onco Targets Ther. 11, 2673–2683. doi: 10.2147/OTT.S159913 Jordão, J. F., Ayala-Grosso, C. A., Markham, K., Huang, Y., Chopra, R., Mclaurin, J., et al. (2010). REFERENCES Kim, M. J., Futai, K., Jo, J., Hayashi, Y., Cho, K., and Sheng, M. (2007). Synaptic accumulation of PSD-95 and synaptic function regulated by phosphorylation of serine-295 of PSD-95. Neuron 56, 488–502. doi: 10.1016/j.neuron.2007.09.007 Armstrong, M. J., and Okun, M. S. (2020). Diagnosis and Treatment of Parkinson Disease: A Review. JAMA 323, 548–560. doi: 10.1001/jama.2019.22360 Kiviniemi, V., Korhonen, V., Kortelainen, J., Rytky, S., Keinänen, T., and Tuovinen, T. (2017). Real-time monitoring of human blood-brain barrier disruption. PLoS One 12:e0174072. eCollection 2017 doi: 10.1371/journal.pone.0174072 Chen, L., Liu, X., Wang, H., and Qu, M. (2017). Gastrodin Attenuates Pentylenetetrazole-Induced Seizures by Modulating the Mitogen-Activated Protein Kinase-Associated Inﬂammatory Responses in Mice. Neurosci. Bull. 33, 264–272. doi: 10.1007/s12264-016-0084-z Koppula, S., Alluri, R., and Kopalli, S. R. (2021). Coriandrum sativum attenuates microglia mediated neuroinﬂammation and MPTP-induced behavioral and oxidative changes in Parkinson’s disease mouse model. Excli. J. 20, 835–850. doi: 10.17179/excli2021-3668 Cheng, H. C., Ulane, C. M., and Burke, R. E. (2010). Clinical progression in Parkinson disease and the neurobiology of axons. Ann. Neurol. 67, 715–725. doi: 10.1002/ana.21995 Kumar, H., Kim, I. S., More, S. V., Kim, B. W., Bahk, Y. Y., and Choi, D. K. (2013). Gastrodin protects apoptotic dopaminergic neurons in a toxin-induced Parkinson’s disease model. Evid. Based Complement Altern. Med. 2013:514095. doi: 10.1155/2013/514095 Doo, A. R., Kim, S. N., Hahm, D. H., Yoo, H. H., Park, J. Y., Lee, H., et al. (2014). Gastrodia elata Blume alleviates L-DOPA-induced dyskinesia by normalizing FosB and ERK activation in a 6-OHDA-lesioned Parkinson’s disease mouse model. BMC Complement. Altern. Med. 14:107. doi: 10.1186/1472-6882-14-107 FosB and ERK activation in a 6-OHDA-lesioned Parkinson’s disease mouse model. BMC Complement. Altern. Med. 14:107. doi: 10.1186/1472-6882-14-107 GBD (2018). Global, regional, and national burden of Parkinson’s disease, 1990- 2016: a systematic analysis for the Global Burden of Disease Study 2016. Lancet Neurol. 17, 939–953. doi: 10.1016/S1474-4422(18)30295-3 Kung, Y., Hsiao, M. Y., Yang, S. M., Wen, T. Y., Chen, M., Liao, W. H., et al. (2021). A single low-energy shockwave pulse opens blood-cerebrospinal ﬂuid barriers and facilitates gastrodin delivery to alleviate epilepsy. Ultrason. Sonochem. 78:105730. doi: 10.1016/j.ultsonch.2021.105730 GBD (2018). Global, regional, and national burden of Parkinson’s disease, 1990- 2016: a systematic analysis for the Global Burden of Disease Study 2016. Lancet Neurol. 17, 939–953. doi: 10.1016/S1474-4422(18)30295-3 Li, C., Chen, X., Zhang, N., Song, Y., and Mu, Y. (2012). Gastrodin inhibits neuroinﬂammation in rotenone-induced Parkinson’s disease model rats. Neural. Regen. Res. 7, 325–331. AUTHOR CONTRIBUTIONS LA and MC contributed to the design of the study. YW, KL, and JL carried out the experiment. YW, CL, and W-SC analyzed May 2022 \| Volume 16 \| Article 884788 Frontiers in Cellular Neuroscience \| www.frontiersin.org 11 Wang et al. Noninvasive Drug Delivery Improves PD We owe our thanks to LetPub (https://www.letpub.com) for its linguistic assistance in preparing this manuscript. We owe our thanks to LetPub (https://www.letpub.com) for its linguistic assistance in preparing this manuscript. the data. YW and KL composed the manuscript. All authors contributed to the article and approved the submitted version. FUNDING This work was supported by the National Natural Science Foundation of China (Grant No. 81960421) and National Key R&D Program of China (Grant No. 2018YFC2001600). This work was supported by the National Natural Science Foundation of China (Grant No. 81960421) and National Key R&D Program of China (Grant No. 2018YFC2001600). The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fncel. 2022.884788/full#supplementary-material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fncel. 2022.884788/full#supplementary-material Supplementary Figure 1 \| Behavioral changes of mice in each group at different time points. (A,B) The pole climbing time and grip time of mice in each group were compared at different time points after injection of ACKNOWLEDGMENTS 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Compared to the Sham ∗P 0 05 ∗∗P 0 01 ∗∗∗P 0 001 O ANOVA ith LSD t t 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Compared to the Sham group. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001. One-way ANOVA with LSD test; n = 8 mean ± SEM We wish to acknowledge the illustration support of Qi Sun and We wish to acknowledge the illustration support of Qi Sun and Prof. Hailong Yang, who has supported in the study design. REFERENCES Antibodies targeted to the brain with image-guided focused ultrasound reduces amyloid-beta plaque load in the TgCRND8 mouse model of Alzheimer’s disease. PLoS One 5:e10549. doi: 10.1371/journal.pone.0010549 Lorenzo, M. F., Thomas, S. C., Kani, Y., Hinckley, J., Lee, M., Adler, J., et al. (2019). Temporal Characterization of Blood-Brain Barrier Disruption with High-Frequency Electroporation. Cancers (Basel) 11:E1850. doi: 10.3390/ cancers11121850 Karakatsani, M. E., Wang, S., Samiotaki, G., Kugelman, T., Olumolade, O. O., Acosta, C., et al. (2019). Amelioration of the nigrostriatal pathway facilitated by ultrasound-mediated neurotrophic delivery in early Parkinson’s disease. J. Control. Release 303, 289–301. doi: 10.1016/j.jconrel.2019. 03.030 Luchtman, D. W., Shao, D., and Song, C. (2009). Behavior, neurotransmitters and inﬂammation in three regimens of the MPTP mouse model of Parkinson’s disease. Physiol. Behav. 98, 130–138. doi: 10.1016/j.physbeh.2009.04.021 Luo, K., Wang, Y., Chen, W. S., Feng, X., Liao, Y., Chen, S., et al. (2022). Treatment Combining Focused Ultrasound with Gastrodin Alleviates Memory Deﬁcit and May 2022 \| Volume 16 \| Article 884788 Frontiers in Cellular Neuroscience \| www.frontiersin.org 12 Noninvasive Drug Delivery Improves PD Wang et al. Neuropathology in an Alzheimer’s Disease-Like Experimental Mouse Model. Neural. Plast. 2022:5241449. doi: 10.1155/2022/5241449 Villalba, R. M., and Smith, Y. (2018). Loss and remodeling of striatal dendritic spines in Parkinson’s disease: from homeostasis to maladaptive plasticity? J. Neural. Transm. (Vienna) 125, 431–447. doi: 10.1007/s00702-017-1735-6 Ma, S. Y., Röyttä, M., Rinne, J. O., Collan, Y., and Rinne, U. K. (1997). Correlation between neuromorphometry in the substantia nigra and clinical features in Parkinson’s disease using disector counts. J. Neurol. Sci. 151, 83–87. doi: 10. 1016/s0022-510x(97)00100-7 Wang, Q., Chen, G., and Zeng, S. J. (2009). Study on the Metabolism of Gastrodin in Rat Brain, Liver, Kidney and Diﬀerent Brain Regions Homogenate. Chin. J. Mod. Appl. Pharm. 26, 614–619. Wang, Q. D., Gu, P., and Dong, Q. Y. (2008). The eﬀect of repetitive transcranial magnetic stimulation on the expressions of growth associated protein-43 and synaptophysin in striatum of Parkinson’s disease model mice. Chin. J. Gerontol. 19, 1885–1888. Mead, B. P., Kim, N., Miller, G. W., Hodges, D., Mastorakos, P., Klibanov, A. L., et al. (2017). Novel Focused Ultrasound Gene Therapy Approach Noninvasively Restores Dopaminergic Neuron Function in a Rat Parkinson’s Disease Model. Nano. Lett. 17, 3533–3542. doi: 10.1021/acs.nanolett.7b00616 Ohta, S., Kikuchi, E., Ishijima, A., Azuma, T., Sakuma, I., and Ito, T. (2020). REFERENCES Investigating the optimum size of nanoparticles for their delivery into the brain assisted by focused ultrasound-induced blood-brain barrier opening. Sci. Rep. 10, 18220. doi: 10.1038/s41598-020-75253-9 Wang, X. L., Xing, G. H., Hong, B., Li, X. M., Zou, Y., Zhang, X. J., et al. (2014). Gastrodin prevents motor deﬁcits and oxidative stress in the MPTP mouse model of Parkinson’s disease: involvement of ERK1/2-Nrf2 signaling pathway. Life Sci. 114, 77–85. doi: 10.1016/j.lfs.2014.08.004 Palasz, E., Wysocka, A., Gasiorowska, A., Chalimoniuk, M., Niewiadomski, W., and Niewiadomska, G. (2020). BDNF as a Promising Therapeutic Agent in Parkinson’s Disease. Int. J. Mol. Sci. 21:1170. doi: 10.3390/ijms21031170 Wei, H. J., Upadhyayula, P. S., Pouliopoulos, A. N., Englander, Z. K., Zhang, X., Jan, C. I., et al. (2021). Focused Ultrasound-Mediated Blood-Brain Barrier Opening Increases Delivery and Eﬃcacy of Etoposide for Glioblastoma Treatment. Int. J. Radiat. Oncol. Biol. Phys. 110, 539–550. doi: 10.1016/j.ijrobp.2020.1 2.019 Pardridge, W. M. (2005). The blood-brain barrier: bottleneck in brain drug development. NeuroRx 2, 3–14. doi: 10.1602/neurorx.2.1.3 Park, J., Aryal, M., Vykhodtseva, N., Zhang, Y. Z., and Mcdannold, N. (2017). Evaluation of permeability, doxorubicin delivery, and drug retention in a rat brain tumor model after ultrasound-induced blood-tumor barrier disruption. J. Control Rel. 250, 77–85. doi: 10.1016/j.jconrel.2016.10.011 Wu, S. K., Tsai, C. L., Huang, Y., and Hynynen, K. (2020). Focused Ultrasound and Microbubbles-Mediated Drug Delivery to Brain Tumor. Pharmaceutics 13.:15. doi: 10.3390/pharmaceutics13010015 Yan, J., Yang, Z., Zhao, N., Li, Z., and Cao, X. (2019). Gastrodin protects dopaminergic neurons via insulin-like pathway in a Parkinson’s disease model. BMC Neurosci. 20:31. doi: 10.1186/s12868-019-0512-x J. Control Rel. 250, 77–85. doi: 10.1016/j.jconrel.2016.10.011 Pouliopoulos, A. N., Kwon, N., Jensen, G., Meaney, A., Niimi, Y., Burgess, M. T., et al. (2021). Safety evaluation of a clinical focused ultrasound system for neuronavigation guided blood-brain barrier opening in non-human primates. Sci. Rep. 11:15043. doi: 10.1038/s41598-021-94188-3 Yang, Q., Zhou, Y., Chen, J., Huang, N., Wang, Z., and Cheng, Y. (2021). Gene Therapy for Drug-Resistant Glioblastoma via Lipid-Polymer Hybrid Nanoparticles Combined with Focused Ultrasound. Int. J. Nanomedicine. 16, 185–199. doi: 10.2147/IJN.S286221 Qi, H., Shen, D., Jiang, C., Wang, H., and Chang, M. (2021). Ursodeoxycholic acid protects dopaminergic neurons from oxidative stress via regulating mitochondrial function, autophagy, and apoptosis in MPTP/MPP(+)-induced Parkinson’s disease. Neurosci. Lett. 741, 135493. doi: 10.1016/j.neulet.2020. 135493 Zhang, Q. S., Heng, Y., Mou, Z., Huang, J. Y., Yuan, Y. H., and Chen, N. H. (2017). REFERENCES Reassessment of subacute MPTP-treated mice as animal model of Parkinson’s disease. Acta. Pharmacol. Sin. 38, 1317–1328. doi: 10.1038/aps.2017.49 Rezai, A. R., Ranjan, M., D’haese, P. F., Haut, M. W., Carpenter, J., Najib, U., et al. (2020). Noninvasive hippocampal blood-brain barrier opening in Alzheimer’s disease with focused ultrasound. Proc. Natl. Acad. Sci. U S A 117, 9180–9182. doi: 10.1073/pnas.2002571117 Zhang, S., Gong, P., Zhang, J., Mao, X., Zhao, Y., Wang, H., et al. (2020). Speciﬁc Frequency Electroacupuncture Stimulation Transiently Enhances the Permeability of the Blood-Brain Barrier and Induces Tight Junction Changes. Front. Neurosci. 14:582324. doi: 10.3389/fnins.2020.582324 Rousselet, E., Joubert, C., Callebert, J., Parain, K., Tremblay, L., Orieux, G., et al. (2003). Behavioral changes are not directly related to striatal monoamine levels, number of nigral neurons, or dose of parkinsonian toxin MPTP in mice. Neurobiol. Dis. 14, 218–228. doi: 10.1016/s0969-9961(03)00108-6 Zhou, N. N., Zhu, R., Zhao, X. M., Zhang, J. M., and Liang, P. (2016). [Eﬀect and mechanism of gastrodin inhibiting β-amyloid plaques in brain of mice]. Yao. Xue. Xue. Bao. 51, 588–594. 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. Sen, A., Hongpaisan, J., Wang, D., Nelson, T. J., and Alkon, D. L. (2016). Protein Kinase Cϵ (PKCϵ) Promotes Synaptogenesis through Membrane Accumulation of the Postsynaptic Density Protein PSD-95. J. Biol. Chem. 291, 16462–16476. doi: 10.1074/jbc.M116.730440 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. Su, Y., Sun, B., Gao, X., Dong, X., Fu, L., Zhang, Y., et al. (2020). Intranasal Delivery of Targeted Nanoparticles Loaded With miR-132 to Brain for the Treatment of Neurodegenerative Diseases. Front. Pharmacol. 11:1165. doi: 10.3389/fphar. 2020.01165 Toy, W. A., Petzinger, G. M., Leyshon, B. J., Akopian, G. K., Walsh, J. P., Hoﬀman, M. V., et al. (2014). Treadmill exercise reverses dendritic spine loss in direct and indirect striatal medium spiny neurons in the 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP) mouse model of Parkinson’s disease. Neurobiol. Dis. 63, 201–209. doi: 10.1016/j.nbd.2013.11.017 Copyright © 2022 Wang, Luo, Li, Liao, Liao, Chen, Chen and Ao. Frontiers in Cellular Neuroscience \| www.frontiersin.org REFERENCES 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. Tsai, H. C., Tsai, C. H., Chen, W. S., Inserra, C., Wei, K. C., and Liu, H. L. (2018). Safety evaluation of frequent application of microbubble-enhanced focused ultrasound blood-brain-barrier opening. Sci. Rep. 8, 17720. doi: 10. 1038/s41598-018-35677-w May 2022 \| Volume 16 \| Article 884788 Frontiers in Cellular Neuroscience \| www.frontiersin.org 13
https://openalex.org/W2896092698	https://eprints.soton.ac.uk/425524/2/biosensors_08_00097.pdf	English	null	Rapid Multiplexed Detection on Lateral-Flow Devices Using a Laser Direct-Write Technique	Biosensors	2,018	cc-by	9,586	Received: 26 September 2018; Accepted: 18 October 2018; Published: 20 October 2018 Abstract: Paper-based lateral ﬂow devices (LFDs) are regarded as ideal low-cost diagnostic solutions for point-of-care (POC) scenarios that allow rapid detection of a single analyte within a ﬂuidic sample, and have been in common use for a decade. In recent years, there has been an increasing need for rapid and simultaneous detection of multiple analytes present within a single sample and to facilitate this, we report here a novel solution—detection using a multi-path LFD created via the precise partitioning of the single ﬂow-path of a standard LFD using our previously reported laser direct-write (LDW) technique. The multiple ﬂow-paths allow the simultaneous detection of the different analytes individually within each of the parallel channels without any cross-reactivity. The appearance of coloured test lines in individual channels indicates the presence of the different analytes within a sample. We successfully present the use of a LDW-patterned multi-path LFD for multiplexed detection of a biomarker panel comprising C-reactive protein (CRP) and Serum amyloid A-1 (SAA1), used for the diagnosis of bacterial infections. Overall, we demonstrate the use of our LDW technique in the creation of a novel LFD that enables multiplexed detection of two inﬂammation markers within a single LFD providing a detection protocol that is comparatively more efﬁcient than the standard sequential multiplexing procedure. Keywords: lateral-ﬂow device; multiplexed detection; laser direct-write; biosensors; inﬂammation detection biosensors biosensors biosensors Article Peijun J. W. He , Ioannis N. Katis , Robert W. Eason and Collin L. Sones Optoelectronics Research Centre, University of Southampton, Southampton SO17 1BJ, UK; I.Katis@soton.ac.uk (I.N.K.); rwe@orc.soton.ac.uk (R.W.E.); cls@orc.soton.ac.uk (C.L.S.) Correspondence: P.He@soton.ac.uk; Tel.: +44-2380599091 www.mdpi.com/journal/biosensors 1. Introduction Across a wide range of ﬁelds that include not just clinical diagnostics but also areas such as food safety testing and environmental assessment etc., the need for simple point-of-care (POC) testing solutions has become increasingly evident in recent years [1–3]. The World Health Organisation (WHO) has also deﬁned a set of essential requirements to which POC diagnostic tools/tests, developed for the challenging needs of under-resourced countries, should adhere. These well-deﬁned criteria are summarized through the ‘ASSURED’ (Affordable, Sensitive, Speciﬁc, User-friendly, Robust, Equipment-free, Deliverable) acronym [4]. The objective therefore for all developers has been to develop POC tests that are not only reasonably affordable but are also sensitive and speciﬁc enough for adoption in routine diagnostics. Signiﬁcant effort has therefore been made to develop such biosensors and one such example is paper-based microﬂuidic devices, which promise to satisfy these requirements. Their inherent advantages such as low-cost, ease of use, portability, requiring small sample volumes and no additional need for laboratory equipment and trained personnel have attracted attention within the diagnostics research domain as a low-cost alternative to conventional POC diagnostic tools [5–7]. Rapid Diagnostic Test (RDT) strips, also known as lateral ﬂow devices (LFDs) are one of the simplest and most established formats of paper-based devices that allow the detection of an analyte Biosensors 2018, 8, 97; doi:10.3390/bios8040097 www.mdpi.com/journal/biosensors 2 of 11 Biosensors 2018, 8, 97 through the testing of a complex bodily ﬂuid such as urine, blood, saliva or even sweat [8–11]. The underlying principle of an LFD is relatively simple: a liquid sample containing the analyte of interest is moved via the paper-enabled capillary action through various zones within a paper strip. During the transport, the analyte interacts with antibodies that have been pre-deposited onto the strip and consequently gets captured at the detection sites namely the test line and the control line. The read-out is normally displayed within 5–30 min, and this is an indication of the detection of the analyte and manifests itself as an appropriate colour-based response at the test line, while a colorimetric response at the control line indicates that sample ﬂow has correctly occurred through the strip and that the device has worked correctly [12]. Low development costs and ease of production of these LFDs have resulted in the expansion of applications across multiple test-sites where rapid tests are required, such as hospitals, physician’s ofﬁces, clinical laboratories and even in the patient’s home [13]. 1. Introduction These tests are therefore now widely used as routine tools at the POC as part of an early-stage detection/treatment protocol, a common example of which is a pregnancy dip-stick [14]. y g p p p g y p Multiplexing, which is deﬁned as simultaneous analysis of multiple analytes under the same set of conditions, is a critical parameter for increasing diagnostic efﬁciency [15]. An obvious example of such a need is in clinical diagnosis, where multiple analytes which are inter-dependent need to be detected and quantiﬁed to allow informed decisions to be made concerning the progression or stage of a particular disease [16]. In recent years, therefore, there has been an increasing demand for POC multiplexed diagnostic assays. The strategies that enable such simultaneous analysis of multiple analytes are largely based on the use of the following underlying diagnostic techniques, i.e., 96-well microtiter plate-based enzyme linked immunosorbent assays (ELISA) [17], real-time polymerase chain reactions (PCR) [18,19], and microarrays or bead-based methods [20,21]. Although all these methods could enable high-throughput and low volume processing, they require expensive non-portable equipment and trained personnel for their operation. Consequently, LFDs are regarded as one of the most competitive candidates for POC settings, due to their unique advantages that have been described earlier. Currently, LFDs that provide such multiplexing are constructed either by laminating or shaping together different individual LFDs [22,23] or alternatively, by multiplexing within a single ﬂow-path [24,25]. Such devices have inherent drawbacks such as, for the former, increased device dimensions and therefore need for larger sample volumes, and, for the latter, an undesired interference between different detection sites, i.e., the inﬂuence of each of the previous test-lines on subsequent lines positioned further along the ﬂow-path. As a result, few commercial devices employing such multiplexing methodologies are available so far. In this article, we propose a novel solution to overcome both of these limitations—a multi-path LFD created via the precise partitioning of the ﬂow-path of a single LFD using a laser direct-write (LDW) technique. The multiple ﬂow-paths allow individual detection of different analytes in each of the separated channels. We successfully demonstrate the use of these LDW-fabricated multi-path LFDs for simultaneous detection of a biomarker panel comprising C-reactive protein (CRP) and Serum amyloid A-1 (SAA1), commonly used for the diagnosis of bacterial infections. 2.1. Device Fabrication Setup 2.1. Device Fabrication Setup 2.1. Device Fabrication Setup As shown in the schematic in Figure 1, the multi-ﬂow path LFD was fabricated via precise partitioning of the nitrocellulose membrane (i.e., the reaction pad) of the single ﬂow channel of a standard LFD into multiple separated ﬂow-paths. As with the standard LFD, the sample dispensed onto the sample pad will ﬂow further via capillary action into the conjugate pad where it picks up the pre-deposited reagent and then splits to ﬂow into individual separated ﬂow-paths created within the reaction pad allowing multiplexed detection without any interference or cross-reactions. As shown in the schematic in Figure 1, the multi-flow path LFD was fabricated via precise partitioning of the nitrocellulose membrane (i.e. the reaction pad) of the single flow channel of a standard LFD into multiple separated flow-paths. As with the standard LFD, the sample dispensed onto the sample pad will flow further via capillary action into the conjugate pad where it picks up the pre-deposited reagent and then splits to flow into individual separated flow-paths created within the reaction pad allowing multiplexed detection without any interference or cross-reactions. As shown in the schematic in Figure 1, the multi-flow path LFD was fabricated via precise partitioning of the nitrocellulose membrane (i.e. the reaction pad) of the single flow channel of a standard LFD into multiple separated flow-paths. As with the standard LFD, the sample dispensed onto the sample pad will flow further via capillary action into the conjugate pad where it picks up the pre-deposited reagent and then splits to flow into individual separated flow-paths created within the reaction pad allowing multiplexed detection without any interference or cross-reactions. Figure 1. Schematic of a multiple (six) flow-path lateral flow device (LFD). Figure 1. Schematic of a multiple (six) ﬂow-path lateral ﬂow device (LFD). Figure 1. Schematic of a multiple (six) flow-path lateral flow device (LFD). Figure 1. Schematic of a multiple (six) flow-path lateral flow device (LFD). Figure 1. Schematic of a multiple (six) ﬂow-path lateral ﬂow device (LFD). Figure 1. Schematic of a multiple (six) flow-path lateral flow device (LFD). The boundaries that separate these individual channels were created using our previously reported local-deposition-assisted LDW technique [26–28]. The schematic of this local-deposition-assisted LDW setup is shown in Figure 2. A photo-polymer was first locally deposited onto the nitrocellulose reaction pad with a deposition nozzle at locations pre-defined by the device design. 2.1. Device Fabrication Setup 2.1. Device Fabrication Setup 2.1. Device Fabrication Setup A laser beam that follows the deposition head subsequently illuminated the deposited polymer pattern inducing photo-polymerisation. These laser-cured patterns that extend through the thickness of the nitrocellulose membrane define the solid walls of the multiple fluidic channels that confine and transport individual liquid flows. The boundaries that separate these individual channels were created using our previously reported local-deposition-assisted LDW technique [26–28]. The schematic of this local-deposition- assisted LDW setup is shown in Figure 2. A photo-polymer was ﬁrst locally deposited onto the nitrocellulose reaction pad with a deposition nozzle at locations pre-deﬁned by the device design. A laser beam that follows the deposition head subsequently illuminated the deposited polymer pattern inducing photo-polymerisation. These laser-cured patterns that extend through the thickness of the nitrocellulose membrane deﬁne the solid walls of the multiple ﬂuidic channels that conﬁne and transport individual liquid ﬂows. The boundaries that separate these individual channels were created using our previously reported local-deposition-assisted LDW technique [26–28]. The schematic of this local-deposition-assisted LDW setup is shown in Figure 2. A photo-polymer was first locally deposited onto the nitrocellulose reaction pad with a deposition nozzle at locations pre-defined by the device design. A laser beam that follows the deposition head subsequently illuminated the deposited polymer pattern inducing photo-polymerisation. These laser-cured patterns that extend through the thickness of the nitrocellulose membrane define the solid walls of the multiple fluidic channels that confine and transport individual liquid flows. Figure 2. Schematic of the laser-based direct-write setup which shows the local deposition of photo-polymer that is subsequently illuminated by exposure from a 405nm c.w. laser. Figure 2. Schematic of the laser-based direct-write setup which shows the local deposition of photo-polymer that is subsequently illuminated by exposure from a 405nm c.w. laser. Figure 2. Schematic of the laser-based direct-write setup which shows the local deposition of photo-polymer that is subsequently illuminated by exposure from a 405 nm c.w. laser. Figure 2. Schematic of the laser-based direct-write setup which shows the local deposition of photo-polymer that is subsequently illuminated by exposure from a 405nm c.w. laser. Figure 2. Schematic of the laser-based direct-write setup which shows the local deposition of photo-polymer that is subsequently illuminated by exposure from a 405nm c.w. laser. Figure 2. Schematic of the laser-based direct-write setup which shows the local deposition of photo-polymer that is subsequently illuminated by exposure from a 405 nm c.w. laser. 1. Introduction Unlike other multiplexing methods, our approach that is presented here, allows the creation of multiple individual ﬂow-paths inside a ‘single’ LFD without increasing its original size. These multiplexed tests do not require multiple inlets and an increased sample volume, and, most importantly, eliminate the interference between individual detection sites positioned within the same channel. Overall, we believe that our LDW approach provides a novel and easy-implementable solution for enabling multiplexed detection via LFDs that hugely improves the detection efﬁciency and will therefore lead to an increase in the current market-size of LFD-based testing within in-vitro diagnostics. 3 of 11 3 of 11 3 f 11 Biosensors 2018, 8, 97 Bi 2018 8 FO Biosensors 2018, 8, 97 Bi 8 FO 3. Results and Discussion Prior to developing the Prior to developing the multiplexed LFD, we ﬁrst demonstrated the successful implementation of single assays on a standard LFD using a sandwich ELISA for the two identiﬁed biomarkers (CRP and SAA1) commonly used as indicators of an inﬂammation. The protocol for the sandwich-ELISA we have used is described in Figure 3a. The target analyte (CRP or SAA1 protein) is sandwiched due to antigen–antibody binding between the capture and the detection antibody a, which is pre-tagged with an Au-nanoparticle via a biotin-streptavidin binding. This binding and the resultant accumulation of Au-nanoparticles at the test line produce the appearance of a red colour at these lines. p g p p of single assays on a standard LFD using a sandwich ELISA for the two identified biomarkers (CRP and SAA1) commonly used as indicators of an inflammation. The protocol for the sandwich-ELISA we have used is described in Figure 3a. The target analyte (CRP or SAA1 protein) is sandwiched due to antigen–antibody binding between the capture and the detection antibody a, which is pre-tagged with an Au-nanoparticle via a biotin-streptavidin binding. This binding and the resultant accumulation of Au-nanoparticles at the test line produce the appearance of a red colour at these lines. The arrangement of a simpliﬁed standard lateral-ﬂow strip that we used for these single detections is as shown in Figure 3b and only consists of a reaction pad made of nitrocellulose membrane and an absorbent pad that is made of cellulose paper. For these proof-of-principle test to ease with the assay procedure, we simpliﬁed the design of the LFD by eliminating the sample pad and conjugate pad. These single LFDs have a dimension of 3 mm in width and 5 cm in length, which are chosen according to the standard commercially available LFDs in market. The capture antibody was pre-deposited and immobilised at the test line and a mixture of the sample and the Au-nanoparticle tagged detection antibody was introduced directly via the reaction pad. The arrangement of a simplified standard lateral-flow strip that we used for these single detections is as shown in Figure 3b and only consists of a reaction pad made of nitrocellulose membrane and an absorbent pad that is made of cellulose paper. For these proof-of-principle test to ease with the assay procedure, we simplified the design of the LFD by eliminating the sample pad and conjugate pad. 2.1. Device Fabrication Setup 2.1. Device Fabrication Setup 2.1. Device Fabrication Setup The laser used for this LDW process was a 405 nm continuous wave (c.w.) diode laser (MLDTM 405 nm, Cobolt AB, Stockholm, Sweden) with a maximum output power of 110 mW). The photopolymer used for creating the boundary walls between individual channels was DeSolite® 3471-3-14 from DSM Desotech, Inc., Elgin, IL, USA. The dispenser platform used for the local deposition of the photopolymer onto the various substrates was a PICO® Pµlse™ dispensing system from Nordson EFD, UK. A different reagent-dispensing system was used for local deposition of antibodies onto the reaction pad for creation of test lines and control line and that was the XYZ3210 dispense platform from Biodot, Irvine, CA, USA. The laser used for this LDW process was a 405 nm continuous wave (c.w.) diode laser (MLDTM 405 nm, Cobolt AB, Stockholm, Sweden) with a maximum output power of 110 mW). The photopolymer used for creating the boundary walls between individual channels was DeSolite® 3471-3-14 from DSM Desotech, Inc., Elgin, IL, USA. The dispenser platform used for the local deposition of the photopolymer onto the various substrates was a PICO® Pµlse™ dispensing system from Nordson EFD, UK. A different reagent-dispensing system was used for local deposition of antibodies onto the reaction pad for creation of test lines and control line and that was the XYZ3210 dispense platform from Biodot, Irvine, CA, USA. The laser used for this LDW process was a 405 nm continuous wave (c.w.) diode laser (MLDTM 405 nm, Cobolt AB, Stockholm, Sweden) with a maximum output power of 110 mW). The photopolymer used for creating the boundary walls between individual channels was DeSolite® 3471-3-14 from DSM Desotech, Inc., Elgin, IL, USA. The dispenser platform used for the local deposition of the photopolymer onto the various substrates was a PICO® Pµlse™dispensing system from Nordson EFD, UK. A different reagent-dispensing system was used for local deposition of antibodies onto the reaction pad for creation of test lines and control line and that was the XYZ3210 dispense platform from Biodot, Irvine, CA, USA. 4 of 11 Biosensors 2018, 8, 97 2 2 Reagents and M 2.2. Reagents and Materials The nitrocellulose me Sartorius and the absorben The nitrocellulose membrane used as the reaction pad was UniSart CN95 purchased from Sartorius and the absorbent pad was cellulose ﬁlter papers (CF1) from GE Healthcare. For the CRP assay, the capture and detection antibodies used were mouse anti-human CRP antibody (MAB17071) and biotinylated mouse anti-human CRP antibody (BAM17072) from R&D Systems, Abingdon, UK. The protein standard was recombinant human CRP expressed in E. coli (Sigma Aldrich C1617). For the SAA1 assay, both the capture and detection antibodies and also the protein standard used were from a DuoSet ELISA kit (DY3019) from R&D Systems. The control line antibody was anti-mouse Goat IgG (AF007) from R&D Systems. The streptavidin-conjugated gold nanoparticles (with an optical density of 10) were obtained from BBI solutions (BA.STP40). The diluent used for preparing all the antibody and sample solutions was 1% BSA in PBS. The bovine serum albumin (BSA) (A2058) and phosphate buffered saline (PBS) (P3831) used were obtained from Sigma Aldrich, Gillingham, UK. assay, the capture and detection antibodies used were mouse anti-human CRP antibody (MAB17071) and biotinylated mouse anti-human CRP antibody (BAM17072) from R&D Systems, Abingdon, UK. The protein standard was recombinant human CRP expressed in E. coli (Sigma Aldrich C1617). For the SAA1 assay, both the capture and detection antibodies and also the protein standard used were from a DuoSet ELISA kit (DY3019) from R&D Systems. The control line antibody was anti-mouse Goat IgG (AF007) from R&D Systems. The streptavidin-conjugated gold nanoparticles (with an optical density of 10) were obtained from BBI solutions (BA.STP40). The diluent used for preparing all the antibody and sample solutions was 1% BSA in PBS. The bovine serum albumin (BSA) (A2058) and phosphate buffered saline (PBS) (P3831) used were obtained from Sigma Aldrich, Gillingham, UK. 3 R lt d Di i 3.1. Standard Single LFD for Detection of CRP We ﬁrst studied the detection of CRP using single LFDs. The capture antibody (mouse anti-human CRP at 1 mg/mL) was locally deposited onto the reaction pad as a line using the BioDot dispenser and left to dry and immobilise overnight. A mixture of the sample (containing the analyte, a recombinant human CRP protein in various concentrations ranging from 10 µg/mL to 50 ng/mL), the detection antibody (biotinylated mouse anti-human CRP at 1.6 µg/mL), and the streptavidin-modiﬁed 40 nm Au-nanoparticles in a volumetric ratio of 3:2:4 (values which we had optimized through previous experiments) was ﬁrst prepared immediately prior to the testing with the single LFDs. To perform the test, 15 µL of the mixture was added into a well of a 96-well microtiter plate and then the LFD was dipped into the sample solution. The device was left to run for 3 min to allow all of the solution in the well to be wicked into the strip. The device was subsequently dipped in a chase buffer of PBS solution for another 5 min to allow the entire reagent to be pushed through to the reaction pad. The result was then immediately captured using a scanner (Epson Perfection V800 Photo A4 Flatbed Scanner). The resultant, appearance of the red-coloured test lines are as shown in Figure 4a. For samples with concentration of 50 ng/mL, the red-coloured test lines however appear within a larger white band around them. The formation of the white bands is a consequence of the blocking of that area/band around the test line by the BSA contained within the capture antibody solution previously dispensed to produce the test line. BSA is a reagent most commonly used to block unwanted nonspeciﬁc binding sites on the nitrocellulose membrane 29. A similar white band, though smaller in size, is also visible for devices tested for higher analyte concentrations. The images captured using the scanner were processed with the ImageJ software (National Institutes of Health, Bethesda, MD, USA) to extract the respective colour intensities of the red colour produced at the test lines. The resultant plot in Figure 4c shows the relationship between the respective test-line colour intensities for each of the individual devices and the corresponding concentrations for the samples used to test each of the devices. The results above show the successful detection of CRP using a standard single ﬂow-path LFD. 3.1. Standard Single LFD for Detection of CRP From the plot in Figure 4c, we observe that the intensity of the test line shows a monotonic increase as a function of concentration of the sample, which shows its potential usefulness in quantitative analysis. With these standard single-channel LFDs, we have shown the capability for detection of CRP with a concentration down to 50 ng/mL, which is comparable with commercial ELISA assays performed on microtiter plates. 3. Results and Discussion Prior to developing the These single LFDs have a dimension of 3 mm in width and 5 cm in length, which are chosen according to the standard commercially available LFDs in market. The capture antibody was pre-deposited and immobilised at the test line and a mixture of the sample and the Au-nanoparticle tagged detection antibody was introduced directly via the reaction pad. Figure 3. Schematic of (a) the sandwich enzyme linked immunosorbent assays (ELISA) protocol, (b) the simplified LFD we used for detection of C-reactive protein (CRP) and Serum amyloid A-1 (SAA1), and (c) detection operation method. Figure 3. Schematic of (a) the sandwich enzyme linked immunosorbent assays (ELISA) protocol, (b) the simpliﬁed LFD we used for detection of C-reactive protein (CRP) and Serum amyloid A-1 (SAA1), and (c) detection operation method. Figure 3. Schematic of (a) the sandwich enzyme linked immunosorbent assays (ELISA) protocol, (b) the simplified LFD we used for detection of C-reactive protein (CRP) and Serum amyloid A-1 (SAA1), and (c) detection operation method. Figure 3. Schematic of (a) the sandwich enzyme linked immunosorbent assays (ELISA) protocol, (b) the simpliﬁed LFD we used for detection of C-reactive protein (CRP) and Serum amyloid A-1 (SAA1), and (c) detection operation method. The operation of this LFD is described in Figure 3c. When the strip is dipped into a mixture of the sample containing the analyte and the Au-nanoparticle tagged detection antibodies, an analyte-antibody-Au nanoparticle complex is formed via the specific chemical binding between the The operation of this LFD is described in Figure 3c. When the strip is dipped into a mixture of the sample containing the analyte and the Au-nanoparticle tagged detection antibodies, an analyte-antibody-Au nanoparticle complex is formed via the speciﬁc chemical binding between the analyte and the tagged-antibody. This complex then ﬂows laterally via capillary action towards the 5 of 11 Biosensors 2018, 8, 97 absorbent pad that is positioned at the other end of the lateral-ﬂow strip. As the complex migrates through the reaction pad, the analyte binds to the capture antibody that is pre-immobilized in the reaction zone and the excess sample-reagent mixture then wicks further and ﬁnally gets collected by the absorbent pad. The accumulation of the Au-nanoparticle tagged antibody-analyte complex produces the appearance of a red colour test line that indicates the presence of the target analyte in the sample. 3.2. Standard Single LFD for Detection of SAA1 Following the experiments for the CRP detection using standard single-channel LFDs, in a similar manner, we then tested their use for detection of SAA1. These LFDs had the same device geometry as those used previously for the CRP testing. The capture antibody used was mouse anti-human SAA1 at 1 mg/mL. The sample mixture was prepared by mixing of the sample (containing the analyte, a recombinant human SAA1 protein with various concentrations ranging from 10 µg/mL to 10 pg/mL), the detection antibody (biotinylated mouse anti-human SAA1 at 12 µg/mL) and the Au-nanoparticles (streptavidin-modiﬁed 40 nm gold nanoparticles) in a volumetric ratio of 3:2:4. The tests were performed in the same way as described for CRP testing by dipping the LFDs into the sample-containing well followed by dipping in a chase buffer. The results are shown in the scanned image in Figure 4b. The image was processed using ImageJ and the data is plotted in Figure 4d. Biosensors 2018, 8, 97 scanned image in 6 of 11 Figure As seen in Figure 4b, for samples with concentrations below 1µg/mL, once again the red-coloured test lines appear within a larger white band. 4d. As seen in Figure 4b, for samples with concentrations below 1µg/mL, once again the red-coloured test lines appear within a larger white band. From both the images in Figure 4b and the plot in Figure 4d, we can see that although the test From both the images in Figure 4b and the plot in Figure 4d, we can see that although the test line does appear for concentrations of 50,000, 1000, 100 and 10 pg/mL, the colour intensity level is low and fairly constant, which thereby limits any quantitative analysis. However, our results show the capability of a qualitative (YES/NO type) detection of SAA1 for concentrations as low as 10 pg/mL, which is more than three orders of magnitudes lower than what can be measured using commercially available ELISA kits. g g p g g line does appear for concentrations of 50,000, 1000, 100 and 10 pg/mL, the colour intensity level is low and fairly constant, which thereby limits any quantitative analysis. However, our results show the capability of a qualitative (YES/NO type) detection of SAA1 for concentrations as low as 10 pg/mL, which is more than three orders of magnitudes lower than what can be measured using commercially available ELISA kits. 3.2. Standard Single LFD for Detection of SAA1 The clinically relevant ranges for CRP and SAA1 are 50 ng/mL to 500 µg/mL [29] and 1 µg/mL The clinically relevant ranges for CRP and SAA1 are 50 ng/mL to 500 µg/mL [29] and 1 µg/mL to 5 mg/mL [30] respectively, and the results above show that while our standard single LFDs cover a section of the relevant range they also span across the comparatively challenging lower concentration range indicating their lower limit-of-detection. We chose not to work across the high concentration ranges as such levels are only observed in patients having acute infection and such concentrations can be detected simply via pre-dilution of the sample, which is a routinely applied procedure when the concentration of antigen in the sample potentially exceeds the highest point of the standard curve [31]. y g g/ µg/ [ ] µg/ to 5 mg/mL [30] respectively, and the results above show that while our standard single LFDs cover a section of the relevant range they also span across the comparatively challenging lower concentration range indicating their lower limit-of-detection. We chose not to work across the high concentration ranges as such levels are only observed in patients having acute infection and such concentrations can be detected simply via pre-dilution of the sample, which is a routinely applied procedure when the concentration of antigen in the sample potentially exceeds the highest point of the standard curve [31]. Figure 4. Scanned images of sets of the standard single (a) CRP and (b) SAA1 LFDs with various CRP and SAA1 analyte concentrations, in each case per ml. (c,d) show calibration curve constructed using the grayscale intensity values taken from the image shown in (a,b). Error bars indicate the standard deviation for 3 individual measurements. Figure 4. Scanned images of sets of the standard single (a) CRP and (b) SAA1 LFDs with various CRP and SAA1 analyte concentrations, in each case per ml. (c,d) show calibration curve constructed using the grayscale intensity values taken from the image shown in (a,b). Error bars indicate the standard deviation for 3 individual measurements. Figure 4. Scanned images of sets of the standard single (a) CRP and (b) SAA1 LFDs with various CRP and SAA1 analyte concentrations, in each case per ml. (c,d) show calibration curve constructed using the grayscale intensity values taken from the image shown in (a,b). Error bars indicate the standard deviation for 3 individual measurements. Figure 4. 3.2. Standard Single LFD for Detection of SAA1 Scanned images of sets of the standard single (a) CRP and (b) SAA1 LFDs with various CRP and SAA1 analyte concentrations, in each case per ml. (c,d) show calibration curve constructed using the grayscale intensity values taken from the image shown in (a,b). Error bars indicate the standard deviation for 3 individual measurements. Figure 4. Scanned images of sets of the standard single (a) CRP and (b) SAA1 LFDs with various CRP and SAA1 analyte concentrations, in each case per ml. (c,d) show calibration curve constructed using the grayscale intensity values taken from the image shown in (a,b). Error bars indicate the standard deviation for 3 individual measurements. Figure 4. Scanned images of sets of the standard single (a) CRP and (b) SAA1 LFDs with various CRP and SAA1 analyte concentrations, in each case per ml. (c,d) show calibration curve constructed using the grayscale intensity values taken from the image shown in (a,b). Error bars indicate the standard deviation for 3 individual measurements. 3.3. Multiplexed Detection of CRP and SAA1 in Single-Channel LFDs 3.3. Multiplexed Detection of CRP and SAA1 in Single-Channel LFDs 3.3. Multiplexed Detection of CRP and SAA1 in Single-Channel LFDs 3.3. Multiplexed Detection of CRP and SAA1 in Single-Channel LFDs p f g After demonstrating the successful detection of the above two analytes in separate LFDs, we then attempted their combined detection using a single-channel multiplexed LFD via the introduction of multiple detection lines within the common reaction zone of a standard single LFD After demonstrating the successful detection of the above two analytes in separate LFDs, we then attempted their combined detection using a single-channel multiplexed LFD via the introduction of multiple detection lines within the common reaction zone of a standard single LFD. introduction of multiple detection lines within the common reaction zone of a standard single LFD. As shown in Figure 5a, to perform this multiplexed detection, we used a multiple test line format. Along the direction of the flow, the test line for SAA1 was positioned 3mm before the test line for CRP, and finally, a control line was positioned 4 mm after the CRP test line. The sample was a pre-prepared mixture of CRP and SAA1 in a 1:1 ratio, with analyte concentrations of 100 ng/mL and 1 µg/mL respectively As shown in Figure 5a, to perform this multiplexed detection, we used a multiple test line format. Along the direction of the ﬂow, the test line for SAA1 was positioned 3mm before the test line for CRP, and ﬁnally, a control line was positioned 4 mm after the CRP test line. The sample was a pre-prepared mixture of CRP and SAA1 in a 1:1 ratio, with analyte concentrations of 100 ng/mL and 1 µg/mL respectively. and 1 µg/mL respectively. Based on the results from our previous section, for these experiments we chose to test for concentrations of 100 ng/mL and 1 µg/mL for CRP and SAA1 respectively, which as shown in Figure 4a and 4b have relatively comparable colour intensity levels Based on the results from our previous section, for these experiments we chose to test for concentrations of 100 ng/mL and 1 µg/mL for CRP and SAA1 respectively, which as shown in Figure 4a,b, have relatively comparable colour intensity levels. 4a and 4b, have relatively comparable colour intensity levels. To perform the tests, we have use an identical volume of 15 µL for the sample-reagent mixture and the results are shown in the scanned image in Figure 5b. 3.3. Multiplexed Detection of CRP and SAA1 in Single-Channel LFDs 3.3. Multiplexed Detection of CRP and SAA1 in Single-Channel LFDs ImageJ was used to extract the colour intensity 7 of 11 mixture colour Biosensors 2018, 8, 97 To perform t d th lt of both the test lines corresponding to CRP and SAA1and then these values were compared with the values previously obtained from the calibration curves in Figure 4c,d. We found that the intensity of the SAA1 test line that is located before the CRP test line stays at a level similar to what was achieved using a standard single-path SAA1-LFD, while the intensity of the CRP test line dropped signiﬁcantly (more than threefold) when compared with the result for the standard single-path CRP-LFD. intensity of both the test lines corresponding to CRP and SAA1and then these values were compared with the values previously obtained from the calibration curves in Figure 4c,d. We found that the intensity of the SAA1 test line that is located before the CRP test line stays at a level similar to what was achieved using a standard single-path SAA1-LFD, while the intensity of the CRP test line dropped significantly (more than threefold) when compared with the result for the standard single-path CRP-LFD. Based on these results we can infer that the second test line is affected by the presence of a ﬁrst test line positioned prior to it in the ﬂow-path. To further conﬁrm this hypothesis, we then used the same devices and reversed the position of the two test lines, as shown in Figure 5c, where the test line for CRP precedes the SAA1 test line. The devices were tested by dipping into the same 15 µL of sample-reagent mixture used for the previous experiments and the result is shown in Figure 5d. When compared with the image shown in Figure 5b, we can clearly observe that the test line for the SAA1 is almost invisible, whereas the intensity of the CRP test line positioned prior to the SAA1 test line shows a signiﬁcant enhancement. Again by extracting the colour intensity using ImageJ, we found that the CRP test line has the same level of intensity as for the standard single LFD and the intensity for the SAA1 test line dropped by almost 7 times. g p Based on these results we can infer that the second test line is affected by the presence of a first test line positioned prior to it in the flow-path. 3.3. Multiplexed Detection of CRP and SAA1 in Single-Channel LFDs 3.3. Multiplexed Detection of CRP and SAA1 in Single-Channel LFDs To further confirm this hypothesis, we then used the same devices and reversed the position of the two test lines, as shown in Figure 5c, where the test line for CRP precedes the SAA1 test line. The devices were tested by dipping into the same 15 µL of sample-reagent mixture used for the previous experiments and the result is shown in Figure 5d. When compared with the image shown in Figure 5b, we can clearly observe that the test line for the SAA1 is almost invisible, whereas the intensity of the CRP test line positioned prior to the SAA1 test line shows a significant enhancement. Again by extracting the colour intensity using ImageJ, we found that the CRP test line has the same level of intensity as for the standard single LFD and the intensity for the SAA1 test line dropped by almost 7 times. Figure 5. Schematic of a single-channel multiplexed LFD with multiple test lines along the same flow-path for multiplexed detection of CRP and SAA1, (a) with the SAA1 test line being before that for CRP and (c) with the CRP test line before that for SAA1. (b,d) are scanned image shows the results for three such LFDs described in (a,c). Figure 5. Schematic of a single-channel multiplexed LFD with multiple test lines along the same ﬂow-path for multiplexed detection of CRP and SAA1, (a) with the SAA1 test line being before that for CRP and (c) with the CRP test line before that for SAA1. (b,d) are scanned image shows the results for three such LFDs described in (a,c). Figure 5. Schematic of a single-channel multiplexed LFD with multiple test lines along the same flow-path for multiplexed detection of CRP and SAA1, (a) with the SAA1 test line being before that for CRP and (c) with the CRP test line before that for SAA1. (b,d) are scanned image shows the results for three such LFDs described in (a,c). Figure 5. Schematic of a single-channel multiplexed LFD with multiple test lines along the same ﬂow-path for multiplexed detection of CRP and SAA1, (a) with the SAA1 test line being before that for CRP and (c) with the CRP test line before that for SAA1. (b,d) are scanned image shows the results for three such LFDs described in (a,c). 3.3. Multiplexed Detection of CRP and SAA1 in Single-Channel LFDs 3.3. Multiplexed Detection of CRP and SAA1 in Single-Channel LFDs These results clearly confirmed here the problem of undesired interference between sequential detection sites positioned within a common flow-path, namely, the influence of a test-line on subsequent lines positioned further along the flow-path. This represents a key barrier to the use of such a single-channel LFD with sequentially positioned test lines as an appropriate tool for multiplexed detection. An alternative, as proposed by others to achieve multiplexed LFDs is to laminate or shape together different individual LFDs. However, this leads to an increased device footprint and therefore requires larger sample volumes, which is also undesirable, especially when working with samples such as blood or saliva etc. that normally are available in limited volumes. These results clearly conﬁrmed here the problem of undesired interference between sequential detection sites positioned within a common ﬂow-path, namely, the inﬂuence of a test-line on subsequent lines positioned further along the ﬂow-path. This represents a key barrier to the use of such a single-channel LFD with sequentially positioned test lines as an appropriate tool for multiplexed detection. An alternative, as proposed by others to achieve multiplexed LFDs is to laminate or shape together different individual LFDs. However, this leads to an increased device footprint and therefore requires larger sample volumes, which is also undesirable, especially when working with samples such as blood or saliva etc. that normally are available in limited volumes. 3 4 Integrated Dual-Channel LFDs for Multiplexed Detection of CRP and SAA1 3.4. Integrated Dual-Channel LFDs for Multiplexed Detection of CRP and SAA1 Based on the simultaneous appearance/absence of test lines in these separated channels, we can then identify the sample to be either CRP/SAA1 positive or positive/negative for both. As can be seen in the image (Figure 6b), both the test lines, for CRP in the left channel and SAA1 in the right channel have almost equal intensities to those for corresponding test lines within the standard single LFDs (Figure 4a,b) that we have previously tested. p y p p g LFD by dipping the strip into a 15 µL sample-reagent mixture and the results are shown in Figure 6b. Based on the simultaneous appearance/absence of test lines in these separated channels, we can then identify the sample to be either CRP/SAA1 positive or positive/negative for both. As can be seen in the image (Figure 6b), both the test lines, for CRP in the left channel and SAA1 in the right channel have almost equal intensities to those for corresponding test lines within the standard single LFDs (Figure 4a,b) that we have previously tested. To further confirm the above observations, the colour intensities of the test lines in these To further conﬁrm the above observations, the colour intensities of the test lines in these LDW-patterned dual-channel LFDs were measured and then compared with those acquired for both the standard single LFDs and single-channel multiplexed LFDs. The three concentrations used for these comparisons were 50 ng/mL, 100 ng/mL and 1 µg/mL for both CRP and SAA1. These results are shown in the plots in Figure 6c,d. For CRP (Figure 6c), at the same concentration, the intensity of the test line in a single-channel multiplexed LFD with the SAA1 test line before that for CRP dropped signiﬁcantly to less than half of that for a standard single LFD. The colour intensity of the test line for both the single-channel multiplexed LFD with CRP test line before that for SAA1 and our LDW patterned dual-channel LFD are at the same level as for the standard single LFD. Furthermore, the trend for the colour intensity levels for SAA1 (Figure 6d) is also the same as that observed for CRP. LDW-patterned dual-channel LFDs were measured and then compared with those acquired for both the standard single LFDs and single-channel multiplexed LFDs. The three concentrations used for these comparisons were 50 ng/mL, 100 ng/mL and 1 µg/mL for both CRP and SAA1. 3 4 Integrated Dual-Channel LFDs for Multiplexed Detection of CRP and SAA1 3.4. Integrated Dual-Channel LFDs for Multiplexed Detection of CRP and SAA1 3.4. Integrated Dual Channel LFDs for Multiplexed Detection of CRP and SAA1 To solve this problem of interference, we propose herein the use of our LDW method for creation of multiple flow-paths in a standard single LFD that allows isolation of each of those different detection sites without increasing the overall dimension of the original device To solve this problem of interference, we propose herein the use of our LDW method for creation of multiple ﬂow-paths in a standard single LFD that allows isolation of each of those different detection sites, without increasing the overall dimension of the original device. different detection sites, without increasing the overall dimension of the original device. As shown in the schematic in Figure 6a, a hydrophobic photopolymer barrier was patterned along the middle of the (reaction pad) nitrocellulose membrane to partition the original single flow channel into two parallel isolated flow-paths where individual test lines can be incorporated for As shown in the schematic in Figure 6a, a hydrophobic photopolymer barrier was patterned along the middle of the (reaction pad) nitrocellulose membrane to partition the original single ﬂow channel into two parallel isolated ﬂow-paths where individual test lines can be incorporated for detection within these separated channels. Here, we use this device with two ﬂow-paths as an example to implement multiplexed detection of CRP and SAA1. The capture antibodies for both test lines were Biosensors 2018, 8, 97 detection within example to implem 8 of 11 as an th test locally deposited into individual channels, as shown in the schematic with the left channel for CRP and the right for SAA1. The appearance or absence of either test line signiﬁes the presence or absence of either marker in the sample. lines were locally deposited into individual channels, as shown in the schematic with the left channel for CRP and the right for SAA1. The appearance or absence of either test line signifies the presence or absence of either marker in the sample. The tests were performed in the same way as for the previous multiplexed test single-channel The tests were performed in the same way as for the previous multiplexed test single-channel LFD by dipping the strip into a 15 µL sample-reagent mixture and the results are shown in Figure 6b. 3 4 Integrated Dual-Channel LFDs for Multiplexed Detection of CRP and SAA1 3.4. Integrated Dual-Channel LFDs for Multiplexed Detection of CRP and SAA1 These results are shown in the plots in Figure 6c,d. For CRP (Figure 6c), at the same concentration, the intensity of the test line in a single-channel multiplexed LFD with the SAA1 test line before that for CRP dropped significantly to less than half of that for a standard single LFD. The colour intensity of the test line for both the single-channel multiplexed LFD with CRP test line before that for SAA1 and our LDW patterned dual-channel LFD are at the same level as for the standard single LFD. Furthermore, the trend for the colour intensity levels for SAA1 (Figure 6d) is also the same as that observed for CRP. Figure 6. (a) Schematic of the laser direct-write (LDW) patterned multiplexed LFD with multiple channels for multiplexed detection of CRP and SAA1; (b) scanned image shows the multiplexed detection of CRP and SAA1 using LDW patterned dual-channel LFDs described in (a); (c,d) plots showing direct comparison of the measured colour intensities of test lines that appeared in standard single LFDs, single-channel multiplexed LFDs and our LDW-patterned dual-channel LFDs for detection of CRP & SAA1 respectively at concentrations of 50 ng/mL, 100 ng/mL and 1 µg/mL. Error bars indicate the standard deviation for 3 individual measurements. Figure 6. (a) Schematic of the laser direct-write (LDW) patterned multiplexed LFD with multiple channels for multiplexed detection of CRP and SAA1; (b) scanned image shows the multiplexed detection of CRP and SAA1 using LDW patterned dual-channel LFDs described in (a); (c,d) plots showing direct comparison of the measured colour intensities of test lines that appeared in standard single LFDs, single-channel multiplexed LFDs and our LDW-patterned dual-channel LFDs for detection of CRP & SAA1 respectively at concentrations of 50 ng/mL, 100 ng/mL and 1 µg/mL. Error bars indicate the standard deviation for 3 individual measurements. Figure 6. (a) Schematic of the laser direct-write (LDW) patterned multiplexed LFD with multiple channels for multiplexed detection of CRP and SAA1; (b) scanned image shows the multiplexed detection of CRP and SAA1 using LDW patterned dual-channel LFDs described in (a); (c,d) plots showing direct comparison of the measured colour intensities of test lines that appeared in standard single LFDs, single-channel multiplexed LFDs and our LDW-patterned dual-channel LFDs for detection of CRP & SAA1 respectively at concentrations of 50 ng/mL, 100 ng/mL and 1 µg/mL. Error bars indicate the standard deviation for 3 individual measurements. Figure 6. 4. Conclusions In conclusion, we have proposed a novel solution to achieve multiplexed diagnosis in LFDs that overcomes the limitations of current techniques including either interference of multiple test sites positioned in the same ﬂow-path or increased device dimensions that require larger sample volumes. We have developed a LDW technique that allows creation of multiple ﬂow-paths in a single LFD simply via the precise partition of the ﬂow-path of a single LFD. The multiple isolated parallel ﬂow-paths then allow individual detection of the different analytes in each of the separated channels without interference or cross-reaction. As a proof of principle, we have shown the successful use of a dual-channel LFD for multiplexed detection of a biomarker panel comprising CRP and SAA1, used commonly for the diagnosis of bacterial infections. The results show that our laser-patterned LFDs performed equally well as the single LFDs and do not need increased device dimensions or additional sample volumes. Overall, we believe that when compared to the current multiplexed detection procedures our technique offers a better solution for multiplexed detection within LFDs. Author Contributions: Conceptualization, P.J.W.H. and C.L.S.; Methodology, P.J.W.H.; Validation, P.J.W.H. and I.N.K.; Writing-Original Draft Preparation, P.J.W.H.; Writing-Review & Editing, P.J.W.H., I.N.K., R.W.E., C.L.S.; Supervision, R.W.E., C.L.S. Funding: This research was funded by the Engineering and Physical Sciences Research Council (EPSRC) Grant Nos. EP/N004388/1, EP/P025757/1 and EP/M027260/1. Funding: This research was funded by the Engineering and Physical Sciences Research Council (EPSRC) Grant Nos. EP/N004388/1, EP/P025757/1 and EP/M027260/1. Acknowledgments: The authors acknowledge the funding received via the Engineering and Physical Sciences Research Council (EPSRC) Grant Nos. EP/N004388/1, EP/P025757/1 and EP/M027260/1. The underpinning RDM data for this paper can be found at https://dor.org/10.5258/SOTON/D0454. Acknowledgments: The authors acknowledge the funding received via the Engineering and Physical Sciences Research Council (EPSRC) Grant Nos. EP/N004388/1, EP/P025757/1 and EP/M027260/1. The underpinning RDM data for this paper can be found at https://dor.org/10.5258/SOTON/D0454. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 3 4 Integrated Dual-Channel LFDs for Multiplexed Detection of CRP and SAA1 3.4. Integrated Dual-Channel LFDs for Multiplexed Detection of CRP and SAA1 (a) Schematic of the laser direct-write (LDW) patterned multiplexed LFD with multiple channels for multiplexed detection of CRP and SAA1; (b) scanned image shows the multiplexed detection of CRP and SAA1 using LDW patterned dual-channel LFDs described in (a); (c,d) plots showing direct comparison of the measured colour intensities of test lines that appeared in standard single LFDs, single-channel multiplexed LFDs and our LDW-patterned dual-channel LFDs for detection of CRP & SAA1 respectively at concentrations of 50 ng/mL, 100 ng/mL and 1 µg/mL. Error bars indicate the standard deviation for 3 individual measurements. 9 of 11 Biosensors 2018, 8, 97 Biosensors 2018, 8, 97 Overall, as anticipated, these results conclusively conﬁrm that there is no interference between two test lines placed within the individual channels of the dual-channel LFD, because both detections are implemented independently in parallel channels rather than in series within a single channel. Additionally, by patterning more than a single barrier in the reaction pad more ﬂow-paths can be created hence allowing multiplexing of more than this proof-of-principle dual assay. In the current dual-channel LFDs, the laser-patterned barrier walls have dimensions of ~500 µm, however, this can be reduced to have dimensions of ~200 µm if desired. It is therefore then possible to pattern a greater number of ﬂuidic ﬂow-paths while maintaining the widths of our LFDs to be similar to that of a standard single LFD (that has a width of ~5mm)—allowing detection of not two, but a greater number of analytes from within the same sample. References 1. Gubala, V.; Harris, L.F.; Ricco, A.J.; Tan, M.X.; Williams, D.E. Point of care diagnostics: Status and future. Anal. Chem. 2011, 84, 487–515. [CrossRef] [PubMed] 2. Safavieh, R.; Juncker, D. Capillarics: Pre-programmed, self-powered microﬂuidic circuits built from capillary elements. Lab Chip 2013, 13, 4180–4189. [CrossRef] [PubMed] 3. Foudeh, A.M.; Didar, T.F.; Veres, T.; Tabrizian, M. Microﬂuidic designs and techniques using lab-on-a-chip devices for pathogen detection for point-of-care diagnostics. Lab Chip 2012, 12, 3249–3266. [CrossRef] [PubMed] 4. Wu, G.; Zaman, M.H. Low-cost tools for diagnosing and monitoring hiv infection in low-resource settings. Bull. World Health Organ. 2012, 90, 914–920. [CrossRef] [PubMed] 5. Martinez, A.W.; Phillips, S.T.; Whitesides, G.M.; Carrilho, E. Diagnostics for the developing world: Microﬂuidic paper-based analytical devices. Anal. Chem. 2010, 82, 3–10. [CrossRef] [PubMed] 6. Hu, J.; Wang, S.; Wang, L.; Li, F.; Pingguan-Murphy, B.; Lu, T.J.; Xu, F. Advances in paper-based point-of-care diagnostics. Biosens. Bioelectron. 2014, 54, 585–597. [CrossRef] [PubMed] 1. Gubala, V.; Harris, L.F.; Ricco, A.J.; Tan, M.X.; Williams, D.E. Point of care diagnostics: Status and future. Anal. Chem. 2011, 84, 487–515. [CrossRef] [PubMed] 1. Gubala, V.; Harris, L.F.; Ricco, A.J.; Tan, M.X.; Williams, D.E. Point of care diagnostics: Status and future. Anal. Chem. 2011, 84, 487–515. [CrossRef] [PubMed] 2. Safavieh, R.; Juncker, D. Capillarics: Pre-programmed, self-powered microﬂuidic circuits built from capillary elements. Lab Chip 2013, 13, 4180–4189. [CrossRef] [PubMed] 3. Foudeh, A.M.; Didar, T.F.; Veres, T.; Tabrizian, M. Microﬂuidic designs and techniques using lab-on-a-chip devices for pathogen detection for point-of-care diagnostics. Lab Chip 2012, 12, 3249–3266. [CrossRef] [PubMed] 3. Foudeh, A.M.; Didar, T.F.; Veres, T.; Tabrizian, M. Microﬂuidic designs and techniques using lab-on-a-chip devices for pathogen detection for point-of-care diagnostics. Lab Chip 2012, 12, 3249–3266. [CrossRef] [PubMed] 4. Wu, G.; Zaman, M.H. Low-cost tools for diagnosing and monitoring hiv infection in low-resource settings. Bull. World Health Organ. 2012, 90, 914–920. [CrossRef] [PubMed] 5. Martinez, A.W.; Phillips, S.T.; Whitesides, G.M.; Carrilho, E. Diagnostics for the developing world: Microﬂuidic paper-based analytical devices. Anal. Chem. 2010, 82, 3–10. [CrossRef] [PubMed] 6. Hu, J.; Wang, S.; Wang, L.; Li, F.; Pingguan-Murphy, B.; Lu, T.J.; Xu, F. Advances in paper-based point-of-care diagnostics. Biosens. Bioelectron. 2014, 54, 585–597. [CrossRef] [PubMed] 10 of 11 Biosensors 2018, 8, 97 7. Wei, X.; Tian, T.; Jia, S.; Zhu, Z.; Ma, Y.; Sun, J.; Lin, Z.; Yang, C.J. Target-responsive DNA hydrogel mediated “stop-ﬂow” microﬂuidic paper-based analytic device for rapid, portable and visual detection of multiple targets. References Anal. Chem. 2015, 87, 4275–4282. [CrossRef] [PubMed] 8. Magambo, K.A.; Kalluvya, S.E.; Kapoor, S.W.; Seni, J.; Choﬂe, A.A.; Fitzgerald, D.W.; Downs, J.A. Utility of urine and serum lateral ﬂow assays to determine the prevalence and predictors of cryptococcal antigenemia in HIV-positive outpatients beginning antiretroviral therapy in mwanza, tanzania. J. Int. AIDS Soc. 2014, 17, 19040. [CrossRef] [PubMed] 9. De Lourdes Moreno, M.; Cebolla, Á.; Muñoz-Suano, A.; Carrillo-Carrion, C.; Comino, I.; Pizarro, Á.; León, F.; Rodríguez-Herrera, A.; Sousa, C. Detection of gluten immunogenic peptides in the urine of patients with coeliac disease reveals transgressions in the gluten-free diet and incomplete mucosal healing. Gut 2015, 66, 250–257. [CrossRef] [PubMed] 10. Carrio, A.; Sampedro, C.; Sanchez-Lopez, J.L.; Pimienta, M.; Campoy, P. Automated low-cost smartphone-based lateral ﬂow saliva test reader for drugs-of-abuse detection. Sensors 2015, 15, 29569–29593. [CrossRef] [PubMed] 11. Paciﬁci, R.; Farré, M.; Pichini, S.; Ortuño, J.; Roset, P.N.; Zuccaro, P.; Segura, J.; de la Torre, R. Sweat testing of MDMA with the drugwipe® analytical device: A controlled study with two volunteers. J. Anal. Toxicol. 2001, 25, 144–146. [CrossRef] [PubMed] Koczula, K.M.; Gallotta, A. Lateral ﬂow assays. Essays Biochem. 2016, 60, 111–120. [CrossRef] [PubMed] y y 13. Ngom, B.; Guo, Y.; Wang, X.; Bi, D. Development and application of lateral ﬂow test strip technology for detection of infectious agents and chemical contaminants: A review. Anal. Bioanal. Chem. 2010, 397, 1113–1135. [CrossRef] [PubMed] 14. Butler, S.A.; Khanlian, S.A.; Cole, L.A. Detection of early pregnancy forms of human chorionic gonadotropin by home pregnancy test devices. Clin. Chem. 2001, 47, 2131–2136. [PubMed] 15. Panhotra, B.; Hassan, Z.; Joshi, C.; Bahrani, A. Visual detection of multiple viral amplicons by dipstick assay: Its application in screening of blood donors a welcome tool for the limited resource settings. J. Clin. Microbiol. 2005, 43, 6218–6219. [CrossRef] [PubMed] 16. Corstjens, P.L.; Claudia, J.; van der Ploeg-van, J.J.; Wiesmeijer, K.C.; Riuttamäki, T.; van Meijgaarden, K.E.; Spencer, J.S.; Tanke, H.J.; Ottenhoff, T.H.; Geluk, A. Lateral ﬂow assay for simultaneous detection of cellular-and humoral immune responses. Clin. Biochem. 2011, 44, 1241–1246. [CrossRef] [PubMed] 17. Mendoza, L.; McQuary, P.; Mongan, A.; Gangadharan, R.; Brignac, S.; Eggers, M. High-throughput microarray-based enzyme-linked immunosorbent assay (ELISA). Biotechniques 1999, 27, 778–789. [CrossRef] [PubMed] 18. Markoulatos, P.; Siafakas, N.; Moncany, M. Multiplex polymerase chain reaction: A practical approach. J. Clin. Lab. Anal. 2002, 16, 47–51. [CrossRef] [PubMed] 19. Pérez-Pérez, F.J.; Hanson, N.D. References Detection of plasmid-mediated AmpC β-lactamase genes in clinical isolates by using multiplex PCR. J. Clin. Microbiol. 2002, 40, 2153–2162. [CrossRef] [PubMed] 20. Spiro, A.; Lowe, M.; Brown, D. A bead-based method for multiplexed identiﬁcation and quantitation of DNA sequences using ﬂow cytometry. Appl. Environ. Microbiol. 2000, 66, 4258–4265. [CrossRef] [PubMed] 20. Spiro, A.; Lowe, M.; Brown, D. A bead-based method for multiplexed identiﬁcation and quantitation of DNA sequences using ﬂow cytometry. Appl. Environ. Microbiol. 2000, 66, 4258–4265. [CrossRef] [PubMed] 21. Huang, X.H.; Wang, Q.; Chen, J.S.; Fu, X.H.; Chen, X.L.; Chen, L.Z.; Li, W.; Bi, J.; Zhang, L.J.; Fu, Q. Bead-based microarray analysis of microRNA expression in hepatocellular carcinoma: miR 338 is downregulated 21. Huang, X.H.; Wang, Q.; Chen, J.S.; Fu, X.H.; Chen, X.L.; Chen, L.Z.; Li, W.; Bi, J.; Zhang, L.J.; Fu, Q. Bead-based microarray analysis of microRNA expression in hepatocellular carcinoma: miR-338 is downregulated. Hepatol. Res. 2009, 39, 786–794. [CrossRef] [PubMed] p 22. Martinez, A.W.; Phillips, S.T.; Butte, M.J.; Whitesides, G.M. Patterned paper as a platform for inexpensive, low-volume, portable bioassays. Angew. Chem. Int. Ed. 2007, 46, 1318–1320. [CrossRef] [PubMed] 23. Fenton, E.M.; Mascarenas, M.R.; López, G.P.; Sibbett, S.S. Multiplex lateral-ﬂow test strips fabricated by two-dimensional shaping. ACS Appl. Mater. Interfaces 2008, 1, 124–129. [CrossRef] [PubMed] 23. Fenton, E.M.; Mascarenas, M.R.; López, G.P.; Sibbett, S.S. Multiplex lateral-ﬂow test strips fabricated by two-dimensional shaping. ACS Appl. Mater. Interfaces 2008, 1, 124–129. [CrossRef] [PubMed] 24. Li, J.; Macdonald, J. Multiplex lateral ﬂow detection and binary encoding enables a molecular colorimetric 7-segment display. Lab Chip 2016, 16, 242–245. [CrossRef] [PubMed] 24. Li, J.; Macdonald, J. Multiplex lateral ﬂow detection and binary encoding enables a molecular colorimetric 7-segment display. Lab Chip 2016, 16, 242–245. [CrossRef] [PubMed] 25. Dineva, M.A.; Candotti, D.; Fletcher-Brown, F.; Allain, J.-P.; Lee, H. Simultaneous visual detection of multiple viral amplicons by dipstick assay. J. Clin. Microbiol. 2005, 43, 4015–4021. [CrossRef] [PubMed] 25. Dineva, M.A.; Candotti, D.; Fletcher-Brown, F.; Allain, J.-P.; Lee, H. Simultaneous visual detection of multiple viral amplicons by dipstick assay. J. Clin. Microbiol. 2005, 43, 4015–4021. [CrossRef] [PubMed] 26. Sones, C.L.; Katis, I.N.; He, P.J.W.; Mills, B.; Namiq, M.F.; Shardlow, P.; Ibsen, M.; Eason, R.W. Laser-induced photo-polymerisation for creation of paper-based ﬂuidic devices. Lab Chip 2014, 14, 4567–4574. [CrossRef] [PubMed] 26. Sones, C.L.; Katis, I.N.; He, P.J.W.; Mills, B.; Namiq, M.F.; Shardlow, P.; Ibsen, M.; Eason, R.W. Laser-induced photo-polymerisation for creation of paper-based ﬂuidic devices. References Lab Chip 2014, 14, 4567–4574. [CrossRef] [PubMed] 11 of 11 11 of 11 Biosensors 2018, 8, 97 27. He, P.J.W.; Katis, I.N.; Eason, R.W.; Sones, C.L. Engineering ﬂuidic delays in paper-based devices using laser direct-writing. Lab Chip 2015, 15, 4054–4061. [CrossRef] [PubMed] 28. He, P.J.W.; Katis, I.N.; Eason, R.W.; Sones, C.L. Laser-based patterning for ﬂuidic devices in nitrocellulose. Biomicroﬂuidics 2015, 9, 26503. [CrossRef] [PubMed] 29. Pepys, M.B.; Hirschﬁeld, G.M. C-reactive protein: A critical update. J. Clin. Investig. 2003, 111, 1805–1812. [CrossRef] [PubMed] 30. Juul-Madsen, H.R.; Viertlböeck, B.; Härtle, S.; Smith, A.L.; Göbel, T.W. Chapter 7—Innate Immune Responses. In Avian Immunology, 2nd ed.; Kaspers, B., Kaiser, P., Eds.; Academic Press: Boston, MA, USA, 2014; pp. 121–147. 31. Scientiﬁc, T. Elisa technical guide and protocols. Manual 2010, 747, 815. 31. Scientiﬁc, T. Elisa technical guide and protocols. Manual 2010, 747, 815. © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W2782930256	https://journals.muni.cz/socialni_studia/article/download/5520/4618	Czech	null	Exkluze v liberálním státě: případ politiky imigrace a občanství	Sociální studia	2,006	cc-by	9,024	Exkluze v liberálním státě: Případ politiky imigrace a občanství Exclusion in the Liberal State. The Case of Immigration and Citizenship Policy Christian Joppke Sociální studia. Fakulta sociálních studií Masarykovy univerzity , 1/2006. S. 55–72. ISSN 1212-813X. Překlad převzat z: Joppke, Ch. 2005. „Exclusion in the Liberal State: The Case of Immigration and Citizenship Policy“. European Journal of Social Theory, 8 (1): 43–61. © SAGE Publications, 2005. Publikováno se svolením Sage Publications Ltd., www.sagepublications.com Publications, 2005. Publikováno se svolením Sage Publications Ltd., www.sagepublications.com ABSTRACT Recent literature on the “exclusions” of the modern nation-state has missed a major transformation in the legitimate mode of excluding, from group- to individual-based. This transformation is explored in a discussion of universalistic trends in contemporary Western states’ immigration and citizenship policies. Conflicting with the notion of a “nation-state” owned by a particular ethnic group or nation, these trends are better captured in terms of a “liberal state” that has self-limited its sovereign prerogatives by constitutional principles of equality and individual rights. Key words: citizenship, discrimination, immigration, liberalism, nations and nationalism. ABSTRACT Citizenship, discrimination, imigration, liberalism, nations and nationalism KEY WORDS Citizenship, discrimination, imigration, liberalism, nations and nationalism KEY WORDS Převládající model organizování národního státu v moderní době – západní národní stát – je poznamenán napětím mezi univerzalistickým liberalismem a partikulárním nacionalismem. Univerzalistický liberalismus usiluje o rovná práva a svobody pro všechny své členy, parti- kulární nacionalismus se snaží zabránit přístupu k výsadám všem, kdo nejsou členy. Již od slavného postřehu Johna Stuarta Milla (1991: 310), že „existence svobodných institucí je v ze- mi tvořené různými národnostmi téměř nemožná“, se často zdůrazňuje, že se nejedná pouze o volnou soutěž protikladných a neslučitelných sil: vznešená fasáda rovnosti a svobody údaj- ně spočívá na nečestných základech nacionalismu, který poskytuje stejnorodost a prostorovou ohraničenost, bez níž by liberalismus nemohl vzkvétat. Někteří se pokusili o nemožné skrze „liberální nacionalismus“ (Tamir, 1993; Miller, 1995), zatímco další ponechali tento rozpor rozporem a namísto toho odhalili liberalismus liberálů jako příklad zlého úmyslu (nejnověji Wimmer, 2002 a Marx 2003 a níže). Pozoruhodným faktem zůstává, že rozhodnutí určující příslušenství k danému státu předchází, a je tedy imunní vůči silám liberalismu a má tendenci být sdíleno napříč politicko- -filosofickými pozicemi. Joseph Carens (1987: 252) pranýřoval z kosmopolitní perspektivy občanství v západních liberálních demokraciích jako „moderní obdobu feudálních výsad“, protože je zpravidla přiděleno při narození, spíše než aby bylo svobodně zvolenou charak- teristikou; Michael Walzer (1983: 61 a dále) obhajoval z komunitaristické perspektivy „při- SOCIÁLNÍ STUDIA 1/2006 dělování členství“ jako akt „nepodléhající vše-prostupujícímu nátlaku spravedlnosti“, pro- tože jinak by nemohla existovat morálně vyspělá společenství (communities of character); Claus Offe (1998: 117) ze sociálně-demokratické perspektivy shledává, že „demokratická teorie nebo ústavní doktrína neposkytují dostatečné důvody k tomu, proč by sociální dimen- ze měla být tím, čím ve skutečnosti je“, a domnívá se, že je to tak v pořádku, protože pouze danost „území“ nebo „národa“ může poskytnout záruku „stejnorodosti“, která je nezbytným předpokladem liberální demokracie; a Will Kymlicka (1995:124) z liberálně nacionalistické perspektivy odvozuje legitimitu práv menšin od předpokládaného neomezeného „práva států rozhodovat o tom, kdo má občanství“, čímž považuje práva menšin za jistou kompenzaci axi- omaticky přepokládaného partikularistického budování národa v moderních státech.1 Inherentní partikulárnost národního státu je často shrnována v pojetí „vlastnictví“ státu jistou národní nebo etnickou skupinou za cenu „exkluze“ všech skupin, které nejsou národní či etnické (nejradikálněji Wimmer 2002; historicky a geograficky citlivěji odstíněno v Marx 2002, 2003). 1 V pozdější stati (2002) Kymlicka rozpracoval názor více zohledňující jemné odlišnosti vztahující se k „sebe-limitujícímu“ budování národa v současných liberálních státech, uhýbá však před nepří- jemnými implikacemi vztahujícími se na jeho ospravedlnění práv menšin. 2 Ze stejného důvodu je následující analýza omezena na formální úroveň zákonů státu a jeho po- litik. Tím není popíráno, že „nacionalistické“ nebo „rasistické“ uvažování nebo jednání nemůže být nepříjemně útočné v jiných sektorech společnosti, dokonce i mezi (některými) elitami státu. Avšak takové uvažování nebo jednání zůstává nutně skryté a zcela ve shodě s formální fasádou „nediskriminace“. V tomto minimálním významu je formální fasáda nediskriminace „skutečná ve svých důsledcích“ a nemůže být zcela odmítnuta jako pouhá propaganda nebo předstírání. Pokud je však vzneseno obvinění z předstírání, nutně se vystavuje protiútoku vytvoření konspirace, která se vymyká reálným rozměrům. 3 Přestože je formálně mimo dosah Mezinárodní konvence OSN o Odstranění všech forem rasové diskriminace (v článku 1.3), podléhá oblast imigrace a občanství některým omezením mezinárod- ního práva (viz Goodwin-Gill 1978: 5. kapitola). Národní stát a jeho exkluze Andreas Wimmer (2002) se znovu vypořádává s paradoxem politické modernity, kdy univerzální principy demokracie a občanství vznikaly pouze v partikulárním prostředí, za vyloučení všech, kteří nesdíleli jisté etnické nebo národní znaky: „Moderní principy zahrnu- tí jsou úzce propojené s etnickými a národními formami vyloučení“ (2002: 1). Stržení verti- kálních nerovností, formálně vepsaných mezi feudální stavy a vrstvy, se uskutečnilo za cenu vztyčení nových horizontálních nerovností mezi vnitřně homogenními etnickými skupinami a národy, z nichž každý usiluje o výlučné propojení se státem za vyloučení všech ostatních, kteří jsou na základě těchto připsaných označení považováni za nepřináležející ke státu. Podle Wimmera je novým zhoubným pojmem, vzniknuvším kombinací nacionalismu a demokracie, „vlastnictví“ státu partikulárním národem nebo etnickou skupinou, jež činí etnický konflikt, xenofobii a rasismus „integrální součástí“ moderních národních států (2002: 5). Přestože Wimmer nahlíží na pojem „vlastnictví“ jako na univerzální pojem politické modernity, rozlišuje mezi dvěma cestami vedoucími k takové modernitě. První cestou je „poli- tizace etnické příslušnosti“, v níž jedna etnická skupina stát monopolizuje, ale zároveň se jí nedaří zevšeobecnit svůj „etnický“ otisk do „národního“ otisku, a tak uvízne v etnickém klien- telismu a zvýhodňování. V tomto případě, který je pravděpodobně charakteristický pro většinu méně rozvinutých nezápadních společností, je tento tlak na exkluzi nasměrován k potlačení nároků vnitřních etnických soupeřů. Druhou cestou je „plná nacionalizace státu a společnosti“, která nastává tehdy, pokud jsou stát i společnost silní, a má za následek úplnou národnostní homogenizace společnosti. V tomto případě, charakteristickém pro rozvinutý Západ, je tlak na exkluzi stále přítomen, ale je nasměrován na vnější „cizince“ a „imigranty“. Jedná se o poněkud neumělou teorii politické modernizace (co například Kanada, Belgie či Španělsko?) a Wimmer vzápětí sám připouští, že jeho zájem je zaměřen méně na příčiny a vzá- jemné srovnání a spíše na obecně použitelné důsledky toho, co nazývá „model národního státu“: nástup „nových forem exkluze založených na etnicko-národnostních principech“ (2002: 81). Jeho hlavním tvrzením je, že tyto „etnicko-národnostní“ exkluze jsou v etnických a nacionálních variantách politické modernizace „strukturálně obdobné“ (2002: 268): „Osud tzv. failií Kurdů v Iráku, kteří byli v 70. letech 20. století zbaveni iráckého občanství a vyhnáni do Iránu…, je podobný nejisté právní situaci imigrantů v plně nacionalizovaných státech“ (2002: 222). Zatímco byl zjevně vytvořen s cílem provokovat, tento antropologický pohled založený na „pohledu z odstupu“ stírá některá základní rozlišení, bez nichž se jen stěží obejdeme. Citizenship, discrimination, imigration, liberalism, nations and nationalism KEY WORDS V tomto článku zastávám názor, že všeobecně dostupná literatura o národech a nacionalismu neodpovídá politické a právní skutečnosti.2 Přinejmenším pro současný západ- ní stát v „severoatlantické bezpečností zóně“ (Deutsch 1957) navrhuji místo toho více odpo- vídající obraz státu jakožto „liberálního státu“, který omezil své nároky na suverenitu ústav- ním zavázáním se k principům rovnosti a práv jednotlivce. Podporuji tento názor empirickým označením domovské půdy pojmu výhradní „vlastnictví“: oblasti imigrační politiky a politiky občanství, skrze které je stanoveno členství ve státě. Měli bychom tento konstrukt zavrhnout, pokud jej nemůžeme nalézt ve zde uvedeném nejpravděpodobnějším případě platnosti tohoto konstruktu „vlastnictví“ (King a spol. 1994: 209). Avšak zdá se, že alternativní obraz liberálního státu omezeného ústavou vytváří paradox: jak mohou politiky, které upravují provždy partikulární hranice určité společnosti, být nosi- teli anonymních znaků univerzalismu a nediskriminace; nejsou partikularismus a diskrimina- ce vepsány do samotných teorií těchto politik, jak je to uznáno v konstruktu mezinárodního práva v (téměř)3 neomezené státní suverenitě v záležitostech imigrační politiky a národnostní- ho zákona? V první části přistupuji k těmto námitkám záporně, přičemž poukazuji na některé 1 V pozdější stati (2002) Kymlicka rozpracoval názor více zohledňující jemné odlišnosti vztahující se k „sebe-limitujícímu“ budování národa v současných liberálních státech, uhýbá však před nepří- jemnými implikacemi vztahujícími se na jeho ospravedlnění práv menšin. 3 Přestože je formálně mimo dosah Mezinárodní konvence OSN o Odstranění všech forem rasové diskriminace (v článku 1.3), podléhá oblast imigrace a občanství některým omezením mezinárod- ního práva (viz Goodwin-Gill 1978: 5. kapitola). 56 Christian Joppke: Exkluze v liberálním státě obtíže, které vyvstanou lpěním na paradigmatu „vlastnictví“ či „národního státu“. V druhé části se k těmto námitkám stavím kladně, odhalujíc některé univerzalistické a nediskriminační znaky současných politik imigrace a občanství v dnešních západních státech. Jak ukazuji ve třetí a závěrečné části, národní státy budou samozřejmě nadále existovat, avšak národní parti- kularismus již nemůže být vynucován skrze jejich politiky členství. V míře, v jaké se odkazy na národní prvky v těchto politikách stále objevují, jeví se tyto politiky pouze lokálními ver- zemi liberálně demokratického přesvědčení o rovnosti a právech jedince. 4 Poznamenejme, že vnější exkluze je srovnatelná s vnitřní exkluzí vzhledem k potenciálním ško- dám, které může přivodit jedinci. Exkluze uprchlíků byla zařazena pod přísnější právní omezení, v pojmech mezinárodního režimu uprchlíků v principu „nezahnání“, nebo dokonce v některých evropských státech (jako Francie či Německo) pod ústavní omezení. Avšak ústavní omezení byla zmírněna nebo zrušena v reakci na masivní nárůst žádostí o azyl na konci 80. let 20. století. 5 Tento způsob argumentace je v souladu s deﬁ nicí „rasové diskriminace“ v Mezinárodní konven- ci o odstranění všech forem rasové diskriminace OSN z roku 1966. V článku 1.1 tato konvence deﬁ nuje „rasovou diskriminaci“ „jako rozlišování, exkluzi, omezování nebo upřednostňování na základě rasy, barvy pleti, národního nebo etnického původu, která má za cíl nebo za důsledek zru- šení nebo oslabení uznání, požívání nebo uplatňování lidských práv a základních svobod na základě rovného zacházení“. Následující pododstavec (1.2) z rovného zacházení vylučuje všechny „rozdíly, exkluze, omezení nebo upřednostňování… mezi občany a cizími státními příslušníky“. Národní stát a jeho exkluze Jedním z těchto rozlišení je rozlišování mezi různými místy exkluze, vnitřními nebo vnějšími; druhým je rozlišování mezi způsoby exkluze, exkluzí skupinovou či individuální. S ohledem na místo 57 SOCIÁLNÍ STUDIA 1/2006 exkluze probíhá exkluze osoby, která se snaží začlenit se do státu (vnější exkluze), na zcela jiné úrovni než exkluze někoho, kdo již ve státě pobývá a nemá jiný stát, u kterého by hledal ochranu (vnitřní exkluze); rozdíl je přinejmenším v tom, že první je reaktivní, zatímco druhá je aktivní.4 „Diskriminaci“ v plném smyslu slova představuje pouze aktivní exkluze, a to tím, že odpírá něko- mu základní „právo na místo“ (Walzer 1983) na základě vrtkavého připsání členství ve skupině.5 p ( ) p p p S ohledem na způsob exkluze fungují pouze některé exkluze na základě etnické přísluš- nosti, národního původu nebo rasy a následně – v současném diskurzu o minoritách a lidských právech – mohou být označeny jako „diskriminační“. Zjevným příkladem je asijská exklu- ze, která byla součástí imigračních politik před šedesátými lety 20. století ve státech obsaze- ných anglicky mluvícími osadníky. V návaznosti na přelomové období v oblasti lidských práv v USA v 60. letech 20. století a na celosvětové posílení kultury lidských práv byly kolektivní a připsané způsoby exkluze nahrazeny individuálními způsoby exkluze, založenými na (nedo- statečných) rodinných vazbách a dovednostech. Politiky imigrace (stejně jako občanství) se staly explicitně „nediskriminačními“.6 Existuje základní rozdíl mezi bezpodmínečným skupi- novým a individuálním vyloučením, a tím, že obě formy propojíme, mnoho nezískáme. Na pozadí tohoto propojení je problematické položit rovnítko mezi exkluzi jako tako- vou a diskriminaci podle následujícího sylogismu vštěpovaného „zdravým rozumem“: politi- ka imigrace (a občanství) je vylučující, exkluze nutně znamená rozlišování a takové politiky tedy následně nemohou nic jiného než „diskriminovat“. V pojmech politických důsledků má tento sylogismus Janusovu tvář: může přinést buď odmítnutí, nebo odsouhlasení jakékoli imi- grační politiky. Cesta k utopii bez hranic pro některé jedince může být snadno přetvořena do hyper-realistické obrany diskriminace. Podle formulace tajemného kritika asijské imigrace do Austrálie Goffrey Blaineyho (1984: 162) imigrační politika z definice „diskriminuje“ – tak proč selektivně neomezit vstup Asiatů, a navrátit tak Austrálii bílým Australanům? 5 Tento způsob argumentace je v souladu s deﬁ nicí „rasové diskriminace“ v Mezinárodní konven- ci o odstranění všech forem rasové diskriminace OSN z roku 1966. Národní stát a jeho exkluze V článku 1.1 tato konvence deﬁ nuje „rasovou diskriminaci“ „jako rozlišování, exkluzi, omezování nebo upřednostňování na základě rasy, barvy pleti, národního nebo etnického původu, která má za cíl nebo za důsledek zru- šení nebo oslabení uznání, požívání nebo uplatňování lidských práv a základních svobod na základě rovného zacházení“. Následující pododstavec (1.2) z rovného zacházení vylučuje všechny „rozdíly, exkluze, omezení nebo upřednostňování… mezi občany a cizími státními příslušníky“. 5 Tento způsob argumentace je v souladu s deﬁ nicí „rasové diskriminace“ v Mezinárodní konven- ci o odstranění všech forem rasové diskriminace OSN z roku 1966. V článku 1.1 tato konvence deﬁ nuje „rasovou diskriminaci“ „jako rozlišování, exkluzi, omezování nebo upřednostňování na základě rasy, barvy pleti, národního nebo etnického původu, která má za cíl nebo za důsledek zru- šení nebo oslabení uznání, požívání nebo uplatňování lidských práv a základních svobod na základě rovného zacházení“. Následující pododstavec (1.2) z rovného zacházení vylučuje všechny „rozdíly, exkluze, omezení nebo upřednostňování… mezi občany a cizími státními příslušníky“. K tématu zemí osídlených anglicky mluvícími osadníky viz Joppke (2005b). Připusťme, že v Ev- ropě je rétorika „nediskriminace“ méně výrazná. Avšak kdykoli byla vznesena obvinění z etnické diskriminace proti některému z evropských států, příslušná vláda existenci takové diskriminace vždy popřela. Výmluvným příkladem je diskuse okolo klauzule o „patriotismu“ Imigračního záko- na v Británii z roku 1971, která byla oponenty (Labour Party a aktivisty z celého světa) napadána jako rasově diskriminační, avšak která byla vládou Toryů hájena na základě uznání neetnických termínů „rodinných vztahů“. K této diskusi viz Joppke (2004b). 58 Christian Joppke: Exkluze v liberálním státě Christian Joppke: Exkluze v liberálním státě Christian Joppke: Exkluze v liberálním státě Je zajímavé, že Wimmer si správně všímá toho, že očividný „rasismus a xenofobie“ jsou v současných západních společnostech přisuzovány „mobilitně sestupujícím“, zatímco „stát… se zjevně zdráhá jednat“ (2002: 215). Avšak Wimmer již není schopen své tvrzení teoreticky zdů- vodnit. Naopak rigidita jeho „modelu národního státu“ ho vede ke zlehčování či zkreslování uni- verzalismu na úrovni národního modelu. Obzvláště zjevné to je v jeho rozboru imigrační politiky ve Švýcarsku, která má být příkladem vnějškově orientované „exkluze ve znacionalizovaných státech“. Jak vyžaduje „model národního státu“, krouží kapitola okolo „systematické diskrimina- ce mezi cizinci a státními příslušníky, vepsané do zákonů o občanství a zvláště do institucionál- ní mašinerie, která kontroluje a omezuje migrační přílivy“ (2002: 222). Návrh, který se zjevně nabízí, zní, že již právní rozlišování mezi občany a nelegálními přistěhovalci představuje „dis- kriminaci“. Národní stát a jeho exkluze Ale jaké označení bychom tedy měli zvolit pro vrtošivé popření práv rodiny a práv na svobodnou volbu pobytu a zaměstnání, které byly znakem původního režimu „hostujících pracovníků“? S ohledem na režim hostujících pracovníků Wimmer uznává, že tento byl následně nahrazen režimem liberálnějším, který status imigrantů v mnoha ohledech přiblížil statusu obča- nů. Tuto změnu není možné vysvětlit v rámci „modelu národního státu“ a sám Wimmer vzápětí upouští od dalšího promýšlení této proměny završením švýcarského příběhu návratem k „restrik- tivní politice“ v roce 1970. Bez povšimnutí tak přechází fakt, že politika kvót z roku 1970 ome- zuje pouze počet imigrantů, a je tedy méně destruktivní než dřívější politika, která nově přícho- zím upírala základní rodinná nebo socio-ekonomická práva. Ve skutečnosti bylo striktní omezení počtu příchozích možná cenou za vnitřní začlenění hostujících pracovníků, kteří se stali imigran- ty. To naznačuje i sám Wimmer (2002: 262): „Tato částečná integrace, která následovala dialek- tiku inkluze a exkluze, šla ruku v ruce s dalším omezením přístupu na státní území. Postupně byl zaveden velmi přísný systém imigračních kvót, kontrolovaných státem.“ Naopak pokud uzavření se před další imigrací bylo výsledkem rostoucí snahy o vnitřní inkluzi, pak touha být začleňují- cí nemohla být motivována pouze „rostoucím soutěžením“ o migrační pracovníky, jak Wimmer podivně jedním dechem prohlašuje (2002: 262) – obě tvrzení jsou logicky neslučitelná. V celém textu je systematicky opouštěna „liberální“ logika (poháněná tlakem soudů či občanské společnosti), jež mohla donutit švýcarský stát k opuštění původní politiky hostují- cích pracovníků (tzv. „gastarbeiterů“), jak tomu bylo ve všech ostatních evropských společ- nostech, které prodělaly ve stejné době podobnou zkušenost s hostujícími pracovníky (viz Guiraudon 2000). Wimmer (2002: 2 a dál) poznamenává na samém začátku svého pojednání, že od 70. let 20. století se „většina západních společností… navrátila k dřívější, liberálnější podobě definice těch, kteří přináleží“, ale uhýbá před zjevným problémem, který tato skuteč- nost přináší pro jeho vylučující „model národního státu“. V kontrastu s Wimmerovým antropologickým a příliš zevšeobecňujícím představením vylučujících „stínů modernity“ politolog Anthony Marx (2002, 2003), který pojednává o stej- ném tématu „národního státu a jeho vyloučeních“ (podobně jako Wimmer, za udivujícího vzá- jemného přehlížení), nahlíží přesněji na příčiny, podoby a načasování národní exkluze. 7 Výčet aktérů a mechanismů, které přivedl k životu „liberální stát“, by přesáhl omezený rozsah tohoto článku. Namísto toho beru existenci liberálního státu za danou a nahlížím pouze na některé jeho důsledky – nebo naopak tyto důsledky (v politikách imigrace nebo občanství) mohou svědčit o existenci liberálního státu. Národní stát a jeho exkluze Zatímco potenciál k exkluzi je nacionalismu vnitřně vlastní, jak je to shrnuto v Yeatsově výroku „je více podstaty v našem nepřátelství než v naší lásce“ (citováno v Marx, 2003: 29), bývá jasně vyjádřen pouze ve specifických situacích – zvláště tehdy, pokud se elity státu snaží zacelit vnitřní roze- pře selektivním vyloučením třetí strany. Marx zmiňuje možnost vnějších exkluzí pouze stručně a celá jeho pozornost je příhodně věnována stěžejnímu bodu, jímž je exkluze vnitřní skupiny. 59 SOCIÁLNÍ STUDIA 1/2006 Pokud přehlédneme jím uváděné příklady, které sahají od náboženských pronásledování v ra- ně moderní Evropě až k jihoafrickému apartheidu, všimneme si, že se z velké části odehrávají zejména v době utváření států a jsou namířeny k odstranění nebo alespoň ke zmírnění a potla- čení historické paměti vnitřní logikou demokracie a „občanské“ státnosti, jež je také ustavována ve vznikajícím národním státě. Zejména jeho analýza „původu exkluze nacionalismu“ v raně moderním Španělsku, Anglii a Francii (Marx 2003) je vedena politickým poselstvím, podle nějž údajně „občanský“ nacionalismus Západu nebyl o nic méně brutální, sektářský nebo vylučují- cí než „etnický“ nacionalismus Východu. Avšak text obsahuje také poselství, že nacionalismus zdaleka není stálým „stínem modernity“, nýbrž že se vyčerpává svými vlastními úspěchy a je vyvažován jinými principy, zejména liberální demokracií a občansko-územní státností. Od skupinové k individuální exkluzi v liberálním státě Stát „vlastněný“ určitým národem, nebo naopak státními elitami, které aktivně struktu- rují společnost k obrazu jistého národa, je adekvátním paradigmatem pro politiky imigrace a občanství prosazované klasickým národním státem od pozdního 19. do raného 20. století. Z vnitrostátního hlediska se jednalo o stát „rozvinuté modernity“ (Scott 1998), zaměřený na vytvoření „dokonale čitelné populace s registrovanými, jedinečnými jmény a adresami prová- zanými se sídly; vykonávajícími identifikovatelné zaměstnání; a jejichž všechny transakce jsou zdokumentovány podle naplánovaného vzorce a v úředním jazyce“ (1998: 82). Z mezinárodního hlediska se jednalo o imperialistický stát, uzavřený do anarchistického a v konečném výsledku násilného boje o přežití vedeného proti ostatním státům. Nacionalismus a rasismus poskytly vhodné fráze sloužící dvěma cílům, vytvoření čitelných a zároveň bojovných společností, neboť líčí kolektivity jako útvary tvořené identickými, navzájem podobnými jednotkami, zároveň však též jako útvary ostře oddělené od ostatních kolektivit. Podle příhodného Taguieffova vyjádře- ní (2001: 202 a dále), v klasických národních státech byly kolektivy individualizovány, zatím- co jedinci byli kolektivizováni. Politiky imigrace a občanství tak byly ve službách reprodukce vnitřně homogenních, avšak vnějškově jasně ohraničených kolektivit, čehož bylo docíleno tří- děním nově příchozích na základě jejich etnické příslušnosti, rasy nebo národního původu. Toto bylo jak archaické, odsunujíce jedince ke statusu kopií nebo navzájem si podobných jednotek uvnitř připsaných skupin, a zároveň v přehnané míře moderní, hnané plánem racionálně řídit společnost shora. „Vědecký plán na udržení Ameriky americkou“ (Warner Parker, 1924: 740) – tento dobový popis imigrační politiky USA roku 1924, založené na kvótách „národního půvo- du“, vyjadřuje vhodně dualitu oné modernisticko-archaické politiky. Ať už přivodilo vznik „liberálního státu“ cokoli (což je mimo rozsah této diskuse)7, je jeho znakem potlačení partikularistického budování národa ve prospěch univerzálních principů lid- ských práv. Status quo ante je drasticky shrnut ve vzpomínce osvětimského vězně Primo Leviho na setkání s Dr. Pannwitzem, který si tohoto vězně vyhlédl pro možnou službu v táboře, jež by 60 Christian Joppke: Exkluze v liberálním státě ho ušetřila jisté smrti: „Tento pohled nebyl pohledem dvou lidí; jakoby si ho mezi skleněnou stěnou akvária vyměnily dvě bytosti, které obývají odlišné světy“ (citováno v Ignatieff 2001: 3). Znakem liberálního státu je rozbití těchto nacionalistických a rasistických stěn a institucionali- zování minima sdíleného lidství. Klíčovým dokumentem je Všeobecná deklarace lidských práv OSN z roku 1948, která se od té doby stala „lingua franca celosvětového morálního myšlení“ (Ignatieff 2001: 53), vnucujíce strukturální překážky v rámci toho, co státy mohou podniknout ve jménu „svého“ lidu. Od skupinové k individuální exkluzi v liberálním státě Jako odpověď na ztrátu ospravedlnění krajního nacionalismu a rasismu holocaustem se západní poválečné státy staly státy ústavními, ve kterých jsou práva většiny ohraničena principy univerzálních lidských práv, vepsaných do vnitrostátních ústav, které již nebyly omezeny na vytyčení vnitřního projektu státu, ale předepisovaly nyní hranice pro zachá- zení státu s jednotlivci (o Evropě viz Stone-Sweet 2000). Fakt, že sdílená lidskost již nemůže být upřena nečlenům národní nebo etnické skupiny, která je ztotožňována se státem, zásadně proměnila význam slova „exkluze“. Toto budu dis- kutovat v následující části, za použití příkladů politik imigrace a občanství. Imigrační politika Imigranti se zpravidla nacházejí na křižovatce vnější a vnitřní exkluze. S ohledem na vněj- ší exkluzi jsou předmětem státní „imigrační politiky“, která upravuje přístup na území státu a k vybraným sektorům společnosti, zejména k trhu práce. Pokud zkoumáme vývoj imigračních politik západních států od jejich prvních systematických vypracování na počátku 20. století, všimneme si jejich vzrůstajícího univerzalismu a snižujícího se rozsahu připsaných skupinových rozdílů v těchto politikách. Kdysi bylo jednoznačně ustaveno, že imigranti jistého etnického nebo národního původu mají být upřednostňováni před jinými, a imigranti jistých nežádoucích „ras“ byli dokonce vyloučeni úplně. Ospravedlněním byl odkaz na jejich odlišnou schopnost „asimilace“ do přijímající společnosti. Jde o překvapivý vývoj, kdy tyto připsané skupinové rozdíly teoreticky vymizely z imigračních politik, jež obecně přešly k hodnocení na základě individuálních kritérií dovedností a rodinných vztahů. Jediným ospravedlnitelným skupinovým rozlišením, které přetrvává, je rozlišování mezi „občany“, kteří mají právo vstoupit a nemohou být vyhoštěni, a „cizími státními příslušníky“, kteří žádné z těchto práv nemají a kteří jsou před- mětem zájmu imigračních a cizineckých státních politik. Zatímco „cizí státní příslušníci“ jsou nadále předmětem exkluze, je tato exkluze zásadně odlišná od exkluze nacionalistické či etnické. Etnická exkluze funguje na základě zvláštních skupinových charakteristik, kdy neexistuje možnost přejít z jedné skupiny do druhé. Naopak cizí státní příslušník není národnostní nebo etnickou kategorií, nýbrž kategorií formálně práv- ní – cizí státní příslušník je vyloučen nikoli proto, že je členem zvláštní skupiny, ale proto, že není občanem. Status cizího státního příslušníka je tedy vymezen záporně a reziduálně. Navíc pokud existuje možnost, že se cizí státní příslušník stane občanem nejprve na základě pobytu a později skrze „naturalizaci“ v novém státě, jedná se o individuální a nikoli skupinové roz- lišení. Dokonce i když mají státy formální svobodu odepřít vstup cizímu státnímu příslušní- kovi, mezinárodní právo zakazuje odmítnutí cizího státního příslušníka na vrtkavém zákla- dě etnického či rasového původu – pravidlo nediskriminace je uplatňováno dokonce i tam, kde se zdá, že státní suverenita dosahuje svého vrcholu (viz Goodwin-Gill 1978: kapitola 5). 61 SOCIÁLNÍ STUDIA 1/2006 Samozřejmě že stejná nediskriminační pravidla upravují v současném liberálním státě práv- ní přechod od vstupu přes pobyt až k občanství; až na výjimku úzce vymezených „etnických migrantů“ (Joppke 2005a) by tak mělo být překročení hranic stejné pro všechny cizí státní příslušníky, bez ohledu na jejich etnický, rasový nebo národní původ. Jediné omezení, které má současný liberální stát k dispozici, je omezení počtu. Imigrační politika Zatímco veřejný odpor proti imigraci je zcela jistě podmíněn tím, kdo migruje, vstřícná reakce libe- rálního státu může spočívat pouze v uzavření se veškeré imigraci, nikdy však v zastavení této partikulární imigrace. Všimněme si, že ačkoli pozastavení náboru do západoevropských států následující po první ropné krizi v roce 1973 nezohledňovalo státní příslušnost, a to i přes zjev- nou převahu imigrantů jistého národnostního a regionálního původu (Turci v Německu a Švý- carsku, Alžířané či Severoafričané ve Francii), bylo jablkem sváru. Restriktivní opatření mohou být specifická pro určité státy či skupiny pouze jako výsledek zvláštního režimu nebo za úzce vymezených podmínek (zejména pokud je exis- tence těchto podmínek před veřejností utajena). Příkladem zvláštního režimu jsou bilate- rální dohody s vysílajícími státy, které samozřejmě umožňují cílenou inkluzi nebo exkluzi svých občanů. Například mezinárodní právní status alžírských imigrantů poskytl konzer- vativnímu francouzskému prezidentovi příležitost se jich na konci 70. let 20. století zba- vit jednoduše neobnovením jejich zvláštního desetiletého práva pobytu, jež bylo založeno na bilaterální smlouvě z roku 1968 a jež tedy bylo mimo kompetence vnitrostátního imig- račního zákona (který od svého zavedení v roce 1945 úmyslně opomíjel rozdíly ve státní příslušnosti). Tento zákon nápadně selhal kvůli „tradiční mobilizaci“ církví, odborářských svazů, levicových stran a Nejvyššího správního soudu (Weil 1995: 99) – přestože existova- la formální možnost diskriminačního opatření, selhala z důvodů značných nerovností, které by tato opatření vytvořila. Druhý zvláštní režim představuje manipulace s neetnickými kategoriemi výběru na základě dovedností a rodinných vztahů s cílem dosáhnout žádaného etnického výsledku. Zvláště v zemích anglicky mluvících osadníků bylo jednoznačné upřednostňování kvalifiko- vaných imigrantů po dlouhou dobu považováno za skryté upřednostňování evropských imig- rantů. A naopak kritika přijímání osob na základě rodinných vztahů byla brána za odmítnutí neevropské imigrace. Avšak proměňující se demografický profil současné imigrace, zvláš- tě nárůst kvalifikace u neevropských imigrantů, odstranil důvod uchylovat se k úskočnému etnickému výběru (a jeho kontrole; viz Joppke, 2005b). Kdekoli přesto dochází k etnickému výběru, je jeho minimální podmínkou, aby zůstal utajen před veřejností, což je často možné skrze vyčerpávající administrativní zdrženlivost, kterou každé zákonodárství – a u imigrace zvláště – ponechává státu při jeho uplatňování. Toto je očividně způsob, jakým se západnímu Německu, ve kterém byl jakýkoli etnický nebo nacistický diskurz kvůli zkušenosti s nacismem formálně postaven mimo zákon, podařilo v populaci hostujících pracovníků v 50. a 60. letech 20. století snížit přítomnost tzv. „Afro- Asiatů“ na minimum (viz poučná Schönwälderova studie 2004). Imigrační politika Jestliže posuzujeme imigrační politiky současných západních států, rozpoznáme, že již nejsou ve službách reprodukce jisté národnosti. Pokud takové státy umožňují nebo nabí- zejí imigraci, nevyhnutelně tím riskují etnickou či rasovou proměnu svých společností. Nacionalistické a xenofobní tendence přítomné v těchto společnostech již nejsou ve sku- 62 Christian Joppke: Exkluze v liberálním státě Christian Joppke: Exkluze v liberálním státě tečnosti institucionalizovány ve státních politikách imigrace (jako tomu bylo na počátku 20. století), ale jsou nyní mimořádnou nebo proti-institucionální odpovědí politikám, které jsou nevyhnutelně nediskriminační, pokud jde o etnický, národnostní či rasový původ migrantů. 8 Chae Chan Ping vs. Spojené státy, 130 USA 581 (1889), s. 606. 9 Graham vs. Richardson, 403 USA 365 (1971), s. 376. 8 Chae Chan Ping vs. Spojené státy, 130 USA 581 (1889), s. 606. 9 Graham vs. Richardson, 403 USA 365 (1971), s. 376. Politika občanství Richardson o 82 let později (který postavil mimo zákon diskri- minace prováděné vládami jednotlivých států federace na základě cizí státní příslušnosti): „Cizí státní příslušníci, podobně jako naši občané, platí daně a mohou být povoláni do služ- by v branných složkách… [Cizí státní příslušníci] mohou po mnoho let ve státě žít, pracovat a přispívat k jeho ekonomickému růstu.“9 63 SOCIÁLNÍ STUDIA 1/2006 Tendence k univerzalismu a k individuální, v protikladu ke skupinově založené, inkluzi či exkluzi je nejvíce viditelná v přístupu k občanství, a tedy v samotné „politice občanství“. Anthony Marx (2002: 113 a dále) tajemně poznamenal, že státu vlastní potenciál pro exkluzi může být někdy orientován externě (namísto interně), a v tomto případě se vztahuje na „cizin- ce“, a to v podobě „selektivního občanství“. Bohužel však nevysvětlil význam slova „selek- tivní“. Celkem vzato však v této oblasti existují opět dva způsoby „selektivity“: skupinová selektivita, v níž připsané charakteristiky (na kterých jedinec nic nemůže změnit) buď oteví- rají, nebo zavírají dveře k občanství; a individuální selektivita, podle které stát neumožňuje získání občanství nově příchozím, je však alespoň nucen být v tomto ohledu stejně vylučující ke každému bez rozdílu. Je zajímavé, že se oba způsoby „selektivity“ v oblasti občanství významně uvolnily. Skupinová selektivita buď zcela vymizela, tak jako rasová exkluze, která poskvrňovala pravi- dla americké naturalizace až do počátku 50. let 20. století; nebo přetrvávají ve velmi zvlášt- ních, pozitivně diskriminačních podobách, jako je samozřejmé přidělení občanství Židům v Izraeli nebo Němcům v Německu. Důležitou součástí těchto pozitivně-diskriminační selek- tivit je, že nevyčerpávají možnosti získání izraelského nebo německého občanství, ale jsou dodatečnými procesy naturalizace, které jsou stejnou měrou přístupné komukoli, nezávisle na jeho původu. S ohledem na individuální selektivitu je velmi zajímavý nejnovější vývoj, přinášejí- cí obrat v získávání občanství postupující od situace, kdy je státu přenechán volný prostor pro uvážení, až k právu na straně cizího státního příslušníka (jež je definováno jako délka pobytu nebo narození na jistém území), s tím, že stát opouští explicitně dané (a individuál- ně ověřované) požadavky na asimilaci v této oblasti. Zejména s ohledem na naturalizaci je objevení se „práva“ na občanství pozoruhodným vývojem, protože v této oblasti dosahova- lo jednání státu na základě uvážení svého vrcholu. Politika občanství Po překonání obtíží spojených se vstupem na území (tj. potenciální vnější exkluze) se imigranti stávají možnými objekty vnitřní exkluze. Avšak možnosti pro jejich vnitřní exkluzi jsou ještě více omezené než pro exkluzi vnější, která nadále zůstává legitimní pod záštitou státních imigračních a občanských politik. Nyní do hry vstupuje demokratická logika, podle které se imigranti stávají spolupracujícími členy společnosti, jimž je stát, pod jehož stře- chu přišli pobývat, povinován stejným zacházením a na jehož ochraně jsou nyní závislí (viz Rubio-Marin 2000). Tento fakt se stal předmětem skutečné ústavní revoluce napříč západní- mi státy. V klasickém národním státě dokonce i dobrovolně najímaní migranti zůstávali pře- devším členy vzájemně neslučitelných a často ponižovaných kolektivit; zůstávali vyloučeni ze základního sociálního zabezpečení a péče; jejich celá existence v přijímajícím státě byla nejistá a byla vystavena riziku nečekaného vyhoštění (o vyhoštění Severoafričanů v mezi- válečné Francii viz Lewis 2000; o vyhoštění Poláků z Wilhelmovského Německa viz Bade 1984: 462–71). Poválečný liberální stát zaručuje lidská práva ústavou, v níž jsou lidská práva nejčastěji formulována odděleně od statusu občanství, avšak omezuje rozmary výkonné moci, a tak se ve skutečnosti dlouhodobě usazení imigranti přiblížili občanům. Toto přiblížení bylo přede- vším výsledkem ústavní politiky nezávislých soudů, které se dnes považují za ochránce jedin- ců oproti přílišnému dosahu státní moci (viz Joppke 2001; pro alternativní náhled zdůrazňující faktory globální úrovně viz Soysal 1994). Měnící se ontologie společnosti byla tímto vývojem jak doprovázena, tak také poháněna: společnost již nevystupuje jako kolektivní osobnost, uvíznuvší v boji o přežití s ostatními kolek- tivními osobnostmi, ale jako místo obchodu a spolupráce mezi svobodnými a rovnými jedinci, pro které je jejich vlastní národnost v konečném důsledku nedůležitá. Kanonicky byl tento názor vyjádřen Nejvyšším soudem Spojených států v případu vyloučení Číňanů z roku 1889: Pokud [Kongres] považuje přítomnost cizinců jiné rasy odmítající asimilaci do naší společnosti za hrozbu míru a bezpečnosti, jejich exkluze nebude strpěna… a je nezvratná s výkonem soudní moci.8 Druhý, novější názor byl podobně vyjádřen Nejvyšším soudem Spojených států ve slav- ném rozhodnutí Graham vs. Politika občanství Naturalizace byla tradičně ponechává- na na uvážení nejen v tom smyslu, že zákonodárce měl svobodu utvářet příslušná pravidla podle toho, jak uznal za vhodné, ale také v daleko silnějším významu, kdy i poté, co byly ze strany žadatele splněny všechny formální požadavky pro naturalizaci, mohl stát stále říci „ne“. Například podle starých německých administrativních pravidel byl brán v úvahu jen „veřejný zájem“ státu a nikdy osobní zájem kandidáta na občanství. Na počátku 90. let 20. století byla tato naturalizace podle uvážení státu v případě cizích státních příslušníků splňujících jisté požadavky na pobyt (a další požadavky) nahrazena „právem“ na získání občanství. Kulturní „asimilace“, jakožto předpoklad k získání občanství, byla explicitně odmítnuta a nahrazena měkčím požadavkem na „integraci“ (Hailbronner a Renner 2001: 659). Po reformě zákona o občanství z roku 1999 byla tato „integrace“ definována „dosta- tečnou znalostí německého jazyka“, stejně jako písemným stvrzením „závazku k liberální- mu demokratickému řádu“ německého státu. Nezaměňujme však rozlišování mezi skupinovou a individuální selektivitou s rozliše- ním jus sanguinis (zákon krve) a jus soli (zákon půdy) vztahujícím se k občanství při narození, které bylo od vlivné Brubakerovy (1992) práce obecně bráno za hlavní rozlišení mezi způsoby „uzavírání se“ moderního národního státu. Jus sanguinis v sobě takovou skupinovou selektivitu nezahrnuje. Je proto nesprávné definovat jus sanguinis jako „systém, díky kterému se člověk stane občanem tím, že se narodí někomu z etnické skupiny, která v jisté zemi převažuje (napří- 64 Christian Joppke: Exkluze v liberálním státě klad tím, že se narodí etnickému Němci v Německu)“ (Fetzer 2000: 218). To sice odpovídá stereotypu, podle kterého je etnické občanství „rasistické“ (např. Hampton 1995), ale již to neodpovídá právní skutečnosti. Už za platnosti Reichs- und Staatsangehörigkeitsgesetz z roku 1913 se člověk stal německým občanem při narození jednoduše tím, že se narozdil německému rodiči, bez ohledu na jeho „etnický původ“. Německý zákon o občanství nebyl nikdy založen na právní definici německé etnické příslušnosti (viz Silagi 1999:79–83). Jakkoli to tehdy bylo nesnadné, vždy existovala možnost získat německé občanství naturalizací. Jak vhodně pozna- menává Dieter Gosenwinkel (2001: 236), pokud tato možnost existovala, ona „krev“ v prin- cipu „jus sanguinis“ byla formální a instrumentální, nikoli substanciální. Tím však nechceme popřít, že německé občanství bylo v minulosti spojeno s inkluzí a exkluzí na základě skupinové příslušnosti. Politika občanství Nebylo však vůbec propojeno s občanstvím via jus sanguinis, ale s administrativ- ním uskutečněním naturalizace (které v meziválečném období upřednostňovalo německý původ žadatelů před ruskými a vylučovalo východní Židy (viz Gosenwinkel 2001: kapitola 7). p ý y ý y ( p ) Položení rovnítka mezi občanství jus sanguinis a „rasistickou“ skupinovou exkluzi také opomíjí to, že od dob Francouzské revoluce odpovídá jus sanguinis veskrze modernímu poje- tí členství, podle kterého je členství právo jedince, které je předáváno – podobně jako rodné příjmení – následující generaci; podle tohoto chápání již jedinec není nahlížen jako vlastnictví feudálního pána, který vlastnil všechny plody své země, včetně lidí – tento feudální význam vlastnictví půdy a lidí stál u zrodu tradice jus soli. V odezvě na migrační vlny konce 19. a průběhu 20. století však došlo k přehodnocení relativní modernosti obou způsobů přiznává- ní občanství při narození, nejprve ve Francii v roce 1889 a s (předčasným) ukončením v Ně- mecku v roce 1999, kdy jus soli převažuje nad jus sanguinis. Většina evropských států nyní doplňuje své tradiční zákony občanství jus sanguinis prvky jus soli (viz Weil 2001). Jeden autor zabývající se tématem práva (Orentlicher, 1998) dokonce zašel tak daleko, že prohlá- sil občanství na základě území upřednostňovanou formou občanství v mezinárodním právu. Následkem toho se občanství stalo méně vylučujícím a zvenku více přístupným. V kombinaci s nediskriminačními přístupy a postupy, které umožňují udělení trvalého pobytu, občanství podle jus soli zaručuje, že občanství bude automaticky (nebo později volbou) uděleno komu- koli, bez ohledu na jeho etnický, rasový nebo národní původ. Výše uvedené musí mít za následek oddělení občanstva od konkrétního národa nebo etnické skupiny a oslabení celého konstruktu „vlastnictví“ státu jistou skupinou. Oddělení státu a národa Obraz, který se vynořuje, je obrazem oddělení státu a národa v politikách členství v libe- rálním státě. Státy jsou zjevně i nadále „národními státy“, identifikovatelnými podle jména jako jedinci, ztělesňující jedinečnou historii a pospolitost. Státy se však stávají široce otevřenými nově příchozím, kteří už nemohou být začleněni nebo vyloučeni na základě připsaných skupinových charakteristik, ale pouze jako jedinci. Liberální a nediskriminační normy spolu s normami lidských práv zabrzdily partikularistické možnosti „budování národa“ státem. Politiky členství mohou být stále formálně zaměřené na „budování národa“, ale jen v obecně použitelném smyslu vytvoře- ní neetnických, liberálně-demokratických kolektivit, které však nejsou odlišné od těch ostatních. Právě v těchto politikách, které jsou zaměřené na střežení hranic určitých společností, byla uplat- 65 SOCIÁLNÍ STUDIA 1/2006 ňována většina partikularismů (přinejmenším rasové, etnické nebo národnostní povahy). Pokud současné západní národní státy vylučují, činí tak individuálním a nediskriminujícím způsobem, který nemůže být vysvětlen kolektivistickým jazykem etnické příslušnosti a nacionalismu. Jeden ze způsobů, jak objasnit návrat od kolektivistických forem inkluze a exkluze k for- mám individuálním, spočívá v systémově-teoretických pojmech (viz Luhmann 1982). Národní stát byl nakažen univerzalistickou logikou, která vládne v diferencovaných sférách moderní společnosti. V segmentované diferenciaci tradiční společnosti přináleželi jedinci ke společnosti přímo svým začleněním, jako celé bytosti spadající do specifického klanu nebo segmentu. Naopak funkční diferenciace moderní společnosti vede k dvojímu důsledku vyloučení jedince z celku společnosti a jeho začlenění pouze v určitých ohledech, avšak jako „svobodného a rovného“ ve všech diferencovaných sférách, od vzdělání k právu, politickému zřízení a ekonomice. Klasický národní stát dlouho představoval výjimku z této logiky, vylučoval a začleňoval jedince jako celé osobnosti, a dokonce se snažil vytyčit umělé národní hranice okolo jednotlivých diferencovaných sfér společnosti, jakou představuje „národní ekonomie“ stárnoucí populace.10 Přesně tento důsle- dek globalizačních a transnacionalizačních procesů osvobodil společenské subsystémy od jejich starých národních omezení a umožnil jim propojit se s jejich ekvivalenty za hranicemi (do těchto procesů jsou dokonce zapojeny i vládní agentury; viz Slaughter 2004). Normy nediskriminace a lidských práv uštědřily velkou ránu poslední (přestože zároveň první) baště nacionalismu. Tváří v tvář vzrůstající pluralizované a autonomní společnosti vymazává současný stát množství etnic- kých a kulturních nacionalismů ze svých zákonů a politik a vydává se směrem k „veřejné neutra- litě“, odpovídající představám liberálních teoretiků a teoretiček (například Dworkin 1978). Vnitřní liberalizace poválečných západních společností, tj. 11 Je zajímavé, že druhou zbývající oblastí segmentované inkluze je rodina. Není proto divu, že národ- ní příslušnost byla často formulována v rodinných termínech (jako v Zolbergově pojmu moderních národů jako „rodinně podobných uskupení“). 10 Pro získání představy viz Weberova neblaze proslulá inaugurační přednáška na Freiburské univer- zitě „Der Nationalstaat und die Volkswirtschaftspolitik“ (1895). Oddělení státu a národa rozpouštění formálních (a často také substanciálních) nerovností, které se ustavují jako důsledek rasy, genderu, nábo- ženství a třídy, ovlivnila dokonce způsob nakládání s nejtrvalejší a nejlegitimnější ze všech skupinových distinkcí, distinkcí mezi „občanem“a „cizím státním příslušníkem“. Pokud bylo „občanství“ přesvědčením, které mělo překonat, nebo alespoň zkrotit tato stará skupinová rozdělení, nemohl být z obroušení těchto ostrých hran vyjmut ani status cizího státního pří- slušníka. Je to paradoxní, neboť existence „občana“ je postavena na předpokladu (rozdílně situovaného) „cizího státního příslušníka“. Nakažlivá logika, která za výše uvedeným stojí, je nepřekonatelným způsobem vyjádřena v rozhodnutí Nejvyššího soudu Spojených států (výše citovaný případ Graham vs. Richardson), které prohlásilo diskriminaci na úrovni jednotlivých států na základě cizí státní příslušnosti za „podezřelou klasifikaci“, podobnou rase nebo gen- deru, protože na cizí státní příslušníky bylo nahlíženo jako na vytvářející jedince „oddělené ostrůvky menšin“ (viz Rosberg 1983). Následně došlo k liberálnímu rozvolnění jedné z posledních spíše „segmentovaných“ než „funkčních“ forem inkluze do moderní společnosti – členství v národním státě.11 Podobně jako v případě mnohonásobné a pouze částečné inkluze do různých sfér společ- 66 Christian Joppke: Exkluze v liberálním státě nosti můžeme stále častěji náležet do různých států zároveň (viz Hansen a Weil 2002); a neetnický individualismus vládne od teritoriálního vstupu až k udělení nejvyššího statku členství, které je stále častěji vyjadřováno jako právo jednotlivce dané splněním jistých podmínek splněny. Samotné vyloučení etnické příslušnosti a nacionalismu z formálních zákonů státu a institucí však neznamená, že tyto vymizely. Naopak etnická příslušnost a nacionalismus se staly neukotvenými možnostmi v jiných sektorech společnosti (jak to Wimmer empiricky potvrdil, avšak teoreticky nezohlednil, Wimmer 2002: 215). Je pode- zřelé, že současně došlo jednak k od-etnizování státu a k nástupu multikulturalismu na levici, jednak k nástupu xenofobního populismu na pravici.12 S ohledem na nástup multi- kulturalismu Daniel Bell již před čtvrtstoletím poznamenal, že právě tehdy, když byl stá- tem vedený nacionalismus na Západě na ústupu, došlo k šíření „etnické příslušnosti“ na úrovni nižší než státní, která umožňovala kombinaci „zájmu a emocionální vazby“ (Bell 1975: 169). Dokonce i nejlepší teoretikové multikulturalismu (jako např. Kymlicka 1995) přehlédli, že multikulturní politika „uznání“ nastoupila právě tehdy, když se začaly vytrá- cet praktiky bezpodmínečné exkluze a vnucování kultury přistěhovalcům a etnickým men- šinám; tj. právě v té době, kdy na migranty nebo etnika existovaly minimální nároky zpět- ně „uznat“ zvláštnosti této země. 12 Připouštím, že následující postřeh o důsledcích od-etnizování státu pro politickou mobilizaci spo- lečnosti je spekulativní; vyjádřeno kladně, mapuji „zvolené příbuzné rysy“ mezi pozorovanými je- vy, zatímco otázka kauzality zůstává nepotvrzena. Stejně tak je pravdou, že tato pozorování mohou obsahovat jádro širší empirické výzkumné agendy. Oddělení státu a národa Z toho vyplývá, že přinejmenším stejnou měrou, jakou byl multikulturalismus tlačen pociťovaným „utlačováním“ (Young 1990) nebo aspirací na kompenzaci za opakující se budování národa (Kymlicka 1995), byl multikulturalismus zároveň tažen vzduchoprázdnem, které nastalo po ústupu státu od etnického a nacionalis- tického partikularismu. Na straně druhé můžeme namítat, že do té míry, do jaké nespoutaný nacionalismus vymizel ze státních politik a institucí a v níž elity hlavního proudu převzaly „proti-po- pulistické normy“ (Freeman 1995), byl takovýto nacionalismus k dispozici politickým podnikavcům extrémní pravice. Navíc fakt, že se nástup xenofobního nacionalismu pra- vice mírně opožďuje za nástupem multikulturalismu, podporuje názor, že xenofobie byla reakcí na multikulturalismus. Svébytná logika pravicového populismu, která jej odlišu- je od ostatních podobných hnutí působících v minulosti, je heslovitě shrnuta v Le Pe- nově pirátském přivlastnění levicového multikulturního sloganu droit à la difference. Přinejmenším v tomto zvláštním případě je současný pravicový populismus podvojnou odpovědí na od-etnizování státu a „menšinovou“ odvetu etnické příslušnosti, která pře- šla pod označení „multikulturalismus“. Pokud to mohou mít „oni“, tak proč ne „my“, národní většina, jejíž zájmy a přání již nejsou adekvátně reprezentovány liberálním stá- tem, zbaveným své tváře a odvahy? Logickou odpovědí na „etnické upřednostňování“, skandované (francouzskými) multikulturalisty, bylo následně ještě hlasitější skandování „národního upřednostňování“, které se stalo hlavním sloganem Národní fronty od roku 1985 (Taguieff 2001: 171). 67 SOCIÁLNÍ STUDIA 1/2006 SOCIÁLNÍ STUDIA 1/2006 V imigračních politikách západních států, které byly zahnány do úzkých multikulturalis- mem levice a xenofobním populismem pravice, došlo nedávno k občansko-nacionalistickému obratu, od vzkříšení nadlouho opuštěného pojmu „amerikanizace“ až k revalorizaci občanství a občanské integrace v mnoha evropských státech (k amerikanizaci viz Pickus 2001; k in- tegraci viz Joppke 2004a). Podnětem k občansko-nacionalistickému obratu je snaha ovlád- nout odstředivé síly pluralizující imigrantské společnosti centristickým přesvědčením, stejně jako zajistit lidem, narozeným v dané zemi, že jejich přirozené prostředí nebude k nepoznání pozměněno imigranty, kteří nemohou být zastaveni právními způsoby, což bude ještě posíleno z ekonomických a demografických důvodů. ý g ý Avšak občansko-nacionalistický obrat čelí všude stejnému dilematu: není v něm možné pojmenovat partikulárnost „zde, a nikoli jinde“. Zatímco cílem je přizpůsobit nově příchozí „americkým“, „holandským“ nebo „britským“ zvyklostem, skutečné vyřčení těchto zvyklos- tí vede pouze k lokálním verzím stejného liberálně-demokratického přesvědčení. 13 Více na toto téma viz Dietrich (2000). Oddělení státu a národa Například Komise pro reformu imigrace (1997) definovala ve Spojených státech „amerikanizaci“ jako „kultivaci sdíleného závazku k americkým hodnotám svobody, demokracie a rovných příle- žitostí“ (1997: 26) společně s důrazem na „rychlé nabytí znalosti anglického jazyka“ (1997: 37) – a dokonce ani s ohledem na anglický jazyk zde nespatřujeme nic specificky amerického. V Británii se nejnovější posun „za multikulturalismus“, oznámený vládou Spojeného králov- ství v Bílé knize Zajištěné hranice, bezpečný ráj (2002), snaží zavázat imigranty k revalorizo- vanému britskému občanství a národní identitě zavedením naturalizační přísahy, přísnějšími testy z anglického jazyka při naturalizaci a povinným vyučováním „občanství a demokracie“ na britských školách. Avšak pokud byla Bílá kniha nucena vyjádřit, co představuje „základní principy“ britského občanství, mohla pouze uvést: „že respektujeme lidská práva a svobody, prosazujeme demokratické hodnoty, svědomitě dodržujeme zákony a plníme své povinnos- ti a závazky“ (2002: 34). Opět v tom nebylo nic specificky „britského“. Ve zvláštním sporu o „dominantní německou kulturu [Leitkultur], v roce 2000 bezúspěšně podněcovaném opo- zičními konzervativními stranami, bylo jediným neprocedurálním prvkem této kultury (mimo jazykových požadavků, bez nichž se neobejde žádný stát) zavázání se ke „křesťansko-západní kultuře“ (CDU 2000), jež zahrnovalo odkazy na „judaismus“, „antickou filosofii“, „humanis- mus“ a „římské právo“, ale očividně nikoli na „islám“. Avšak jakkoli může být tato implicitní exkluze ožehavá, nebylo nic specificky „německého“ v kultuře, kterou byli imigranti žádáni sdílet; neúspěšně, jak se posléze ukázalo. j Během celého občansko-nacionalistického obratu tak zůstávají konkrétní národy, do kte- rých mají být imigranti socializováni, nepojmenovány. Jediným legitimním nacionalismem v západních státech je „liberální nacionalismus“, ale i toto spojení je pojmovou kontradik- cí, neboť musí být definováno v univerzalistických pojmech, jež se neliší „tady“ a „tam“. Komentátor diskuse o německé Leitkultur zachytil dilema liberálního státu následovně: „Jediným přijatelným požadavkem integrace je ve skutečnosti respekt k liberálně demokra- tickému řádu. Avšak pokud je zachována pouze formální svoboda, nikoli však její konkrétní obsah, co tedy zbývá z německých zvláštností?“13 Nemožnost vnucení zvláštních „americ- kých“, „holandských“, „britských“ nebo „německých“ způsobů symbolizuje odloučení státu 68 Christian Joppke: Exkluze v liberálním státě a národa ve státních politikách členství, dokonce i v každém pokusu o centristické přibrzdění odstředivého multikulturalismu a obranu před zuřivým nacionalismem populistické pravice. Jediný diskriminující potenciál inherentní občansko-nacionalistickému obratu, zvláště po teroristických útocích ve Spojených státech v září 2001, spočívá v možnosti zkomplikovat požadavky na integraci, učinit liberální stát pouze pro liberálně smýšlející. Oddělení státu a národa Celkově různoro- dá reakce západních států na teroristické útoky, kdy jejich čelní představitelé pokaždé zdů- razňovali, že restriktivní opatření jsou součástí „války proti terorismu“, ale nejsou zamířeny na „muslimy“ jako takové, tento strach nepotvrzuje. Avšak dokonce liberalistické reductio ad absurdum, jeho proměna do podoby zapřísáhlého boje proti exkluzi, v sobě ukrývá odkaz na „stín modernity“, který je zcela beze vztahu k „národně-státní“ formě. Závěr: „Neustávající nárůst národního státu“ Cíl předcházející debaty byl skromný a omezený: ukázat, jak liberálně-univerzalistická logika práv jedince a nediskriminace vpadla do oblasti vytyčování hranic a definice členství, které jsou často líčeny pod vlivem partikularistického budování národa. Současné západní státy samozřejmě pokračují v „exkluzi“, avšak individuálním a sebe-limitujícím způsobem, který se značně liší od otevřeně diskriminačních exkluzí na úrovni skupiny, uplatňovaných v minulosti. Toto stěží představuje nový náhled: političtí sociologové globálních lidských práv obhajují tento argument již nejméně po jedno desetiletí (Soyosal 1994, Jacobson 1996). Jedná se však o natolik závažnou záležitost, že je vhodné ji znovu vyjádřit a s požadovanou detailností čelit překvapivě vytrvalému obrazu národů a nacionalismu v literatuře, obrazu „nacionální exkluze“ a „skupinového vlastnictví“ státu jako v podstatě neměnného „stínu modernity“ (Wimmer 2002). Nejedná se však o obhajování naivní liberální utopie sebe-naplňujících se práv a zani- kajících států. Naopak dokonce „severní-západní státy“, které se staly „skromnějšími“ a sa- my sebe limitují s ohledem na morální a kulturní regulace a které slábnou pod tíhou ekono- mické globalizace, den ze dne narůstají, svazují, regulují a prostupují své společnosti jako nikdy předtím (Mann 1997). Jak dále Michael Mann zdůrazňuje, „neustávající nárůst“ je dokonce způsoben stejnými faktory, kterým je obecně připisováno oslabování státu, jako je šíření nároků na „práva“ obklopující etnickou příslušnost, sexualitu a gender nebo ekologii: „Potřebujeme zákony upravující všechny tyto záležitosti a složité nároky na sociální zajiš- tění, které implikují“ (1997: 492). Poznamenejme však, že „národní“ komponent tohoto druhu expanzivního „národního státu“ je spíše metaforou pro jeho rozsah a infrastrukturu, velmi málo se podobající vylučující, homogenní a personifikované kolektivitě minulosti. Přeložila Alice Navrátilová Poděkování Toto je poslední ze série několika přednášek, jež jsem napsal jako hostující profesor na Russel Sage Foundation v New Yorku v letech 2002–2003. S lítostí musím prohlásit, že se zároveň jedná o poslední veřejnou možnost poděkovat Eriku Wannerovi a jeho velice vstříc- ným kolegům. Přeložila Alice Navrátilová Přeložila Alice Navrátilová 69 SOCIÁLNÍ STUDIA 1/2006 Literatura Bade, K. J. 1984. Auswanderer, Wanderarbeiter, Gastarbeiter. Ostﬁ ldern: Scripta Mercaturae. Bell, D. 1975. „Ethnicity and Social Change.“ In N. Glazer, D. P. Moynihan (ed Bell, D. 1975. „Ethnicity and Social Change.“ In N. Glazer, D. P. Moynihan (eds.) Ethnicity, Cambridge, MA: Harvard University Press. Cambridge, MA: Harvard University Press. Cambridge, MA: Harvard University Press. Blainey, G. 1984. All for Australia. North Ryde: Methuen Hayes. Brubaker, R. 1992. Nationhood and Citizenship in France and Germany. Cambridge, MA: Harvard University Press. Carens, J. 1987. „Aliens and Citizens: The Case for Open Borders.“ Review of Politics, 49 (2): 251–73. CDU (Christlich-Demokratische Union Deutschlands) 2000. Arbeitsgrundlage für die Zuwan- derungs-Kommission der CDU Deutschlands. Berlin, November. Online dokument do- stupný z: <http://www.cdu.de> Deutsch, K. W. 1957. Political Community and the North Atlantic Area. Princeton, NJ: Prin- ceton University Press. Dietrich, S. 2000. „Die Inländerfrage.“ Frankfurter Allgemeine Zeitung, 25 October, s. 1. Dworkin, R. 1978. „Liberalism.“ In S. Hampshire (ed.) Public and Private Morality, Cam- bridge: Cambridge University Press. Fetzer, J. S. 2000. Public Attitudes toward Immigration in the United States, France, and Ger- many. New York: Cambridge University Press. Freeman, G. 1995. „Modes of Immigration Politics in Liberal Democratic States.“ Internatio- nal Migration Review, 29 (4): 881–902. Goodwin-Gill, G. 1978. International Law and the Movement of Persons between States. Ox- ford: Clarendon Press. Gosewinkel, D. 2001. Einbürgern und Ausschliessen. Göttingen: Vandenhoeck a Gosewinkel, D. 2001. Einbürgern und Ausschliessen. Göttingen: Vandenhoeck and Ruprecht. Guiraudon, V. 2000. Les Politiques d’immigration en Europe. Paris: L’Harmattan. Gosewinkel, D. 2001. Einbürgern und Ausschliessen. Göttingen: Vandenhoeck and Ruprecht. Guiraudon, V. 2000. Les Politiques d’immigration en Europe. Paris: L’Harmattan. Hailbronner, K.; Renner, G. 2001. Staatsangehörigkeitsrecht. Munich: Beck. Hampton, J. 1995. „Immigration, Identity, and Justice.“ In W. Schwartz (ed.) Justice in Immi- gration, New York: Cambridge University Press. Hansen, R.; Weil, P. (eds.) 2002. Dual Citizenship, Social Rights, and Federal Citizenship in the US and Europe. Oxford: Berghahn. , ; , ( ) p, g , p the US and Europe. Oxford: Berghahn. Ignatieff, M. 2001. Human Rights as Politics and Idolatry. Princeton, NJ: Princeton University Press. Jacobson, D. 1996. Rights across Borders. Baltimore, MD: Johns Hopkins University Press. p g Ignatieff, M. 2001. Human Rights as Politics and Idolatry. Princeton, NJ: Princeton University Press. Jacobson, D. 1996. Rights across Borders. Baltimore, MD: Johns Hopkins University Press. 2001. Human Rights as Politics and Idolatry. Princeton, NJ: Princeton University Press. Ignatieff, M. 2001. Literatura Human Rights as Politics and Idolatry. Princeton, NJ: Princeton University Press. J b D 1996 Ri h B d B lti MD J h H ki U i it P . 1996. Rights across Borders. Baltimore, MD: Johns Hopkins University Press. Joppke, Ch. 2001. „The Legal-Domestic Sources of Immigrant Rights.“ Comparative Political Studies, 34 (4): 339–66. Joppke, Ch. 2004a. „The Retreat of Multiculturalism in the Liberal State.“ British Journal of Sociology, 55 (2): 237–57. Joppke, Ch. 2004b. „Primordial Beliefs and Immigration Policy: The Case of Britain’s Pat- rials.“ In J. Alexander, G. Marx, C. Williams (eds.) Self, Social Structure, and Beliefs: Exploration in Sociology, Berkeley: University of California Press. Joppke, Ch. 2005a. Selecting by Origin: Ethnic Migration in the Liberal State. Cambridge, MA: Harvard University Press. Joppke, Ch. 2005b. „Are Non-discriminatory Immigration Policies Reversible? Evidence from the United States and Australia.“ Comparative Political Studies, 38 (1). 70 Christian Joppke: Exkluze v liberálním státě King, G.; Keohane, R. O.; Verba, S. 1994. Designing Social Inquiry. Princeton, NJ: Princeton University Press. Kymlicka, W. 1995. Multicultural Citizenship. Oxford: Oxford University Press. Kymlicka, W. 2002. „Territorial Boundaries.“ (Nepublikovaný rukopis.) Lewis, M. D. 2000. The Company of Strangers: Immigration and Citizenship in Interwar Lyon and Marseille. Disertační práce, obor „History“, New York University. Luhmann, N. 1982. The Differentiation of Society. New York: Columbia Universi Mann, M. 1997. „Has Globalization Ended the Rise and Rise of the Nation-State?“ Review of International Political Economy, 4 (3): 472–96. Marx, A. 2002. „The Nation-State and its Exclusions.“ Political Science Quarterly, 117 (1): 103–26. Marx, A. 2003. Faith in Nation: Exclusionary Origins of Nationalism. New York: Oxford University Press. Mill, J. S. 1991. Considerations on Representative Government. Buffalo, NY: Prometheus Books. Miller, D. 1995. On Nationality. Oxford: Clarendon Press. Offe, C. 1998. „ ,Homogeneity‘ and Constitutional Democracy.“ Journal of Political Philosophy, 6 (2): 113–41. Orentlicher, D. 1998. „Citizenship and National Identity.“ In D. Wippmann (ed.) International Law and Ethnic Conﬂ ict, Ithaca, NY: Cornell University Press. Pickus, N. 2001. „Which America? Nationalism among the Nationalists.“ (Nepublikovaný rukopis). Rosberg, G. 1983. „Discrimination against the ,Nonresident‘ Alien.“ University of Pittsburgh Law Review, 44: 399–407. Rubio-Marin, R. 2000 Immigration as a Democratic Challenge. Cambridge: Cambridge Uni- versity Press. Schoenwaelder, K. 2004. „Why Germany’s Guestworkers Were Largely Europeans.“ Ethnic and Racial Studies, 27 (2): 248–65. Scott, J. C. 1998. Seeing Like a State. New Haven, CT: Yale University Press. Silagi, M. 1999. Literatura Vertreibung und Staatsangehörigkeit. Bonn: Kulturstiftung der deutschen Vertriebenen. Slaughter, A.-M. 2004. „Disaggregated Sovereignty.“ Government and Opposition, 39 (2): 159–90. Soysal, Y. 1994. Limits of Citizenship. Chicago: University of Chicago Press. Stone-Sweet, A. 2000. Governing with Judges. Oxford: Oxford University Press. T i ff P A 2001 Th F f P j di Mi li U i it f Mi t P Stone-Sweet, A. 2000. Governing with Judges. Oxford: Oxford University Press. Taguieff, P.-A. 2001. The Force of Prejudice. Minneapolis: University of Minnesota Pr Stone-Sweet, A. 2000. Governing with Judges. Oxford: Oxford University Press. Taguieff, P.-A. 2001. The Force of Prejudice. Minneapolis: University of Minneso g f j p y Tamir, Y. 1993. Liberal Nationalism. Princeton, NJ: Princeton University Press. Tamir, Y. 1993. Liberal Nationalism. Princeton, NJ: Princeton University Press. US Commission on Immigration Reform 1997. Becoming an American. Washin Tamir, Y. 1993. Liberal Nationalism. Princeton, NJ: Princeton University Press. US Commission on Immigration Reform 1997. Becoming an American. Washington, DC: Government Printing Ofﬁ ce. US Commission on Immigration Reform 1997. Becoming an American. Washington, DC: Government Printing Ofﬁ ce. Walzer, M. 1983. Spheres of Justice. New York: Basic Books. Warner Parker, A. 1924. „The Quota Provisions of the Immigration Act of 1924.“ American Journal of International Law, 18 (4): 737–54. Weber, M. 1971 [1895]. „Der Nationalstaat und die Volkswirtschaftspolitik.“ Obnovené vydá- ní in M. Weber Gesammelte Politische Schriften. Tübingen: Mohr. 71 SOCIÁLNÍ STUDIA 1/2006 Weil, P. 1995. „Racisme et discrimination dans la politique française de l’immigration.“ Ving- tieme siecle, July–Sept.: 77–102. Weil, P. 2001. „Access to Citizenship.“ In A. Aleinikoff , D. Klusmeyer (eds.) Citizenship Today. Washington, DC: Carnegie Endowment for International Peace. White Paper (UK Government) 2002. Secure Borders, Safe Haven. London: The Stationery Ofﬁ ce. Wimmer, A. 2002. Nationalist Exclusion and Ethnic Conﬂ ict: Shadows of Modernity. Cam- bridge: Cambridge University Press. Young, I. M. 1990. Justice and the Politics of Difference. Princeton, NJ: Princeton University Press. Zolberg, A. 1999. „Matters of State: Theorizing Immigration Policy.“ In C. Hirschman, P. Ka- sinitz, J. DeWind (eds.) The Handbook of International Migration. New York: Russell Sage Foundation. Autor Christian Joppke je profesorem sociologie na Mezinárodní univerzitě v Brémách. Je autorem četných knih a publikací o sociálních hnutích, imigraci, občanství, státu, nejnověji Třídění podle původu: etnická migrace v liberálním státě (Harvard University Press, 2005). Kontakt: c.joppke@iu-bremen.de 72
https://openalex.org/W2553231196	https://www.freidok.uni-freiburg.de/dnb/download/12627	English	null	Access, timing and frequency of very early stroke rehabilitation – insights from the Baden-Wuerttemberg stroke registry	BMC neurology	2,016	cc-by	7,813	Reuter et al. BMC Neurology (2016) 16:222 DOI 10.1186/s12883-016-0744-7 Reuter et al. BMC Neurology (2016) 16:222 DOI 10.1186/s12883-016-0744-7 © The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: bjoern.reuter@uniklinik-freiburg.de 1Department of Neurology and Neurophysiology, University Hospital Freiburg, Breisacher Straße 64, 79106 Freiburg, Germany Full list of author information is available at the end of the article Access, timing and frequency of very early stroke rehabilitation – insights from the Baden-Wuerttemberg stroke registry Björn Reuter1* , Christoph Gumbinger2, Tamara Sauer3, Horst Wiethölter4, Ingo Bruder5, Curt Diehm6, Peter A. Ringleb2, Werner Hacke2, Michael G. Hennerici3, Rolf Kern7 and and Stroke Working Group of Baden-Wuerttemberg Background therapy vs. out-of bed mobilization) in everyday clinical practice [16]. Based on the authors clinical impressions we hypothesized a positive correlation between stroke symp- tom severity and VER at higher frequency, which might need reevaluation after publication of the AVERT trial. With a descriptive analysis of the Baden-Wuerttemberg (BW) stroke registry we thus aimed to provide a more detailed insight into the application of PT, OT, and ST in patients with IS and ICH in clinical practice. Organized stroke unit care has proven to be more effect- ive than treatment on general wards only [1–3]. A sys- tematic review of the Stroke Unit Trialists Collaboration demonstrated that professional stroke unit care is par- ticularly superior because it prevents immobility-related complications. Further, physical therapy (PT), occupa- tional therapy (OT) and speech therapy (ST) including are regarded to significantly account for this effect [4]. Dysphagia is a common finding in stroke and associated with a higher risk for pneumonia, malnutrition and death, thus making a swallowing assessment necessary immediately after admission by trained nursing staff and/or speech therapists [5, 6]. Recently a Cochrane review confirmed the general effectiveness of physical rehabilitation on stroke outcome and also provided some evidence for beneficial effects when treatment was initi- ated early, although a lack of high quality studies was noted [7]. Very early rehabilitation (VER) is thought to prevent inactivity-associated complications of multiple biological systems, i.e. the respiratory system (pneumo- nia, atelectases), blood circulation (deep vein throm- bosis, pulmonary embolism), immunosuppression, bedsores and catabolism/muscle atrophy. Further favourable effects are hypothesized to comprise of stim- ulated neuronal plasticity and a reduced risk for mood disorders associated with stroke [8–11]. The benefits of VER are however accompanied by potentially harmful side effects as arterial blood pressure oscillation during exercise and/or out of bed mobilization with a higher risk for hematoma growth in intracerebral hemorrhage (ICH), collapsing collateral blood supply of the penum- bra in ischemic stroke (IS), and secondary ICH in suc- cessfully recanalized IS patients [12–14]. However, the evidence for both the precise beneficial and harmful ef- fects of VER remains poor [15]. Abstract Background: While the precise timing and intensity of very early rehabilitation (VER) after stroke onset is still under discussion, its beneficial effect on functional disability is generally accepted. The recently published randomized controlled AVERT trial indicated that patients with severe stroke might be more susceptible to harmful side effects of VER, which we hypothesized is contrary to current clinical practice. We analyzed the Baden-Wuerttemberg stroke registry to gain insight into the application of VER in acute ischemic stroke (IS) and intracerebral hemorrhage (ICH) in clinical practice. Methods: 99,753 IS patients and 8824 patients with ICH hospitalized from January 2008 to December 2012 were analyzed. Data on the access to physical therapy (PT), occupational therapy (OT), and speech therapy (ST), the time from admission to first contact with a therapist and the average number of therapy sessions during the first 7 days of admission are reported. Multiple logistic regression models adjusted for patient and treatment characteristics were carried out to investigate the influence of VER on clinical outcome. Results: PT was applied in 90/87% (IS/ICH), OT in 63/57%, and ST in 70/65% of the study population. Therapy was mostly initiated within 24 h (PT 87/82%) or 48 h after admission (OT 91/89% and ST 93/90%). Percentages of patients under therapy and also the average number of therapy sessions were highest in those with a discharge modified Rankin Scale score of 2 to 5 and lowest in patients with complete recovery or death during hospitalization. The outcome analyses were fundamentally hindered due to biases by individual decision making regarding the application and frequency of VER. Conclusions: While most patients had access to PT we noticed an undersupply of OT and ST. Only little differences were observed between patients with IS and ICH. The staff decisions for treatment seem to reflect attempts to optimize resources. Patients with either excellent or very unfavorable prognosis were less frequently assigned to VER and, if treated, received a lower average number of therapy sessions. On the contrary, severely disabled patients received VER at high frequency, although potentially harmful according to recent indications from the randomized controlled AVERT trial. Keywords: Acute stroke, Rehabilitation, Physical therapy, Occupational therapy, Speech therapy, Stroke unit concept Reuter et al. BMC Neurology (2016) 16:222 Page 2 of 10 Page 2 of 10 Background In April 2015 the phase III randomized controlled “Efficacy and safety of very early mobilization within 24 h of stroke onset (AVERT)” trial was published and demonstrated a lesser chance to achieve functional independence 3 months after stroke in the earlier and more intensively treated stroke group, defined as a modified Rankin scale (mRS) of 0–2. Sub- group analyses indicated that patients with severe stroke and/or ICH might be more susceptible to harmful side effects [16]. Multiple factors might have contributed to these findings. The treatment group was mobilized earl- ier and received twice as much daily sessions of out of bed therapy, which took three times longer than for the control group. Despite to this uncertainty regarding the harmful factor, it is somewhat unclear to what extend regular stroke care will have to change in light of the AVERT findings. This moreover since there is a lack of description of interdisciplinary therapy content (type, frequency and intensity) and therapy strategy (in-bed Methods We performed a retrospective analysis of patients with acute IS and ICH prospectively registered in a large and consecutive hospital-based stroke registry in central Eur- ope. Permission to analyze the registry was obtained by the governing board of the office for quality assurance in hospitals (Geschäftsstelle für Qualitätssicherung im Krankenhaus, GeQik). Setting, eligibility criteria and study size BW is Germany’s third largest federal state regarding size and population with 35,742 km2 and 10.8 million inhabi- tants. In 1998 BW implemented a structured three-level medical concept for the treatment of stroke [17]. Today about 140 hospitals are involved in acute stroke care, comprising of stroke centers, hospitals with regional or local stroke units, and hospitals without a specific stroke unit. The quality of acute stroke treatment is monitored since 2004. Transfer of pseudonymized data on baseline and treatment characteristics to the Office for Quality Assurance in Hospitals is stipulated by federal state law. Data covering a period of 5 years, from January 1, 2008 to December 31, 2012 were analyzed in the present study. 99,753 patients with IS and 8824 patients with ICH were selected out of 122,394 patients with discharge ICD10 diagnosis I61 (nontraumatic ICH) or I63 (IS). 9180 patients with IS and 4637 patients with ICH were excluded from our analysis according to the following criteria, making sufficient participation in VER unlikely (Fig. 1): diagnosis and treatment in emergency depart- ments only (N = 1841 [IS] and N = 1132 [ICH]); discharge from in-hospital care or death within 24 h (N = 2767 [IS] and N = 1513 [ICH]); patients requiring mechanical venti- lation, reported as “during hospitalization” from 2008 to 2009 and reported as “within 24 h after admission” from 2010 to 2012 (N = 4511 [IS] and N = 1969 [ICH]); and first rehabilitation therapy > 7 days of admission for other reasons (N = 61 [IS] and N = 23 [ICH]). Baseline charac- teristics and data on PT, OT, and ST of the remaining patients are described in detail. Variables and statistical analysis Documentation includes patient demographic data, medical history on cardiovascular risk factors (arterial Reuter et al. BMC Neurology (2016) 16:222 Page 3 of 10 Fig. 1 Study cohort selection flow diagram Fig. 1 Study cohort selection flow diagram as (4:6)*7 = 4.7, rounded five sessions). The registry col- lects no data on the amount of therapy in minutes or the specific type of therapy, i.e. in-bed or out of-bed therapy. hypertension and hypercholesterolemia documented since 2010) and previous cerebrovascular events, hospital admission time, level of hospital care, admitting department and ward, nature and timing of diagnostic procedures, intravenous thrombolysis, in-hospital com- plications, discharge information, and hospital mortality. Stroke severity at admission was assessed using the National Institute of Health Stroke Scale (NIHSS) score and modified Rankin Scale (mRS) score. A pre-stroke mRS score was documented at admission to estimate acute deterioration of functional ability. At discharge a final mRS score was determined. Data on paresis, aphasia, and dysarthria was collected based on clinical examination at admission and discharge. We used standard descriptive statistics to explore pa- tient characteristics and access to VER stratified by dis- charge diagnosis IS and ICH and functional disability at admission and discharge. Functional disability was mea- sured as paresis, aphasia, and dysarthria at admission and discharge, and mRS score at discharge. Multiple lo- gistic regression models were then used to assess the as- sociation between VER and clinical outcomes. The selected outcome parameters were (A) the mRS score at discharge (ordinal logistic regression analysis), (B) the full recovery from specific disabilities at discharge (par- esis, dysarthria, aphasia; binary logistic regression analysis), (C) the risk to develop pneumonia during the hospitalization period (binary logistic regression ana- lysis), and finally (D) the mortality rate (binary logistic regression analysis). The analyses were performed for the stroke etiologies IS and ICH independently and the models were fitted stratified either for the time interval from admission to first therapy session or for the num- ber of therapy sessions during the first 7 days of hospitalization. All analyses were adjusted for patient characteristics (age, sex, pre-stroke and admission mRS scores, NIHSS score at admission, prior stroke event, First contact to a therapist was documented in the data collection form in three time intervals for PT (no treatment, <24 h, 24–48 h, >48 h after admission) and two time intervals for OT and ST (no treatment, <48 h, ≥48 h after admission). Timing, numbers of therapy sessions and functional disability at discharge 90% of the IS patients were attended at least once by a physiotherapist, 63% by an occupational therapist and 70% by a speech therapist (Table 2). When looking ex- clusively on the treated IS patients 87% received their first PT session within 24 h after admission, 91% their first OT session and 93% their first ST session within 48 h after admission, respectively. The median of ther- apy sessions during the first 7 days after admission was five for all therapies. The mean numbers provide a more detailed insight, with PT being applied most frequently with 4.9 sessions (standard deviation (SD) 1.8), followed by OT with 4.2 (SD 1.8) units and ST with 4.0 (SD 1.9) sessions. Further stratification according to the mRS score at discharge demonstrated that percentages of treated patients were highest for those with a mRS score of 2 to 5 both regarding the general access to treatment and the average number of treatment sessions (Fig. 3a and c). Table 1 Patient characteristics and general stroke care Variable IS ICH Patients, n 99.753 8.824 Female sex, % 50 49 Age, median (IQR) 76 (68, 83) 76 (68, 83) NIHSS, median (IQR) 4 (2, 9) 7 (3, 14) Missing data on NIHSS at admission, % 16 18 Paresis at admission, % 66 67 Missing data on paresis at admission, % 1 3 Aphasia at admission, % 30 35 Missing data on aphasia at admission, % 3 9 Dysarthria at admission, % 40 42 Missing data on dysarthria at admission, % 5 10 Level of stroke care, % Center 23 30 Regional 19 18 Local 38 33 Other 21 19 Admitting ward, % Stroke unit 71 61 Intensive care unit 8 23 General ward 22 16 Pneumonia, % 5 10 Median length of stay in days (IQR) 9 (6, 13) 11 (7, 16) Information on arterial hypertenstion and hypercholesterolemia was not The proportion of ICH patients being attended by a therapist were 3% less for PT, 6% less for OT, and 5% less for ST, respectively (Table 2). In addition the pro- portion of patients receiving treatment within the first 24 h (PT) or 48 h after admission (OT, ST) was simi- larly lower. However, compared to IS the mean num- ber of applied therapy sessions was higher for all therapies (PT 5.2 (SD 1.8) units, OT 4.4 (SD 1.8) units, and ST 4.2 (SD 1.8) units). Patient baseline characteristics Patient characteristics of the study cohorts are shown in Table 1. The sex ratio and age were equally balanced in stroke patients suffering IS or ICH with approximately 50% being women and a median age of 76 years (inter- quartile range (IQR) 68 to 83 years). The median NIHSS score in IS was 4 (IQR 2 to 9) and 7 (IQR 3 to 14) in ICH. About two thirds of patients with both IS and ICH presented with a paresis at admission. Aphasia was re- ported in 30% (IS) and 35% (ICH) and dysarthria in 40% (IS) and 42% (ICH). In both groups 86% were function- ally independent previous to acute stroke with a mRS score of 0 to 2 and this number declined to 58% in IS and 36% in ICH at discharge (Fig. 2). Patients with ICH Variables and statistical analysis The number of therapy sessions (PT, OT, and ST) was collected for the first 7 days of hospitalization. Zero therapy sessions were documented for patients being attended once by a therapist but with- out need for treatment. In case of discharge home, transfer to other facilities or death within 7 days of ad- mission a calculated score had to be extrapolated by the admitting hospital. To provide an example, four therapy sessions within 6 days of hospitalization are calculated Reuter et al. BMC Neurology (2016) 16:222 Page 4 of 10 Page 4 of 10 diabetes, atrial fibrillation), admitting facility, IV thromb- olysis, and length of hospital-stay. All statistical tests were two-sided, and p values of <0.05 were considered to be statistically significant. The analyses were carried out using SAS 9.3 (SAS Institute Inc., Cary, NC, USA). had a higher likelihood to be treated in stroke centers providing maximum care. Nevertheless, approximately 20% of the patients with IS or ICH were treated in hos- pitals without a stroke unit. Percentages of patients be- ing admitted to specialized stroke units were 71% in patients with IS and 61% in patients with ICH, while 8% of the patients with IS were treated on intensive care units compared to 23% with ICH. Pneumonias occurred in 5% of the patients with IS and 10% with ICH. The median length of hospital stay for IS was 9 days com- pared to 11 days for ICH. Timing, numbers of therapy sessions and functional disability at discharge Stratification accord- ing to the mRS score at discharge showed a similar distribution pattern as in IS patients, but revealed lower percentages in particular in those ICH patients with either a very favourable or a very unfavourable outcome (mRS 0 or 6, Fig. 3b and d). Access to therapy in specific disabilities related to stroke The analysis of VER with respect to specific stroke symptoms at admission revealed that ≥95% of the pa- tients with IS or ICH and paresis were treated with PT, while OT was applied less frequently with approximately 70% (Table 3). Compared to IS a lower percentage was Information on arterial hypertenstion and hypercholesterolemia was not routinely documented over the entire study period and is therefore missing for N = 37,846 patients. Abbreviations: IQR interquartile range, NIHSS National Institutes of Health stroke scale Reuter et al. BMC Neurology (2016) 16:222 Page 5 of 10 Fig. 2 Scores on mRS prior to stroke, at admission, and at discharge for patients with IS and ICH. The mRS score at discharge was missing for N = 4221 (4%) of the patients with IS and N = 629 (7%) of the patients with ICH Fig. 2 Scores on mRS prior to stroke, at admission, and at discharge for patients with IS and ICH. The mRS score at discharge was missing for N = 4221 (4%) of the patients with IS and N = 629 (7%) of the patients with ICH Fig. 2 Scores on mRS prior to stroke, at admission, and at discharge for patients with IS and ICH. The mRS score at disch N = 4221 (4%) of the patients with IS and N = 629 (7%) of the patients with ICH mRS prior to stroke, at admission, and at discharge for patients with IS and ICH. The mRS score at discharge was missing fo he patients with IS and N = 629 (7%) of the patients with ICH observed for ST in patients with aphasia or dysarthria suffering from ICH (87–88% vs. 83–84%). patients with rapid improvement of stroke symptoms received therapy less frequently, many patients with foreseeable very poor outcome were either not attended by a therapist or received less therapy sessions, and patients at high risk to develop pneumonia were attended by a speech therapist more frequently. As a Information on units are given only for those patients being attended by a therapist. Numbers of units are reported for the first 7 da patient was hospitalized for less than 7 days an extrapolated and rounded 7 day-frequency was reported Effectiveness and mortality The outcome analyses we hindered by heavily interfering individual decision making of the hospital staff, i.e. Table 2 General access to VER, time from admission to first therapy session and average num eneral access to VER, time from admission to first therapy session and average number of therapy sessions Table 2 General access to VER, time from admission to first therapy session and average number of therapy sessions IS (N = 99.753) ICH (N = 8.824) Variable physical therapy, % occupational therapy, % speech therapy, % physical therapy, % occupational therapy, % speech therapy, % No 10 37 30 13 43 35 Yes 90 63 70 87 57 65 < =24 h 87 - - 82 - - 24–48 h 9 - - 13 - - < =48 h - 91 93 - 89 90 > 48 h 4 9 7 5 11 10 Units median (IQR) 5 (4, 6) 5 (3, 5) 5 (2, 5) 5 (5, 6) 5 (3, 5) 5 (3, 5) mean (SD) 4.9 (1.8) 4.2 (1.8) 4.0 (1.9) 5.2 (1.8) 4.4 (1.8) 4.2 (1.8) Information on units are given only for those patients being attended by a therapist. Numbers of units are reported for the first 7 days of hospital stay. In case the patient was hospitalized for less than 7 days an extrapolated and rounded 7 day-frequency was reported Information on units are given only for those patients being attended by a therapist. Numbers of units are reported for the first 7 days of hospital stay. In case the patient was hospitalized for less than 7 days an extrapolated and rounded 7 day-frequency was reported Page 6 of 10 Reuter et al. BMC Neurology (2016) 16:222 Fig. 3 Abbrevations: PT, physical therapy; OT, occupational therapy; ST, speech therapy ig. 3 Abbrevations: PT, physical therapy; OT, occupational therapy; ST, speech therapy application of VER unlikely. In our study population ap- proximately 80% of the patients admitted for IS or ICH were treated on stroke units or intensive care units with a correspondingly high level of expertise. About 90% of the study cohorts had access to PT and this number was ≥95% for patients with motor deficits at admission. OT was applied in approximately 60% of the total study co- horts. 26% (IS) and 28% (ICH) of the patients with motor deficits at admission were not attended by an occupational therapist. Effectiveness and mortality 70% (IS) and 65% (ICH) of the total study population were evaluated and/or treated by speech therapists. For patients with speech or language disorders at admission access to ST was higher, suggest- ing a moderate undersupply of 10% (IS) to 14% (ICH). The majority of patients with IS and, to a slightly minor degree, ICH received their first PT session within 24 h and OT or ST session within 48 h of hospitalization. consequence and although we`ve adjusted the statistical model for patient characteristics, hospital and treatment parameters as successfully done previously we were finally unable to produce meaningful results (data not shown) [18–20]. Discussion This opinion is supported by observations from a post-hoc analysis of the INTERACT 2 trial that exces- sive blood pressure variability as it may occur during Table 3 Access to therapy for patients with specific disabilities at admission IS, % ICH, % Paresis at admission Physical therapy 96 95 Occupational therapy 71 69 Aphasia at admission Speech therapy 88 83 Dysarthria at admission Speech therapy 87 84 Data on therapy was missing in IS (group total N = 99.753) for N = 7.496 (paresis), N = 4.186 (aphasia), N = 4.619 (dysarthria) and in ICH (group total N = 8.824) for N = 1.308 (paresis), N = 709 (aphasia) and N = 771 (dysarthria) Table 3 Access to therapy for patients with specific disabilities at admission Data on therapy was missing in IS (group total N = 99.753) for N = 7.496 (paresis), N = 4.186 (aphasia), N = 4.619 (dysarthria) and in ICH (group total N = 8.824) for N = 1.308 (paresis), N = 709 (aphasia) and N = 771 (dysarthria) Page 7 of 10 Reuter et al. BMC Neurology (2016) 16:222 Page 7 of 10 patients participated in the trial, pre-specified subgroup analyses were still underpowered to draw robust conclu- sions. Very recent data from AVERT indicates that short and frequent out-of bed mobilization is superior to an increased amount of time spent with out-of bed activity [35]. In this instable phase VER for patients with severe disability might also be applied in a neurosensorial approach, verticalisation by robotic devices, repetitive treatment by motocycle in bed, swallowing and respiratory treatment [36]. While most clinical trials and observational studies focused on mobilization and physical rehabilitation approaches, the impact of ST was less frequently investigated. ST does not require mobilization and thus is not associated with several po- tentially harmful side effects of out-of bed PT. Two small clinical trials with in total 59 patients suggested that early ST for mild to moderate aphasia within 3 days of stroke onset results in improved outcome after 6 months [37, 38]. Moreover, very early swallowing assessment by trained nurses or speech therapists is of high value to identify patients at risk for aspiration pneumonia and malnutrition [39]. out-of bed mobilization seems to be associated with poorer outcome [22]. Discussion In our analysis a discharge mRS score of 2 to 5 was associated with the highest applica- tion of VER both regarding its general access and the average number of sessions for all three types of therapy. This observation was first reported for stroke patients in the 1970s, albeit the availability of PT, OT and ST was much less and the timing from onset to therapy was reported in weeks rather than days [23]. It might also ex- plain the higher average number of therapy sessions in patients with ICH, with patients being more severely dis- abled compared to IS. We performed multiple logistic regression analyses to investigate the influence of VER on stroke outcome but the results were fundamentally contradictory, most likely because we were finally unable to statistically compensate biases by individual decision making. We thus believe that stroke registries are not suitable to investigate the influence of VER on stroke outcome. Although the incidence of ischemic and hemorrhagic strokes is high with millions affected worldwide annu- ally, the optimal dose and timing for very early stroke rehabilitation are entirely unclear [24]. Data from animal stroke models revealed a potentially NMDA-mediated increased cell death rate within the infarct and peri- infarct area under very early and intense training, which was counterbalanced by an improved motor perform- ance when therapy started at the earliest 5 days post- stroke [25–29]. However, the intensity of training in ani- mal models is much higher than compared to humans [28]. A trial comparable to animal studies used constraint-induced movement therapy and observed a worse functional day 90 outcome when therapy started at day 10 after stroke onset [30]. VER within 24 h of stroke onset was investigated in three randomized con- trolled trials of limited size which were published be- tween 2008 and 2010. A systematical review in 2015 did not demonstrate superiority of VER compared to stand- ard care [31–34]. In April 2015 the large phase III AVERT trial was published and to the surprise of the community reported a less favourable 3 month stroke outcome of the earlier and higher-dose treated group. Discussion Despite to the fact that presumably most hospitalized stroke patients receive VER in a widely varying amount, surprisingly little is known about its current stage in clinical routine [16]. We report data from the Baden-Wuerttemberg stroke registry on the general access to VER, the timing from admission to first physical, occupational, and speech therapy and the average numbers of therapy sessions. All admitted patients with the ICD10 diagnoses I61 and I63 from a 5 year period were taken into analysis, except for patients with either early discharge within 24 h of ad- mission or mechanical ventilation, thereby making the Based on our data VER might be less frequently ap- plied to patients with ICH compared to IS and also the proportion of patients with ICH and immediate access to therapy was observed to be lower. Differences were small but consistent throughout the analysis. Two pos- sible factors might have contributed to this finding. First, patients with fatal ICH were more frequently denied therapy compared to IS patients with the same outcome. This might be due to earlier decision making for pallia- tive care in ICH than in IS. Second, a survey from 2011 indicated that stroke professionals seem to be more re- strained regarding early mobilization in patients with ICH for concerns regarding blood pressure oscillation, which also may account for the observed differences [21]. Discussion Although the multiple differences between the study groups (earlier mobilization at higher frequency and for a longer period of time) impair the identification of the actually harmful factor, the AVERT trial broke with the widely-accepted assumption that very early and intense mobilization and rehabilitation represents the best med- ical therapy to promote recovery in all patients with acute cerebrovascular disease [16]. Further stroke sub- group analyses hypothesized that in particular patients with ICH and/or severe disability might benefit from de- layed mobilization and rehabilitative treatment at lower dosage. However, even although more than 2000 Obviously, interdisciplinary treatment in the acute phase has to be coordinated and adapted to the acute diagnostic investigations and treatments, otherwise VER might not be applied in the adequate moment [36]. It thus seems very likely that specific subgroups concern- ing stroke etiology, severity of stroke symptoms, comor- bidities, and acute stroke therapy like hematoma evacuation, IV thrombolysis, or thrombectomy need to be taken into account for an individualized rehabilitation protocol [40]. An overview on worldwide national stroke guidelines was presented 2015 by Bernhardt et al and demonstrated a widely recommended early mobilization regime (22 out of 30 national guidelines, of these eight recommended mobilization within 24 h after stroke on- set). Early rehabilitation is endorsed in 11 national guidelines, i.e. Canada, Croatia, Finland, Germany, Japan, New Zealand, Norway, Scotland, Singapore, South Africa, and the USA [15]. Current US-American guide- lines state that VER in the acute care hospital is op- tional, but all patients should at least undergo rehabilitative assessment prior to discharge to an inpatient rehabilitation care unit [41, 42]. The 2015 up- dated Canadian stroke rehabilitation guideline was the first to have incorporated findings from the AVERT trial and does no longer recommend frequent out-of bed activity within 24 h after stroke onset [43]. As a supra- national committee, the European Stroke Organization guidelines for the management of ischemic stroke from 2008 recommend an early initiation of rehabilitation therapy but do not provide precise time frames [44]. In Germany, treatment on specialized stroke units or inten- sive care units is remunerated with the procedure code Reuter et al. BMC Neurology (2016) 16:222 Page 8 of 10 Page 8 of 10 8–981.x. This includes daily PT, OT, and ST for patients with treatable disabilities and therapy has to be com- menced not later than the next day after admission. Acknowledgments g The authors wish to thank Christian Stock, Institute of Medical Biometry and Informatics, University of Heidelberg, for the fundamental statistical support and all collaborators in the hospitals of Baden-Wuerttemberg for providing data. All participating hospitals are listed at http://www.geqik.de/ fssa2011.php. Stroke working group of Baden-Wuerttemberg: Katrin Schoser (Klinikum Tut- tlingen, Germany), Michael Daffertshofer (Department of Neurology, Stadtkli- nik Baden-Baden, Germany), Curt Diehm (Department of Internal/Vascular Medicine, Max-Grundig-Klinik, Bühl, Germany), Stephan Neumaier (Medical services of health insurance Freiburg, Germany), Horst Wiethölter (formerly affiliated to Bürgerhospital Stuttgart, Germany), Elke Drewitz (Klinikum Stutt- gart, Germany). Discussion within less than 7 days of admission a calculated score had to be extrapolated by the admitting hospital and it is regarded very plausible that the complexity of data input will negatively affect its accuracy. Based on the lower interquartile range of hospitalization time (7 days in ICH and 6 days in IS) approximately 25% of the ICH and IS patients were discharged after less than 7 days of admission. Thus for this fraction the registry has no ac- cess to the raw data and data quality cannot be compar- ably assured. Thirdly, missing data on functional disability at admission might bias the results and thus interpretation of data. y Based on the general access to VER and the average numbers of therapy sessions our data from routine hospital care demonstrates that severely disabled stroke patients receive training at high frequency, most likely under the assumption that they will benefit most. We re- gard our data robust even though the “admission to treatment time” and not “onset to treatment time” was measured, since there is a correlation between stroke se- verity and lesser onset to hospital admission time [18, 45]. However, the minimal demand of rehabilitation therapy is at least not to be harmful to the treated subject. The results of the AVERT trial should therefore be taken into account for the application of VER in clinical routine to avoid overly intense therapy in severely disabled patients, as done in the current Canadian stroke rehabilitation practice guidelines [43]. One further important lesson from AVERT is the ur- gent need for further clinical trials which will have to focus on stroke subgroups instead of the entire spectrum of IS and nontraumatic ICH. Otherwise conclusions as from AVERT lead to misunderstanding. The condition to meas- ure the outcome of the rehabilitation treatment needs to in- clude shaping, that means (as shown in the Baden- Wuerttemberg stroke registry) the treatment has to be adapted to the degree of disability of the patient and his rehabilitation potential. It moreover might be best evalu- ated by individual goal assessment based for instance on the International Classification of Functioning, Disability and Health [46]. In the meantime a temporary international consensus for severely disabled stroke patients and patients with ICH is regarded necessary. Abbrevations ICH I b ICH: Intracerebral hemorrhage; IS: Ischemic stroke; OT: Occupational therapy; PT: Physical therapy; ST: Speech therapy; VER: Very early rehabilitation Conclusion Patients admitted for IS or ICH in Baden-Wuerttemberg have excellent access to PT, while an approximately 12– 17% undersupply of ST and more in particular a 30% un- dersupply of OT in patients with treatable disabilities was observed. When stratified according to the mRS at discharge, we observed that in acute stroke care the staff decision for VER and its frequency seem to reflect at- tempts to optimize resources, i.e. those with either very good or very unfavorable prognosis received therapy less frequently. Severely disabled stroke patients received VER at the earliest and treatment sessions were applied at highest frequency. Since there are current concerns how to perform rehabilitation therapy in severely dis- abled ischemic stroke patients and patients with ICH in the very early, vulnerable phase, a temporary consensus for this subgroup is regarded necessary. Future clinical trials are needed to investigate the response of specific stroke subgroups to VER and the methodology of these trials will have to be fundamentally reconsidered Specific limitations of our study need to be discussed. Firstly, since the data on therapy was collected from a stroke registry, no quantitative (amount of PT, OT, and ST sessions in minutes) or qualitative (in-bed or out of- bed therapy) information was available. This limits the ability to directly compare our results to the AVERT trial. Moreover, in the BW stroke registry the documen- tation on timing of OT and ST (≤48 h, >48 h) was less precise compared to PT (≤24 h,>24–48 h, >48 h), which limits a detailed comparison for the first 48 h after ad- mission. Secondly, in large stroke registries the general accuracy of results strongly depends on the accuracy of data input at the participating hospitals. In the Baden- Wuerttemberg stroke registry data documentation is usually performed by a physician, diagnosis related groups (DRG) coordinator or trained study nurse, which should ensure a sufficient data quality. The provided data is validated centrally by predefined logic and range checks and annual performance reports including feed- back on data quality are provided for each participating hospital. Nevertheless, for patients being discharged Abbrevations ICH: Intracerebral hemorrhage; IS: Ischemic stroke; OT: Occupational therapy; PT: Physical therapy; ST: Speech therapy; VER: Very early rehabilitation Funding g The study was supported by an internal grant from the Dept. of Neurology, Medical Faculty Mannheim, University of Heidelberg, Germany. The source of funding did not influence the study design, the collection, analysis, and interpretation of data, the writing of the report, and the decision to submit the article for publication. Page 9 of 10 Page 9 of 10 Reuter et al. BMC Neurology (2016) 16:222 Competing interests All authors declare: no support from any organisation for the submitted work. CG holds a scholarship from the “Nachwuchsakademie Versorgungsforschung” (a health service research body) for a programme in Baden-Wuerttemberg. R.K. has received speaker’s honoraria from Boehringer Ingelheim that where unrelated to this study, Pfizer and Philips Healthcare. P.R. has received lecture fees and travel compensation from Boehringer- Ingelheim that where unrelated to this study, Ferrer, Paion, Bayer, and Sanofi. W.H. reported honoraria from Johnson & Johnson, Bayer and advisory board fees from Boehringer Ingelheim that where unrelated to this study. M.G.H. has received research grant support from Pfizer. 12. Aries MJ, Elting JW, Stewart R, De Keyser J, Kremer B, Vroomen P. Cerebral blood flow velocity changes during upright positioning in bed after acute stroke: an observational study. BMJ Open. 2013;3:e002960. 13. Treger I, Shafir O, Keren O, Ring H. Orthostatic hypotension and cerebral blood flow velocity in the rehabilitation of stroke patients. Int J Rehabil Res. 2006;29(4):339–42. 14. Olavarria VV, Arima H, Anderson CS, Brunser AM, Munoz-Venturelli P, Heritier S, Lavados PM. Head position and cerebral blood flow velocity in acute ischemic stroke: a systematic review and meta-analysis. Cerebrovasc Dis. 2014;37(6):401–8. 15. Bernhardt J, English C, Johnson L, Cumming TB. Early mobilization after stroke: early adoption but limited evidence. Stroke. 2015;46(4):1141–6. Authors’ contributions BR designed the study, analyzed data, and wrote the paper. CG, TS, HW, CD, PR, WH, and MGH were involved in planning of analysis and in interpretation of data. IB was involved in preparation of source data, RK supervised the research and revised the paper. All authors read and approved the final manuscript. 10. Cumming TB, Collier J, Thrift AG, Bernhardt J. The effect of very early mobilisation after stroke on psychological well-being. J Rehabil Med. 2008;40(8):609–14. 11. Diserens K, Moreira T, Hirt L, Faouzi M, Grujic J, Bieler G, Vuadens P, Michel P. Early mobilization out of bed after ischaemic stroke reduces severe complications but not cerebral blood flow: a randomized controlled pilot trial. Clin Rehabil. 2012;26(5):451–9. Availability of data and materials and mobility following stroke. Cochrane Database Syst Rev. 2014;4, CD001920. and mobility following stroke. Cochrane Database Syst Rev. 2014;4, CD001920. and mobility following stroke. Cochrane Database Syst Rev. 2014;4, CD001920. The statistical code used in this analysis is available from the corresponding author. The source data of the stroke registry are not publicly available due to privacy policy regulations. 8. Zeiler SR, Krakauer JW. The interaction between training and plasticity in t poststroke brain. Curr Opin Neurol. 2013;26(6):609–16. 8. Zeiler SR, Krakauer JW. The interaction between training and plasticity in the poststroke brain. Curr Opin Neurol. 2013;26(6):609–16. 9. Biernaskie J, Chernenko G, Corbett D. Efficacy of rehabilitative experience declines with time after focal ischemic brain injury. J Neurosci. 2004;24(5):1245–54. Ethics approval and consent to participate The study was approved by the ethics committee of the Medical Faculty, University of Heidelberg (S339-2012) and by the governing board of the office for quality assurance in hospitals (Geschäftsstelle für Qualitätssicherung im Krankenhaus, GeQiK). 17. Gumbinger C, Reuter B, Wietholter H, Bruder I, Rode S, Drewitz E, Habscheid W, Daffertshofer M, Diehm C, Neumaier S, et al. A consecutive and prospective stroke database covers the state of Baden-Wuerttemberg with 10.8 million inhabitants in Germany. Neuroepidemiology. 2013;41(3-4):161–8. Author details 1 1Department of Neurology and Neurophysiology, University Hospital Freiburg, Breisacher Straße 64, 79106 Freiburg, Germany. 2Department of Neurology, University Hospital Heidelberg, Heidelberg, Germany. 3Department of Neurology, Universitätsmedizin Mannheim, University of Heidelberg, Mannheim, Germany. 4formerly affiliated to Department of Neurology, Bürgerhospital, Stuttgart, Germany. 5Office for Quality Assurance in Hospitals (GeQiK), Baden-Wuerttembergische Hospital Association, Stuttgart, Germany. 6Department of Internal/Vascular Medicine, Max-Grundig-Klinik, Bühl, Germany. 7Department of Neurology, Klinikum Kempten-Oberallgaeu, Kempten, Germany. 18. Gumbinger C, Reuter B, Stock C, Sauer T, Wietholter H, Bruder I, Rode S, Kern R, Ringleb P, Hennerici MG, et al. Time to treatment with recombinant tissue plasminogen activator and outcome of stroke in clinical practice: retrospective analysis of hospital quality assurance data with comparison with results from randomised clinical trials. BMJ. 2014;348:g3429. 19. Reuter B, Gumbinger C, Sauer T, Wietholter H, Bruder I, Rode S, Ringleb PA, Kern R, Hacke W, Hennerici MG, et al. Intravenous thrombolysis for acute ischaemic stroke in the elderly: data from the Baden-Wuerttemberg stroke registry. Eur J Neurol. 2016;23(1):13–20. y Max-Grundig-Klinik, Bühl, Germany. 7Department of Neurology, Klinikum Kempten-Oberallgaeu, Kempten, Germany. 20. Reuter B, Gumbinger C, Sauer T, Wietholter H, Bruder I, Diehm C, Ringleb PA, Kern R, Hacke W, Hennerici MG, et al. Intravenous thrombolysis is effective in young adults: results from the Baden-Wuerttemberg stroke registry. Front Neurol. 2015;6:229. Received: 28 May 2016 Accepted: 8 November 2016 Received: 28 May 2016 Accepted: 8 November 2016 21. Sjoholm A, Skarin M, Linden T, Bernhardt J. Does evidence really matter? Professionals' opinions on the practice of early mobilization after stroke. J Multidiscip Healthc. 2011;4:367–76. Consent for publication Not applicable. 16. group ATC, Bernhardt J, Langhorne P, Lindley RI, Thrift AG, Ellery F, Collier J, Churilov L, Moodie M, Dewey H, et al. Efficacy and safety of very early mobilisation within 24 h of stroke onset (AVERT): a randomised controlled trial. Lancet. 2015;386(9988):46–55. References 1. Collaborative systematic review of the randomised trials of organised inpatient (stroke unit) care after stroke. Stroke Unit Trialists' Collaboration. BMJ. 1997;314:1151-9. 22. Manning L, Hirakawa Y, Arima H, Wang X, Chalmers J, Wang J, Lindley R, Heeley E, Delcourt C, Neal B, et al. Blood pressure variability and outcome after acute intracerebral haemorrhage: a post-hoc analysis of INTERACT2, a randomised controlled trial. Lancet Neurol. 2014;13(4):364–73. 2. Stroke Unit Trialists C. Organised inpatient (stroke unit) care for stroke. Cochrane Database Syst Rev 2013;9:CD000197 2. Stroke Unit Trialists C. Organised inpatient (stro Cochrane Database Syst Rev. 2013;9:CD000197. Cochrane Database Syst Rev. 2013;9:CD000197. 23. Brocklehurst JC, Andrews K, Richards B, Laycock PJ. How much physical therapy for patients with stroke? Br Med J. 1978;1(6123):1307–10. 3. Saposnik G, Fang J, O'Donnell M, Hachinski V, Kapral MK, Hill MD, Investigators of the Registry of the Canadian Stroke Network for the Stroke Outcome Research Canada Working G. Escalating levels of access to in- hospital care and stroke mortality. Stroke. 2008;39(9):2522–30. 24. Lang CE, Lohse KR, Birkenmeier RL. Dose and timing in neurorehabilitation: prescribing motor therapy after stroke. Curr Opin Neurol. 2015;28(6):549–55. 25. Humm JL, Kozlowski DA, Bland ST, James DC, Schallert T. Use-dependent exaggeration of brain injury: is glutamate involved? Exp Neurol. 1999;157(2):349–58. 4. Govan L, Langhorne P, Weir CJ, Stroke Unit Trialists C. Does the prevention of complications explain the survival benefit of organized inpatient (stroke unit) care?: further analysis of a systematic review. Stroke. 2007;38(9):2536–40. 26. Farrell R, Evans S, Corbett D. Environmental enrichment enhances recovery of function but exacerbates ischemic cell death. Neuroscience. 2001;107(4):585–92. 5. Geeganage C, Beavan J, Ellender S, Bath PM. Interventions for dysphagia and nutritional support in acute and subacute stroke. Cochrane Database Syst Rev. 2012;10, CD000323. 27. Wahl AS, Schwab ME. Finding an optimal rehabilitation paradigm after stroke: enhancing fiber growth and training of the brain at the right moment. Front Hum Neurosci. 2014;8:381. 6. Martino R, Foley N, Bhogal S, Diamant N, Speechley M, Teasell R. Dysphagia after stroke: incidence, diagnosis, and pulmonary complications. Stroke. 2005;36(12):2756–63. 28. Krakauer JW, Carmichael ST, Corbett D, Wittenberg GF. Getting neurorehabilitation right: what can be learned from animal models? Neurorehabil Neural Repair. 2012;26(8):923–31. 7. Pollock A, Baer G, Campbell P, Choo PL, Forster A, Morris J, Pomeroy VM, Langhorne P. Physical rehabilitation approaches for the recovery of function Page 10 of 10 Reuter et al. BMC Neurology (2016) 16:222 29. References Bland ST, Schallert T, Strong R, Aronowski J, Grotta JC, Feeney DM. Early exclusive use of the affected forelimb after moderate transient focal ischemia in rats : functional and anatomic outcome. Stroke. 2000;31(5):1144–52. 30. Dromerick AW, Lang CE, Birkenmeier RL, Wagner JM, Miller JP, Videen TO, Powers WJ, Wolf SL, Edwards DF. Very early constraint-induced movement during stroke rehabilitation (VECTORS): a single-center RCT. Neurology. 2009;73(3):195–201. 31. Bernhardt J, Dewey H, Thrift A, Collier J, Donnan G. A very early rehabilitation trial for stroke (AVERT): phase II safety and feasibility. Stroke. 2008;39(2):390–6. 32. Sundseth A, Thommessen B, Ronning OM. Outcome after mobilization within 24 h of acute stroke: a randomized controlled trial. Stroke. 2012;43(9):2389–94. 33. Langhorne P, Stott D, Knight A, Bernhardt J, Barer D, Watkins C. Very early rehabilitation or intensive telemetry after stroke: a pilot randomised trial. Cerebrovasc Dis. 2010;29(4):352–60. 34. Lynch E, Hillier S, Cadilhac D. When should physical rehabilitation commence after stroke: a systematic review. Int J Stroke. 2014;9(4):468–78. 35. Bernhardt J, Churilov L, Ellery F, Collier J, Chamberlain J, Langhorne P, Lindley RI, Moodie M, Dewey H, Thrift AG, et al. Prespecified dose-response analysis for A Very Early Rehabilitation Trial (AVERT). Neurology. 2016. 36. Berney L, Wasserfallen JB, Grant K, Levivier M, Simon C, Faouzi M, Paillex R, Schweizer V, Diserens K. Acute neurorehabilitation: does a neurosensory and coordinated interdisciplinary programme reduce tracheostomy weaning time and weaning failure? NeuroRehabilitation. 2014;34(4):809–17. 37. Godecke E, Ciccone NA, Granger AS, Rai T, West D, Cream A, Cartwright J, Hankey GJ. A comparison of aphasia therapy outcomes before and after a very early rehabilitation programme following stroke. Int J Lang Commun Disord. 2014;49(2):149–61. 38. Mattioli F, Ambrosi C, Mascaro L, Scarpazza C, Pasquali P, Frugoni M, Magoni M, Biagi L, Gasparotti R. Early aphasia rehabilitation is associated with functional reactivation of the left inferior frontal gyrus: a pilot study. Stroke. 2014;45(2):545–52. 39. Suntrup S, Meisel A, Dziewas R, Ende F, Reichmann H, Heuschmann P, Ickenstein GW. Dysphagia diagnostics and therapy of acute stroke: federal survey of certified stroke units. Nervenarzt. 2012;83(12):1619–24. 40. Arnold SM, Dinkins M, Mooney LH, Freeman WD, Rawal B, Heckman MG, Davis OA. Very early mobilization in stroke patients treated with intravenous recombinant tissue plasminogen activator. J Stroke Cerebrovasc Dis. 2015;24(6):1168–73. 41. Winstein CJ, Stein J, Arena R, Bates B, Cherney LR, Cramer SC, Deruyter F, Eng JJ, Fisher B, Harvey RL, et al. 44. European Stroke Organisation Executive C, Committee ESOW. Guidelines for management of ischaemic stroke and transient ischaemic attack 2008. Cerebrovasc Dis. 2008;25(5):457–507. 43. Hebert D, Lindsay MP, McIntyre A, Kirton A, Rumney PG, Bagg S, Bayley M, Dowlatshahi D, Dukelow S, Garnhum M, et al. Canadian stroke best practice recommendations: Stroke rehabilitation practice guidelines, update 2015. Int J Stroke. 2016;11(4):459–84. References Guidelines for adult stroke rehabilitation and recovery: a guideline for healthcare professionals from the American Heart Association/American Stroke Association. Stroke. 2016;47(6):e98–169. 42. Miller EL, Murray L, Richards L, Zorowitz RD, Bakas T, Clark P, Billinger SA, American Heart Association Council on Cardiovascular N, the Stroke C. Comprehensive overview of nursing and interdisciplinary rehabilitation care of the stroke patient: a scientific statement from the American Heart Association. Stroke. 2010;41(10):2402–48. 43. Hebert D, Lindsay MP, McIntyre A, Kirton A, Rumney PG, Bagg S, Bayley M, Dowlatshahi D, Dukelow S, Garnhum M, et al. Canadian stroke best practice recommendations: Stroke rehabilitation practice guidelines, update 2015. Int J Stroke. 2016;11(4):459–84. 44. European Stroke Organisation Executive C, Committee ESOW. Guidelines for management of ischaemic stroke and transient ischaemic attack 2008. Cerebrovasc Dis. 2008;25(5):457–507. Submit your next manuscript to BioMed Central and we will help you at every step: Submit your next manuscript to BioMed Central and we will help you at every step: 45. Puolakka T, Vayrynen T, Happola O, Soinne L, Kuisma M, Lindsberg PJ. Sequential analysis of pretreatment delays in stroke thrombolysis. Acad Emerg Med. 2010;17(9):965–9. Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit and we will help you at every step: 46. Cerniauskaite M, Quintas R, Boldt C, Raggi A, Cieza A, Bickenbach JE, Leonardi M. Systematic literature review on ICF from 2001 to 2009: its use, implementation and operationalisation. Disabil Rehabil. 2011;33(4):281–309.
https://openalex.org/W4231825123	https://www.repository.cam.ac.uk/bitstreams/d1d5cc6c-2c6e-4ad6-9340-f800e54ec69f/download	English	null	Decreasing hospital burden of COVID-19 during the first wave in Regione Lombardia: an emergency measures context	Research Square (Research Square)	2,021	cc-by	6,189	Decreasing hospital burden of COVID-19 during the first wave in Regione Lombardia: an emergency measures context Francesca Maria Grosso1†, Anne Margaret Presanis2†, Kevin Kunzmann2, Chris Jackson2, Alice Corbella2,3, Giacomo Grasselli4, Aida Andreassi5, Annalisa Bodina5, Maria Gramegna5, Silvana Castaldi1,4, Danilo Cereda5†, Daniela De Angelis2† and Covid-19 Lombardy Working Group RESEARCH Open Access Correspondence: francesca.grosso@unimi.it * Correspondence: francesca.grosso@unimi.it †Grosso Francesca Maria, Presanis Anne Margaret, Cereda Danilo and De Angelis Daniela contributed equally to this work. 1Postgraduate School of Public Health, Department of Biomedical Sciences for Health, Via Pascal 36, University of Milan, Milan, Italy Full list of author information is available at the end of the article * Correspondence: francesca.grosso@unimi.it †Grosso Francesca Maria, Presanis Anne Margaret, Cereda Danilo and De Angelis Daniela contributed equally to this work. 1Postgraduate School of Public Health, Department of Biomedical Sciences for Health, Via Pascal 36, University of Milan, Milan, Italy Full list of author information is available at the end of the article Grosso et al. BMC Public Health (2021) 21:1612 https://doi.org/10.1186/s12889-021-11669-w Grosso et al. BMC Public Health (2021) 21:1612 https://doi.org/10.1186/s12889-021-11669-w Abstract Background: The aim of this study is to quantify the hospital burden of COVID-19 during the first wave and how it changed over calendar time; to interpret the results in light of the emergency measures introduced to manage the strain on secondary healthcare. Methods: This is a cohort study of hospitalised confirmed cases of COVID-19 admitted from February–June 2020 and followed up till 17th July 2020, analysed using a mixture multi-state model. All hospital patients with confirmed COVID-19 disease in Regione Lombardia were involved, admitted from February–June 2020, with non-missing hospital of admission and non-missing admission date. Results: The cohort consists of 40,550 patients hospitalised during the first wave. These patients had a median age of 69 (interquartile range 56–80) and were more likely to be men (60%) than women (40%). The hospital-fatality risk, averaged over all pathways through hospital, was 27.5% (95% CI 27.1–28.0%); and steadily decreased from 34.6% (32.5–36.6%) in February to 7.6% (6.3–10.6%) in June. Among surviving patients, median length of stay in hospital was 11.8 (11.6–12.3) days, compared to 8.1 (7.8–8.5) days in non-survivors. Averaged over final outcomes, median length of stay in hospital decreased from 21.4 (20.5–22.8) days in February to 5.2 (4.7–5.8) days in June. Conclusions: The hospital burden, in terms of both risks of poor outcomes and lengths of stay in hospital, has been demonstrated to have decreased over the months of the first wave, perhaps reflecting improved treatment and management of COVID-19 cases, as well as reduced burden as the first wave waned. The quantified burden allows for planning of hospital beds needed for current and future waves of SARS-CoV-2 i. Study participants The Covid-19 Regional Database (Additional file 1: Ap- pendix A.1.3) is an integrated database collecting data from laboratories, hospitals and Local Healthcare Agen- cies and comprises detailed, but pseudo-anonymised, retrospective individual-level data on the cohort of all individuals in Lombardia diagnosed with Covid-19 dur- ing the first wave from February to June 2020 [8–10]. In Lombardia, seventeen “first-responder hub hospi- tals” were selected to be part of the Infectious Disease Hospital Network and charged with admission of sus- pected cases, because of their expertise in infectious dis- eases or their membership of the Venous-venous ECMO Respiratory Failure Network [2]. General Practitioners and Family Pediatricians received training on surveil- lance activities [3]; temperature scanners were set up in airports to screen passengers from in-bound flights; and an Operations Room for Emergencies was organised to manage emergency calls from suspected Covid-19 pa- tients [4]. Data, as extracted on 5th August 2020, include 95,777 records, representing 95,354 individuals with confirmed COVID-19 disease in 2020, observed from 1st December 2019 to 17th July 2020. The dataset records age, gender, Local Healthcare Agency district of Lombardia, co- morbidities, symptoms, whether the individual is a healthcare worker or care home resident, whether or not the individual was hospitalised, and details of the admit- ting hospital if the individual was hospitalised. For each individual, dates of symptom onset, positive laboratory test, hospital admission, ICU admission, ICU discharge, hospital discharge, recovery and death are recorded. Ex- cluding duplicate records and records with inconsistent, missing or invalid dates leaves 94,474 individuals, of whom 46,609 were hospitalised. Finally, excluding the 12.4% of hospitalised patients with missing hospital of admission leaves 40,550 individuals in the dataset. [ ] On 20/02/2020 the first Italian patient was diagnosed with Covid-19 in the Lombardia town of Codogno, which soon became the Italian epicenter of the pan- demic. The number of confirmed infected patients rose to 403 by the following week, with 213 patients admitted to hospitals [5]. This abrupt rise in the number of con- firmed cases, peaking eventually on the 20th of March 2020, put a large strain on the healthcare system in Lombardia [6], an Italian region of approximately 10 million inhabitants of whom 41% over 55 years of age (Additional file 1: Appendix A.1.1). © The Author(s). 2021, corrected publication 2021. 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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. Grosso et al. BMC Public Health (2021) 21:1612 Page 2 of 9 Introduction of the next waves, currently in progress. The burden is quantified, using a multi-state modelling approach [7], in terms of risks of progression through hospitals of Covid-19 patients, together with lengths of stay in hos- pital and ICU. On the 9th of January 2020, the health authorities in China reported that a novel strain of coronavirus, later named SARS-CoV-2, was the causative agent for many of the severe acute respiratory syndromes occurring in the area of Wuhan [1]. In response to the emerging situ- ation, several pre-pandemic measures were implemented by the Italian Ministry of Health and by Regione Lombardia. Study participants The growing need for hospital capacity led to non-urgent procedures being cancelled (48 h after the first case) and to expanding the number of beds, especially in the ICU wards, where the pre-crisis capacity was around 720, immediately raised to 861 [2]. Regional Coordination Units were created (Additional file 1: Appendix A.1.3) to manage the crisis. The Regional Unit of Coordination for Hospital Admis- sion collected data on the number of vacant beds daily and redirected patients with the purpose of redistribut- ing the burden equally among the 17 first-responder hub hospitals (the “hub-and-spoke” model) especially in- volved during the initial phase of the pandemic and then among all hospitals in the region. A total of 482 ICU beds were made available over 18 days from the 20th February [2]. Multi-state model The progression of patients through hospital can be rep- resented by a multi-state model with states Hospital (ward entered on admission date), ICU (entered at ICU admission), Post-ICU (entered after ICU discharge), Dis- charge (entered on date of hospital discharge), and Death (entered on date of death) (Additional file 1: Ap- pendix Fig. A.2). The outcomes of interest are: the prob- abilities of entering each next state (a “next event”) given the current state; the probabilities of entering a final state (either a Death or Discharge event) given hospital admission (“hospital-fatality risk”, defined in Additional file 1: Appendix A.4) or given the current state; corre- sponding times to each next event, conditional on ex- periencing the next event, i.e. the lengths of stay in each state; and the total length of stay (LoS) in hospital. All of the enacted emergency measures likely impacted on the ability of the healthcare system of Lombardia to provide care. The aims of this study are: to estimate how mortality risk and progression of patients through hos- pital changed through the first wave; to interpret these changes in relation to the emergency measures imple- mented; and to inform management and relevant models The multi-state model is implemented as a “mixture model” [7, 11], combining multinomial or binomial lo- gistic regression of probabilities of different pathways through hospital on covariates with parametric time-to- event analyses for the time taken to move from one state Grosso et al. BMC Public Health (2021) 21:1612 Page 3 of 9 to the next (each LoS), conditional on the next event oc- curring. Analyses are carried out in R 3.6.3 using the flexsurv package [7]. morbidity; 4% were care home residents; 4% were healthcare workers; and most (71%) were admitted to hospitals with a large bed capacity. Further covariate summaries for these patients are shown in Additional file 1: Appendix (Table A.4). f g In the Covid-19 Regional Database, outcomes/next events are missing for < 1% of individuals in the Hospital and ICU states, and for 15% of individuals in the Post- ICU state (Additional file 1: Appendix Fig. A.1). It is un- known why outcomes are missing, i.e. whether they had not happened by the end-date of the data, 17th July, (“right-censoring”), or whether they had not been re- corded (“missing data/loss to follow-up”). By month of admission Figure 2 shows the estimated odds ratios of risk of next events by month compared to a baseline of March, to- gether with corresponding predicted probabilities of next events, demonstrating the overall decreasing trend in se- vere events. The probability of ICU admission from a hospital ward decreased from 14.3% (13.0–16.0%) in February to 2.6% (1.7–3.6%) in June; the hospital-fatality risk without ICU decreased from 25.7% (24.1–27.6%) in February to 6.6% (5.0–8.0%) in June; and the ICU- fatality risk decreased from 46.0% (41.5–52.8) in Febru- ary to 26.1% (17.6–36.4%) in May–June. These models do not account for individual patient char- acteristics, which are explored in a companion paper [12]. All methods were performed in accordance with the Declaration of Helsinki. Overall results h Figure 1 shows the specific hospital-fatality risks from each state: 23.1% (22.7–23.5%) from hospital without ICU; 42.5% (40.8–43.9%) from ICU; and 9.8% (9.0– 11.0%) from post-ICU. When averaged over all pathways through hospital, the overall hospital-fatality risk was 27.5% (95% CI 27.1–28.0%). The overall median (95% CI of median) LoS in hos- pital, averaged over all pathways through hospital and final outcomes was 10.4 (10.1–10.9) days. Among surviv- ing patients, median LoS in hospital was 11.8 (11.6– 12.3) days, while those whose final outcome was death had a median time to death of 8.1 (7.8–8.5) days. The probability of ICU admission was 9.2% (9.0–9.5%) and the corresponding median time from hospital ad- mission to ICU admission was 3.5 (3.3–3.6) days. The median LoS in ICU was 11.0 (10.7–11.6) days: 12.6 (12.2–13.2) days for survivors and 9.6 (9.3–10.1) days for non-survivors. The median LoS in a post-ICU ward was 18.0 (17.1–18.7) days: 18.8 (18.2–19.5) days for survivors and 10.1 (8.9–11.9) days for non-survivors. By pathway through hospital, median LoS in hospital was: 10.8 (10.3–10.9) days for surviving patients who were not ad- mitted to ICU (hospital-discharge); 40.7 (40.1–41.8) days for surviving patients who were admitted ICU (hospital- ICU-postICU-discharge); 6.9 (6.6–7.1) days for non- survivors who were not admitted to ICU (hospital- death); 15.1 (14.8–15.9) days for patients who died in ICU (hospital-ICU-death); and 31.7 (30.0–33.0) days for patients who died in a post-ICU ward (ward-ICU-post- ICU-death). We consider four models with different covariate com- binations: (a) no covariates; (b) month of hospital admis- sion; (c) hospital bed capacity; (d) both month of hospital admission and hospital bed capacity. Bed cap- acity is defined in terms of numbers of both hospital and ICU beds (Additional file 1: Appendix Table A.3). Point estimates and 95% confidence intervals (CIs) are re- ported for each probability of interest, as well as for the median and interquartile ranges (IQRs) of each time-to- event distribution. The confidence intervals represent parameter uncertainty, whereas the median and IQRs summarise the heterogeneity across individuals in the time-to-event distributions. Multi-state model Since it is im- possible to distinguish between these possibilities, two alternative assumptions are made: (1) the missing out- comes are ignorable, i.e. individuals with missing out- comes have the same distribution of outcomes as those observed; (2) these individuals are right-censored at a time t after their last observed event. In this second case, the parametric time-to-event models account for the censoring, and typically t would be the length of time till the end of the dataset, i.e. till 17th July. However, we judge that it is implausible that all of those with missing outcomes are still in hospital by mid-July, so instead make the conservative assumption that t is 1 day. The results are very similar under the two assumptions (Add- itional file 1: Appendix A.3), so in what follows, only the results under assumption [2] are reported. As we con- sider discharge a final outcome, we ignore the 2.02% of discharged patients who are observed to die after dis- charge (Additional file 1: Appendix Fig. A.1). Dataset description The cohort consists of 40,550 individuals who under- went hospitalization during the first wave. Their median age was 69 (interquartile range [56–80]); the proportion of men (60%) was higher than women (40%); most (69%) were admitted in March; 64% had at least one co- The trend in fatality risk is more uncertain from the post-ICU state, with only February (at 30.6% [23.5– 37.6%]) significantly higher than March (8.4% [7.6– 9.7%]), and the post-ICU-fatality risk estimated to be Grosso et al. BMC Public Health (2021) 21:1612 Page 4 of 9 Fig. 1 Multi-state model with estimated risks (point estimate and 95% CI in brackets) of moving from current states to next events. The numbers in each state in square brackets are the observed numbers of patients reaching each state. These observed numbers do not include the numbers of patients with missing next events (< 1% in the Hospital and ICU states, 15% in the Post-ICU state), nor the ignored 2% of patients who died after being discharged from hospital. In contrast, the estimated risks account for the missing next events, assuming they are censoring at 1 day after the patients’ last observed events Fig. 1 Multi-state model with estimated risks (point estimate and 95% CI in brackets) of moving from current states to next events. The numbers in each state in square brackets are the observed numbers of patients reaching each state. These observed numbers do not include the numbers of patients with missing next events (< 1% in the Hospital and ICU states, 15% in the Post-ICU state), nor the ignored 2% of patients who died after being discharged from hospital. In contrast, the estimated risks account for the missing next events, assuming they are censoring at 1 day after the patients’ last observed events 15.1% (7.8–29.9%) in May–June. Assuming the same risks of death from ICU or post-ICU in May and June, the overall hospital-fatality risk, regardless of path through hospital, is estimated to have steadily decreased from 34.6% (32.5–36.6%) in February to 29.8% (29.3– 30.3%) in March, 22.0% (21.1–23.0%) in April, 14.7% (13.3–15.9%) in May and 7.6% (6.3–10.6%) in June. There is no clear trend in non-survivors who are not admitted to ICU: the median time to death is largest in February, at 18.0 (16.4–19.8) days, dropping to 5.5 (5.4– 5.6) days in March, but then doesn’t appear to change significantly from March through to June. Dataset description Similarly, the times to death from ICU and from a post-ICU stay do not appear to vary much with month of admission. The overall median LoS in hospital, regardless of out- come, decreased from 21.4 (20.5–22.8) days in February to 5.2 (4.7–5.8) days in June. Among survivors, median LoS in hospital, whether or not they had an ICU admis- sion, decreased from 24.6 (22.8–26.1) days in February to 5.1 (4.8–5.8) days in June. Non-survivors had slightly shorter median LoS, decreasing from 17.8 (17.1–19.9) days in February to 4.7 (3.3–6.0) days in June. Effect of hospital bed capacity Note also that odds ratios are presented for 2/3 or 1/2 of the next events, since the probability of the remaining event (discharge in all three columns) is just defined as 1 minus the other probabilities and 28.4% (27.9–28.9%) in medium and large hospitals respectively. survivors: time to death is 7.0 (6.6–7.3), 7.5 (6.7–7.6) and 7.6 (6.8–8.0) days respectively. When adjusting for both hospital bed capacity and month of admission simultaneously, our findings are similar to the univariable results in Sections 3.3 and 3.4 (Additional file 1: Appendix A.3.4). LoS for survivors in hospital without ICU and in a post-ICU ward become shorter as hospital bed capacity becomes larger, with small but significant effects (Fig. 5, top-left panel), whereas LoS in ICU does not vary signifi- cantly (Fig. 5, bottom-left panel). The effect of hospital bed capacity on time to death from hospital without ICU is marginally significant, but with only a small effect resulting in only a day’s difference between small and large hospitals. In ICU and post-ICU wards, the time to death does not vary by bed capacity (Fig. 5, bottom-right panel). Averaged over all pathways through hospital, me- dian LoS is 13.7 (13.2–14.2) days in small hospitals, 10.4 (10.1–10.7) days in medium hospitals, and 9.7 (9.4–9.9) days in large hospitals. This effect of hospital bed cap- acity appears significant for survivors (11.1 [10.8–11.7], 13.0 (12.3–13.3) and 16.6 (16.0–17.8) days respectively for large, medium and small hospitals), but not for non- Effect of hospital bed capacity Hospital size, in terms of bed capacity, has a (un- adjusted) significant effect on the probabilities of next events from hospital admission: the smallest hospitals have both the lowest risk of ICU admission (odds ratio 0.450 [0.400–0.506] relative to the largest) and a slightly lower risk of death without ICU admission (odds ratio 0.773 [0.723–0.827]) (Fig. 4). These lower risks corres- pond to a higher probability of discharge among small hospitals (Fig. 4). However, note that the majority of the smallest hospitals have zero ICU beds, so the lower risk of ICU admission may be an artefact of not having the capacity. The risk of death in ICU is also significantly smaller in the smallest hospitals compared to the largest (odds ratio 0.292 [0.220–0.386]), but may again be an artefact of the lower ICU capacity. Hospital bed capacity has no significant effect on risks of next events after a post-ICU stay. These risks combine to result in a hospital-fatality risk that is lowest in small hospitals: 21.4% (20.6–22.3%) compared to 30.2% (29.0–31.5%) The median times spent by survivors in different stages of hospital (pre-ICU, ICU, and post-ICU) also ap- pear to have decreased with calendar month of admis- sion (Fig. 3). The median time to ICU admission decreases from 2.8 (2.5–3.3) days in February to 1.0 (0.6–1.6) days in June. Median LoS in hospital among survivors who were not admitted to ICU reduced from 22.4 (21.1–23.6) days in February to 5.1 (4.7–5.6) days in June; median LoS in ICU among survivors reduced from 10.3 (9.1–13.0) days in February to 7.5 (6.6–9.7) days in May–June, although it was longest in March, at 12.8 (12.3–13.4) days. Heterogeneity amongst survivors in their lengths of stay also appears to have decreased with month of admission (dashed lines in Fig. 3). Grosso et al. BMC Public Health (2021) 21:1612 Page 5 of 9 Fig. 2 Estimated odds ratios (odds of next event in each month compared to the odds of next event in March) and predicted probabilities of moving from the current state (columns) to next events (colours). Points are point estimates and vertical bars represent 95% confidence intervals. Note that for the ICU and post-ICU states, the observed numbers of events in June were small, so the “May” month includes events from both May and June combined. Discussion 3), the time to discharge, both from ordinary ward and from post-ICU, is the measure changing most substantially over time. These decreases, altogether, suggest an improvement in patient management, supported by the progressive in- crease in clinical knowledge of COVID-19 and a less se- vere disease presentation at hospital admission, resulting from prolonged and strict lockdown measures over the course of 3 months. In contrast, there is less evidence of any change in the lengths of stay for non-survivors and furthermore, the effect of bed capacity on LoS is not sig- nificant for non-survivors. This finding may indicate that more frail patients were unfortunately largely impacted by their condition and thus less responsive to the pro- gressive amelioration of care, although no specific cure has been found yet. The long LoS for patients on the pathway hospital-admission-to-ICU-to-PostICU-to-dis- charge (40.7 days [40.1–41.8]) may be affected by the lack of downstream beds: during the first wave of the pandemic, hospitals struggled to find facilities for post- hospitalization care and rehabilitation. This shortage At the peak of the first wave in March, the number of hospitalized patients rose to 7387, 37.15% of the re- ported positive cases (19,884, 10), although this ratio is affected by a selection bias resulting from the early pol- icy of preferential testing of symptomatic cases. Prelim- inary work on extending the multi-state model to estimate hospital admission risk amongst cases with symptoms suggests 45.7% (45.2–46.1%) of symptomatic cases in Milano ATS required hospital admission in the first wave. Making the admittedly strong assumption that these results are applicable more widely to Lombar- dia, 45 beds might be expected to be needed for every 100 symptomatic cases. Moreover, our results suggest that the majority of beds (almost 70% of the total hos- pital admissions) should be planned for wards, while around 10% of hospital admissions are expected to re- quire ICU beds; post-ICU beds should number at least 60% of ICU beds (Fig. 1). Length of stay (LoS) in hospital is also an important measure to factor in when planning for an emergency. Our estimates of both overall median length of stay in hospital (10.4 days (10.1–10.9)) and in ICU (11.0 days (10.7–11.6)) are comparable with the LoS reported in the systematic review of Rees et al. [14] covering publi- cations between 1 January 2020 and 12 April 2020. Discussion This analysis has demonstrated substantial changes in the hospital burden of COVID-19 disease in Lombardia over the first wave. Quantifying burden is paramount for contingency planning in terms of beds, equipment and staff needed in hospitals. Figure 1 shows the proportion of patients that will most likely experience each outcome after admission, that is 9.2% (9.0–9.5%) will undergo ICU admission, a proportion that is much lower than the 24% reported by Boelle et al. [13]. In contrast, 67.8% (67.4–68.0%) will be discharged without ICU admission, a number that is comparable to the 63% found by Boelle Grosso et al. BMC Public Health (2021) 21:1612 Page 6 of 9 Fig. 3 Summaries of the distributions of times from current state (columns) to next events (colours), by calendar month of admission. The 95% CI of the median times (solid lines) represent uncertainty in the estimate, whereas the inter-quartile range of the distribution (dashed lines) represents heterogeneity in the population Fig. 3 Summaries of the distributions of times from current state (columns) to next events (colours), by calendar month of admission. The 95% CI of the median times (solid lines) represent uncertainty in the estimate, whereas the inter-quartile range of the distribution (dashed lines) represents heterogeneity in the population Fig. 3 Summaries of the distributions of times from current state (columns) to next events (colours), by calendar month of admission. The 95% CI of the median times (solid lines) represent uncertainty in the estimate, whereas the inter-quartile range of the distribution (dashed lines) represents heterogeneity in the population et al. The outcome of death without ICU admission is also slightly higher (23.1% [22.7–23.5%]) when compared to Boelle et al. (13%). whose final outcome was death, seems consistent with observations in the review but only for overall median length of hospitalization. Decreases in the risks of severe events such as ICU ad- mission and mortality have been estimated from Febru- ary to June 2020, with corresponding increases in the risks of the positive outcomes of discharge from either ICU or hospital. Total LoS in hospital, averaged over final outcome, has decreased over the same months. Similarly, lengths of stay pre-ICU, in ICU, in hospital overall for those not admitted to ICU, and in post-ICU wards, among survivors who are eventually discharged, have reduced over time. Moreover (Fig. Discussion The authors found a hospital LoS of 4 days (1–9) outside China compared to 14 days (10–19) in China; and an ICU LoS of 7 days (4–11 days) outside China compared to 8 days (5–13) in China. The longer estimated LoS we found for surviving patients, when compared to patients Grosso et al. BMC Public Health (2021) 21:1612 Page 7 of 9 Fig. 4 Estimated odds ratios (relative to large hospitals) and predicted probabilities of moving from the current state (columns) to next events (colours) Fig. 4 Estimated odds ratios (relative to large hospitals) and predicted probabilities of moving from the current state (columns) to next events (colours) might have affected discharges from hospitals, as also hypothesized by Boelle et al. [13]. Regione Lombardia has a high proportion of individuals older than 65 (Add- itional file 1: Appendix A.1.1), including those resident in care homes and long-term facilities, explaining the scarcity of available post-hospital beds. Increasing the number of beds in both long-term facilities and in post- ICU wards where only low-grade assistance is needed, would relieve hospitals and increase patient turnover. transferred from smaller, less resourced hospitals to spe- cialist, highly skilled hospitals with ICU beds, supporting the “high case volume-better performances” model [15]. The evidence of hospital size effect is very important, as it is a proxy of the effective impact of the emergency mea- sures on hospital management and on patients’ lives, help- ing inform the management of further pandemic waves. Some assumptions and limitations to this analysis are inev- itable. The 12% of cases without information on their admit- ting hospital were excluded. The analysis assumes this missingness is ignorable, i.e. that cases with missing hospital information were similar to those with observed hospitals of admission. Similarly, excluding patients with inconsistent data on ICU admissions and discharges is assumed not to have biased results in any way. Hospital bed capacity was de- fined in terms of a combination of numbers of hospital and ICU beds (see Additional file 1: Appendix Table A.3). Bed capacity could have been defined in different ways, so results could depend on this definition. Supplementary Information Supplementary Information y The online version contains supplementary material available at https://doi. org/10.1186/s12889-021-11669-w. The online version contains supplementary material available at https://doi. org/10.1186/s12889-021-11669-w. Authors’ contributions Dr. Grosso FM. and Dr. Presanis AM served as co-first authors and contributed equally to the work. Dr. Cereda D and Professor De Angelis D served as co- last authors and contributed equally to the work. Professor De Angelis D and Dr. Cereda D had full access to all the data in the study and take responsibil- ity for the integrity of the data and the accuracy of the data analysis. Dr. Grosso FM, Dr. Presanis AM, Professor De Angelis D and Dr. Cereda D con- ceptualized, made substantial contributions to data analysis, and wrote the first draft of the manuscript. Dr. Castrofino A, Dr. Del Castillo G performed the literature search. Crottogini L, Dr. Andreassi A, Dr. Salmoiraghi M, Dr. Bodina A collected all data. Dr. Kunzman K, Dr. Presanis AM, Dr. Corbella A, Dr. Jackson C, Professor De Angelis D performed data analysis. Dr. Grosso FM, Dr. Presanis AM, Dr. Cereda D, Professor De Angelis D made substantial contribution to interpretation of data. Dr. Corbella A, Professor Castaldi S, Dr. Zanella A, Dr. Grasselli G, Dr. Tirani M critically reviewed the initial manuscript. The final version of the manuscript was critically revised and finally approved as submitted by all the authors. Nevertheless, the presented estimates give crucial evi- dence to support planning hospital care for the current and any potential future wave of infection. Discussion Such an assumption implies individuals being alive/next event-free for a longer period of time than a single day, so es- timates of times to next events would be expected to be lar- ger, although estimated risks would be expected to be similar to those under the 1-day censoring assumption, due to low proportions censored. Nevertheless, the presented estimates give crucial evi- dence to support planning hospital care for the current and any potential future wave of infection. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12889-021-11669-w. Additional file 1. Acknowledg Not applicab Authors’ con Dr. Grosso FM equally to th last authors a Dr. Cereda D ity for the int Grosso FM, D ceptualized, first draft of t the literature Bodina A col Dr. Jackson C Dr. Presanis A contribution Zanella A, Dr The final vers as submitted Funding This work ha Jackson, Pres (Presanis, De Availability The data tha Lombardia b admission in small hospitals. Finally, the analysis presented, assuming individuals with missing outcomes are censored 1 day after their last observed event, was demonstrated to be very similar to assuming the missingness is ignorable. The results might not have been as similar if a different censoring assumption had been chosen, such as censoring individuals with missing outcomes at the date of the data (17th July). Such an assumption implies individuals being alive/next event-free for a longer period of time than a single day, so es- timates of times to next events would be expected to be lar- ger, although estimated risks would be expected to be similar to those under the 1-day censoring assumption, due to low proportions censored. Funding g This work has been funded by the Medical Research Council (De Angelis, Jackson, Presanis, Unit programme number MC UU 00002/11); the UKRI-MRC (Presanis, De Angelis, grant no MC_PC_19074). Discussion The fact that a large propor- tion of hospitals (over 50%) had zero ICU beds may also have caused artefactual results in terms of the risk of ICU Hospital size appears associated with length of stay, with shorter stays in larger hospitals, at least for survivors, with small but significant effects. This finding remains both when adjusting for month of admission and when adjust- ing additionally for patient characteristics such as age, gender and co-morbidities [12]. The largest hospitals have more beds, more patients and more skilled staff, and most of them were among the 17 hospitals in the Covid-19 Net- work. While the association may be confounded with the different case-mix in smaller versus larger hospitals, nevertheless, we posit that our finding reflects the imple- mentation of the hub-and-spoke model: high-risk patients Grosso et al. BMC Public Health (2021) 21:1612 Page 8 of 9 Fig. 5 Summaries of distributions of lengths of stay in hospital, by current state (y-axis), next event (panels) and hospital bed capacity (colours). The 95% CI of the median times (solid lines) represent uncertainty in the estimate, whereas the inter-quartile range of the distribution (dashed lines) represents heterogeneity in the population Fig. 5 Summaries of distributions of lengths of stay in hospital, by current state (y-axis), next event (panels) and hospital bed capacity (colours). The 95% CI of the median times (solid lines) represent uncertainty in the estimate, whereas the inter-quartile range of the distribution (dashed lines) represents heterogeneity in the population Fig. 5 Summaries of distributions of lengths of stay in hospital, by current state (y-axis), next event (panels) and hospital bed capacity (colours). The 95% CI of the median times (solid lines) represent uncertainty in the estimate, whereas the inter-quartile range of the distribution (dashed lines) represents heterogeneity in the population admission in small hospitals. Finally, the analysis presented, assuming individuals with missing outcomes are censored 1 day after their last observed event, was demonstrated to be very similar to assuming the missingness is ignorable. The results might not have been as similar if a different censoring assumption had been chosen, such as censoring individuals with missing outcomes at the date of the data (17th July). Ethics approval consent to partecipate Data collection and analysis were part of outbreak investigations during a public health emergency. Processing of COVID-19 data is necessary for rea- sons of public interest in the area of public health, such as protecting against serious cross-border threats to health or ensuring high standards of quality and safety of health care, and therefore exempted from institutional review board approval (Regulation EU 2016/679 GDPR). 13. Boëlle P, Delory T, Maynadier X, et al. Trajectories of Hospitalization in COVID-19 patients: an observational study inFrance. J Clin Med. 2020;9(10): 3148. https://doi.org/10.3390/jcm9103148. 14. Rees EM, Nightingale ES, Jafari Y, Waterlow NR, Clifford S, B. Pearson CA, et al. COVID-19 length of hospital stay: a systematic review and data synthesis. BMC Med. 2020;18(1):270. https://doi.org/10.1186/s12916-020-0172 6-3. This study is a retrospective cohort analysis. The research question, design and data collection were motivated by the public health emergency response to the COVID-19 epidemic early in March 2020. Regione Lombardia collected data during the emergency using the existing information system for infectious diseases, regulated by a 2004 regional decree, that was then updated in May 2020 to regulate the COVID-19 data flows. Data are pseudo- anonymized. Consent for this data collection at patient level is given in hos- pitals and laboratories, which send the data to Regione Lombardia. Although patients were not directly involved in the study design, the experiences of clinicians and public health officials interacting with patients were crucial to the design of the data collection. Permission to use data has been obtained by the Covid-19 Research Committee of Regione Lombardia on the 12th of May 2020. 15. O’Brien SM, DeLong ER, Peterson ED. Impact of case volume on hospital performance assessment. Arch Intern Med. 2008;168(12):1277–84. https:// doi.org/10.1001/archinte.168.12.1277. Availability of data and materials The data that support the findings of this study are available from Regione Lombardia but restrictions apply to the availability of these data, which were Additional file 1. Page 9 of 9 Page 9 of 9 Grosso et al. BMC Public Health (2021) 21:1612 Grosso et al. BMC Public Health (2021) 21:1612 used under license for the current study, and so are not publicly available. Data are however available from the authors upon reasonable request and with permission of Covid-19 Research Committee of Regione Lombardia. 10. Welfare Directorate of Regione Lombardia, Regional Decree N°XI/3114; 07 July 2020. 11. Larson MG, Dinse GE. A mixture model for the regression analysis of competing risks data. J Royal Stat Soc Series C (Applied Statistics). 1985; 34(3):201–11 JSTOR, www.jstor.org/stable/2347464. Accessed 30 Dec. 2020. Declarations 12. Presanis AM, Kunzmann K, Grosso F, Jackson C, Corbella A, Gramegna M, et al. Risk factors for severe hospital burden during the first wave of COVID- 19 disease in Regione Lombardia. In preparation; 2021. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Consent for publication Not applicable as data are pseudo anonymized. Author details 1 1Postgraduate School of Public Health, Department of Biomedical Sciences for Health, Via Pascal 36, University of Milan, Milan, Italy. 2MRC Biostatistics Unit, University of Cambridge, East Forvie Building, Robinson way, Cambridge, UK. 3Department of Statistics, University of Warwick, Mathematical Sciences Building, Academic Loop Road, Warwick, UK. 4Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, University of Milan, Via Francesco Sforza 28, Milan, Italy. 5Welfare General Directorate, Regione Lombardia, Piazza Città di Lombardia 1, Milan, Italy. Received: 1 March 2021 Accepted: 12 August 2021 References 1. European Center of Disease Control. Timeline of ECDC’s response to COVID- 19. https://www.ecdc.europa.eu/en/covid-19/timeline-ecdc-response. Accessed 8 Dec 2020. 2. Grasselli G, Pesenti A, Cecconi M. Critical care utilization for the COVID-19 outbreak in Lombardy, Italy: early experience and forecast during an emergency response. JAMA. 2020;323(16):1545–6. https://doi.org/10.1001/ja ma.2020.4031. 3. Welfare Directorate of Regione Lombardia, Protocol G1.2020.0003279, 28 January 2020; 4. Welfare Directorate of Regione Lombardia, Protocol G1.2020.0005384; 07 February 2020; 5. Regione Lombardia; Dashboard Covid-19; https://www.regione.lombardia.it/ wps/portal/istituzionale/HP/servizi-e-informazioni/cittadini/salute-e- prevenzione/coronavirus/dashboard-covid19; Accessed 16 Dec 2020. 6. Armocida B, Formenti B, Ussai S, Palestra F, Missoni E, The Italian Health System and the COVID-19 challenge. [published online March 25, 2020] The Lancet Public Health; doi: https://doi.org/10.1016/S2468-2667(20)30074-8. 7. Jackson CH, Tom BDM, Kirwan PD, Mandal S, Seaman SR, Kunzmann K, et al. A comparison of two frameworks for multi-state modelling, applied to outcomes after hospital admissions with COVID-19. Submitted; 2020. 8. Welfare Directorate of Regione Lombardia, Regional Decree N° XI/3019; 30 March 2020. 9. Welfare Directorate of Regione Lombardia, Regional Decree N° VII/18853; 30 September 2004.
https://openalex.org/W4365816910	https://archive.logos-science.com/index.php/conference-proceedings/article/download/628/644	Ukrainian	null	НАДІЙНІСТЬ ФУНКЦІОНУВАННЯ МАТРИЧНИХ СПЕЦПРОЦЕСОРІВ РЕАЛЬНОГО ЧАСУ	GRUNDLAGEN DER MODERNEN WISSENSCHAFTLICHEN FORSCHUNG	2,023	cc-by-sa	1,093	31. März, 2023 • Zürich, Schweizerische Eidgenossenschaft • 97 31. März, 2023 • Zürich, Schweizerische Eidgenossenschaft • 97 DOI 10.36074/logos-31.03.2023.30 DOI 10.36074/logos-31.03.2023.30 НАДІЙНІСТЬ ФУНКЦІОНУВАННЯ МАТРИЧНИХ СПЕЦПРОЦЕСОРІВ РЕАЛЬНОГО ЧАСУ Благодарний Микола Петрович кандидат технічних наук, доцент, професор кафедри мехатроніки та електротехніки Національний аерокосмічний університет “Харківський авіаційний інститут” УКРАЇНА де W(t) –продуктивність МСП в поточний час; W0 – продуктивність абсолютно надійного МСП. ( ) р у W0 – продуктивність абсолютно надійного МСП. W0 – продуктивність абсолютно надійного МСП. Значенння W(t) в загальному випадку залежить від параметрів Т0 , tа, d та параметрів потоків збоїв та відмов, які діють на елементи МСП та методів забезпечення відмовостійкості [1]. Таким чином характерними рисами функціонування МСП є наступні: − ефективне функціонування в реальному масштабі часу; − використання в якості структурного базису НВІС-архітектур на кристалах і напівпровідникових пластинах[1]; − використання в якості структурного базису НВІС-архітектур на кристалах і напівпровідникових пластинах[1]; − високі вимоги до надійносних характеристик (безвідмовне функціонування на всьому інтервалі застосування за призначенням або безпечне закінчення функціонування (можливість функціонування зі зниженим рівнем якості) при настанні фатальних відмов)[2]; − істотне перевищення інтенсивності збоїв λс над інтенсивністю відмов λо (λс = β λо, β = 10 ÷100)[3]; − істотне перевищення інтенсивності збоїв λс над інтенсивністю відмов λо (λс = β λо, β = 10 ÷100)[3]; ( β , β )[ ]; − кластерізація (групування) відмов і збоїв[3]; − кластерізація (групування) відмов і збоїв[3]; − функціонування МСП на активних часових інтервалах і простої ̶ на пасивних часових інтервалах застосування[2]. − функціонування МСП на активних часових інтервалах і простої ̶ на пасивних часових інтервалах застосування[2]. Порівняльний аналіз значень Кs(t) показує наявність значного запасу ефективності МСП, який неможливо використовувати, реалізуючи тількі відомі методи забезпечення відмово стійкості [1, 3]. Почергове знаходження МСП на активних часових інтервалах тривалістю ta (ta = tio – tiH, i = 1,2, …, де tiH ̶ момент початку i-го активного часового інтервалу, tio ̶ момент закінчення i-го активного часового інтервалу) і на пасивних часових інтервалах тривалістю tП (tП = t(i+1)Н – tiО, i = 1,2, …)) вимагає уточнення поняття відмови МСП. Відмова МСП настає в таких випадках [2]: – протягом часового інтервалу ∆ta (допустимий часовий інтервал збільшення значення ta, ∆ta ≪ ta) в МСП не можуть бути усунені наслідки відмов (збоїв) ПМ, що виникають на активному часовому інтервалі; − на інтервалі (tнi, toi) функціонування не забезпечується постійне значення KS(t), тобто ) , ( oi t t t i    : const )t( Ks = , Кs(t)  Kтр. Вимоги до безвідмовного функціонування МСП визначимо системою нерівностей: { 𝒍(𝒕𝒂)𝝉𝒑≤∆𝒕𝒂; 𝑲𝒔(𝒕𝑯𝒊) →𝒎𝒂𝒙; 𝑷МСП(𝒕) ≥𝑷МСП тр (𝒕), 𝒕∈(𝒕𝑯𝒊 , 𝒕𝑶𝒊), (1) { 𝒍(𝒕𝒂)𝝉𝒑≤∆𝒕𝒂; 𝑲𝒔(𝒕𝑯𝒊) →𝒎𝒂𝒙; 𝑷МСП(𝒕) ≥𝑷МСП тр (𝒕), 𝒕∈(𝒕𝑯𝒊 , 𝒕𝑶𝒊), (1) { 𝒍(𝒕𝒂)𝝉𝒑≤∆𝒕𝒂; 𝑲𝒔(𝒕𝑯𝒊) →𝒎𝒂𝒙; 𝑷МСП(𝒕) ≥𝑷МСП тр (𝒕), 𝒕∈(𝒕𝑯𝒊 , 𝒕𝑶𝒊), (1) (1) де W(t) –продуктивність МСП в поточний час; W0 – продуктивність абсолютно надійного МСП. де W(t) –продуктивність МСП в поточний час; УКРАЇНА УКРАЇНА У більшості застосувань матричні спецпроцесори (МСП) функціонують циклічно або неперервно [1-3]. Охарактеризуємо умови застосування МСП точками тривимірного простору (Т0, ta, q), де Т0, ta, q – відповідно тривалість застосування МСП за призначенням, тривалість активного інтервалу ta, шпаруватість застосування МСП за призначенням q (q = tn/ta, де tn – тривалість пасивного інтервалу) і діаграмою функціонування, наведеними на рис.1. Наприклад, множина точок циліндра 1 може характеризувати режими функціонування систем навігації, множина точок циліндра 2 – режими функціонування критичних об’єктів тривалого застосування, множина точок циліндра 3 – функціонування невідновлюваних систем [2]. Рис. 1. Умови застосування та режими роботи МСП Параметри Т0, ta, q визначають умови використання методів забезпечення відмовостійкості МСП і накладають обмеження на їх характеристики. Для сучасних МСП реального часу (РЧ) характерними є такі вимоги: − можливість збереження працездатності при їх застосуванні в агресивних середовищах; − можливість обробки даних у реальному часі. Рис. 1. Умови застосування та режими роботи МСП Параметри Т0, ta, q визначають умови використання методів забезпечення відмовостійкості МСП і накладають обмеження на їх характеристики. Для сучасних МСП реального часу (РЧ) характерними є такі вимоги: − можливість збереження працездатності при їх застосуванні в агресивних середовищах; − можливість обробки даних у реальному часі. р − можливість обробки даних у реальному часі. 98 98 • Grundlagen der modernen wissenschaftlichen Forschung Надійність функціонування МСП доцільно оцінювати комплексним показником – коефіцієнтом збереження Кs(t) [2]. Коефіциент Кs(t) визначає відносний об’єм та корисність виконуваних МСП функцій в поточний момент часу у порівнянні с гранично можливими ,)t( W )t( W K 0 s = (1) (1) ,)t( W )t( W K 0 s = де W(t) –продуктивність МСП в поточний час; де РМСП (t) – ймовірність безвідмовної роботи МСП. де РМСП (t) – ймовірність безвідмовної роботи МСП. Розв’язок системи (1) нерівностей визначає умови забезпечення максимальної ефективності функціонування МСП. 31. März, 2023 • Zürich, Schweizerische Eidgenossenschaft 99 Високі вимоги до значень КS(t) і РМСП(t) не можуть бути задоволені при використанні відомих методів забезпечення активної відмовостійкості для МПС з наступних причин [1-3]: − мала кратність ковзного резервування робочих ПМ МСП; − низька ефективність використання частково-працездатних ПМ для забезпечення ефективного функціонування МСП; − низька ефективність використання частково-працездатних ПМ для забезпечення ефективного функціонування МСП; − неможливість використання відомих методів реконфігурації при функціонуванні МСП в реальному масштабі часу; − неможливість використання відомих методів реконфігурації при функціонуванні МСП в реальному масштабі часу; − неможливість повторного використання на активних часових інтервалах функціонування МСП в робочих конфігурацій ПМ, які відновилися після дії збоїв; − неможливість повторного використання на активних часових інтервалах ціонування МСП в робочих конфігурацій ПМ, які відновилися після дії збоїв; − відсутність властивості адаптації структури МСП до особливостей застосування за призначенням (зміни параметрів tа, tп, Т0, інтенсивностей відмов і збоїв, вимог до продуктивності та відмовостійкості). − відсутність властивості адаптації структури МСП до особливостей застосування за призначенням (зміни параметрів tа, tп, Т0, інтенсивностей відмов і збоїв, вимог до продуктивності та відмовостійкості). Забезпечення надійності функціонування матричних спецпроцесорів реального часу повинно досягатися розв’язанням наступних завдань: − розробки і дослідження методів реконфігурації МСП, використання яких дозволить здійснювати реконфігурацію в реальному часі і оптимально витрачати резервні ПМ; − об'єднання частково-працездатні ПМ в еквіваленти справних ПМ з подальшим їх використанням за призначенням; − здійснення перед активними часовими інтервалами застосування МСП деградації або редеградаціі їх структури при збереженні необхідного рівня реконфігуроздатності. − здійснення перед активними часовими інтервалами застосування МСП деградації або редеградаціі їх структури при збереженні необхідного рівня реконфігуроздатності. Список використаних джерел: Список використаних джерел: р р [1] Кун С. Матричные процессоры на СБИС: Пер. с англ. – М.: Мир, 1991. ̶ 672 с. [2] Н. П. Благодарный. Модели эффективности использования однородных процессорных сред. Радіоелектронні і комп'ютерні системи, науково-технічний журнал, 6(47), Харків "ХАІ”, 2010. ̶ С.229-235. [3] V. S. Kharchenko, V.V. Gostishchev, N.P. Blagodarny, V.A. Melnikov A Reconfigurability of Fault-Tolerant Systems: the Measures, Algorithms and Modeling Technique// Успехи современной радиоэлектроники, 2002. №5. С. 62-72.
https://openalex.org/W3119308075	https://hal.inria.fr/hal-03329290/document	English	null	VoxPopuli: A Large-Scale Multilingual Speech Corpus for Representation Learning, Semi-Supervised Learning and Interpretation	null	2,021	cc-by	9,289	To cite this version: Changhan Wang, Morgane Rivière, Ann Lee, Anne Wu, Chaitanya Talnikar, et al.. VoxPopuli: A Large-Scale Multilingual Speech Corpus for Representation Learning, Semi-Supervised Learning and Interpretation. ACL 2021 - 59th Annual Meeting of the Association for Computational Linguistics, Aug 2021, Bangkok, Thailand. 10.18653/v1/2021.acl-long.80. hal-03831929 HAL Id: hal-03831929 https://inria.hal.science/hal-03831929v1 Submitted on 27 Oct 2022 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Abstract research community has been mostly focused on speech-to-text tasks with English as input. While multilingual ASR (Pratap et al., 2020; Ardila et al., 2020) and ST datasets (Wang et al., 2020b; Iranzo- S´anchez et al., 2020) have recently been made avail- able, the amount of data available quickly drops beyond the top few high-resource languages. We introduce VoxPopuli, a large-scale multi- lingual corpus providing 400K hours of un- labeled speech data in 23 languages. It is the largest open data to date for unsuper- vised representation learning as well as semi- supervised learning. VoxPopuli also con- tains 1.8K hours of transcribed speeches in 15 languages and their aligned oral inter- pretations into 15 target languages totaling 17.3K hours. We provide speech recogni- tion (ASR) baselines and validate the versa- tility of VoxPopuli unlabeled data in semi- supervised ASR and speech-to-text transla- tion under challenging out-of-domain settings. The corpus is available at https://github. com/facebookresearch/voxpopuli. Xiv:2101.00390v2 [cs.CL] 27 Jul 2021 Simultaneous speech translation (interpretation) has witnessed a resurgence with the applications of end-to-end encoder-decoder models. Most of the recent studies focus on text output and leverage ST corpora that are translated ofﬂine in the writ- ten form. There are differences, however, between translationese and interpretese (Sridhar et al., 2013; He et al., 2016), where interpreters develop a vari- ety of strategies to improve simultaneity. Models trained on translation corpora are unlikely to learn from these interpretation skills to achieve better quality-latency trade-offs. Finally, there has been little research (Jia et al., 2019; Tjandra et al., 2019; Zhang et al., 2020a) into speech output due to the lack of open data. Existing corpora (Tohyama et al., 2004; Bendazzoli et al., 2005) are either of limited size or no longer publicly available. arXiv:2101.00390v2 [cs.CL] 27 Ju ⋆Equal contribution. 2.1 Data Acquisition VoxPopuli sources data from 2009-2020 European Parliament (EP) event recordings, which include plenary sessions, committee meetings and other events. In each event, speakers give speeches in turn in different European Union (EU) languages. These speeches are partially transcribed (for ple- nary sessions only) and interpreted into 24 EU lan- guages. The interpretations are only oral without any transcription. In the following part, we refer to the original speech as “source speech” and to the interpreted one as “target speech”. We download audio clips for both source and target speeches from the ofﬁcial website1. We also crawl the transcript, speaker information and starting/ending timestamps for each speech (for plenary sessions only) from that source, with which we later align the speech to its transcript and interpretation ut- terance by utterance. The acquired raw data suf- fers from missing audios, incomplete transcripts and inaccurate timestamps. We build data process- ing pipelines to segment speech paragraphs into utterances and ﬁlter out the ones with erroneous transcriptions. Table 1: Statistics for unlabeled (“Unlab.”) and tran- scribed speech data in VoxPopuli: duration in hours (“Hrs”), number of speakers (“Spkrs”), percentage of female speakers (“F%”) and number of tokens (“Tkns”). Durations are calculated on segmented au- dios where leading and trailing silence is trimmed. The LM data is a combination of VoxPopuli transcription and sentences from EuroParl (Koehn, 2005). Table 1: Statistics for unlabeled (“Unlab.”) and tran- scribed speech data in VoxPopuli: duration in hours (“Hrs”), number of speakers (“Spkrs”), percentage of female speakers (“F%”) and number of tokens (“Tkns”). Durations are calculated on segmented au- dios where leading and trailing silence is trimmed. The LM data is a combination of VoxPopuli transcription and sentences from EuroParl (Koehn, 2005). 1 Introduction Transcribed LM Hrs Hrs Spkrs (F%) Tkns Tkns En 24.1K 543 1313 (29.6) 4.8M 60.1M De 23.2K 282 531 (30.6) 2.3M 50.0M Fr 22.8K 211 534 (38.6) 2.1M 58.6M Es 21.4K 166 305 (40.6) 1.6M 57.4M Pl 21.2K 111 282 (23.7) 802K 13.6M It 21.9K 91 306 (33.8) 757K 52.1M Ro 17.9K 89 164 (27.6) 739K 10.3M Hu 17.7K 63 143 (30.3) 431K 13.0M Cs 18.7K 62 138 (24.9) 461K 13.5M Nl 19K 53 221 (39.3) 488K 54.6M Fi 14.2K 27 84 (56.8) 160K 34.5M Hr 8.1K 43 83 (33.1) 337K 285K Sk 12.1K 35 96 (33.8) 270K 13.3M Sl 11.3K 10 45 (43.9) 76K 12.6M Et 10.6K 3 29 (43.7) 18K 11.3M Lt 14.4K 2 21 (14.8) 10K 11.5M Pt 17.5K - - - - Bg 17.6K - - - - El 17.7K - - - - Lv 13.1K - - - - Mt 9.1K - - - - Sv 16.3K - - - - Da 13.6K - - - - All 384K 1791 4295 15M 467M Es, Pl, It, Ro, Hu, Cs, Nl, Fi, Sk, Sl, Lt and Da) totaling 17.3K hours. We describe our corpus creation methodology in Section 2 and analyze the created corpus in Sec- tion 3. We provide ASR baselines and demonstrate the value of our multilingual unlabeled data as well as weakly labeled data on several non-English lan- guages in Section 4. 2.2.2 Transcribed Speech The VoxPopuli transcribed set comes from aligning the full-event source speech audio with the tran- scripts for plenary sessions. Ofﬁcial timestamps are available for locating speeches by speaker in the full session, but they are frequently inaccurate, resulting in truncation of the speech or mixture of fragments from the preceding or the succeed- ing speeches. To calibrate the original timestamps, we perform speaker diarization (SD) on the full- session audio using pyannote.audio (Bredin et al., 2020) and adopt the nearest SD timestamps (by L1 distance to the original ones) instead for seg- mentation. Full-session audios are segmented into speech paragraphs by speaker, each of which has a transcript available. 1https://multimedia.europarl.europa.eu 1https://github.com/amsehili/auditok 1 Introduction Recent progress in speech-to-text tasks such as automatic speech recognition (ASR) and speech translation (ST) has been achieved by the devel- opment and application of unsupervised speech pre-training methods (Oord et al., 2018; Schnei- der et al., 2019; Baevski et al., 2020; Conneau et al., 2020; Wu et al., 2020; Nguyen et al., 2020), with semi-supervised learning (self-training) (Kahn et al., 2020a; Pino et al., 2020; Zhang et al., 2020b; Xu et al., 2020) or a combination of both meth- ods (Xu et al., 2020). This line of research lever- ages large amounts of unlabeled English speech data (Kahn et al., 2020b) that enable improve- ments in English ASR or out-of-English ST. Large amounts of multilingual audio data are needed in order to achieve similar progress for multilingual ASR and ST. Similarly, most ASR and ST research is currently conducted on the LibriSpeech (Panay- otov et al., 2015) and MuST-C benchmarks (Cattoni et al., 2020; Di Gangi et al., 2019). As a result, the In this paper, we introduce VoxPopuli, a large- scale multilingual speech corpus for representation learning, semi-supervised learning and interpreta- tion. It contains the largest open unlabeled speech data to date, totaling 400K hours in 23 languages: Bulgarian (Bg), Czech (Cs), Croatian (Hr), Dan- ish (Da), Dutch (Nl), English (En), Estonian (Et), Finnish (Fi), French (Fr), German (De), Greek (El), Hungarian (Hu), Italian (It), Latvian (Lv), Lithua- nian (Lt), Maltese (Mt), Polish (Pl), Portuguese (Pt), Romanian (Ro), Slovak (Sk), Slovene (Sl), Spanish (Es) and Swedish (Sv). VoxPopuli also provides a total of 1.8K hours of transcribed speech in 16 languages (En, De, Fr, Es, Pl, It, Ro, Hu, Cs, Nl, Fi, Hr, Sk, Sl, Et and Lt) and their aligned oral interpretations into 15 target languages (En, De, Fr, Unlab. 2.2.1 Unlabeled Speech We construct VoxPopuli unlabeled set from all source and target speeches in 23 EU languages (excluding Irish because of very limited data avail- ability). We segment full-event audios into short clips of 15-30 seconds using an energy-based voice activity detection (VAD) algorithm1. Each audio clip has a maximum of 2 seconds of continuous silence, and silent clips are discarded. Around 16% of the data is dropped after silence removal, which leads to a ﬁnal overall duration of around 400K hours. The speech paragraphs have an average dura- tion of 197 seconds, which leads to signiﬁcant memory usage and prevents efﬁcient parallelism (batching) during model training. We hence further segment these paragraphs into utterances with a maximum duration of 20 seconds. 2.2.1 Unlabeled Speech We leverage Source Target (Oral Interpretation) En De Fr Es Pl It Ro Hu Cs Nl Fi Sk Sl Lt Da Total En - 463 427 441 432 461 457 382 427 400 442 433 434 398 370 6.0K De 187 - 196 204 214 217 198 205 214 196 217 208 218 164 179 2.8K Fr 169 187 - 187 172 197 195 144 170 158 168 168 156 139 134 2.3K Es 130 138 135 - 118 148 128 93 118 115 124 114 108 83 86 1.6K Pl 68 66 54 55 - 67 55 43 67 42 55 62 57 50 34 775 It 69 77 76 79 72 - 75 61 68 64 71 66 70 53 60 961 Ro 60 59 59 58 49 61 - 38 50 43 48 50 46 38 29 688 Hu 30 38 25 27 29 30 27 - 27 20 31 29 26 21 18 378 Cs 39 35 29 30 36 32 31 23 - 23 29 55 29 25 18 434 Nl 31 43 35 29 27 38 24 25 25 - 32 25 23 19 25 401 Fi 15 18 15 13 13 13 13 12 13 11 - 14 12 11 9 182 Hr 31 27 27 24 27 28 24 22 24 22 24 26 37 21 20 384 Sk 21 22 14 16 19 16 16 14 32 13 16 - 17 13 10 239 Sl 6 6 4 5 5 6 5 4 5 4 5 6 - 4 3 68 Lt 1 1 1 1 1 1 1 1 1 1 1 1 1 - 0 13 Total 857 1.2K 1.1K 1.2K 1.2K 1.3K 1.2K 1.1K 1.2K 1.1K 1.3K 1.3K 1.2K 1.0K 995 17.3K Table 2: Duration statistics (hours) of aligned speech-to-speech data in VoxPopuli between 15 source languages and 15 target languages. strategy is to align source and target at the sentence level using ASR. speech recognition (ASR) systems to force-align speech paragraphs to the given transcripts and cut the utterances by ending punctuation or the longest silence inside the sentence if it exceeds 20 seconds. The ASR systems are TDS models (Hannun et al., 2019) trained with ASG criterion (Collobert et al., 2016) on audio tracks from in-house de-identiﬁed video data. The resulting utterance segments may have incorrect transcriptions due to incomplete raw transcripts or inaccurate ASR force-alignment. 2.2.1 Unlabeled Speech We use the predictions from the same ASR systems as references and ﬁlter the candidate segments by a maximum threshold of 20% character error rate (CER). We ﬁrst compare the spectrogram of the source and the target speech to remove the identical parts and segment the target speech into paragraphs. These identical speech are due to either the short delay between the time the source speaker and the interpreter started, or the fact that the source lan- guage is the same as the target one, and thus no interpretation is needed. For long target paragraphs, we further segment them by silence into audio clips of at most 15 minutes long. We use the same ASR model described in Section 2.2.2 and a language model (Section 2.2.4) to decode the segmented tar- get audio. The decoded text is also forced aligned with the target audio, so that we have the times- tamps of every decoded word. We split the ﬁltered utterances into train, devel- opment and test sets with disjoint speakers and target duration ratio (18:1:1). To determine the assignments, we group utterances by speaker and sort them by overall duration in ascending order. We assign the sorted groups to the test set in or- der until it reaches 20 speakers or the target dura- tion (whichever comes later). The same process is repeated on the remaining utterance groups to construct the development set (with minimum 10 speakers instead). Finally, the rest of utterances make up the train set. This approach ensures higher speaker diversity in the test and development sets. For each source segment produced in Sec- tion 2.2.2, we locate all decoded words that are within a window of ﬁve seconds to its start and end. A set of candidate target segments can be generated from all possible combinations of the starting and ending decoded words. We compute the cosine similarity between the LASER represen- tation (Artetxe and Schwenk, 2019) of the source text and each decoded text in the candidate set to ﬁnd the best target segment, i.e. the one with the highest score. We ﬁrst carry out this process for all source segments, respectively, and then ﬁnetune the boundaries of overlapping target segments for consecutive source segments. Finally, a threshold of 0.75 is applied on the similarity score to ﬁlter out low-quality alignments, which can be due to ASR errors. In addition to ASR output, we also collect human transcription on 400 hours of English target speech. The human annotators were asked to provide times- tamps for each word while transcribing, and thus we can apply the same alignment process described above on human transcription and generate a set of ground truth speech-to-speech alignment data. Speech-to-speech alignment The statistics of the speech-to-speech alignment between all source languages and 15 target languages are shown in Table 2. Compared with the total amount of data available for each source language (“Transcribed hours” in Table 1), we obtain target alignments for more than 70% of the source sentences in En, De, Fr, Es and It, more than 50% for Pl, Ro, Cs, Nl and Hr, and the rest has at least 40% of source seg- ments aligned. To examine the quality of our ASR system, we align the ASR output with the human transcription we collect on English target speech and see a word error rate (WER) of 31.7. With the human transcription, we can produce ground truth speech-to-speech alignment data that is 1.1 times larger than the size of the alignment data cre- ated from using ASR output, indicating that around 12% of the low-quality alignments are ﬁltered due to ASR errors. If we compare the ASR-based and the ground truth alignment data, there is on average a 0.75-second shift in the target segment bound- aries. As a by-product from this alignment process, source text and target speech is aligned, which pro- vides speech-to-text “translation” data in the re- versed direction. This data is weakly labeled—the label (text) may contain more information than the speech data (interpretation is likely to drop unim- portant details) and hence is not exact. However, it is still useful for ST model training as an addition to labeled data. 2.2.3 Speech-To-Speech Alignment Even though every source speech is associated with corresponding simultaneous interpretations in tar- get languages, considerable preprocessing and ﬁl- tering is necessary to make this dataset usable. Our Original (French) Vous le savez tous, la forˆet recule. Toutes les deux secondes dans le monde, c’est l’´equivalent d’un terrain de football qui est d´etruit, c’est en un an l’´equivalent du terri- toire de la Gr`ece qui est d´eforest´e et c’est ´evidemment dramatique. Trans- lation As you all know, the forest is receding. Every two seconds, across the world, the equivalent of a football pitch is destroyed; within a year, an area the size of Greece is deforested. Clearly, this is a tragic situation. Inter- pretation You all know that we are losing forests ev- ery second, the surface the size area of a football ﬁeld is lost in the forest. This is really tragic. Table 3: An example from VoxPopuli for interpretese vs. translationese. Translationese is verbatim and exact, while interpretese tends to be more general and summa- rizing with unimportant details dropped. 3 Data Analysis Unlabeled speech As we can see from Table 1, VoxPopuli has a total of 400K hours of unlabeled data well-distributed across 23 EU languages, re- sulting in 8K-24K hours of data for each language. This ensures adequate data on languages with lower ASR resource, which are likely to beneﬁt more from semi-supervised learning. It also facilitates multilingual model training since there is not much data imbalance and little need for tuning data sam- pling strategy. Transcribed speech The VoxPopuli transcribed data contains 16 languages totaling 1.8K hours and 4.3K speakers, whose detailed statistics can be found in Table 1, including duration (hours) by language, number of speakers, percentage of fe- male speakers and number of tokens. The data distribution is imbalanced and reﬂects the natural distribution of the number of native speakers. The remaining 7 languages (Pt, Bg, El, Lv, Mt, Sv and Da) are not covered due to either limited data vol- ume or the availability of processing pipelines. Table 3: An example from VoxPopuli for interpretese vs. translationese. Translationese is verbatim and exact, while interpretese tends to be more general and summa- rizing with unimportant details dropped. Table 3: An example from VoxPopuli for interpretese vs. translationese. Translationese is verbatim and exact, while interpretese tends to be more general and summa- rizing with unimportant details dropped. 4.1 Experimental Setup For representation learning, we perform speaker diarization before VAD-based segmentation so that each utterance contains exactly one speaker. We augment the data with time dropout, pitch modiﬁ- cation and reverberation (Kharitonov et al., 2020) during model training. For non-wav2vec models, we extract 80- dimensional log-mel ﬁlterbank speech features with 25ms windows size and 10ms shift. We apply per-utterance CMVN (cepstral mean and variance normalization) to the extracted features. For GPU memory efﬁciency, we remove training samples that have more than 60 seconds of speech or have more than 1024 characters. Table 5: Phoneme discriminability of unsupervised features across languages. We report ABX discrim- inability score on the 10s test set from ZeroSpeech 2017† for English (“En”), French (“Fr”) and Man- darin (“Zh”). We compare our models with the MFCC baseline, the supervised topline and the state-of-the- art monolingual (English) model‡. We measure the generality of the representations by standard deviation (“Std.”) of the scores across the 3 languages. We see that multilingual representations generalize better and are more robust on unseen languages. † Dunbar et al. (2017). ‡ Riviere and Dupoux (2020). Table 5: Phoneme discriminability of unsupervised features across languages. We report ABX discrim- inability score on the 10s test set from ZeroSpeech 2017† for English (“En”), French (“Fr”) and Man- darin (“Zh”). We compare our models with the MFCC baseline, the supervised topline and the state-of-the- art monolingual (English) model‡. We measure the generality of the representations by standard deviation (“Std.”) of the scores across the 3 languages. We see that multilingual representations generalize better and are more robust on unseen languages. † Dunbar et al. (2017). ‡ Riviere and Dupoux (2020). We train wav2vec 2.0 (Baevski et al., 2020) mod- els with original hyper-parameter settings using fairseq (Ott et al., 2019), except for Table 7 where we use wav2letter (Pratap et al., 2018) and fol- low Talnikar et al. (2020) to do ﬁnetuning using both supervised CTC (Graves et al., 2006) loss and unsupervised wav2vec 2.0 loss. The largest model (“VP-100K”) takes 10 days on 128 V100 GPUs for 1M updates. For non-wav2vec models, we train Transformer (Vaswani et al., 2017) with cross-entropy criterion using fairseq S2T (Wang et al., 2020a). For Section 4.2 and Section 4.4.1, we use phoneme vocabularies for models that we evaluate with PER (phone error rate) and character vocabularies for the other. 4.1 Experimental Setup For Section 4.4.2, we use Unigram (Kudo and Richardson, 2018) vocabu- laries with 2K subwords for all models. To improve ST model training, we pre-train the encoder on the LibriSpeech (Panayotov et al., 2015) ASR task. tends to be more general and summarizing with unimportant details dropped. Human interpreters regularly apply these tactics to make better quality- latency trade-offs. Speech-to-speech translation models may beneﬁt from these tactics if they are trained on interpretation data that VoxPopuli pro- vides. 2.2.4 Language Modeling Data We measure the generality of the representations by standard deviation (“Std.”) of the scores across the 3 languages. We see that multilingual representations generalize better and are more robust on unseen languages. † Dunbar et al. (2017). ‡ Riviere and Dupoux (2020). 2.2.4 Language Modeling Data Bottom: wav2vec 2.0 Base model pre-trained on 10K-hour VoxPopuli unlabeled data (23 languages) and ﬁne-tuned on Vox- Populi ASR data. As we can see, pre-training with in-domain unlabeled data substantially improves performance especially for low-resource languages. Table 4: VoxPopuli ASR baselines and in-domain unsupervised pre-training. We report VoxPopuli dev and test WER for languages with ≥10 hours of data. Top: supervised monolingual Transformer baselines. Bottom: wav2vec 2.0 Base model pre-trained on 10K-hour VoxPopuli unlabeled data (23 languages) and ﬁne-tuned on Vox- Populi ASR data. As we can see, pre-training with in-domain unlabeled data substantially improves performance especially for low-resource languages. Table 4: VoxPopuli ASR baselines and in-domain unsupervised pre-training. We report VoxPopuli dev and test WER for languages with ≥10 hours of data. Top: supervised monolingual Transformer baselines. Bottom: wav2vec 2.0 Base model pre-trained on 10K-hour VoxPopuli unlabeled data (23 languages) and ﬁne-tuned on Vox- Populi ASR data. As we can see, pre-training with in-domain unlabeled data substantially improves performance especially for low-resource languages. Within/Across Speaker ↓ En Fr Zh Std. ↓ MFCC 12.1/23.4 12.6/25.5 11.5/21.3 - Sup.† 6.2/8.0 8.7/10.8 7.9/10.3 - LL-6K‡ 4.5/6.2 8.4/12.7 8.2/8.2 1.8/2.7 VoxPopuli En-500 6.9/9.9 9.6/14.5 8.7/9.7 1.1/2.2 Fr-500 8.1/12.1 9.1/13.8 9.2/10.1 0.5/1.5 En+Fr-500 6.9/9.8 9.0/13.1 8.6/9.6 0.9/1.6 Table 5: Phoneme discriminability of unsupervised features across languages. We report ABX discrim- inability score on the 10s test set from ZeroSpeech 2017† for English (“En”), French (“Fr”) and Man- darin (“Zh”). We compare our models with the MFCC baseline, the supervised topline and the state-of-the- art monolingual (English) model‡. We measure the generality of the representations by standard deviation (“Std.”) of the scores across the 3 languages. We see that multilingual representations generalize better and are more robust on unseen languages. † Dunbar et al. (2017). ‡ Riviere and Dupoux (2020). Within/Across Speaker ↓ En Fr Zh Std. ↓ MFCC 12.1/23.4 12.6/25.5 11.5/21.3 - Sup.† 6.2/8.0 8.7/10.8 7.9/10.3 - LL-6K‡ 4.5/6.2 8.4/12.7 8.2/8.2 1.8/2.7 VoxPopuli En-500 6.9/9.9 9.6/14.5 8.7/9.7 1.1/2.2 Fr-500 8.1/12.1 9.1/13.8 9.2/10.1 0.5/1.5 En+Fr-500 6.9/9.8 9.0/13.1 8.6/9.6 0.9/1.6 Table 5: Phoneme discriminability of unsupervised features across languages. We report ABX discrim- inability score on the 10s test set from ZeroSpeech 2017† for English (“En”), French (“Fr”) and Man- darin (“Zh”). We compare our models with the MFCC baseline, the supervised topline and the state-of-the- art monolingual (English) model‡. 2.2.4 Language Modeling Data To train language models (LM) for ASR decoding, we combine VoxPopuli transcription in the training set with the EuroParl corpus (Koehn, 2005), which is from the proceedings of the European Parlia- ment from 1996 to 2011. To process the EuroParl data, we ﬁrst apply the sentence segmentation tool provided with the corpus. We remove all texts in the parentheses, replace hyphens and slashes with space, and remove all other punctuation except apostrophes. All digits are converted into words, and all texts are normalized into lowercase. Table 1 shows the statistics of the LM data. Interpretese vs. translationese We exemplify the differences between simultaneous oral interpre- tation and ofﬂine written translation using VoxPop- uli in Table 3. The latter is verbatim and exact compared to the original speech, while the former En De It Fr Es Pl Ro Hu Nl Cs Sl Fi Hr Sk Avg. ↓ Sup. Dev 30.1 29.0 41.6 28.6 27.4 27.1 28.5 27.4 35.7 27.8 95.7 45.7 44.9 30.2 37.1 baseline Test 30.0 29.3 45.2 30.5 31.4 25.6 27.7 27.9 38.3 27.7 96.5 41.6 40.2 32.7 37.5 VP-10K Dev 15.5 17.2 19.1 13.9 8.6 12.8 8.3 11.5 18.5 11.1 20.6 21.1 15.6 10.4 14.6 + FT Test 16.2 16.2 21.5 15.4 11.0 12.5 9.4 12.0 19.7 11.8 26.1 17.1 14.1 11.1 15.3 Table 4: VoxPopuli ASR baselines and in-domain unsupervised pre-training. We report VoxPopuli dev and test WER for languages with ≥10 hours of data. Top: supervised monolingual Transformer baselines. Bottom: wav2vec 2.0 Base model pre-trained on 10K-hour VoxPopuli unlabeled data (23 languages) and ﬁne-tuned on Vox- Populi ASR data. As we can see, pre-training with in-domain unlabeled data substantially improves performance especially for low-resource languages. En De It Fr Es Pl Ro Hu Nl Cs Sl Fi Hr Sk Avg. ↓ Sup. Dev 30.1 29.0 41.6 28.6 27.4 27.1 28.5 27.4 35.7 27.8 95.7 45.7 44.9 30.2 37.1 baseline Test 30.0 29.3 45.2 30.5 31.4 25.6 27.7 27.9 38.3 27.7 96.5 41.6 40.2 32.7 37.5 VP-10K Dev 15.5 17.2 19.1 13.9 8.6 12.8 8.3 11.5 18.5 11.1 20.6 21.1 15.6 10.4 14.6 + FT Test 16.2 16.2 21.5 15.4 11.0 12.5 9.4 12.0 19.7 11.8 26.1 17.1 14.1 11.1 15.3 Table 4: VoxPopuli ASR baselines and in-domain unsupervised pre-training. We report VoxPopuli dev and test WER for languages with ≥10 hours of data. Top: supervised monolingual Transformer baselines. 4 Experiments & Results We provide VoxPopuli ASR baselines and vali- date the versatility of VoxPopuli unlabeled data in unsupervised representation learning and semi- supervised learning for ASR as well as ST. We also evaluate the quality of speech-to-speech align- ment indirectly via the weakly labeled ST data it produces. We use the best checkpoint by validation loss for evaluation, except for Section 4.4.2 where we average the 10 best checkpoints. We build n-gram language models for decoding (when speciﬁed) us- ing KenLM (Heaﬁeld, 2011). PT PT Langs. PER ↓(VoxPopuli Langs.) PER ↓(Other Langs.) PER Domain Hours In Out Es Fr It Nl Sv Ky Ru Tr Tt Zh Avg. ↓Std. ↓ m-CPC† Out 60K 0 1 36.4 44.3 37.8 43.1 46.5 37.5 42.4 45.7 40.6 53.2 42.7 4.8 wav2vec 2.0 Base (95M) XLSR-Mono‡ In <0.4K 1 0 6.8 10.4 10.9 37.4 63.6 29.6 11.6 44.0 21.4 31.4 26.7 17.2 XLSR-10‡ In 1.4K 10 1 9.4 13.4 13.8 16.3 21.0 8.6 11.2 11.7 8.3 24.5 13.8 5.1 VP-Mono-5K Out 4.5K 1 0 6.8 8.6 7.5 9.7 9.3 - - - - - - - VP-10K Out 10K 5 18 8.5 11.9 11.0 13.6 15.0 10.9 12.4 13.1 8.8 19.3 12.5 3.0 VP-100K Out 100K 5 18 7.6 10.3 9.7 12.2 13.0 9.4 10.7 11.7 8.0 17.5 11.0 2.7 wav2vec 2.0 Large (317M) XLSR-10‡ In 1.4K 10 1 7.9 12.6 11.7 14.0 20.6 7.0 9.3 9.7 7.2 22.8 12.3 5.2 XLSR-53‡ In+Out 56K 10 43 2.9 5.0 6.7 5.8 12.2 6.1 8.1 7.1 5.1 18.3 7.6 4.2 VP-Mono-5K Out 4.5K 1 0 5.5 7.0 6.1 7.2 6.3 - - - - - - - VP-10K Out 10K 5 18 6.3 8.9 7.9 9.3 9.7 9.3 9.2 11.3 7.6 18.8 9.8 3.2 VP-100K Out 100K 5 18 5.4 7.7 6.5 8.0 8.3 8.5 8.0 9.8 6.9 17.3 8.6 3.1 Table 6: Few-shot ASR with out-of-domain out-of-language unsupervised pre-training. We adopt the Com- mon Voice (CV) few-shot phoneme recognition setup† and report test PER (phone error rate). Our wav2vec 2.0 models are pre-trained on VoxPopuli (out-of-CV-domain) either with 4.5K-hour monolingual data (“VP-Mono- 5K”) or 10K-hour/100K-hour multilingual data (“VP-10K” and “VP-100K”). Pre-training languages may include the ones being evaluated (“In”) and others (“Out”). Our models outperform XLSR-Mono and XLSR-10 (same architecture as ours but using in-domain CV data) on most languages with out-of-domain and (partially) out-of- language pre-training. 4 Experiments & Results (2020) Train Hours Test WER ↓ De Fr Es De Fr Es Baseline† 1582 787 660 12.8 19.4 16.5 VP-50K 314 364 203 17.0 18.8 11.9 + LM (20%) (46%) (31%) 7.8 9.6 10.0 Table 7: ASR with out-of-domain unsupervised pre- training and less supervision. We report test WER on Common Voice (CV). Top: supervised baseline trained on the combination of an extended CV train set and sev- eral other corpora (decoding with LM). Bottom: our wav2vec 2.0 Base model pre-trained on 50K-hour Vox- Populi data (out-of-CV-domain) and ﬁne-tuned on the standard CV train set (a subset of the baseline’s one). We optionally use 4-gram LMs trained on CV for de- coding. Our model outperforms the baseline (even without LM) while using less supervised train data. †Deepspeech Polyglot. Train Hours Test WER ↓ De Fr Es De Fr Es Baseline† 1582 787 660 12.8 19.4 16.5 VP-50K 314 364 203 17.0 18.8 11.9 + LM (20%) (46%) (31%) 7.8 9.6 10.0 4.3 Unsupervised Representation Learning We follow the setting in Rivi`ere et al. (2020) to evaluate unsupervised speech representations by phoneme discriminability on 3 languages (English, French and Mandarin), and report ABX discrim- inability score (Schatz et al., 2013) on the 10s test set from ZeroSpeech 2017 (Dunbar et al., 2017). Standard deviation (“Std.”) of the scores across the 3 languages is also reported as a measure for the generality of the representations. As previous studies focus on monolingual representations, we explore multilingual representations and examine their generality across languages. We train CPC- based models (Riviere and Dupoux, 2020) on 500- hour English and 500-hour French unlabeled data from VoxPopuli, respectively. And we combine En- glish and French data with 50% sampling (so that the total duration remains the same) for the multi- lingual setting. We observe from Table 5 that the multilingual model (“En+Fr-500”) performs com- parably to the monolingual ones (“En-500” and “Fr-500”) on their seen languages and performs bet- ter on unseen language (“Zh”). Its scores vary less across languages (lower “Std.”) compared to “En- 500”. The variance of the scores is comparable to “Fr-500” while the average is lower. We conclude that multilingual representations generalize better across languages and are more robust on unseen Table 7: ASR with out-of-domain unsupervised pre- training and less supervision. We report test WER on Common Voice (CV). Top: supervised baseline trained on the combination of an extended CV train set and sev- eral other corpora (decoding with LM). Bottom: our wav2vec 2.0 Base model pre-trained on 50K-hour Vox- Populi data (out-of-CV-domain) and ﬁne-tuned on the standard CV train set (a subset of the baseline’s one). We optionally use 4-gram LMs trained on CV for de- coding. Our model outperforms the baseline (even without LM) while using less supervised train data. †Deepspeech Polyglot. 4 Experiments & Results Our best model (VP-100K Large) performs competitively to XLSR-53, which leverages 52K-hour out-of-CV-domain data in addition to the CV data. † Rivi`ere et al. (2020) ‡ Conneau et al. (2020) PT PT Langs. PER ↓(VoxPopuli Langs.) PER ↓(Other Langs.) PER Domain Hours In Out Es Fr It Nl Sv Ky Ru Tr Tt Zh Avg. ↓Std. ↓ m-CPC† Out 60K 0 1 36.4 44.3 37.8 43.1 46.5 37.5 42.4 45.7 40.6 53.2 42.7 4.8 wav2vec 2.0 Base (95M) XLSR-Mono‡ In <0.4K 1 0 6.8 10.4 10.9 37.4 63.6 29.6 11.6 44.0 21.4 31.4 26.7 17.2 XLSR-10‡ In 1.4K 10 1 9.4 13.4 13.8 16.3 21.0 8.6 11.2 11.7 8.3 24.5 13.8 5.1 VP-Mono-5K Out 4.5K 1 0 6.8 8.6 7.5 9.7 9.3 - - - - - - - VP-10K Out 10K 5 18 8.5 11.9 11.0 13.6 15.0 10.9 12.4 13.1 8.8 19.3 12.5 3.0 VP-100K Out 100K 5 18 7.6 10.3 9.7 12.2 13.0 9.4 10.7 11.7 8.0 17.5 11.0 2.7 wav2vec 2.0 Large (317M) XLSR-10‡ In 1.4K 10 1 7.9 12.6 11.7 14.0 20.6 7.0 9.3 9.7 7.2 22.8 12.3 5.2 XLSR-53‡ In+Out 56K 10 43 2.9 5.0 6.7 5.8 12.2 6.1 8.1 7.1 5.1 18.3 7.6 4.2 VP-Mono-5K Out 4.5K 1 0 5.5 7.0 6.1 7.2 6.3 - - - - - - - VP-10K Out 10K 5 18 6.3 8.9 7.9 9.3 9.7 9.3 9.2 11.3 7.6 18.8 9.8 3.2 VP-100K Out 100K 5 18 5.4 7.7 6.5 8.0 8.3 8.5 8.0 9.8 6.9 17.3 8.6 3.1 Table 6: Few-shot ASR with out-of-domain out-of-language unsupervised pre-training. We adopt the Com- mon Voice (CV) few-shot phoneme recognition setup† and report test PER (phone error rate). Our wav2vec 2.0 models are pre-trained on VoxPopuli (out-of-CV-domain) either with 4.5K-hour monolingual data (“VP-Mono- 5K”) or 10K-hour/100K-hour multilingual data (“VP-10K” and “VP-100K”). Pre-training languages may include the ones being evaluated (“In”) and others (“Out”). Our models outperform XLSR-Mono and XLSR-10 (same architecture as ours but using in-domain CV data) on most languages with out-of-domain and (partially) out-of- language pre-training. Our best model (VP-100K Large) performs competitively to XLSR-53, which leverages 52K-hour out-of-CV-domain data in addition to the CV data. † Rivi`ere et al. (2020) ‡ Conneau et al. 4.2 Speech Recognition (ASR) Baselines Fr→En ↑ Es→En ↑ De→En ↑ Fr ↓ Es ↓ De ↓ Train hours (EP+CV) 38+264 32+113 42+184 38+264 32+113 42+184 Test set EP CV EP CV EP CV EP CV EP CV EP CV (Cascaded) Baseline† 25.4 27.6 26.5 27.4 21.3 21.0 24.3 18.3 15.0 21.4 19.8 16.0 Our end-to-end baseline 24.5 27.0 20.5 26.6 17.5 20.0 20.8 18.8 17.2 14.1 23.2 18.4 With 800h self-training 26.7 28.6 22.4 26.8 18.8 20.1 19.5 17.3 15.6 13.7 21.8 17.5 With 3000h self-training 27.4 28.9 22.7 27.3 19.6 20.0 19.0 17.0 15.3 13.2 21.4 17.3 400h weakly labeled 22.9 10.1 22.2 10.9 18.0 8.8 + labeled 31.1 30.3 28.4 29.7 24.4 23.4 Table 8: ST and ASR using VoxPopuli data for self-training or weak supervision. Left: test BLEU for ST models. Right: test WER for ASR models. We evaluate in-VoxPopuli-domain performance with EuroParl-ST (EP) and the out-of-domain performance with CoVoST 2 (CV). We combine both corpora to train our baseline and pseudo-label 3K-hour monolingual VoxPopuli unlabeled data for self-training. For ST training with weak supervision, we combine EP, CV and 300h weakly labeled data from VoxPopuli. Both approaches for leveraging VoxPopuli data improve in-domain (EP) and out-of-domain (CV) performance simultaneously. † EP baselines from Iranzo-S´anchez et al. (2020) and CV baselines from Wang et al. (2020b). Table 8: ST and ASR using VoxPopuli data for self-training or weak supervision. Left: test BLEU for ST models. Right: test WER for ASR models. We evaluate in-VoxPopuli-domain performance with EuroParl-ST (EP) and the out-of-domain performance with CoVoST 2 (CV). We combine both corpora to train our baseline and pseudo-label 3K-hour monolingual VoxPopuli unlabeled data for self-training. For ST training with weak supervision, we combine EP, CV and 300h weakly labeled data from VoxPopuli. Both approaches for leveraging VoxPopuli data improve in-domain (EP) and out-of-domain (CV) performance simultaneously. † EP baselines from Iranzo-S´anchez et al. (2020) and CV baselines from Wang et al. (2020b). on the full 400K-hour data to future work, which is supposed to achieve even better performance. languages. For quick exploration, we leverage only part of the VoxPopuli unlabeled data and leave the validation on more data to future work. In-domain pre-training We examine the con- ventional in-domain pre-training setting on the Vox- Populi ASR benchmark. We evaluate the VP-10K model, where the pre-training data is ﬁltered so that it has no overlaps with the transcribed development and test set. 4.2 Speech Recognition (ASR) Baselines From table 4, we see that pre-training using unlabeled data brings signiﬁcant gains to all the languages (average 59% test WER reduction). The gains are most signiﬁcant on the low-resource languages, where improvements are qualitative (for example, from nearly 100% test WER on Sl down to around 30%). 4.4 Semi-Supervised Learning We explore two semi-supervised learning settings for the application of VoxPopuli unlabeled data: unsupervised pre-training followed by supervised ﬁne-tuning for ASR and self-training for ASR as well as ST. 1wav2vec 2.0 Base (95M) unless speciﬁed otherwise. 4.2 Speech Recognition (ASR) Baselines We provide monolingual Transformer baselines for the 14 languages that have more than 10 hours of transcribed data (see Table 1). Both development and test WER are reported in Table 4. We see that several low-resource languages (Fi, It, Hr, Sk and Sl) suffer from high recognition errors (>40% WER) due to the lack of training data. Even the highest resource one (En) has a high WER of around 30%. Fr→En ↑ Es→En ↑ De→En ↑ Fr ↓ Es ↓ De ↓ Train hours (EP+CV) 38+264 32+113 42+184 38+264 32+113 42+184 Test set EP CV EP CV EP CV EP CV EP CV EP CV (Cascaded) Baseline† 25.4 27.6 26.5 27.4 21.3 21.0 24.3 18.3 15.0 21.4 19.8 16.0 Our end-to-end baseline 24.5 27.0 20.5 26.6 17.5 20.0 20.8 18.8 17.2 14.1 23.2 18.4 With 800h self-training 26.7 28.6 22.4 26.8 18.8 20.1 19.5 17.3 15.6 13.7 21.8 17.5 With 3000h self-training 27.4 28.9 22.7 27.3 19.6 20.0 19.0 17.0 15.3 13.2 21.4 17.3 400h weakly labeled 22.9 10.1 22.2 10.9 18.0 8.8 + labeled 31.1 30.3 28.4 29.7 24.4 23.4 Table 8: ST and ASR using VoxPopuli data for self-training or weak supervision. Left: test BLEU for ST models. Right: test WER for ASR models. We evaluate in-VoxPopuli-domain performance with EuroParl-ST (EP) and the out-of-domain performance with CoVoST 2 (CV). We combine both corpora to train our baseline and pseudo-label 3K-hour monolingual VoxPopuli unlabeled data for self-training. For ST training with weak supervision, we combine EP, CV and 300h weakly labeled data from VoxPopuli. Both approaches for leveraging VoxPopuli data improve in-domain (EP) and out-of-domain (CV) performance simultaneously. † EP baselines from Iranzo-S´anchez et al. (2020) and CV baselines from Wang et al. (2020b). 4.4.1 ASR with Unsupervised Pre-Training 4.4.1 ASR with Unsupervised Pre Training Self-supervised (unsupervised) pre-training such as wav2vec 2.0 (Baevski et al., 2020) substan- tially reduces the need of labeled data in ASR. Furthermore, multilingual pre-training (Conneau et al., 2020) allows cross-lingual transfer, which brings extra gains especially to low-resource lan- guages. Pre-training wav2vec 2.0 models is, how- ever, resource-intensive and hence re-training mod- els for each task with different domains is imprac- tical. With the large-scale multilingual data in VoxPopuli, we explore if scaling multilingual pre- training can take us towards the one-model-ﬁts-all paradigm by alleviating the impacts of domain or language mismatch between pre-training and ﬁne- tuning. We train wav2vec 2.0 models 1 on 10K- hour, 50K-hour and 100K-hour VoxPopuli data in 23 languages (denoted as “VP-10K”, “VP-50K” and “VP-100K”, respectively). We also train mod- els with 4.5K-hour monolingual data (denoted as “VP-Mono-5K”) for comparison. For quick veriﬁ- cation, we use only part of the VoxPopuli unlabeled data for pre-training. We leave training the models Out-of-domain pre-training We examine the out-of-domain pre-training setting using the Com- mon Voice (CV) ASR corpus (Ardila et al., 2020). In contrast with the political domain oral speech in VoxPopuli, they are more ﬂuent read speech of no copyright sentences (for example, Wikipedia articles). We adopt the few-shot phoneme recog- nition setup on CV v3 from Rivi`ere et al. (2020), with which domain adaptation is limited during ﬁne-tuning due to the small data volume — it has 1-hour train set, 20-minute development set and 1-hour test set for 10 languages including 5 VoxPopuli ones. We present the performance of VP-Mono-5K, VP-10K and VP-100K with the m- CPC (Rivi`ere et al., 2020) and XLSR (Conneau et al., 2020) baselines in Table 6, where phone error rate (PER) is reported. The XLSR baselines share the same wav2vec 2.0 architecture as our models but are trained with in-domain CV data. VP-Mono- 5K outperforms XLSR-Mono and XLSR-10 on all 5 VoxPopuli languages (except for a tie on Es with XLSR-Mono). VP-100K outperforms XLSR-10 on 8 (9) out of the 10 languages. VP-100K (Large) overall performs competitively to XLSR-53, which leverages 52K-hour out-of-domain data in addition to the in-domain CV data. Notably, it outperforms XLSR-53 on Zh, which is covered by XLSR-53 but remote from the EU languages in VP-100K. This suggests the high generality of the speech represen- tations VP-100K learned. 5 VoxPopuli languages (except for a tie on Es with XLSR-Mono). 1https://www.caito.de/2019/01/the-m-ailabs-speech- dataset 1http://www.voxforge.org 4.4.1 ASR with Unsupervised Pre-Training VP-100K outperforms XLSR-10 on 8 (9) out of the 10 languages. VP-100K (Large) overall performs competitively to XLSR-53, which leverages 52K-hour out-of-domain data in addition to the in-domain CV data. Notably, it outperforms XLSR-53 on Zh, which is covered by XLSR-53 but remote from the EU languages in VP-100K. This suggests the high generality of the speech represen- tations VP-100K learned. data in self-training so that it has the same duration as the unlabeled one. We observe from Table 8 that self-training on VoxPopuli improves both in- domain (“EP”) and out-of-domain (“CV”) perfor- mance with similar magnitude most of the time. For ST, self-training helps to narrow the gap be- tween end-to-end models and the cascaded ones (more labeled data available) without the addition of expensive labeled data. 4.5 Weakly Supervised ST We also evaluate our multilingual model (VP- 50K) under the normal setup (CV v5.1) and report test WER in Table 7. They are compared with supervised baselines from DeepSpeech-Polyglot1, which leverage extended CV train sets and several other corpora for training as well as LM for de- coding. Our model outperforms the baseline with ﬁne-tuning on the standard CV train set (a subset of the baseline’s one), even when not using LM in decoding. We evaluate the quality of the weakly labeled ST data from our speech-to-speech alignment on the same benchmark as the self-training experiments. This also provides an indirect evaluation for our alignment pipeline since imprecise alignments hurt the ST label quality. We examine the performance of weakly supervised training as well as joint train- ing using both labeled and weakly labeled data. We see from Table 8 that the former is on par with (or better than) the supervised baseline in the VoxPop- uli domain (“EP”) with 0.3x-1.8x more training data than the baseline. Joint training brings sub- stantial gains to both in-domain (“EP”) and out-of- domain (“CV”) performance, and it outperforms self-training. This suggests that our weakly labeled data (0.4K hours) is much more informative and efﬁcient than the pseudo-labeled data (3K hours) when combined with labeled data. Out-of-language pre-training In the few-shot phoneme recognition setup (Table 6), VP-100K does not cover 5 of the 10 CV languages (Ky, Ru, Tr, Tt and Zh) in pre-training, but leverages data from 18 additional EU languages. It outperforms the in-domain in-language XLSR baselines on most of the uncovered languages (except Ky which is a remote central Asian language). Moreover, it per- forms more stably across all the 10 languages with a smaller variance (standard deviation) on PER. 1https://gitlab.com/Jaco-Assistant/deepspeech-polyglot 4.4.2 Self-Training for ASR and ST Multilingual speech corpora LibriLight (Kahn et al., 2020b) currently represents the largest scale unlabeled speech corpus but it is limited to English. MLS (Pratap et al., 2020) is a recently released large-scale multilingual corpus of read speech in 8 languages, derived from LibriVox. MAILABS1 is also derived from Librivox and has about 1000 hours available in 9 languages. While MLS and MAILABS are derived from audiobooks, Vox- Forge1 and Common Voice (Ardila et al., 2020) gather data via crowd-sourcing. VoxForge col- lected data in about 15 different languages with about 300 hours of speech in total; Common Voice currently supports 60 languages for a total of 7327 validated hours available. The CMU Wilderness dataset (Black, 2019) collects readings from the New Testament, with 700 different languages avail- Self-training (Scudder, 1965) is a classical semi- supervised learning approach, where unlabeled data is equipped with pseudo-labels from a su- pervised model and then combined with labeled data for model training. We use the combination of EuroParl-ST (Iranzo-S´anchez et al., 2020) and CoVoST 2 (Wang et al., 2020b) for both ASR and ST labeled data in 3 languages (directions). The former is created from 2009-2012 EP plenary ses- sions and hence has the same domain as VoxPop- uli. The latter is based on Common Voice v4, which has different domain than VoxPopuli and dominates the combined train set. We train Trans- former Base (Vaswani et al., 2017) supervised base- lines and use 0.8K/3K-hour monolingual VoxPop- uli unlabeled data (from 2013-2020 sessions only to avoid overlaps with EuroParl-ST) to self-train Transformer Large models. We upsample labeled 1http://www.voxforge.org 8 Ethical Considerations able. IARPA Babel program1 collected data for 24 languages, mostly from conversational telephone speech. The dataset is however not released and under an open license, and focused on low-resource languages, with labeled data ranging between 25 to 65 hours per language. able. IARPA Babel program1 collected data for 24 languages, mostly from conversational telephone speech. The dataset is however not released and under an open license, and focused on low-resource languages, with labeled data ranging between 25 to 65 hours per language. We acknowledge the European Union (EU) for cre- ating and publishing the materials used by Vox- Populi. We will add citations as well as acknowl- edgements in our release. We paid the market price to transcription vendors for the human an- notations we collected. VoxPopuli includes all available speeches from the 2009-2020 EP events without any selections on the topics or speakers. The speech contents represent the standpoints of the speakers in the EP events, many of which are EU ofﬁcials. Speech-to-Text and Speech-to-Speech Transla- tion Apart from machine translation (Koehn, 2005), the European Parliament open data has fos- tered the development of corpora for speech-to- text translation and for simultaneous interpreta- tion. EuroParl-ST (Iranzo-S´anchez et al., 2020) is a multilingual speech-to-text translation corpus with translations between 6 European languages (En, Fr, De, Es, It and Pt). Similarly, EPIC (Bendazzoli et al., 2005) is derived from the European Parlia- ment with simultaneous interpretation speeches in Italian, English and Spanish. CIAIR (Tohyama et al., 2004) and STC (Shimizu et al., 2014) are si- multaneous interpretation corpora between English and Japanese with a total of about 180 hours for the former, while the latter is currently unavailable for download. The MaSS dataset (Zanon Boito et al., 2020) also provides speech to speech alignments for about 8k utterances across 8 languages, for a total of about 23h of speech. 6 Conclusion Alexei Baevski, Yuhao Zhou, Abdelrahman Mohamed, and Michael Auli. 2020. wav2vec 2.0: A frame- work for self-supervised learning of speech represen- tations. Advances in Neural Information Processing Systems, 33. In this paper, we introduce a large-scale multilin- gual speech corpus, VoxPopuli, for representation learning, semi-supervised learning and interpreta- tion. VoxPopuli provides the largest open unla- beled speech data to date, which has broad applica- tions including unsupervised pre-training and self- training. VoxPopuli is also the ﬁrst corpus for large amounts of open speech-to-speech interpretation data. We provide VoxPopuli ASR baselines and val- idate the versatility of VoxPopuli unlabeled data in semi-supervised learning under challenging out-of- domain settings. The corpus is available at https: //github.com/facebookresearch/voxpopuli. Claudio Bendazzoli, Annalisa Sandrelli, et al. 2005. An approach to corpus-based interpreting studies: developing epic (european parliament interpreting corpus). Proceedings of Challenges of Multidimen- sional Translation. Alan W Black. 2019. Cmu wilderness multilingual speech dataset. In ICASSP 2019-2019 IEEE Interna- tional Conference on Acoustics, Speech and Signal Processing (ICASSP), pages 5971–5975. IEEE. Herv´e Bredin, Ruiqing Yin, Juan Manuel Coria, Gre- gory Gelly, Pavel Korshunov, Marvin Lavechin, Diego Fustes, Hadrien Titeux, Wassim Bouaziz, and Marie-Philippe Gill. 2020. pyannote.audio: neural building blocks for speaker diarization. In ICASSP 2020, IEEE International Conference on Acoustics, Speech, and Signal Processing, Barcelona, Spain. 1https://www.iarpa.gov/index.php/research- programs/babel References Rosana Ardila, Megan Branson, Kelly Davis, Michael Kohler, Josh Meyer, Michael Henretty, Reuben Morais, Lindsay Saunders, Francis Tyers, and Gre- gor Weber. 2020. Common voice: A massively- multilingual speech corpus. In Proceedings of The 12th Language Resources and Evaluation Confer- ence, pages 4218–4222, Marseille, France. Euro- pean Language Resources Association. Mikel Artetxe and Holger Schwenk. 2019. Mas- sively multilingual sentence embeddings for zero- shot cross-lingual transfer and beyond. Transac- tions of the Association for Computational Linguis- tics, 7:597–610. 7 Acknowledgements We thank Gabriel Synnaeve, Tatiana Likhoma- nenko, Jade Copet, Vineel Pratap, Jiatao Gu and Alexis Conneau for helpful discussions on the project. Roldano Cattoni, Mattia Antonino Di Gangi, Luisa Bentivogli, Matteo Negri, and Marco Turchi. 2020. Must-c: A multilingual corpus for end-to-end speech translation. Computer Speech & Language, 66:101155. Ronan Collobert, Christian Puhrsch, and Gabriel Syn- naeve. 2016. Wav2letter: an end-to-end convnet- based speech recognition system. arXiv preprint arXiv:1609.03193. Jacob Kahn, Ann Lee, and Awni Hannun. 2020a. Self-training for end-to-end speech recognition. In ICASSP 2020-2020 IEEE International Confer- ence on Acoustics, Speech and Signal Processing (ICASSP), pages 7084–7088. IEEE. Alexis Conneau, Alexei Baevski, Ronan Collobert, Ab- delrahman Mohamed, and Michael Auli. 2020. Un- supervised cross-lingual representation learning for speech recognition. Jacob Kahn, Morgane Rivi`ere, Weiyi Zheng, Evgeny Kharitonov, Qiantong Xu, Pierre-Emmanuel Mazar´e, Julien Karadayi, Vitaliy Liptchinsky, Ronan Collobert, Christian Fuegen, et al. 2020b. Libri-light: A benchmark for asr with limited or no supervision. In ICASSP 2020-2020 IEEE International Conference on Acoustics, Speech and Signal Processing (ICASSP), pages 7669–7673. IEEE. Mattia A Di Gangi, Roldano Cattoni, Luisa Bentivogli, Matteo Negri, and Marco Turchi. 2019. Must-c: a multilingual speech translation corpus. In 2019 Con- ference of the North American Chapter of the Asso- ciation for Computational Linguistics: Human Lan- guage Technologies, pages 2012–2017. Association for Computational Linguistics. Eugene Kharitonov, Morgane Rivi`ere, Gabriel Syn- naeve, Lior Wolf, Pierre-Emmanuel Mazar´e, Matthijs Douze, and Emmanuel Dupoux. 2020. Data augmenting contrastive learning of speech representations in the time domain. Ewan Dunbar, Xuan Nga Cao, Juan Benjumea, Julien Karadayi, Mathieu Bernard, Laurent Besacier, Xavier Anguera, and Emmanuel Dupoux. 2017. The zero resource speech challenge 2017. In 2017 IEEE Automatic Speech Recognition and Understanding Workshop (ASRU), pages 323–330. IEEE. Philipp Koehn. 2005. Europarl: A parallel corpus for statistical machine translation. In MT summit, vol- ume 5, pages 79–86. Citeseer. Alex Graves, Santiago Fern´andez, Faustino Gomez, and J¨urgen Schmidhuber. 2006. Connectionist temporal classiﬁcation: Labelling unsegmentedse- quence data with recurrent neural networks. ICML 2006 - Machine Learning, Proceedings of the Twenty-Third International Conference. Taku Kudo and John Richardson. 2018. SentencePiece: A simple and language independent subword tok- enizer and detokenizer for neural text processing. In Proceedings of the 2018 Conference on Empirical Methods in Natural Language Processing: System Demonstrations, pages 66–71, Brussels, Belgium. Association for Computational Linguistics. Awni Hannun, Ann Lee, Qiantong Xu, and Ronan Col- lobert. 2019. Sequence-to-sequence speech recogni- tion with time-depth separable convolutions. CoRR, abs/1904.02619. 7 Acknowledgements Ha Nguyen, Fethi Bougares, N. Tomashenko, Y. Est`eve, and L. Besacier. 2020. Investigating self-supervised pre-training for end-to-end speech translation. In INTERSPEECH. He He, Jordan Boyd-Graber, and Hal Daum´e III. 2016. Interpretese vs. translationese: The uniqueness of human strategies in simultaneous interpretation. In Proceedings of the 2016 Conference of the North American Chapter of the Association for Computa- tional Linguistics: Human Language Technologies, pages 971–976. Aaron van den Oord, Yazhe Li, and Oriol Vinyals. 2018. Representation learning with contrastive pre- dictive coding. arXiv preprint arXiv:1807.03748. Myle Ott, Sergey Edunov, Alexei Baevski, Angela Fan, Sam Gross, Nathan Ng, David Grangier, and Michael Auli. 2019. fairseq: A fast, extensible toolkit for sequence modeling. In Proceedings of NAACL-HLT 2019: Demonstrations. Kenneth Heaﬁeld. 2011. Kenlm: Faster and smaller language model queries. In Proceedings of the sixth workshop on statistical machine translation, pages 187–197. Javier Iranzo-S´anchez, Joan Albert Silvestre-Cerd`a, Javier Jorge, Nahuel Rosell´o, Adri`a Gim´enez, Al- bert Sanchis, Jorge Civera, and Alfons Juan. 2020. Europarl-st: A multilingual corpus for speech trans- lation of parliamentary debates. In ICASSP 2020- 2020 IEEE International Conference on Acoustics, Speech and Signal Processing (ICASSP), pages 8229–8233. IEEE. Vassil Panayotov, Guoguo Chen, Daniel Povey, and Sanjeev Khudanpur. 2015. Librispeech: an asr corpus based on public domain audio books. In 2015 IEEE International Conference on Acoustics, Speech and Signal Processing (ICASSP), pages 5206–5210. IEEE. J. Pino, Qiantong Xu, Xutai Ma, Mohammad Javad Dousti, and Yun Tang. 2020. Self-training for end- to-end speech translation. In INTERSPEECH. Ye Jia, Ron J. Weiss, Fadi Biadsy, Wolfgang Macherey, Melvin Johnson, Zhifeng Chen, and Yonghui Wu. 2019. Direct speech-to-speech translation with a sequence-to-sequence model. In Interspeech 2019, 20th Annual Conference of the International Speech Communication Association, Graz, Austria, 15-19 September 2019, pages 1123–1127. ISCA. Vineel Pratap, Awni Hannun, Qiantong Xu, Jeff Cai, Jacob Kahn, Gabriel Synnaeve, Vitaliy Liptchin- sky, and Ronan Collobert. 2018. wav2letter++: The fastest open-source speech recognition system. CoRR, abs/1812.07625. you need. In Advances in Neural Information Pro- cessing Systems, volume 30, pages 5998–6008. Cur- ran Associates, Inc. Vineel Pratap, Qiantong Xu, Anuroop Sriram, Gabriel Synnaeve, and Ronan Collobert. 2020. MLS: A Large-Scale Multilingual Dataset for Speech Re- search. In Proc. Interspeech 2020, pages 2757– 2761. Changhan Wang, Yun Tang, Xutai Ma, Anne Wu, Dmytro Okhonko, and Juan Pino. 2020a. fairseq s2t: Fast speech-to-text modeling with fairseq. 7 Acknowledgements In Pro- ceedings of the 2020 Conference of the Asian Chap- ter of the Association for Computational Linguistics (AACL): System Demonstrations. Morgane Riviere and Emmanuel Dupoux. 2020. To- wards unsupervised learning of speech features in the wild. SLT 2020: IEEE Spoken Language Tech- nology Workshop. Changhan Wang, Anne Wu, and Juan Pino. 2020b. Covost 2 and massively multilingual speech-to-text translation. arXiv e-prints, pages arXiv–2007. Morgane Rivi`ere, Armand Joulin, Pierre-Emmanuel Mazar´e, and Emmanuel Dupoux. 2020. Unsuper- vised pretraining transfers well across languages. In ICASSP 2020-2020 IEEE International Confer- ence on Acoustics, Speech and Signal Processing (ICASSP), pages 7414–7418. IEEE. Anne Wu, Changhan Wang, Juan Pino, and Jiatao Gu. 2020. Self-supervised representations improve end- to-end speech translation. In INTERSPEECH. Thomas Schatz, Vijayaditya Peddinti, Francis Bach, Aren Jansen, Hynek Hermansky, and Emmanuel Dupoux. 2013. Evaluating speech features with the minimal-pair abx task: Analysis of the classi- cal mfc/plp pipeline. In INTERSPEECH 2013: 14th Annual Conference of the International Speech Com- munication Association, pages 1–5. Qiantong Xu, Alexei Baevski, Tatiana Likhoma- nenko, Paden Tomasello, Alexis Conneau, Ronan Collobert, Gabriel Synnaeve, and Michael Auli. 2020. Self-training and pre-training are comple- mentary for speech recognition. arXiv preprint arXiv:2010.11430. Marcely Zanon Boito, William Havard, Mahault Gar- nerin, ´Eric Le Ferrand, and Laurent Besacier. 2020. MaSS: A large and clean multilingual corpus of sentence-aligned spoken utterances extracted from the Bible. In Proceedings of the 12th Language Resources and Evaluation Conference, pages 6486– 6493, Marseille, France. European Language Re- sources Association. Steffen Schneider, Alexei Baevski, Ronan Collobert, and Michael Auli. 2019. wav2vec: Unsupervised Pre-Training for Speech Recognition. In Proc. Inter- speech 2019, pages 3465–3469. H Scudder. 1965. Probability of error of some adap- tive pattern-recognition machines. IEEE Transac- tions on Information Theory, 11(3):363–371. Chen Zhang, Xu Tan, Yi Ren, Tao Qin, Kejun Zhang, and Tie-Yan Liu. 2020a. Uwspeech: Speech to speech translation for unwritten languages. arXiv preprint arXiv:2006.07926. Hiroaki Shimizu, Graham Neubig, Sakriani Sakti, Tomoki Toda, and Satoshi Nakamura. 2014. Collec- tion of a simultaneous translation corpus for compar- ative analysis. In LREC, pages 670–673. Citeseer. Yu Zhang, James Qin, Daniel S Park, Wei Han, Chung- Cheng Chiu, Ruoming Pang, Quoc V Le, and Yonghui Wu. 2020b. Pushing the limits of semi- supervised learning for automatic speech recogni- tion. arXiv preprint arXiv:2010.10504. Vivek Kumar Rangarajan Sridhar, John Chen, and Srinivas Bangalore. 2013. Corpus analysis of simul- taneous interpretation data for improving real time speech translation. 7 Acknowledgements In INTERSPEECH, pages 3468– 3472. Chaitanya Talnikar, Tatiana Likhomanenko, Ronan Collobert, and Gabriel Synnaeve. 2020. Joint masked cpc and ctc training for asr. arXiv preprint arXiv:2011.00093. Andros Tjandra, Sakriani Sakti, and Satoshi Nakamura. 2019. Speech-to-speech translation between untran- scribed unknown languages. In 2019 IEEE Auto- matic Speech Recognition and Understanding Work- shop (ASRU), pages 593–600. IEEE. Hitomi Tohyama, Shigeki Matsubara, Koichiro Ryu, N Kawaguch, and Yasuyoshi Inagaki. 2004. Ciair simultaneous interpretation corpus. In Proc. Orien- tal COCOSDA. Ashish Vaswani, Noam Shazeer, Niki Parmar, Jakob Uszkoreit, Llion Jones, Aidan N Gomez, Ł ukasz Kaiser, and Illia Polosukhin. 2017. Attention is all
https://openalex.org/W4236379807	https://zenodo.org/records/3390342/files/12081__1_219955_LE_332564.pdf	Dutch	null	NIEUWS IN ZAKE WETGEVING, RESOLUTIES EN BESLISSINGEN OP HET GEBIED DER BELASTINGEN	MAB	1,938	cc-by	5,900	Het nut van een goede voorraadadministratie. b l i li h i d ij d d ƒ hier nog al stringente eischen stelt, aangezien aan de voorraadadministratie in vele ondernemingen nog al eens het een en ander ontbreekt. Men vergete echter niet, dat elk arrest slechts betrekking heeft op een speciaal geval en in het onderhavige geval, naar de Raad van Beroep vaststelde, „belanghebbende noch voorraadstaten, noch andere behoorlijke specificaties zijner voorraden heeft kunnen toonen en zijn boek houding zoodanig is ingericht, dat elke contrôle uitgesloten”. V l d i f d kt i lijkt hi b g g j g Ten slotte is de verhooging bij navordering teruggebracht tot het viervoud der enkelvoudig verschuldigde belasting ter wijl voor de opgelegde verhooging nu beroep openstaat bij den directeur der invoerrechten en accijnzen, g g g g Vooral de cursief gedrukte overweging lijkt ons hier van be lang; had de belastingplichtige aan de hand van een uit zijn administratie opgestelde normen-calculatie (het middel als een quantitatieve voorraadadministratie ontbreekt) zijn standpunt kunnen verdedigen, dan was wellicht de uitspraak voor hem minder ongunstig geweest. MAANDBLAD VOOR ACCOUNTANCY EN BEDRIJFSHUISHOUDKUNDE MAANDBLAD VOOR ACCOUNTANCY EN BEDRIJFSHUISHOUDKUNDE 180 uitkeering van het leven afhankelijk (zijnde de vier bronnen in art. 4, 2e lid I.B. genoemd) vormt dat goed, dat kapitaal, die arbeid of dat recht op periodieke uitkeering en niet het recht, krachtens hetwelk de opbrengst daarvan rechtstreeks wordt verkregen, de bron van inkomen. Voor de vader-voogd, die een wettelÿk vruchtgenot heeft van het vermogen van zijn kinderen, zijn dus de bestanddeelen van het vermogen de bronnen van inkomen. Aldus besliste de H.R. met zijn arrest dd. 27 April 1938 (no. 7918). H t il k d t d H R i f ijki dan wordt in het algemeen belasting geheven over den werke- lijken verkoopprijs. Yan de aanneemsom van het onroerend goed wordt dus per saldo niet belast: alle kosten om de vooraf gereedgemaakte roerende goederen samen te voegen tot het onroerend goed inclusief de daarop betrekking hebbende alge- meene kosten, loonen voor toezicht e.d. benevens een deel van de winst. De geheele winst kan niet worden vrijgesteld omdat ook in den fictieven verkoopprijs der zelfgemaakte roerende goederen behalve hun kostprijs een winstopslag moet worden opgenomen. Het principe van de wet, dat de oplevering van onroerend goed niet belastbaar is, blijft dus gehandhaafd. Maakt de aannemer van onroerend goed niet eerst zelf roe rende goederen ten behoeve van het aangenomen onroerend goed, dan is hij niet belastingplichtig (bijv. het maken van een centrale verwarming of een electrische, sanitaire of tele fonische installatie). O k d b l b h id h i b ik i d d ( ) Het wil ons voorkomen, dat de H.R. in afwijking van een vroeger door hem ingenomen standpunt met dit arrest het standpunt door Schendstok in zijn openbare les verdedigd tot het zijne maakt. Schendstolc gebruikt in dit verband het beeld van de bedding (i.c. het recht van vruchtgenot) en de bronnen. Een principieel arrest, dat praktische consequenties zal heb ben in verband met het fictie-stelsel vervat in de artt. 13 en 14 W.I.B. g ( g ) Een principieel arrest, dat praktische consequenties zal heb ben in verband met het fictie-stelsel vervat in de artt. 13 en 14 W.I.B. ) Ook de belastbaarheid van het eigen gebruik is veranderd. Onder de oude wet was alleen belast het beschikken door fabri kanten voor eigen gebruik, voor zichzelf en voor hun gezinnen, over goederen uit hun bedrijfsvoorraad. Het nut van een goede voorraadadministratie. b l i li h i d ij d d ƒ Een belastingplichtige meende zijn goederenvoorraad ƒ 6000 lager te moeten waardeeren, waarmede hij zijn winst drukte. De Raad van Beroep overwoog dat, waar door geen enkel con troleerbaar gegeven de opgegeven balanscijfers worden ge rechtvaardigd, belanghebbende niet aannemelijk heeft gemaakt, dat het door hem in mindering van de winst gebrachte bedrag van ƒ 6000 als mindere waarde der goederen is te aanvaarden. Belanghebbende deed nog een beroep op de algemeene be kendheid van de waardedaling der goederen in het betrokken jaar wegens het slechte conjunctuurverloop, doch de Raad verwierp dit ook met de overweging: „dat evenmin het feit, dat de conjunctuur de waarde der goederen in dat jaar heeft doen dalen, waar de oorspronkelijke waarde niet voldoende vaststaat, eenig gewicht in de schaal kan leggen”. Bij d H R h d d b l ti li hti l i D g g gg Bij de H.R. had de belastingplichtige al evenmin succes. De H.R. overwoog, en in deze overweging komt bovenstaand op schrift tot zijn recht, dat op de belastingplichtige de bewijs last voor het geleden verlies rustte en dat een tengevolge van de conjunctuur geleden verlies niet zal kunnen worden be oordeeld en vastgesteld zonder dat betrouwbare gegevens zijn verstrekt, waaruit blijkt de omvang, de samenstelling en het tijdstip van aanschaffing van den voorraad, waarop het ver lies door waardevermindering zou zijn geleden. O l kki b h d i d ij t pp j g Volgens het nieuwe artikel 4bis kan een handelaar als fa brikant worden beschouwd, wanneer hij t.o.v. den fabrikant van wien hij in hoofdzaak zijn goederen betrekt niet als on afhankelijk en wezenlijk zelfstandig kan worden aangemerkt. Hiermede kan worden opgetreden tegen verkoopmaatschap pijen. D ij b li d b l i d i p jDe wijze van betaling der belasting wordt in zooverre ge wijzigd dat wanneer vroeger op een aanslagbiljet moest wor den betaald, thans op het aangiftebiljet zegels tot het bedrag van de verschuldigde belasting moeten worden geplakt. Hierbij mag de gevraagde teruggaaf worden verrekend. Ook mag men het bedrag der verschuldigde belasting storten bij of over maken aan den ontvanger der accijnzen, die daarvoor een ont vangstbewijs verstrekt. Dit ontvangbewijs moet dan in plaats van de zegels aan het aangiftebiljet worden gehecht. T l tt i d h i bij d i t b ht g j g Oppervlakkig beschouwd zou men geneigd zijn te meenen, dat de H.R. Berekening opbrengst vrij beroep k b ik” dt Als „goed koopmansgebruik” wordt voor vrije beroepen aanvaard het belastbaar inkomen vast te stellen aan de hand van de in eenig jaar werkelijk ontvangen bedragen. „Onder handen werk” en nog niet ontvangen reeds gedeclareerde pos ten vallen er dus buiten. Hoe nu als op grond van art. 14 W.I.B. het inkomen moet worden geschat? De R. v. B. te Zwolle meende, dat voor zoo’n „eerste” jaar dit systeem geen Als „goed koopmansgebruik” wordt voor vrije beroepen aanvaard het belastbaar inkomen vast te stellen aan de hand van de in eenig jaar werkelijk ontvangen bedragen. „Onder handen werk” en nog niet ontvangen reeds gedeclareerde pos ten vallen er dus buiten. Hoe nu als op grond van art. 14 W.I.B. het inkomen moet worden geschat? De R. v. B. te Zwolle meende, dat voor zoo’n „eerste” jaar dit systeem geen (Bijdragen en mededeelingen zende men aan den Secretaris der Redactie) (Bijdragen en mededeelingen zende men aan den Secretaris der Redactie) MAANDBLAD VOOR ACCOUNTANCY EN BEDRIJFSHUISHOUDKUNDE Het nieuwe artikel 3e stelt nu belastbaar het — anders dan als grond- of hulpstof ten behoeve van het bedrijf van fabrikaat — voor eigen gebruik beschikken over goederen. Behalve het zuivere privé-verbruik is dus ook belastbaar het zelf maken van bedrijfsmiddelen, meubelen, drukwerk etc. voor het eigen bedrijf, het maken van goederen welke door den fabrikant zelf worden verhuurd en het aanwenden van zelfgemaakte goederen voor reparatie van roerende goederen toebehoorende aan den fabrikant of aan anderen. Dit laatste brengt mee dat bij reparatie omzetbelas ting moet worden geheven over de onderdeden welke daarbij worden aangebracht aan het te repareeren goed en welke door den fabrikant zelf zijn gemaakt. Het aanbrengen van deze onderdeden is vrij zoodat van de prijs voor de reparatie een deel belastbaar is. Dit deel wordt gevormd door de fictieve verkoopprijs van de zelfgemaakte onderdeden. V l h t i tik l 4bi k h d l l f Opneming van de kasgelden door een groot-aandeelhouder van een N.V., waarin geen winst aanwezig is p j Een hoogst interessante zijde zit aan een dergelijk „opsou peeren” van kapitaal nog vast in verband met het 2e lid van art. 5 AV.D.T.B. t.w. de vraag of wij hier al of niet te doen hebben met winstanticipatic. In opgemeld artikel behandelt Meyburg deze zijde van het vraagstuk ook. Wij volstaan met daarheen te verwijzen. M t i h i t h f t d it k d R d De directeur •—- groot aandeelhouder van een N.Y., waarin geen Avinst aanwezig Avas, had de geAvoonte de inkomsten der N.V., dus het kasgeld, te eigen behoeve aan te wenden. De Inspecteur had het door hem opgenomen kasgeld bij zijn in komen geteld en hem er een aanslag voor opgelegd. De Am- sterdamsche Raad van Beroep kreeg over het geval te oor- deelen en .......... stelde den belastingplichtige in het gelijk in een breedvoerig gemotiveerde uitspraak, die de aandacht ver dient. N t t h bb d t d di t it l i j Men vraagt zich intusschen af wat de uitspraak van de Raad v. Beroep geweest zou zijn als de Inspecteur gesteld had, dat terugbetaling der schuld niet in de bedoeling heeft gelegen en de Raad v. Beroep dit ook feitelijk had uitgemaakt. O.i. had ook in dat geval de Inspecteur in het ongelijk gesteld moeten worden, omdat dat opsoupeeren van het kapitaal er te dik op ligt, maar de motiveering had dan een andere moeten zijn en wellicht hadden Avij een meer prineipieëele uitspraak ge kregen. Na eerst overwogen te hebben, dat de directeur nooit salaris had genoten en daarop geen recht had (was het anders ge weest dan had het opgenomen geld Avellicht als een verkapt salaris beschouwd kunnen worden), stelde de Raad vast dat het beschikken over de kasmiddelen alleen mogelijk was dank zij de machtspositie, die hij als groot-aandeelhouder bekleedde. Het kan in deze dus slechts gaan over de vraag, aldus de Raad, of er opbrengst van aandeelen is genoten. Ware er winst in de N.V. geweest, dan zou zulks te vermoeden zijn; aange zien er in deze echter geen winst aanwezig Avas, kan dit niet vermoed Avorden, daar art. 42e W. v. K. zelfs verbiedt winst uit te deelen als er nog een verlies is. Wettelijk vruchtgenot. k h lk h Voor iemand, die krachtens welk recht ook de opbrengst ver krijgt van eenig goed, kapitaal, arbeid of recht op periodieke MAANDBLAD VOOR ACCOUNTANCY EN BEDRIJFSHUISHOUDKUNDE 181 van art. 41c W. v. K.1), terwijl een dergelijke terugbetaling van kapitaal niet valt onder art. 6 W.I.B. De belastingplich tige werd dus in het gelijk gesteld. D Mi i t i i ti d l idd l van art. 41c W. v. K.1), terwijl een dergelijke terugbetaling van kapitaal niet valt onder art. 6 W.I.B. De belastingplich tige werd dus in het gelijk gesteld. D Mi i t i i ti d l idd l toepassing mocht vinden. De H.R. was van andere meening en bezigde daarvoor als argument, dat het gevolg van de op vatting van den Raad van Beroep zou zijn, dat éénzelfde in komst in het eene jaar als verschuldigd geworden, in het an dere jaar opnieuw als ontvangen zou worden belast. (H.R. dd. 17 Nov. ’37 B 6526). Deze uitspraak voldoet aan ons ge voel voor billijkheid, maar de argumentatie lijkt ons niet juist. Het komt toch geregeld voor bij zoo’n „eerste jaar”, dat een zelfde inkomst eerst als bestanddeel van de begrooting, later als effectief genoten wordt belast. , J i t h d l k l h t Z l h t d g g j g De Minister ging in cassatie en droeg als middel o.m. voor schending van art. 6 W.I.B. en art. 41c W. v. K., daarbij als toelichting gevende, dat hier geen terugbetaling van kapitaal aanwezig kan zijn daar de formaliteiten van art. 41c W. v. K. niet waren vervuld; de bedoeling van belanghebbende kan hierin geen verandering brengen. Men is geneigd op te mer ken, dat dit de omgekeerde wereld is, aangezien doorgaans de fiscus stelt dat niet op het formeel juridische, maar op het materieele, op datgene Avat bedoeld werd en in feite tot stand kwam, gelet dient te worden. D Mi i t h d d hij l ht k t g Juister had ons geleken als het Zwolsche systeem ondeug delijk was verklaard louter op grond van het feit, dat liet met de logica strijdt. g De Minister had geen succes daar hij slechts opkwam tegen een subsidiaire overweging; gesteld al dat het middel deugde lijk was (de H.R. laat er zich niet over uit) dan zou toch altijd nog door den R. v. B. a) Art. 41c W. v. K. schrijft voor kapitaalvermindering voor voor afgaande statutenwijziging en het vervullen van een eenige andere formaliteiten. Afschrijving op een bedrijfsmiddel In zijn arrest dd. 1 Dec. ’37 heeft de H.R. het als zijnde in overeenstemming met goed koopmansgebruik in principe toelaatbaar geacht op een bedrijfsmiddel, indien in een eerste periode na de aanschaffing de bedrijfswaarde ervan sneller daalt dan later, in dat tijdvak daarop meer af te schrijven dan in een later tijdvak. M k i i d it k i i h t j Men kan o.i. in deze uitspraak een species zien van het genus uitspraken, waarbij de H.R. t.a.v. de afschrijvingen een ruimer standpunt inneemt dan vroeger. j De uitspraak lijkt ons juist; Avij hebben hier, zoo het niet in de bedoeling ligt het opgenomen geld ooit terug te betalen, ten slotte te doen met een „opsoupeeren” van kapitaal. Of een dergelijk doen Avellicht kwade civielrechtelijke gevolgen kan hebben kan hier onbesproken blijven. E h t i t t ijd it d lijk Wettelijk vruchtgenot. k h lk h te recht zijn vastgesteld, dat aan de N.V. een schuld voor gelijk bedrag als liet opgenomen geld is ontstaan, waarvan terugbetaling is te verwachten. In de rij van overwinningen, die de fiscus op dit gebied in de laatste jaren heeft weten te behalen, valt deze „nederlaag”, vooral omdat zij in eerste instantie Aroor den Amsterdamschen Raad geleden werd, op. D it k (dd 13 O t b ’37 B 6504) i i Waarde privé-gebruik van auto lli h b d d l i bouwrente of formeele dividend declareering hoewel er geen winst is), maar buiten het civielrechtelijke begrip vruchten kan hetgeen bij ontstentenis van winst uit kapitaal moest stammen nimmer vruchten zijn. D it k lijkt j i t Aij h bb hi h t i t Waarde privé-gebruik van auto lli h b d d l i Bij de vaststelling van het bedrag, dat als privé-gebruik van een auto, die voor zakelijke zoowel als voor privé-doeleinden wordt gebruikt, bij het inkomen moet worden gesteld, moet niet alleen met de z.g. variabele kosten (benzine, olie, banden- slijtage) rekening worden gehouden, maar ook een evenredig deel der z.g. vaste kosten (wegenbelasting, personeele belasting, garagehuur en onderhoud) worden bijgeteld. Aldus H.R. dd. 30 Mrt. ’38 rolno. 7870). g , p De uitspraak (dd. 13 October ’37 B 6504) is in overeen stemming met het standpunt door Meyburg verdedigd in De Naamlooze Vennootschap, aflevering Maart 1936. Deze schrij ver kwam daarbjj tot de conclusie, dat art. 6 W.I.B. alleen vruchten van roerend kapitaal Avil belasten; nu blijve dit wel iswaar niet beperkt tot vruchten in civielrechtelijke zin (bijv. bouwrente of formeele dividend declareering hoewel er geen winst is), maar buiten het civielrechtelijke begrip vruchten kan hetgeen bij ontstentenis van winst uit kapitaal moest stammen nimmer vruchten zijn. De uitspraak lijkt ons juist; Avij hebben hier, zoo het niet in de bedoeling ligt het opgenomen geld ooit terug te betalen, ten slotte te doen met een „opsoupeeren” van kapitaal. Of een dergelijk doen Avellicht kwade civielrechtelijke gevolgen kan hebben kan hier onbesproken blijven. Een hoogst interessante zijde zit aan een dergelijk „opsou peeren” van kapitaal nog vast in verband met het 2e lid van art. 5 AV.D.T.B. t.w. de vraag of wij hier al of niet te doen hebben met winstanticipatic. In opgemeld artikel behandelt Meyburg deze zijde van het vraagstuk ook. Wij volstaan met daarheen te verwijzen. Men vraagt zich intusschen af wat de uitspraak van de Raad v. Beroep geweest zou zijn als de Inspecteur gesteld had, dat terugbetaling der schuld niet in de bedoeling heeft gelegen en de Raad v. Beroep dit ook feitelijk had uitgemaakt. O.i. had ook in dat geval de Inspecteur in het ongelijk gesteld moeten worden, omdat dat opsoupeeren van het kapitaal er te dik op ligt, maar de motiveering had dan een andere moeten zijn en wellicht hadden Avij een meer prineipieëele uitspraak ge kregen. g p De uitspraak (dd. 13 October ’37 B 6504) is in overeen stemming met het standpunt door Meyburg verdedigd in De Naamlooze Vennootschap, aflevering Maart 1936. Deze schrij ver kwam daarbjj tot de conclusie, dat art. 6 W.I.B. alleen vruchten van roerend kapitaal Avil belasten; nu blijve dit wel iswaar niet beperkt tot vruchten in civielrechtelijke zin (bijv. Opneming van de kasgelden door een groot-aandeelhouder van een N.V., waarin geen winst aanwezig is Het ligt dus meer voor de hand om aan te nemen, dat tegenover dit opnemen der kas middelen een vordering op den groot-aandeelhouder ontstaat Avaarvan terugbetaling vei'Avacht mag worden. Zelfs al zou nu moeten worden aangenomen, aldus overAvoog de Raad subsi diair, dat het niet in de bedoeling ligt die schuld ooit terug te betalen, dan nog is er geen opbrengst van aandeelen, aldus de Raad, maar mag men vermoeden, dat het de bedoeling ge weest is kapitaal terug te betalen, zij het dan met A'oorbijgaau Gedeeltelijk niet in gebruik zijnd kantoorgebouw NY b d i k i i h b d ijf Een N.Y. benutte door inkrimping van haar bedrijf ver schillende verspreid door het kantoorgebouw liggende ver trekken niet meer; die vertrekken waren ongemeubileerd. Op grond daarvan meende zij, dat zij niet voor het geheele kan toorgebouw kon worden aangeslagen, maar slechts voor het gedeelte, dat bij haar nog in gebruik was. Zij staafde dit met er op te wijzen, dat zij de niet gemeubelde vertrekken wilde verhuren en zich daartoe reeds in verbinding had ge steld met een makelaarskantoor, dat haar echter bericht had, dat een verhuring binnen afzienbaren tijd wel niet mogelijk zou zijn, daar de stand van het kantoorgebouw daarvoor niet gunstig was. D A t d h R d B f t d t it t 2 g g Is hier het bij art. 14 nog al eens voorkomende „achteraf- praatje” in een in rechte niet toelaatbare wijze aanwezig? De IT.R. meent van niet en zulks op grond van de volgende over weging, waarbij het om een fijne nuanceering gaat: d t d R d i d t di h id d t i d d d i h t g g, j j g g „dat de Raad in de omstandigheid, dat inderdaad in het kalenderjaar 1935 de opbrengst van de bron ƒ 4000.— heeft bedragen, grond vond — en ook mocht vinden — voor het ver moeden, dat er op 1 Mei 1935 voldoende feiten waren, die aan een begrooting op dat bedrag grooter waarschijnlijkheid gaven dan aan een begrooting op een lager bedrag of op nihil, zooals belanghebbende had aangevoerd; ’ ’ d t d i d t b l h bb d i t i l d g g ; „dat de overweging, dat belanghebbende er niet in geslaagd is tegen dat vermoeden tegenbewijs te leveren, is van feite- lijken aard en in cassatie niet met vrucht bestreden kan wor den”. M l k d d H R hi fij j g g De Amsterdamsche Raad van Beroep gaf toe, dat uit art. 2 der Wet op de Personeele Belasting blijkt, dat de wetgever het gebruiken van een gedeelte van een gebouw kent, maar art. 2 geeft geen antwoord op de vraag wanneer zulks het ge val is. Uit art. 3 P.B. Een getorpedeerde stichting bij i l i h i Iemand richtte bij notarieele acte een stichting op. Als doel werd aangegeven: ziekenverpleging; de stichter stelde zich zelf aan tot bestuurder en liet in de statuten den bestuurder de macht toekennen om de statuten te wijzigen; van het kapi taal dat hij inbracht behield hij voor zichzelf en voor een door hem aan te wijzen opvolger het vruchtgebruik voor. Hij stelde zich nu op het standpunt, dat hij niet voor het in de stichting gebrachte vermogen en het inkomen daaruit in de vermogens- resp. de inkomstenbelasting kon worden aangeslagen. De lieger ging niet op; de Inspecte r ontkende de rechts p g g g De H.R. bevestigde de uitspraak als zijnde van fcitelijken aard, maar uit de omstandigheid, dat het den H.R. opportuun lijkt in zijn uitspraak ten overvloede te vermelden, dat van de feitelijkheden „in het bijzonder de aandacht verdient, dat het hier naar ’s Raads beslissing eenige verspreid door het ge bouw gelegen vertrekken betreft” mag men wellicht opmaken, dat de H.R. het hierboven becritiseerde argument van den Raad van Beroep ook niet bijster sterk achtte. O i i d it k i b d t d h h d De vlieger ging niet op; de Inspecteur ontkende de rechts geldigheid van de stichting en de Arnhemsche Raad van Be roep viel gretig bij, terwijl de H.R. zijn fiat aan de uitspraak van den R. v. B. gaf (arrest dd. 20 April ’38 B 6G66). De argumentatie van de uitspraak beoogde aan te toonen dat van een afzondering van vermogen (een vereischte voor de oprich ting van een stichting) in deze feitelijk niet gesproken kan worden. De uitspraak is lezenswaardig, al was het alleen maar om te ervaren, hoe men bij de oprichting van een stichting niet te werk moet gaan. D R d B t d i f it di h t ht p j Overigens is deze uitspraak in verband met de heerschende malaise van practische beteekenis. MAANDBLAD VOOR ACCOUNTANCY EN BEDRIJFSHUISHOUDKUNDE 182 MAANDBLAD VOOR ACCOUNTANCY EN BEDRIJFSHUISHOUDKUNDE de stichter en zijn notaris) mag men zich toch afvragen of het hierboven vermelde punt 2 wel houdbaar is. de stichter en zijn notaris) mag men zich toch afvragen of het hierboven vermelde punt 2 wel houdbaar is. verwachten, dat dc toelage over het belastingjaar 1935/36 dit maximum zoude beloopen, eerder dan dat belanghebbende over dat tijdvak een geringere toelage zou ontvangen”. In aanslui ting daarop overwoog de Raad dat de belastingplichtige er niet in geslaagd was dit vermoeden te ontzenuwen. I hi h t bij t 14 l k d ht f Gedeeltelijk niet in gebruik zijnd kantoorgebouw NY b d i k i i h b d ijf dient een belastingplichtige in deze niet de dupe worden van het feit, dat hij in de op zichzelf reeds ongelukkige omstandigheid verkeert, dat zijn kantoorgebouw op een ongelukkige stand ligt. D H R b i d d i k l ij d f i lijk Van iemand, die de feitelijke beschikking over het geheele gebouw heeft, maar slechts een gedeelte ervan gemeubeld en in feitelijk gebruik heeft, kan niet gezegd worden, dat hij slechts van de gemeubelde gedeelten gebruiker is in den zin der wet. Van afzonderlijk in gebruik zijnde gedeelten van ge bouwen, waarvan art. 2 § 2 P.B. gewaagt, kan slechts sprake zijn als het overige niet in feitelijk gebruik zijnde gedeelte de bestemming heeft om eerlang afzonderlijk van het in feite lijk gebruik zijnde gedeelte in gebruik te worden genomen. Uit de feiten zooals hierboven vermeld kon de Raad echter j g j g g g Uit de feiten zooals hierboven vermeld kon de Raad echter niet concludeeren tot het aanwezig zijn van een dergel ijk af zonderlijk in gebruik zijnd gedeelte. Het bevredigt het rechts gevoel niet, dat men onder de argumenten, op grond waarvan de Raad tot deze feitelijke conclusie kwam, aantreft de over weging, dat uit liet bericht van den makelaar was op te maken, dat in verband met dc slechte stand van het gebouw een ver huring van dc ongemeubelde vertrekken voorshands niet te ver wachten was. O.i. dient een belastingplichtige in deze niet de dupe worden van het feit, dat hij in de op zichzelf reeds ongelukkige omstandigheid verkeert, dat zijn kantoorgebouw op een ongelukkige stand ligt. D H R b ti d d it k l ij d f it lijk Begrooting naar op 1 Mei bekende feiten (art. 14 I.B.) I d h d 1 M i ’35 i b (d f i Iemand had op 1 Mei ’35 een nieuwe bron, (de functie van Directeur van een stichting), waaraan naast een vast salaris een toelage verbonden Avas, die maximum ƒ 4000.— zou kun nen bedragen. Hij had die toelage op nihil begroot, omdat op 1 Mei ’35 niet vast stond, hoeveel die toelage zou bedragen. Achteraf was komen vast te staan, dat hij over het kalender jaar 1935 ƒ 4000.— als toelage had gekregen. De Amsterdam- sche Raad van Beroep vond nu in dit feit grond voor het ver moeden „dat de op 1 Mei ’35 bestaande feiten mochten doen Verkeerde adresseering van een beroepschrift k d d i b h if Door verkeerde adresseering van een beroepschrift was dit stuk wel binnen de wettelijke termijn door de post ter inspectie bezorgd, maar te laat bij den Raad van Beroep. De belasting plichtige werd door den Raad van Beroep niet ontvankelijk verklaard, welke beslissing door den H.R. werd bekrachtigd (arrest dd. 27 April ’38 B 6668). g De Raad van Beroep noemt drie feiten, die voor het rechts geldig bestaan van een stichting aanwezig moeten zijn: 1. een blijvende afzondering van een vermogen; 2. voor een bepaald aangewezen buiten het finantieel belang van den oprichter gelegen doel; 3. met een regeling voor het gebruik en beheer van het vermogen. H l i d f it l di it d bli d g Hoewel, gezien de feiten, zooals die uit de gepubliceerde uitspraak blijken, een iegelijk met het resultaat, waartoe de Raad van Beroep kwam, zal instemmen (behalve dan wellicht Gedeeltelijk niet in gebruik zijnd kantoorgebouw NY b d i k i i h b d ijf blijkt volgens den Raad, dat gedeelte lijk gemeubelde perceelen geacht worden geheel in gebruik te zijn en onder gedeeltelijk gemeubelde perceelen rekent de Raad ook een perceel, waarvan niet alle vertrekken zijn gemeu bileerd. V i d di d f i lijk b hikki h h l Men zal moeten erkennen, dat de H.R. hier fijntjes nuan ceert. Bevredigend wordt de uitspraak daardoor voor ons nog niet. Het achteraf gebleken feit, dat over 1935 inderdaad ƒ 4000.— getoucheerd is, blijft dan toch maar de grond voor een vermoeden, dat er op 1 Mei 1935 voldoende feiten waren etc. Hoe men het ook keert of draait, men zal als men ernst maakt met dc woorden in de wet: „Voor dc vaststelling van dit bedrag wordt uitsluitend op de hij den aanvang van het belastingjaar bestaande gegevens gelet” elk argument ont leend aan achteraf gebleken feiten moeten passeeren. Dit geldt zoowel voor een ruwe argumentatie in den zin van: „achteraf is gebleken dat de opbrengst f. X. was, dus op 1 Mei kon men vermoeden, dat de opbrengst f. X. zou zijn”, als voor de in deze aanwezige fijnere nuanceering, dat achteraf gebleken fei ten het vermoeden rechtvaardigen, dat er op 1 Mei ’35 vol doende feiten waren, die aan een bepaalde begrooting grooter waarschijnlijkheid gaven. , Van iemand, die de feitelijke beschikking over het geheele gebouw heeft, maar slechts een gedeelte ervan gemeubeld en in feitelijk gebruik heeft, kan niet gezegd worden, dat hij slechts van de gemeubelde gedeelten gebruiker is in den zin der wet. Van afzonderlijk in gebruik zijnde gedeelten van ge bouwen, waarvan art. 2 § 2 P.B. gewaagt, kan slechts sprake zijn als het overige niet in feitelijk gebruik zijnde gedeelte de bestemming heeft om eerlang afzonderlijk van het in feite lijk gebruik zijnde gedeelte in gebruik te worden genomen. Uit de feiten zooals hierboven vermeld kon de Raad echter niet concludeeren tot het aanwezig zijn van een dergel ijk af zonderlijk in gebruik zijnd gedeelte. Het bevredigt het rechts gevoel niet, dat men onder de argumenten, op grond waarvan de Raad tot deze feitelijke conclusie kwam, aantreft de over weging, dat uit liet bericht van den makelaar was op te maken, dat in verband met dc slechte stand van het gebouw een ver huring van dc ongemeubelde vertrekken voorshands niet te ver wachten was. O.i. Bescheiden. 11. jou maal vellen (dus losse kasstaten). 12 t tb h id ( l l i t j 12. ontvangstbescheiden (als regel intern). 13 it fb h id ( l l t ) In „Der Buchführer” van 15 October 1938 schrijft E. Loh mann over bovenstaand onderwerp. Waar het bij ons nog wei nig is gekomen tot een afzonderlijke behandeling van dit punt, nemen wij enkele van zijn gedachten over. g ( g 13. uitgaafbeseheiden (als regel extern). 2. Bank. 21 d ld 21. dagsaldobilletten. 22 N t ’ De beteekenis van het belegstuk is: 1 b d b kh di g 22. Nota’s. Hi bjj 22. Nota s. Hierbjj kan worden opgemerkt dat notitie van notanum- mers op dagsaldobilletten wel gewenscht is. Anders kan de gescheiden opberging tot moeilijkheden voeren. g 1. bron van de boekhouding, waarop men teruggrijpt bij ge detailleerde beschouwing der posten. Vooral bij onze mo derne administratie, zonder of bijna zonder omschrijvingen, is deze functie van belang. 2 b ij t kk d b kh di d L h t 3. Postrekening. 31 d ld bill tt g 2. bewijsstukken voor de boekhouding, door Lohmann van te grooter belang geacht naarmate men meer met losbladige boeken en kaarten werkt. g 31. dagsaldobilletten. 32 N t ’ f g 32. Nota’s van af- en bijsch Zie opmerking sub. 22. g 32. Nota’s van af- en bijschrijven. Zi ki b 22 j Zie opmerking sub. 22. Men kan onderscheiden interne en externe bescheiden. De externe worden overgenomen op interne, als voorbereiding voor de boeking. D i t t kk i d t d b ki t 4. Wissels. 41 bl d 41. bladen uit het creatieregister. Zie opmerking sub 5 41. bladen uit het creatieregister. Zie opmerking sub. 5. 41. bladen uit het creatieregister. Zie opmerking sub. 5. g Zie opmerking sub. 5. g Door op interne stukken voor iedere post de boeking aan te geven, is de eigenlijke inschrijving in de administratie terug gebracht tot eenvoudige mechanisch te verrichten arbeid. Dit „mechanisch” zoowel overdrachtelijk als letterlijk op te vatten. 5. Cheques. 51 Bl d q 51. Bladen uit het crcatie-register. Opmerking: Voor 41 en 51 kunnen ook wisselcopievellen en chequecopievellen in de plaats treden. Tenslotte ook copie-advies-brieven. Het aangeven van de boekingen op interne belegstukken, al of niet uitgaande van een extern stuk, wil Lohmann gedecen traliseerd doen verrichten. D.w.z. LASTIGE GEVALLEN. LASTIGE GEVALLEN. Wanneer het initiatief voor een transactie bij de onderne ming zelve ligt, b.v. het afgeven van een cheque, wachte men met de boeking niet totdat een extern belegstuk (banknota) aanwezig is, maar boeke onmiddellijk op grond van een intern formulier. Zoo verdient het aanbeveling ontvangst van goe deren ook reeds te boeken voordat de factuur binnenkomt. D ti i dit l t t tl t L h Ontbreken van een volmacht voor een cassatie-procedure D h i d b h if i i i Door een gemachtigde was een beroepschrift in cassatie in MAANDBLAD VOOR ACCOUNTANCY EN BEDRIJFSHUISHOUDKUNDE 183 gediend zonder dat van een machtiging door den belasting plichtige bleek. De H.R. (arrest dd. 20 April ’38 B 6663) achtte de belastingplichtige op grond daarvan niet ontvan kelijk in zijn beroep. Men dient er dus attent op te zijn. dat hetzij door medeonderteekening door den belastingplichtige van het beroepschrift, hetzij door bijsluiting van een volmacht, de H.R. zich kan overtuigen van het aanwezig zijn eener mach tiging. De volmacht behoeft niet gezegeld te zijn. E TEKENBROEK dern die vereinbarungsgemasz erfolgte und berechnete Liefe rung die vermögenverandernde Tatsache ist.” V h di t d d ht d d d i i g g Voor hem die met de gedachtengang der moderne admini- stratieleer vertrouwd is, zegt deze motiveering al heel weinig. Het criterium „vermogensverandering” als aanleiding voor een boeking is in feite reeds lang losgelaten. De voornaamste reden waarom men, b.v. bij ingang van goederen, niet met de boeking wacht totdat de factuur binnenkomt is deze, dat men die goederen, als zijnde aan het bedrijf toebehoorende waar den, reeds direct in het controlesysteem der administratie wil betrekken. A h t b d b h id ijdt L h t j E. TEKENBROEK UIT HET BUITENLAND AA OSC C AG A Aan het bewaren der bescheiden wijdt Lohmann een aparte paragraaf. Hij voert een decimale indeeling in en wil naar de daarin gegeven groepen de stukken opbergen, binnen iedere groep zelf weer naar nummervolgorde. Zijn indeeling is de volgende: Red.: F. HAARBOSCH, CH. HAGEMAN, Drs A. TH. DE LANGE en Drs W. P. DEN TURK (Bijdragen en mededeelingen zende men aan den Secretaris der Redactie) Red.: F. HAARBOSCH, CH. HAGEMAN, Drs A. TH. DE LANGE en Drs W. P. DEN TURK (Bijd d d li d d S i (Bijdragen en mededeelingen zende men aan den Secretaris der Redactie) (Bijdragen en mededeelingen zende men aan den Secretaris der Redactie) 1. Kas. 11 j Bescheiden. iedere bedrijfsafdeeling „kon tiert ’ ’ haar eigen stukken en geeft ze dan aan de administratie door. Dat geeft tweeërlei voordeel. D di d t ti t t t d b t f h l t t t 6. Binnenkomende rekening. 61 N l f t 6. Binnenkomende rekening. 61 N l f t 6. Binnenkomende rekening. 61. Normale facturen. 62. creditnota ’s. 7. Uitgaande rekeningen. 71. normale copie-facturen. 72. copie-creditnota’s. ' 8. Interne boekingsopdrachten. 81. overboekingen. 82. verrekeningen. 83..................................... enz. d. T. 61. Normale facturen. 62 dit t ’ 62. creditnota ’s. g a. De man die de transacties tot stand brengt of helpt tot stand brengen weet het best hoe ze moeten worden geboekt. Wanneer een centrale instantie ervoor moet zorgen, zal deze toch telkens weer in de afdeelingen moeten informee- ren naar de eigenlijke strekking der doorgegeven beschei den. b D k i k i d i h d h 7. Uitgaande rekeningen. 71 l i f t g g 71. normale copie-facturen. 72 i dit t ’ ' p 72. copie-creditnota’s. 8. Interne boekingsopdrachten. 81 b ki 81. overboekingen. 82 k i b. De kennis van rekeningen, codeering en het daarom heen hangende vaktaaltje is geen monopolie van één of twee menschen, maar wordt door meerdere beoefend. In geval van ziekte of verhindering van één dier velen wordt niet zooveel verwarring gesticht als wanneer een houder van wat Lohmann humoristisch aanduidt als „Kopfmonopol” verhinderd is zijn arbeid te verrichten. b. De kennis van rekeningen, codeering en het daarom heen hangende vaktaaltje is geen monopolie van één of twee menschen, maar wordt door meerdere beoefend. In geval van ziekte of verhindering van één dier velen wordt niet zooveel verwarring gesticht als wanneer een houder van wat Lohmann humoristisch aanduidt als „Kopfmonopol” verhinderd is zijn arbeid te verrichten. g 82. verrekeningen. 83 d. T. Verbod van het verschaffen van inlichtingen door het personeel aan den accountant . Mag het door den directeur van een N.V. gegeven voor schrift, dat alle inlichtingen omtrent bcheersdaden uitsluitend door hem aan den accountant zullen worden verstrekt, voor De motiveering voor dit laatste ontleent Lohmann aan Meltzer, die schrijft dat: „Nicht die Rechnungserteilung, son-
https://openalex.org/W1998758034	http://www.scirp.org/journal/PaperDownload.aspx?paperID=51477	English	null	Performance of Concrete with Fly Ash and Kaolin Inclusion	International journal of geosciences	2,014	cc-by	2,390	Abstract Waste materials of environmental risks, costly landfill disposal can be utilized in cement and con- crete applications. Partial replacement of cement in engineering projects reduces the cost of con- struction with ecological benefits. The type and mix ratio affects the strength and workability of cement-concrete matrices. This research deals with the replacement of Class F fly ash and kaolin with cement used in concrete. Replacement of 10% fly ash with cement improves the comprehen- sive strength of concrete in 28 days period, and the workability is increased by 53.8%. The kaolin replacement reduced both the strength and workability of concrete. Keywords Fly Ash, Kaolin, Strength, Workability Performance of Concrete with Fly Ash and Kaolin Inclusion Afaf Ghais, Duaa Ahmed, Ethar Siddig, Isra Elsadig, Samah Albager Chemical Engineering Department, University of Khartoum, Khartoum, Sudan Email: afafghais2000@gmail.com Received 27 August 2014; revised 23 September 2014; accepted 15 October 2014 Copyright © 2014 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/ International Journal of Geosciences, 2014, 5, 1445-1450 Published Online November 2014 in SciRes. http://www.scirp.org/journal/ij http://dx.doi.org/10.4236/ijg.2014.512118 International Journal of Geosciences, 2014, 5, 1445-1450 Published Online November 2014 in SciRes. http://www.scirp.org/journal/ij http://dx.doi.org/10.4236/ijg.2014.512118 How to cite this paper: Ghais, A., Ahmed, D., Siddig, E., Elsadig, I. and Albager, S. (2014) Performance of Concrete with Fly Ash and Kaolin Inclusion. International Journal of Geosciences, 5, 1445-1450. http://dx.doi.org/10.4236/ijg.2014.512118 1. Introduction Addition of fly ash and kaolin to concrete offers advantages such as performance improvement, reduction of the amount of by-product materials, landfill cost and enables manufacturers to minimize mining and processing vir- gin materials, thereby reducing the emission of carbon dioxide. g y g Four basic ingredients of the conventional concrete are Portland cement, fine and coarse aggregates and water. The production of high performance concrete (HPC) needs to incorporate the supplementary cementations mate- rials such as fly ash and kaolin in the concrete mix [1]. Low calcium fly ash (ASTM Class F) and kaolin have been widely used as a replacement of cement in normal and high strength concrete [2] [3]. In normal strength concrete, the replacement level of fly ash can be more than 50% [4] [5], while in high strength concrete, the replacement level is usually limited to 15% ± 25% [2]. The main objectives of using fly ash in high strength concrete are to reduce heat generation and to obtain better durability properties. For several cases, early strength of concrete was low, particularly where a significant portion, 30 percent or more, of the How to cite this paper: Ghais, A., Ahmed, D., Siddig, E., Elsadig, I. and Albager, S. (2014) Performance of Concrete with Fly Ash and Kaolin Inclusion. International Journal of Geosciences, 5, 1445-1450. http://dx.doi.org/10.4236/ijg.2014.512118 A. Ghais et al. Portland cement was replaced with fly ash [6]. Wild et al., [7] proved that for addition of metakaolin up to 30%, compressive strength at 28 days increased within the range of 1.53% - 35%. However, Priyank, et al., found that the compressive strength decreased when replacement with kaolin percentage increased. The fact is that the type of kaolin affects the concrete strength [8]. The property of concrete which determines the amount of useful internal work necessary to produce full compaction is known as workability. The workability of fresh concrete depends mainly on the material, mix proportion and environmental conditions. The use of pozzolan, such as fly ash, plays an important role in con- tributing to a better workability of HPC [9]. Tanaka et al. [10] found that with 10% lesser water binder ratio, the workability of the concrete increased with an increase in amount of fly ash. Wild et al. found that workability of concrete at 0.45 water cement (w/c) decreased with increase in replacement of cement by metakaolin. 2.2. Experimental Method 2.2. Experimental Method 1. Introduction In the present study, the effect of fly ash and kaolin on concrete strength and workability were studied, and a comparison of the two binders was made. 2.1. Materials Kaolin materials from North Omdurman, Khartoum State and class F fly ash study sample from Road and Building Institute, University of Khartoum with the following compositions were used for the experimental work Table 1. 2.2.2. Comprehensive Strength Compaction of fresh concrete in 6 cubic steel moulds was achieved, applying Thirty-five manual strokes per layer in three equal layers. The cube dimension was 150 mm × 150 mm × 150 mm. For comprehensive strength the cubes were left to dry for one day and then immersed in water, three of the cubes were left for 7 days and the other three were left for 28, the compressive strength was measured in N/mm2, using compressive testing ma- chine Photo 2. 2.2.1. Concrete Mix with Fly Ash and Kaolin Concrete mixtures and cement paste was prepared with (8.75 kg cement,18.75 kg fine aggregate, 28.13 kg coarse aggregate and 5.28 kg water ) in a ratio of (1:2:3). Fly ash was used in the proportions of 10%, 20%, and 30 wt% of the total cementitious materials. The concrete mixtures were proportioned and mixed in a pan mixer, Photo 1. For concrete kaolin mixture, the kaolin to cement replacement ratio was 10%, 20%, and 30 wt%. 1446 Table 1. Fly ash and kaolin chemical composition. Components % (w/w) SiO2 Al2O3 Fe2O3 CaO LOI Fly 86.5 4.24 0.07 1.12 2.5 Kaolin 88.60 0.04 18.07 0.2 - Photo 1. Pan mixer. Table 1. Fly ash and kaolin chemical composition. Components % (w/w) SiO2 Al2O3 Fe2O3 CaO LOI Fly 86.5 4.24 0.07 1.12 2.5 Kaolin 88.60 0.04 18.07 0.2 - Photo 1. Pan mixer. 1446 2.2.3. Workability Test The slump test used to characterize the workability of concrete. Its apparatus consists of a mold in the shape of a frustum of a cone with a base diameter of 8 inches, a top diameter of 4 inches, and a height of 12 inches, Photo 3. The mold was filled with concrete in three layers of equal volume. Each layer was compacted with 25 strokes of a tamping rod. The slump cone mold was lifted vertically upward and the change in height of the concrete was measured. 1447 Photo 2. Comprehensive strength machine. Photo 3. Slump cone mold. Photo 2. Comprehensive strength machine. Photo 2. Comprehensive strength machine. Photo 2. Comprehensive strength machine. Photo 3. Slump cone mold. Photo 3. Slump cone mold. 1447 A. Ghais et al. A. Ghais et al. 4. Conclusions The following conclusions are drawn on the use of fly ash and kaolin in concrete making: 1) The addition of fly ash for long life term improves the concrete strength. 2) Addition of 10% fly ash increases the concrete strength. 2) Addition of 10% fly ash increases the concrete strength. y g ) Higher fly ash cement replacement in concrete reduces the comprehensive strength. 4) The concrete workability is improved with fly ash addition. y p y 5) The addition of Kaolin under study reduces the comprehensive strength and workability. he addition of Kaolin under study reduces the comprehensive strength and workability. 5) The addition of Kaolin under study reduces the comprehensive 6) The fly ash improves the concrete properties better than kaolin. The fact is that the calcium and aluminum content in fly ash are higher than that in kaolin, which enhances the pozzolana reactions when the water is added to the fly ash concrete mix. 6) The fly ash improves the concrete properties better than kaolin. The fact is that the calcium and aluminum content in fly ash are higher than that in kaolin, which enhances the pozzolana reactions when the water is added to the fly ash concrete mix. 3. Results and Discussion Concrete workability with kaolin. 0 10 20 30 40 50 60 70 0 10 20 30 Kaolin % Workability ,mm cement, the calcium content was reduced and concrete strength as well. For kaolin samples there are no calcuim, so the 100% concrete pose highest strength. Since Class F ash reacts slowly with water. The complete pozolana reactions will complete after long time, the strength of concrete cubes were high after 28 days. The results was agreed with other researcher that con- certe class F fly ash have slow strength gain at early ages and delayed setting that results in extended curing time requirements [11]. The concrete workability which represents the mixture ability to fill the mold properly with the desired work, without reducing the concrete’s quality, was increased with the addition of fly ash. The maximum workability was achieved at 30% fly ash. y crete-kaolin mixture the strength was reduced to 40.8% of the ummix concrete with the addition o n in 7 days. For the 28 days the strength reduction percent is 44.7 % with 30% kaolin replacement. For concrete-kaolin mixture the strength was reduced to 40.8% of the ummix concrete with t 30% kaolin in 7 days. For the 28 days the strength reduction percent is 44.7 % with 30% kaolin rep Dramatic reduction in concrete workability was obtained with the increasing kaolin amount. Wi the workability was approximately zero. In general, small particle size of kaolin and fly ash with higher specific surface area are favorable to produce highly dense and impermeable concrete; however, they cause lower concrete workability. Acknowledgements Authors are thankful to the Road and Building Research Institute, University of Khartoum—Sudan, for their great help during the experimental programme. [3] ACI Committee 211 (1993) Guide for Selecting Proportions for High-Strength Concrete with Portland Cement and Fly Ash, ACI 226.4R. ACI Material Journal, 90, 272-283. 3. Results and Discussion The comprehensive strength for 7 days and 28 days, and the workability of concrete with fly ash and kaolin with 10%, 20%, 30% were measured. The results were shown in Figures 1-4. For the sample where the strength was measured after 7 days, the comprehensive strength of concrete was re- duced with the addition of fly ash, the strength was reduced by 10%, 21.2%, 40.8% with the addition of 10%, 20%, 30% respectively. For the long term immeresion, 28 days, the strength increased by 4.99% with 10% fly ash, then the strength was reduced by 14% and 27.5% with 20%, 30% fly ash addition. The fact that for long term process the pozzolana materials in Flass F fly ash, react with water and cement for higher concrete strength. The best result was obtained with 10% fly ash. Fly ash samples under study consist low CaO of 1.12%. The reason that at early strength age (7 days) of 10% fly ash replacement to cement, the calcuim, alumunum, silicon constitutes react with water to form pozzolan complex polymer that enhance the strength of concrete mix. With increasing the replacement ratio of fly ash to 1448 Figure 1. Concrete strength with fly ash. Figure 2. Concrete workability with fly ash. Figure 3. Concrete strength with kaolin. 0 5 10 15 20 25 30 35 0 10 20 30 7 Days 28 Days Fly Ash % Strength,N/mm 2 0 50 100 150 200 250 0 10 20 30 Fly Ash % Workability,mm 0 5 10 15 20 25 30 35 0 10 20 30 7 Days 28 Days Kaolin % Strength ,N/mm2 Figure 1. Concrete strength with fly ash. 0 5 10 15 20 25 30 35 0 10 20 30 7 Days 28 Days Fly Ash % Strength,N/mm 2 0 50 100 150 200 250 0 10 20 30 Fly Ash % Workability,mm 0 5 10 15 20 25 30 35 0 10 20 30 7 Days 28 Days Fly Ash % Strength,N/mm 2 Figure 4. Concrete workability with kaolin. cement, the calcium content was reduced and concrete strength as well. For kaolin samples there are no calcuim, so the 100% concrete pose highest strength. Si Cl F h t l l ith t Th l t l ti ill l t ft l ti 0 10 20 30 40 50 60 70 0 10 20 30 Kaolin % Workability ,mm Figure 4. [2] ACI Committee 226 (1988) Use of Fly Ash in Concrete, ACI 226.3R-87. ACI Material Journal, 85, 381-408. Journal of the Chinese Institute of Engineers, 19, 645-655. http://dx.doi.org/10.1080/02533839.1996.9677830 [2] ACI Committee 226 (1988) Use of Fly Ash in Concrete, ACI 226.3R-87. ACI Material Journal, 85, 381-408. [3] ACI Committee 211 (1993) Guide for Selecting Proportions for High-Strength Concrete with Portland Cement and Fly Ash, ACI 226.4R. ACI Material Journal, 90, 272-283. [1] Chang, T.-P., Chung, F.-C. and Lin, H.-C. (1996) A Mix Proportioning Methodology for High Performance Concrete. Journal of the Chinese Institute of Engineers, 19, 645-655. http://dx.doi.org/10.1080/02533839.1996.9677830 [2] ACI Committee 226 (1988) Use of Fly Ash in Concrete, ACI 226.3R-87. ACI Material Journal, 85, 381-408. [3] ACI Committee 211 (1993) Guide for Selecting Proportions for High-Strength Concrete with Portland Cement and Fly Ash, ACI 226.4R. ACI Material Journal, 90, 272-283. [1] Chang, T.-P., Chung, F.-C. and Lin, H.-C. (1996) A Mix Proportioning Methodology for High Performance Concrete. Journal of the Chinese Institute of Engineers, 19, 645-655. http://dx.doi.org/10.1080/02533839.1996.9677830 References 1449 A. Ghais et al. [4] Langley, W.S., Carette, G.G. and Malhotra, V.M. (1989) Structural Concrete Incorporating High Volumes of ASTM Class F Fly Ash. ACI Material Journal, 86, 507-514. [5] Carette, G., Bilodeau, A., Chevrier, R.L. and Malhotra, V.M. (1993) Mechanicalproperties of Concrete Incorporating High Volumes of Fly Ash from Sources in the U.S. ACI Material Journal, 90, 535-544. [6] (2014) Using Fly Ash in Concrete. http://precast.org/ [7] Wild, S., Khatib, J.M. and Jones, A. (1996) Relative Strength, Pozzolanic Activity and Cement Hydration in Super- plasticized Metakaolin Concrete. Cement and Concrete Research, 26, 1537-1544. http://dx.doi.org/10.1016/0008-8846(96)00148-2 [8] Bhimani, P. and Vyas, C.M. (2013) Performance of Concrete with China Clay (Kaolin) Waste. International Journal of Latest Trends in Engineering and Technology (IJLTET), 2, 49-54. [9] Hwang, C.L., Lee L.S. and Lin, F.Y. (1996) Densified Mixture Design Algorithm and Early Properties of High Per- formance Concrete. Journal of the Chinese Institute of Civil Engineering and Hydraulic Engineering, 8, 217-229. [10] Tanaka, H., Wingnarajah, S., Sugimoto, K., Ukida, K. and Ishii, M. (1992) Characteristics of Concrete Incorporating Sorted Fly Ash. 4th CANMET/ACI International Conference on the Use of Fly Ash, Silica Fume, Slag and Natural Pozzolans in Concrete, Istanbul, 971-986. [11] Ardani, A. (2012) Evaluation of High-Volume, Fly Ash Mixtures (Paste and Mortar Components) Using a Dynamic Shear Rheometer and an Isothermal Calorimeter. FHWA Publication No. FHWA-HRT-12-062, NTIS Accession No. PB2012-112546, HRDI-10, (202) 493-3422.
https://openalex.org/W2786503273	https://biblio.ugent.be/publication/8592300/file/8593137.pdf	English	null	Pseudorapidity distributions of charged hadrons in proton-lead collisions at s N N = 5.02 $$ \sqrt{s_{\mathrm{NN}}}=5.02 $$ and 8.16 TeV	The Journal of high energy physics/The journal of high energy physics	2,018	cc-by	16,335	Pseudorapidity distributions of charged hadrons in proton-lead collisions at √sNN = 5.02 and 8.16 TeV JHEP01(2018)045 The CMS collaboration Received: October 25, 2017 Revised: December 12, 2017 Accepted: December 27, 2017 Published: January 11, 2018 Received: October 25, 2017 Revised: December 12, 2017 Accepted: December 27, 2017 Published: January 11, 2018 Received: October 25, 2017 Revised: December 12, 2017 Accepted: December 27, 2017 Published: January 11, 2018 Open Access, Copyright CERN, for the beneﬁt of the CMS Collaboration. The CMS collaboration E-mail: cms-publication-committee-chair@cern.ch E-mail: cms-publication-committee-chair@cern.ch Abstract: The pseudorapidity distributions of charged hadrons in proton-lead colli- sions at nucleon-nucleon center-of-mass energies √sNN = 5.02 and 8.16 TeV are presented. The measurements are based on data samples collected by the CMS experiment at the LHC. The number of primary charged hadrons produced in non-single-diﬀractive proton- lead collisions is determined in the pseudorapidity range \|ηlab\| < 2.4. The charged- hadron multiplicity distributions are compared to the predictions from theoretical cal- culations and Monte Carlo event generators. In the center-of-mass pseudorapidity range \|ηcm\| < 0.5, the average charged-hadron multiplicity densities ⟨dNch/dηcm⟩ \|ηcm\| < 0.5 are 17.31 ± 0.01 (stat) ± 0.59 (syst) and 20.10 ± 0.01 (stat) ± 0.85(syst) at √sNN = 5.02 and 8.16 TeV, respectively. The particle densities per participant nucleon are compared to similar measurements in proton-proton, proton-nucleus, and nucleus-nucleus collisions. Keywords: Hadron-Hadron scattering (experiments), Heavy-ion collision ArXiv ePrint: 1710.09355 https://doi.org/10.1007/JHEP01(2018)045 Contents 1 Introduction 1 2 The CMS detector 2 3 Event selection 2 4 Data analysis 3 4.1 Systematic uncertainties 5 5 Results 6 6 Summary 9 The CMS collaboration 15 Contents 1 Introduction 2 The CMS detector 3 Event selection 4 Data analysis 4.1 Systematic uncertainties 5 Results 6 Summary The CMS collaboration Contents 1 Introduction 2 The CMS detector 3 Event selection 4 Data analysis 4.1 Systematic uncertainties 5 Results 6 Summary The CMS collaboration Contents 1 Introduction 1 2 The CMS detector 2 3 Event selection 2 4 Data analysis 3 4.1 Systematic uncertainties 5 5 Results 6 6 Summary 9 The CMS collaboration 15 1 2 2 3 5 6 9 15 JHEP01(2018)045 15 1 Introduction Studies of charged-hadron yields have long been a key tool for exploring perturbative and nonperturbative quantum chromodynamics (QCD) phenomena in high-energy particle and nuclear collisions [1]. Measurements in proton-lead (pPb) collisions can shed light on initial- state nuclear eﬀects in these interactions [2]. An example is the nuclear modiﬁcation of parton distribution functions (PDFs) that can be observed in measurements of hadron [3–7] and jet [8–10] production. Such measurements also provide reference data for understand- ing the hot, dense medium produced in nucleus-nucleus (AA) collisions. At the CERN LHC energies, measurements of proton-nucleus (pA) collisions allow studies of the nuclear gluon distributions and parton shadowing eﬀects at very small values (10−4–10−6) of the Bjorken x variable [2, 11]. This provides a crucial test of current theoretical approaches for high-energy QCD [11–13], and yields important constraints on phenomenological models and event generators [14–17]. The number of primary charged hadrons, Nch, is commonly characterized by its pseu- dorapidity density, dNch/dη. The pseudorapidity, η, is deﬁned as −ln[tan θ/2], where θ is the polar angle of the particle with respect to the beam axis. The center-of-mass energy dependence of dNch/dη constrains the theoretical modeling of particle production arising from hard and soft QCD processes in high-energy hadronic interactions. In the presence of the quark-gluon plasma (QGP), the hot medium produced in AA collisions, modiﬁcations of hadron production have been observed. Studying the energy dependence of the pseudo- rapidity density in diﬀerent colliding systems (proton-proton (pp), pA, AA), for both total inelastic and non-single-diﬀractive (NSD) [18–20] collision processes, improves our under- standing of these modiﬁcations in the AA case by identifying nuclear eﬀects present in the initial state. Monte Carlo (MC) event generators, which reproduce the main characteristics – 1 – of experimental results from hadronic collisions at lower energies, can provide predictions for the energy dependence of hadron production using diﬀerent implementations of QCD eﬀects [21]. In this paper, measurements of dNch/dηlab (where the pseudorapidity is measured in the laboratory frame) in the range \|ηlab\| < 2.4 are reported for NSD events in pPb collisions delivered by the LHC in 2016 at √sNN = 5.02 and 8.16 TeV. 1 Introduction Following earlier analyses in pp collisions at √s = 0.9–13 TeV [22–25] and in lead-lead collisions at √sNN = 2.76 TeV [26], Nch is restricted to “primary” charged hadrons, deﬁned to include prompt hadrons as well as decay products of all particles with proper decay length cτ < 1 cm, where τ is the proper lifetime of the particle and c is the velocity of light in vacuum. Contributions from prompt leptons and decay products of longer-lived particles and secondary interactions are excluded. For √sNN = 5.02 (8.16) TeV, the beam energies per nucleon were 4 (6.5) TeV and 1.58 (2.56) TeV for the proton and lead nucleus, respectively. Because the beam energies were asymmetric and the proton was going in the positive ηlab direction, massless particles emitted at midrapidity in the nucleon-nucleon center-of-mass, ηcm = 0, will be detected at ηlab = 0.465. Results are compared to predictions from the KLN model [11], as well as the Epos LHC (v3400) [17, 27], Hijing [14] (versions 1.3 [15] and 2.1 [12]), and Dpmjet- III [16] MC event generators. The √sNN dependence of dNch/dηcm in the region ηcm ≈0 is also presented. JHEP01(2018)045 2 The CMS detector The central feature of the CMS apparatus is a superconducting solenoid of 6 m internal diameter. Within the solenoid volume are a silicon pixel and strip tracker, a lead tungstate crystal electromagnetic calorimeter, and a brass and scintillator hadron calorimeter, each composed of a barrel and two endcap sections. The silicon tracker measures charged par- ticles within the range \|ηlab\| < 2.5. It consists of 1440 silicon pixel detector modules. The barrel region of the pixel detector consists of three layers, which are very close to the beam line. They are located at average radii of 4.3, 7.2, and 11.0 cm, and provide excellent posi- tion resolution with their 150×100 µm pixels. The forward hadron (HF) calorimeter uses steel as an absorber and quartz ﬁbers as the sensitive material. It consists of two halves, each located 11.2 m from the interaction region, and together they provide coverage in the range 3.0 < \|ηlab\| < 5.2. The beam pickup for timing (BPTX) devices were used to trigger the detector readout. They are located around the beam pipe at a distance of 175 m on either side of the interaction point (IP) and are designed to provide precise information on the LHC bunch structure and the timing of the incoming beams. Muons are detected in gas-ionization chambers embedded in the steel ﬂux-return yoke outside the solenoid. A more detailed description of the CMS detector, together with a deﬁnition of the coordinate system used and the relevant kinematic variables, can be found in ref. [28]. 3 Event selection The data used in this analysis were taken with the beam conﬁguration in which the proton beam traveled in the negative pseudorapidity direction, and selected to contain collision – 2 – events recorded during low-intensity beam conﬁgurations, with 0.3–0.6% proton-lead inter- action probability per bunch crossing. The collision events are selected online by requiring a coincidence of signals from both BPTX devices, indicating the presence of both proton and lead ion bunches crossing the IP, and at least one energy deposit above the readout threshold of 3 GeV on either side of the HF. The oﬄine selection of NSD events is accom- plished by requiring that at least one energy deposit greater than 3 GeV is found on each of the two sides of the HF and at least one reconstructed interaction vertex is found. A study of noncolliding bunches shows that these requirements are also suﬃcient to reject all backgrounds not originating from pPb collisions. The probability to select events in the presence of a single (noncolliding) beam is found to be around 2 × 10−5 per bunch cross- ing, to be compared to the average number of collisions per bunch crossing of 4.5 × 10−3. Consequently, the contribution of background events from beam, beam halo, and cosmic ray sources to the observed yields is negligible. The total number of pPb collision events passing the selection criteria is approximately 420 thousand and 3 million at √sNN = 5.02 and 8.16 TeV, respectively. JHEP01(2018)045 The corrections from the detector-level oﬄine event selection to the hadron-level event deﬁnition are derived from MC simulations with the Epos generator. The MC simulations are produced with the same vertex distribution along the interaction region as observed in data. The detector response is simulated with Geant4 [29] and processed through the same event reconstruction chain as the collision data. 4 Data analysis In the presence of a magnetic ﬁeld, charged particles follow curved trajectories, perturbed mostly by multiple Coulomb scattering. The reconstructed pixel clusters (or “hits”) alone are suﬃcient to reconstruct vertices and tracks with high precision and purity. The analysis technique is based on tracklets, pairs of hits from two diﬀerent layers, and relies on the fact that for a primary charged hadron, the diﬀerences in pseudorapidity (∆η) and azimuthal angle (∆φ) between the two hits are small. This method is sensitive to charged hadrons with transverse momenta pT as low as 40 MeV/c. The primary vertex reconstruction is based on pixel hits in the ﬁrst two layers of the detector, as in ref. [26]. In the ﬁrst step, a hit from the ﬁrst layer is selected and a matching hit from the second layer is sought. If the \|∆φ\| of the hits is smaller than 0.05 (optimized to maximize the vertex reconstruction eﬃciency), the z positions of the hits (with the z axis deﬁned to be parallel to the beam axis) are extrapolated linearly and projected onto the beam axis. This procedure is repeated for every hit in the ﬁrst layer, and the projected z positions are saved as vertex candidates. The primary vertex is determined in a second step. If the magnitude of the diﬀerence between the z positions of any two vertex candidates is smaller than 0.12 cm, they are combined into a vertex cluster. The vertex cluster with the highest number of associated vertex candidates is selected as the primary vertex, and the ﬁnal vertex z position, zv, is given by the average z position of the associated vertex candidates. The typical resolution of zv is 0.02–0.04 cm, depending on the number of pixel hits. 4 Data analysis The vertex reconstruction eﬃciency is found to be high even – 3 – 0.1 − 0.05 − 0 0.05 0.1 η ∆ 3 − 10 2 − 10 1 − 10 1 Fraction of tracklets Data LHC POS E 1.3 IJING H = 8.16 TeV NN s pPb CMS a) 0.1 − 0.05 − 0 0.05 0.1 φ ∆ Fraction of tracklets 0.01 0.02 0.03 Data LHC POS E 1.3 IJING H = 8.16 TeV NN s pPb CMS b) 2 − 1 − 0 1 2 φ ∆ 2 − 10 1 − 10 Fraction of tracklets Data LHC POS E 1.3 IJING H = 8.16 TeV NN s pPb CMS c) Figure 1. The ∆η (a) and ∆φ (b, c) distributions of hit pairs for tracklets in pPb collisions at 8.16 TeV (squares) and from MC simulations with the Epos and Hijing 1.3 generators (solid lines). The statistical uncertainties are smaller than the marker sizes for all distributions shown. 0.1 − 0.05 − 0 0.05 0.1 φ ∆ Fraction of tracklets 0.01 0.02 0.03 Data LHC POS E 1.3 IJING H = 8.16 TeV NN s pPb CMS b) 2 − 1 − 0 1 2 φ ∆ 2 − 10 1 − 10 Fraction of tracklets Data LHC POS E 1.3 IJING H = 8.16 TeV NN s pPb CMS c) 0.1 − 0.05 − 0 0.05 0.1 η ∆ 3 − 10 2 − 10 1 − 10 1 Fraction of tracklets Data LHC POS E 1.3 IJING H = 8.16 TeV NN s pPb CMS a) JHEP01(2018)045 Figure 1. The ∆η (a) and ∆φ (b, c) distributions of hit pairs for tracklets in pPb collisions at 8.16 TeV (squares) and from MC simulations with the Epos and Hijing 1.3 generators (solid lines). The statistical uncertainties are smaller than the marker sizes for all distributions shown. for low-multiplicity events with few pixel hits, with around 90 (100)% eﬃciency for events with 4 (10) hits in the ﬁrst layer. The tracklet reconstruction follows a separate algorithm from the vertex reconstruc- tion. There is no requirement on the ∆φ of the hits. Instead, a hit on a given layer is paired with the hit on another layer which is closest in η (where η is measured with respect to the primary vertex) and these two hits form a tracklet. No hit can be used more than once. 4 Data analysis No selection is applied on the hit quality or charge, such that the analysis is rather insensitive to the accuracy of the simulation of pixel cluster charge. Three diﬀerent types of tracklets can be reconstructed, corresponding to diﬀerent combinations of the three pixel detector layers: 1+2, 1+3, and 2+3. The reconstruction eﬃciency, acceptance, fraction of back- ground hits, and sensitivity to particle pT is diﬀerent for each type of tracklet. This serves as a consistency check for the analysis, and reduces systematic biases in the measurement. Figures 1(a) and (b) show the ∆η and ∆φ distributions of reconstructed hit pairs for tracklets in data and simulation. To suppress the combinatorial background, while still including most particles in the analysis, only tracklets with \|∆η\| < 0.1 are considered “signal”. In this kinematic region, there is good agreement between data and simulations with the Epos generator, indicating that the pT distributions of both hard and soft particles in data are described well by this MC generator. The Hijing generator, used in this analysis for systematic studies, gives a poorer description of the distributions, especially for ∆φ . Tracklets corresponding to charged hadrons that originate from the primary vertex have small but nonzero ∆φ due to the magnetic ﬁeld in the detector, while background tracklets from uncorrelated pixel hits form a roughly ﬂat ∆φ spectrum over the entire ∆φ range, as shown in ﬁgure 1(c), where the abscissa is extended to \|∆φ\| < 2. Hence, a sideband region deﬁned by 1 < \|∆φ\| < 2 is used to estimate the background fraction, which is then subtracted from the signal region (\|∆φ\| < 1) to obtain the uncorrected dNch/dηlab [26]. The background estimation and subtraction is performed as a function of ηlab, zv, and tracklet multiplicity. Typical values of the estimated background fraction in the signal region in data increase with \|ηlab\| from 10–25%. The ηlab range is restricted to \|ηlab\| < 2.4 to avoid a large acceptance correction. – 4 – The ﬁnal results need to be corrected for contributions from decaying particles with cτ > 1 cm, particles created in secondary interactions, and prompt leptons. The contribu- tion of these particles to dNch/dηlab is removed using a correction factor found using MC simulations. In addition, corrections are needed to account for the selection, eﬃciency, and acceptance of reconstructed tracklets, as well as trigger and vertexing eﬃciencies. 4 Data analysis The ac- ceptance factor includes the extrapolation down to pT = 0 GeV/c. Correction factors (with a typical total of <15%) are derived using the Epos event generator as a reference and are calculated as a function of ηlab, zv, and tracklet multiplicity, as was done in ref. [26]. To account for the diﬀerences between data and MC in the pixel detector geometry and its alignment conditions, an additional correction is applied as a function of ηlab and zv. This correction is obtained by taking the ratio between data and simulation of the geometrical distribution of tracklets in (ηlab, zv) intervals. The size of this correction ranges from 0 to 5%, where the largest correction factors are associated with the presence of inactive tracker modules. JHEP01(2018)045 4.1 Systematic uncertainties The systematic uncertainties in the ﬁnal results arise from several sources: detector mis- alignment, pixel hit reconstruction ineﬃciency, pixel cluster splitting, background model- ing, selection of signal and sideband regions, parametrization of the correction factors, and the NSD event selection. For each source of uncertainty, that part of the analysis procedure is varied independently and the change is propagated to the ﬁnal results. The individual contributions are then summed in quadrature to give the total systematic uncertainty. To estimate the uncertainty from detector misalignment, each pixel hit is oﬀset by a small distance corresponding to the uncertainty in the alignment of the pixel detectors. The eﬀects of pixel hit reconstruction ineﬃciency are studied by randomly excluding 0.5% of the pixel hits from the analysis. The 0.5% ineﬃciency value is determined by studying tracklets reconstructed from pixel hits in layers 1 and 3, and taking the double ratio in data and simulation of the fraction of tracklets that have no corresponding hit in layer 2. Pixel cluster splitting refers to the situation where the charge deposit in the pixel detector from a single charged particle is reconstructed as two separate pixel clusters. Its eﬀect on the measurement is estimated by randomly splitting pixel clusters with a probability of 1.2%, as determined by previous studies [22]. The contributions from the above three sources are all below 1%. The remaining uncertainties are associated with the MC correction factors. Additional pixel hits, randomly sampled from the hit distributions in data, are added such that the ∆φ sidebands match between data and MC. The percentage of additional pixel hits needed is less than 5%. The variations observed compared to the nominal results are around 1.5– 2.5%. The signal and sideband regions are also varied to \|∆φ\| < 1.5 and 1.5 < \|∆φ\| < 3.0, respectively. A variation of 0.6–1.5% is found as compared to the nominal setting, which is propagated as a systematic uncertainty. 4.1 Systematic uncertainties Diﬀerent multiplicity variables are used to parametrize the correction factors, in addition to the background-subtracted tracklets variable used for the nominal results: number of tracklets (before background subtraction), number of pixel hits in the ﬁrst pixel layer used (layer 1 for tracklet type 1+2 and 1+3, and – 5 – Source Uncertainty [%] 5.02 TeV 8.16 TeV Data and simulation Detector misalignment 0.2 – 1.0 0.2 – 1.0 Pixel hit reconstruction ineﬃciency 1.0 1.0 Pixel cluster splitting 0.3 – 0.8 0.3 – 0.6 MC corrections Background modeling 1.3 – 3.2 1.5 – 2.5 Signal and sideband region selection 0.5 – 1.5 0.6 – 1.5 Choice of parametrization variable 1.6 – 2.5 1.5 – 3.5 NSD selection 1.2 1.2 Total uncertainty 3.0 – 4.3 3.7 – 4.6 JHEP01(2018)045 Table 1. Summary of the systematic uncertainties from various sources, for pPb collisions at 5.02 and 8.16 TeV. The range of values indicates the minimum and maximum uncertainties across the ηlab range. layer 2 for tracklet type 2+3). The maximum deviation in each ηlab interval, 1.5–2.5%, is quoted as an uncertainty. An uncertainty is assigned for the selection of NSD events. The fraction of the single-diﬀractive events removed by the event selection, as determined from the Epos generator, is 16% when the tracklet multiplicity in the event is less than 10, and falls quickly to 0% with increasing tracklet multiplicity. This fraction is varied from 0% to twice the nominal value, and the maximum deviation from the ﬁnal results, 1.2%, is quoted as the uncertainty. A summary of the systematic uncertainties for the measurements at 5.02 and 8.16 TeV is shown in table 1. layer 2 for tracklet type 2+3). The maximum deviation in each ηlab interval, 1.5–2.5%, is quoted as an uncertainty. An uncertainty is assigned for the selection of NSD events. The fraction of the single-diﬀractive events removed by the event selection, as determined from the Epos generator, is 16% when the tracklet multiplicity in the event is less than 10, and falls quickly to 0% with increasing tracklet multiplicity. This fraction is varied from 0% to twice the nominal value, and the maximum deviation from the ﬁnal results, 1.2%, is quoted as the uncertainty. A summary of the systematic uncertainties for the measurements at 5.02 and 8.16 TeV is shown in table 1. 5 Results Pseudorapidity density distributions of charged hadrons in the region \|ηlab\| < 2.4 for NSD pPb collisions are shown in ﬁgure 2. The distributions shown are the average of the measured distributions from the three types of tracklets (1+2, 1+3, and 2+3), which are consistent with each other within 3%. A clear diﬀerence in the particle densities between the lead ion (ηlab < 0) and the proton (ηlab > 0) beam directions is observed. The measured dNch/dηlab distribution at 5.02 TeV agrees with the measurement by the ALICE Collaboration [30]. The multiplicities at 8.16 TeV are signiﬁcantly higher than those at 5.02 TeV. Figure 3 shows a comparison between the measurement at 8.16 TeV and theoretical calculations from the Hijing (versions 1.3 and 2.1), Epos LHC (v3400), and Dpmjet-III MC generators, and the KLN model. The Hijing and Epos generators were tuned to data from RHIC and the LHC, respectively. Calculations from Hijing 2.1, a two-component model that combines perturbative QCD descriptions of hard parton scatterings with a string excitation model for soft interactions, agree with the experimental data in the re- gion −0.5 < ηlab < 1.5 when the nuclear modiﬁcation of the initial parton distributions (shadowing) is included in the calculation. The Hijing 1.3 calculation overpredicts the par- ticle density because it has an older implementation of the gluon shadowing eﬀects. The – 6 – Figure 2. Distributions of the pseudorapidity density of charged hadrons in the region \|ηlab\| < 2.4 in NSD pPb collisions at √sNN = 5.02 (open squares) and 8.16 TeV (full squares). The measurement at 5.02 TeV by the ALICE Collaboration [30] is shown as ﬁlled circles. The shaded boxes indicate the systematic uncertainties which, in the case of the CMS data, are correlated between the two beam energies. The proton beam goes in the positive ηlab direction. JHEP01(2018)045 Figure 2. Distributions of the pseudorapidity density of charged hadrons in the region \|ηlab\| < 2.4 in NSD pPb collisions at √sNN = 5.02 (open squares) and 8.16 TeV (full squares). The measurement at 5.02 TeV by the ALICE Collaboration [30] is shown as ﬁlled circles. The shaded boxes indicate the systematic uncertainties which, in the case of the CMS data, are correlated between the two beam energies. The proton beam goes in the positive ηlab direction. 5 Results importance of shadowing can be assessed using the comparison of Hijing 2.1 simulations generated with and without this physics process included. The results are signiﬁcantly higher than the data when shadowing is disabled. The KLN parton saturation model combines Glauber modeling of the collision geometry with a simple model for the unin- tegrated parton distributions that accounts for the existence of a saturation momentum scale [31, 32]. It describes the particle density accurately for \|ηlab\| < 1 but overall shows a steeper increase of density versus ηlab than observed in the data, similar to what was observed in the comparisons to the PHOBOS deuteron-gold (dAu) data at 200 GeV [33] and ALICE data at 5.02 TeV [30]. The Dpmjet-III generator, commonly used in the de- scription of cosmic ray, nucleon-nucleon, and nucleon-nucleus interactions, is based on the dual parton model [34], which generates soft hadronic interactions by considering the ex- pansion of nonperturbative QCD in the limit where the number of color and ﬂavor states are large [35]. This generator is found to predict both a steeper increase versus ηlab and a higher particle density over the measured ηlab interval. The Epos generator, which is based on the Gribov-Regge theory and includes the eﬀect of collective hadronization in hadron-hadron scattering, was found to describe pp data up to 13 TeV [25], but underpre- dicts the observed dNch/dηlab by a roughly constant factor over the entire measured range for pPb at 8.16 TeV. One of the main goals of the heavy ion studies is to understand hadron production in the extremely dense medium formed in AA collisions. One way to approach this goal is to consider a direct comparison between the charged-hadron multiplicity density in minimum – 7 – Figure 3. Distributions of the pseudorapidity density of charged hadrons in the region \|ηlab\| < 2.4 for NSD pPb collisions at 8.16 TeV (squares) compared to predictions from the MC event generators Epos LHC [17, 27] (v3400), Hijing [14] (versions 1.3 [15] and 2.1 [12]), and Dpmjet-III [16], as well as from the KLN model [11]. The shaded boxes around the data points indicate their systematic uncertainties. The proton beam goes in the positive ηlab direction. JHEP01(2018)045 Figure 3. 5 Results Distributions of the pseudorapidity density of charged hadrons in the region \|ηlab\| < 2.4 for NSD pPb collisions at 8.16 TeV (squares) compared to predictions from the MC event generators Epos LHC [17, 27] (v3400), Hijing [14] (versions 1.3 [15] and 2.1 [12]), and Dpmjet-III [16], as well as from the KLN model [11]. The shaded boxes around the data points indicate their systematic uncertainties. The proton beam goes in the positive ηlab direction. bias pp and pA collisions, reference systems for particle production in the absence of a QGP, and central AA collisions (the most extreme type of collisions with the highest particle multiplicities). The comparison is made by dividing dNch/dηcm by the number of participating nucleons, Npart, determined by a Glauber model calculation [4, 36]. This normalization is the one assumed in two-component models (e.g. Hijing) for the bulk of the particle production. In order to compare particle production in pPb collisions to that in symmetric collision systems such as pp or AA, the rapidity shift due to the asymmetric beam energies must be taken into account. The average charged-hadron multiplicity density at midrapidity in the center-of-mass frame, ⟨dNch/dηcm⟩\|\|ηcm\|<0.5, in pPb collisions is calculated by inte- grating the data in the interval −0.035 < ηlab < 0.965, corresponding to \|ηcm\| < 0.5 for massless particles. A correction is applied to account for the massless assumption entering the calculation of the pseudorapidity shift: 0.1 and 0.2% for the 5.02 TeV and 8.16 TeV analyses, respectively, as obtained from the Epos generator. The 1% variation in the results, obtained when this correction is evaluated from Hijing, is quoted as an addi- tional uncertainty for the ⟨dNch/dηcm⟩\|\|ηcm\|<0.5 results. In the range \|ηcm\| < 0.5, values of 17.31 ± 0.01 (stat) ± 0.59 (syst) and 20.10 ± 0.01 (stat) ± 0.85 (syst) are obtained for pPb collisions at √sNN = 5.02 and 8.16 TeV, respectively. Figure 4 shows the dependence of normalized dNch/dηcm on the collision energy for various collision systems and event selections. 5 Results The NSD pA results are found to be lower than those from central AA collisions [26, 37–50] (s0.158 NN dependence) and NSD pp collisions – 8 – 10 2 10 3 10 4 10 (GeV) NN s 0 1 2 3 4 5 6 7 〉 part N 〈 / 0 ≈ cm η 〉 cm η d / h c N d〈 CMS ) NSD p pp (p CMS ALICE CDF UA5 UA1 STAR 0.110 NN s ∝ ) Inel. p pp (p CMS ALICE UA5 PHOBOS ISR 0.103 NN s ∝ Central AA CMS ALICE ATLAS BRAHMS PHENIX STAR PHOBOS NA50 0.158 NN s ∝ pA NSD pPb CMS pPb ALICE dAu PHOBOS dAu PHENIX (Central) pA Inel. pPb E178 pAu NA35 Figure 4. Comparison of the measured dNch/dηcm at midrapidity, scaled by the number of par- ticipating nucleons (Npart) in pPb [30, 51], pAu [52], dAu [33, 48, 53] and central heavy ion colli- sions [26, 37–50], as well as NSD [22, 23, 50, 54–57] and inelastic [25, 37, 56, 58, 59] pp collisions. The AA data points at √sNN = 2.76 TeV have been shifted horizontally for visibility. The dashed curves, included to guide the eye, correspond to a ﬁt to the data points using the same functional form as in refs. [46, 59]. 10 2 10 3 10 4 10 (GeV) NN s 0 1 2 3 4 5 6 7 〉 part N 〈 / 0 ≈ cm η 〉 cm η d / h c N d〈 CMS ) NSD p pp (p CMS ALICE CDF UA5 UA1 STAR 0.110 NN s ∝ ) Inel. p pp (p CMS ALICE UA5 PHOBOS ISR 0.103 NN s ∝ Central AA CMS ALICE ATLAS BRAHMS PHENIX STAR PHOBOS NA50 0.158 NN s ∝ pA NSD pPb CMS pPb ALICE dAu PHOBOS dAu PHENIX (Central) pA Inel. pPb E178 pAu NA35 JHEP01(2018)045 Figure 4. Comparison of the measured dNch/dηcm at midrapidity, scaled by the number of par- ticipating nucleons (Npart) in pPb [30, 51], pAu [52], dAu [33, 48, 53] and central heavy ion colli- sions [26, 37–50], as well as NSD [22, 23, 50, 54–57] and inelastic [25, 37, 56, 58, 59] pp collisions. The AA data points at √sNN = 2.76 TeV have been shifted horizontally for visibility. 5 Results The dashed curves, included to guide the eye, correspond to a ﬁt to the data points using the same functional form as in refs. [46, 59]. (s0.110 NN dependence) at similar center-of-mass energies, but coincide with the trend observed in inelastic pp collisions (s0.103 NN dependence). While the diﬀerence between the NSD pp and pA results could be attributed to non-QGP nuclear eﬀects, the similarity between the NSD pA and total inelastic pp is yet to be understood. Acknowledgments We congratulate our colleagues in the CERN accelerator departments for the excellent performance of the LHC and thank the technical and administrative staﬀs at CERN and at other CMS institutes for their contributions to the success of the CMS eﬀort. In ad- dition, we gratefully acknowledge the computing centers and personnel of the Worldwide LHC Computing Grid for delivering so eﬀectively the computing infrastructure essential to our analyses. Finally, we acknowledge the enduring support for the construction and operation of the LHC and the CMS detector provided by the following funding agencies: BMWFW and FWF (Austria); FNRS and FWO (Belgium); CNPq, CAPES, FAPERJ, and FAPESP (Brazil); MES (Bulgaria); CERN; CAS, MoST, and NSFC (China); COL- CIENCIAS (Colombia); MSES and CSF (Croatia); RPF (Cyprus); SENESCYT (Ecuador); MoER, ERC IUT, and ERDF (Estonia); Academy of Finland, MEC, and HIP (Finland); CEA and CNRS/IN2P3 (France); BMBF, DFG, and HGF (Germany); GSRT (Greece); OTKA and NIH (Hungary); DAE and DST (India); IPM (Iran); SFI (Ireland); INFN (Italy); MSIP and NRF (Republic of Korea); LAS (Lithuania); MOE and UM (Malaysia); BUAP, CINVESTAV, CONACYT, LNS, SEP, and UASLP-FAI (Mexico); MBIE (New Zealand); PAEC (Pakistan); MSHE and NSC (Poland); FCT (Portugal); JINR (Dubna); MON, RosAtom, RAS, RFBR and RAEP (Russia); MESTD (Serbia); SEIDI, CPAN, PCTI and FEDER (Spain); Swiss Funding Agencies (Switzerland); MST (Taipei); ThEPCenter, IPST, STAR, and NSTDA (Thailand); TUBITAK and TAEK (Turkey); NASU and SFFR (Ukraine); STFC (United Kingdom); DOE and NSF (U.S.A.). JHEP01(2018)045 ( ) ( ) ( ) Individuals have received support from the Marie-Curie program and the European Research Council and Horizon 2020 Grant, contract No. 675440 (European Union); the Leventis Foundation; the A. P. 6 Summary The pseudorapidity distributions of primary charged hadrons have been measured by the CMS experiment at the LHC in proton-lead collisions at √sNN = 5.02 and 8.16 TeV. Based on pairs of pixel clusters from two diﬀerent layers of the barrel region of the CMS pixel de- tector, the distributions have been obtained for NSD pPb events at both collision energies. The measured dNch/dηlab distribution at 5.02 TeV is consistent with published results by the ALICE Collaboration. At 8.16 TeV, the measured dNch/dηlab distribution is higher than the predictions of Epos LHC, but signiﬁcantly lower than the predictions from the Hijing 1.3 and Dpmjet-III event generators. At ηlab ≈0, the measured distributions are in good agreement with calculations from the KLN gluon saturation model and predic- tions from the Hijing 2.1 event generator with the eﬀects of gluon shadowing included. The charged-hadron multiplicity densities in the nucleon-nucleon center-of-mass frame, ⟨dNch/dηcm⟩\|\|ηcm\|<0.5, are 17.31±0.01 (stat)±0.59 (syst) and 20.10±0.01 (stat)±0.85 (syst) at √sNN = 5.02 and 8.16 TeV, respectively. When comparing the average charged-particle – 9 – density per participant nucleon for pp, pA, and AA collisions as a function of collision energy, the pA results are found to be below those in central AA collisions and NSD pp collisions, but coincide with the trend seen in inelastic pp collisions. These results represent the ﬁrst measurement of hadron production at this new center-of-mass energy frontier in nuclear collisions, and provide constraints for the understanding of nonperturbative QCD eﬀects in high-energy nuclear collisions. Acknowledgments Sloan Foundation; the Alexander von Humboldt Founda- tion; the Belgian Federal Science Policy Oﬃce; the Fonds pour la Formation `a la Recherche dans l’Industrie et dans l’Agriculture (FRIA-Belgium); the Agentschap voor Innovatie door Wetenschap en Technologie (IWT-Belgium); the Ministry of Education, Youth and Sports (MEYS) of the Czech Republic; the Council of Science and Industrial Research, India; the HOMING PLUS program of the Foundation for Polish Science, coﬁnanced from European Union, Regional Development Fund, the Mobility Plus program of the Min- istry of Science and Higher Education, the National Science Center (Poland), contracts Harmonia 2014/14/M/ST2/00428, Opus 2014/13/B/ST2/02543, 2014/15/B/ST2/03998, and 2015/19/B/ST2/02861, Sonata-bis 2012/07/E/ST2/01406; the National Priorities Re- search Program by Qatar National Research Fund; the Programa Severo Ochoa del Prin- cipado de Asturias; the Thalis and Aristeia programs coﬁnanced by EU-ESF and the – 10 – Greek NSRF; the Rachadapisek Sompot Fund for Postdoctoral Fellowship, Chulalongkorn University and the Chulalongkorn Academic into Its 2nd Century Project Advancement Project (Thailand); the Welch Foundation, contract C-1845; and the Weston Havens Foun- dation (U.S.A.). Open Access. This article is distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits any use, distribution and reproduction in any medium, provided the original author(s) and source are credited. References JHEP01(2018)045 [1] W. Busza, Review of experimental data on hadron-nucleus collisions at high-energies, Acta Phys. Polon. B 8 (1977) 333 [INSPIRE]. [2] C.A. Salgado et al., Proton-nucleus collisions at the LHC: scientiﬁc opportunities and requirements, J. Phys. G 39 (2012) 015010 [arXiv:1105.3919] [INSPIRE]. [3] CMS collaboration, Nuclear eﬀects on the transverse momentum spectra of charged particles in pPb collisions at √sNN = 5.02 TeV, Eur. Phys. J. C 75 (2015) 237 [arXiv:1502.05387] [INSPIRE]. [4] CMS collaboration, Charged-particle nuclear modiﬁcation factors in PbPb and pPb collisions at √sNN = 5.02 TeV, JHEP 04 (2017) 039 [arXiv:1611.01664] [INSPIRE]. [5] CMS collaboration, Study of B meson production in p+Pb collisions at √sNN = 5.02 TeV using exclusive hadronic decays, Phys. Rev. Lett. 116 (2016) 032301 [arXiv:1508.06678] [INSPIRE]. [6] ALICE collaboration, D-meson production in p-Pb collisions at √sNN = 5.02 TeV and in pp collisions at √s = 7 TeV, Phys. Rev. C 94 (2016) 054908 [arXiv:1605.07569] [INSPIRE]. [7] CMS collaboration, Measurement of prompt and nonprompt J/ψ production in pp and pPb collisions at √sNN = 5.02 TeV, Eur. Phys. J. C 77 (2017) 269 [arXiv:1702.01462] [INSPIRE]. [8] CMS collaboration, Transverse momentum spectra of inclusive b jets in pPb collisions at √sNN = 5.02 TeV, Phys. Lett. B 754 (2016) 59 [arXiv:1510.03373] [INSPIRE]. [9] CMS collaboration, Measurement of inclusive jet production and nuclear modiﬁcations in pPb collisions at √sNN = 5.02 TeV, Eur. Phys. J. C 76 (2016) 372 [arXiv:1601.02001] [INSPIRE]. [10] CMS collaboration, Studies of dijet transverse momentum balance and pseudorapidity distributions in pPb collisions at √sNN = 5.02 TeV, Eur. Phys. J. C 74 (2014) 2951 [arXiv:1401.4433] [INSPIRE]. [11] A. Dumitru, D.E. Kharzeev, E.M. Levin and Y. Nara, Gluon saturation in pA collisions at the LHC: KLN model predictions for hadron multiplicities, Phys. Rev. C 85 (2012) 044920 [arXiv:1111.3031] [INSPIRE]. [12] R. Xu, W.-T. Deng and X.-N. Wang, Nuclear modiﬁcation of high-pT hadron spectra in p+A collisions at LHC, Phys. Rev. C 86 (2012) 051901 [arXiv:1204.1998] [INSPIRE]. [13] J.L. Albacete, A. Dumitru, H. Fujii and Y. Nara, CGC predictions for p + Pb collisions at the LHC, Nucl. Phys. A 897 (2013) 1 [arXiv:1209.2001] [INSPIRE]. – 11 – [14] X.-N. Wang and M. Gyulassy, HIJING: a Monte Carlo model for multiple jet production in pp, pA and AA collisions, Phys. Rev. D 44 (1991) 3501 [INSPIRE]. [15] M. Gyulassy and X.-N. References Wang, HIJING 1.0: A Monte Carlo program for parton and particle production in high-energy hadronic and nuclear collisions, Comput. Phys. Commun. 83 (1994) 307 [nucl-th/9502021] [INSPIRE]. [16] S. Roesler, R. Engel and J. Ranft, The Monte Carlo event generator DPMJET-III, in the proceedings of the Advanced Monte Carlo for radiation physics, particle transport simulation and applications conference (MC2000), October 23–26, Lisbon, Portugal (2000). [17] T. Pierog, I. Karpenko, J.M. Katzy, E. Yatsenko and K. Werner, EPOS LHC: test of collective hadronization with data measured at the CERN Large Hadron Collider, Phys. Rev. C 92 (2015) 034906 [arXiv:1306.0121] [INSPIRE]. JHEP01(2018)045 [18] C. Halliwell et al., Energy dependence of the pseudorapidity distributions in proton-nucleus collisions between 50 GeV/c and 200 GeV/c., Phys. Rev. Lett. 39 (1977) 1499 [INSPIRE]. [19] W. Kittel and E.A. De Wolf, Soft multihadron dynamics, World Scientiﬁc, Singapore (2005 [20] CMS collaboration, Charged particle multiplicities in pp interactions at √s = 0.9, 2.36 and 7 TeV, JHEP 01 (2011) 079 [arXiv:1011.5531] [INSPIRE]. [21] D. d’Enterria, R. Engel, T. Pierog, S. Ostapchenko and K. Werner, Constraints from the ﬁrst LHC data on hadronic event generators for ultra-high energy cosmic-ray physics, Astropart. Phys. 35 (2011) 98 [arXiv:1101.5596] [INSPIRE]. [22] CMS collaboration, Transverse momentum and pseudorapidity distributions of charged hadrons in pp collisions at √s = 0.9 and 2.36 TeV, JHEP 02 (2010) 041 [arXiv:1002.0621] [INSPIRE]. [23] CMS collaboration, Transverse-momentum and pseudorapidity distributions of charged hadrons in pp collisions at √s = 7 TeV, Phys. Rev. Lett. 105 (2010) 022002 [arXiv:1005.3299] [INSPIRE]. [24] CMS and TOTEM collaborations, Measurement of pseudorapidity distributions of charged particles in proton-proton collisions at √s = 8 TeV by the CMS and TOTEM experiments, Eur. Phys. J. C 74 (2014) 3053 [arXiv:1405.0722] [INSPIRE]. [25] CMS collaboration, Pseudorapidity distribution of charged hadrons in proton-proton collisions at √s = 13 TeV, Phys. Lett. B 751 (2015) 143 [arXiv:1507.05915] [INSPIRE]. [26] CMS collaboration, Dependence on pseudorapidity and centrality of charged hadron production in PbPb collisions at a nucleon-nucleon centre-of-mass energy of 2.76 TeV, JHEP 08 (2011) 141 [arXiv:1107.4800] [INSPIRE]. [27] K. Werner, F.-M. Liu and T. Pierog, Parton ladder splitting and the rapidity dependence of transverse momentum spectra in deuteron-gold collisions at RHIC, Phys. Rev. C 74 (2006) 044902 [hep-ph/0506232] [INSPIRE]. [28] CMS collaboration, The CMS experiment at the CERN LHC, 2008 JINST 3 S08004 [INSPIRE]. [29] GEANT4 collaboration, S. Agostinelli et al., GEANT4 — a simulation toolkit, Nucl. Instrum. Meth. References A 506 (2003) 250 [INSPIRE]. [30] ALICE collaboration, Pseudorapidity density of charged particles in p + Pb collisions at √sNN = 5.02 TeV, Phys. Rev. Lett. 110 (2013) 032301 [arXiv:1210.3615] [INSPIRE]. – 12 – [31] D. Kharzeev and E. Levin, Manifestations of high density QCD in the ﬁrst RHIC data, Phys. Lett. B 523 (2001) 79 [nucl-th/0108006] [INSPIRE]. [32] D. Kharzeev, E. Levin and M. Nardi, The onset of classical QCD dynamics in relativistic heavy ion collisions, Phys. Rev. C 71 (2005) 054903 [hep-ph/0111315] [INSPIRE]. [33] PHOBOS collaboration, B.B. Back et al., Pseudorapidity distribution of charged particles in d + Au collisions at √sNN = 200 GeV, Phys. Rev. Lett. 93 (2004) 082301 [nucl-ex/0311009] [INSPIRE]. [34] A. Capella, U. Sukhatme, C.-I. Tan and J. Tran Thanh Van, Dual parton model, Phys. Rept. 236 (1994) 225 [INSPIRE]. [35] G. Veneziano, Regge intercepts and unitarity in planar dual models, Nucl. Phys. B 74 (1974) 365 [INSPIRE]. JHEP01(2018)045 [36] M.L. Miller, K. Reygers, S.J. Sanders and P. Steinberg, Glauber modeling in high energy nuclear collisions, Ann. Rev. Nucl. Part. Sci. 57 (2007) 205 [nucl-ex/0701025] [INSPIRE]. [37] PHOBOS collaboration, B. Alver et al., Phobos results on charged particle multiplicity and pseudorapidity distributions in Au+Au, Cu+Cu, d+Au and p+p collisions at ultra-relativistic energies, Phys. Rev. C 83 (2011) 024913 [arXiv:1011.1940] [INSPIRE]. [38] NA50 collaboration, M.C. Abreu et al., Scaling of charged particle multiplicity in Pb Pb collisions at SPS energies, Phys. Lett. B 530 (2002) 43 [INSPIRE]. [39] STAR collaboration, C. Adler et al., Multiplicity distribution and spectra of negatively charged hadrons in Au+Au collisions at √sNN = 130 GeV, Phys. Rev. Lett. 87 (2001) 112303 [nucl-ex/0106004] [INSPIRE]. [40] BRAHMS collaboration, I.G. Bearden et al., Charged particle densities from Au+Au collisions at √sNN = 130 GeV, Phys. Lett. B 523 (2001) 227 [nucl-ex/0108016] [INSPIRE]. [41] BRAHMS collaboration, I.G. Bearden et al., Pseudorapidity distributions of charged particles from Au+Au collisions at the maximum RHIC energy, Phys. Rev. Lett. 88 (2002) 202301 [nucl-ex/0112001] [INSPIRE]. [42] PHENIX collaboration, K. Adcox et al., Centrality dependence of charged particle multiplicity in Au-Au collisions at √sNN = 130 GeV, Phys. Rev. Lett. 86 (2001) 3500 [nucl-ex/0012008] [INSPIRE]. [43] PHOBOS collaboration, B.B. Back et al., Charged particle multiplicity near mid-rapidity in central Au+Au collisions at √s = 56 A/GeV and 130 A/GeV, Phys. Rev. Lett. 85 (2000) 3100 [hep-ex/0007036] [INSPIRE]. [44] PHOBOS collaboration, B.B. References Back et al., Charged particle pseudorapidity density distributions from Au+Au collisions at √sNN = 130 GeV, Phys. Rev. Lett. 87 (2001) 102303 [nucl-ex/0106006] [INSPIRE]. [45] B.B. Back et al., The Signiﬁcance of the fragmentation region in ultrarelativistic heavy ion collisions, Phys. Rev. Lett. 91 (2003) 052303 [nucl-ex/0210015] [INSPIRE]. [46] ALICE collaboration, Charged-particle multiplicity density at mid-rapidity in central PbPb collisions at √sNN = 2.76 TeV, Phys. Rev. Lett. 105 (2010) 252301 [arXiv:1011.3916] [INSPIRE]. [47] ATLAS collaboration, Measurement of the centrality dependence of the charged particle pseudorapidity distribution in lead-lead collisions at √sNN = 2.76 TeV with the ATLAS detector, Phys. Lett. B 710 (2012) 363 [arXiv:1108.6027] [INSPIRE]. – 13 – [48] PHENIX collaboration, A. Adare et al., Transverse energy production and charged-particle multiplicity at midrapidity in various systems from √sNN = 7.7 to 200 GeV, Phys. Rev. C 93 (2016) 024901 [arXiv:1509.06727] [INSPIRE]. [49] STAR collaboration, B.I. Abelev et al., Identiﬁed particle production, azimuthal anisotropy and interferometry measurements in Au+Au collisions at √sNN = 9.2 GeV, Phys. Rev. C 81 (2010) 024911 [arXiv:0909.4131] [INSPIRE]. [50] STAR collaboration, B.I. Abelev et al., Systematic measurements of identiﬁed particle spectra in pp, d+Au and Au+Au collisions from STAR, Phys. Rev. C 79 (2009) 034909 [arXiv:0808.2041] [INSPIRE]. [51] J.E. Elias et al., An experimental study of multiparticle production in hadron-nucleus interactions at high-energy, Phys. Rev. D 22 (1980) 13 [INSPIRE]. JHEP01(2018)045 JHEP01(2018)045 [52] NA35 collaboration, T. Alber et al., Charged particle production in proton, deuteron, oxygen and sulphur nucleus collisions at 200 GeV per nucleon, Eur. Phys. J. C 2 (1998) 643 [hep-ex/9711001] [INSPIRE]. [53] PHENIX collaboration, C. Aidala et al., Measurements of azimuthal anisotropy and charged-particle multiplicity in d+Au collisions at √sNN = 200, 62.4, 39 and 19.6 GeV, Phys. Rev. C 96 (2017) 064905 [arXiv:1708.06983] [INSPIRE]. [54] UA1 collaboration, C. Albajar et al., A study of the general characteristics of p¯p collisions at √s = 0.2 TeV to 0.9 TeV, Nucl. Phys. B 335 (1990) 261 [INSPIRE]. [55] UA5 collaboration, K. Alpgard et al., Particle multiplicities in ¯pp interactions at √s = 540 GeV, Phys. Lett. 121B (1983) 209 [INSPIRE]. [56] UA5 collaboration, G.J. Alner et al., Scaling of pseudorapidity distributions at c.m. energies up to 0.9 TeV, Z. Phys. C 33 (1986) 1 [INSPIRE]. [57] CDF collaboration, F. Abe et al., Pseudorapidity distributions of charged particles produced in ¯pp interactions at √s = 630 GeV and 1800 GeV, Phys. Rev. References D 41 (1990) 2330 [INSPIRE]. [58] Aachen-CERN-Heidelberg-Munich collaboration, W. Thome et al., Charged particle multiplicity distributions in pp collisions at ISR energies, Nucl. Phys. B 129 (1977) 365 [INSPIRE]. [59] ALICE collaboration, Charged-particle multiplicity measurement in proton-proton collisions at √s = 0.9 and 2.36 TeV with ALICE at LHC, Eur. Phys. J. C 68 (2010) 89 [arXiv:1004.3034] [INSPIRE]. – 14 – The CMS collaboration Vrije Universiteit Brussel, Brussel, Belgium S. Abu Zeid, F. Blekman, J. D’Hondt, I. De Bruyn, J. De Clercq, K. Deroover, G. Flouris, D. Lontkovskyi, S. Lowette, S. Moortgat, L. Moreels, Q. Python, K. Skovpen, S. Tavernier, W. Van Doninck, P. Van Mulders, I. Van Parijs D. Beghin, H. Brun, B. Clerbaux, G. De Lentdecker, H. Delannoy, B. Dorney, G. Fasanella, L. Favart, R. Goldouzian, A. Grebenyuk, G. Karapostoli, T. Lenzi, J. Luetic, T. Maer- schalk, A. Marinov, T. Seva, E. Starling, C. Vander Velde, P. Vanlaer, D. Vannerom, R. Yonamine, F. Zenoni, F. Zhang2 Ghent University, Ghent, Belgium Ghent University, Ghent, Belgium A. Cimmino, T. Cornelis, D. Dobur, A. Fagot, M. Gul, I. Khvastunov3, D. Poyraz, C. Roskas, S. Salva, M. Tytgat, W. Verbeke, N. Zaganidis Universit´e Catholique de Louvain, Louvain-la-Neuve, Belgium H. Bakhshiansohi, O. Bondu, S. Brochet, G. Bruno, C. Caputo, A. Caudron, P. David, S. De Visscher, C. Delaere, M. Delcourt, B. Francois, A. Giammanco, M. Komm, G. Krintiras, V. Lemaitre, A. Magitteri, A. Mertens, M. Musich, K. Piotrzkowski, L. Quertenmont, A. Saggio, M. Vidal Marono, S. Wertz, J. Zobec Yerevan Physics Institute, Yerevan, Armenia A.M. Sirunyan, A. Tumasyan Institut f¨ur Hochenergiephysik, Wien, Austria W. Adam, F. Ambrogi, E. Asilar, T. Bergauer, J. Brandstetter, E. Brondolin, M. Drag- icevic, J. Er¨o, M. Flechl, M. Friedl, R. Fr¨uhwirth1, V.M. Ghete, J. Grossmann, J. Hrubec, M. Jeitler1, A. K¨onig, N. Krammer, I. Kr¨atschmer, D. Liko, T. Madlener, I. Mikulec, E. Pree, N. Rad, H. Rohringer, J. Schieck1, R. Sch¨ofbeck, M. Spanring, D. Spitzbart, W. Waltenberger, J. Wittmann, C.-E. Wulz1, M. Zarucki JHEP01(2018)045 Institute for Nuclear Problems, Minsk, Belarus V. Chekhovsky, V. Mossolov, J. Suarez Gonzalez Universiteit Antwerpen, Antwerpen, Belgium E.A. De Wolf, D. Di Croce, X. Janssen, J. Lauwers, M. Van De Klundert, H. Van Haevermaet, P. Van Mechelen, N. Van Remortel E.A. De Wolf, D. Di Croce, X. Janssen, J. Lauwers, M. Van De Klundert, H. Va Haevermaet, P. Van Mechelen, N. Van Remortel Vrije Universiteit Brussel, Brussel, Belgium The CMS collaboration Yerevan Physics Institute, Yerevan, Armenia A.M. Sirunyan, A. Tumasyan Universit´e de Mons, Mons, Belgium N. Beliy Centro Brasileiro de Pesquisas Fisicas, Rio de Janeiro, Brazil W.L. Ald´a J´unior, F.L. Alves, G.A. Alves, L. Brito, M. Correa Martins Junior, C. Hensel, A. Moraes, M.E. Pol, P. Rebello Teles – 15 – Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil E. Belchior Batista Das Chagas, W. Carvalho, J. Chinellato4, E. Coelho, E.M. Da Costa, G.G. Da Silveira5, D. De Jesus Damiao, S. Fonseca De Souza, L.M. Huertas Guativa, H. Malbouisson, M. Melo De Almeida, C. Mora Herrera, L. Mundim, H. Nogima, L.J. Sanchez Rosas, A. Santoro, A. Sznajder, M. Thiel, E.J. Tonelli Manganote4, F. Torres Da Silva De Araujo, A. Vilela Pereira Universidade Estadual Paulista a, Universidade Federal do ABC b, S˜ao Paulo, Brazil S. Ahujaa, C.A. Bernardesa, T.R. Fernandez Perez Tomeia, E.M. Gregoresb, P.G. Mercadanteb, S.F. Novaesa, Sandra S. Padulaa, D. Romero Abadb, J.C. Ruiz Vargasa JHEP01(2018)045 Institute for Nuclear Research and Nuclear Energy of Bulgaria Academy of Sciences A. Aleksandrov, R. Hadjiiska, P. Iaydjiev, M. Misheva, M. Rodozov, M. Shopova, G. Sul- tanov University of Soﬁa, Soﬁa, Bulgaria University of Soﬁa, Soﬁa, Bulgaria A Dimitrov I Glushkov L Litov B Pavlov P Petkov University of Soﬁa, Soﬁa, Bulgaria University of Soﬁa, Soﬁa, Bulgaria A. Dimitrov, I. Glushkov, L. Litov, B. Pavlov, P. Petkov A. Dimitrov, I. Glushkov, L. Litov, B. Pavlov, P. Petkov Beihang University, Beijing, China W. Fang6, X. Gao6, L. Yuan Beihang University, Beijing, China W. Fang6, X. Gao6, L. Yuan Institute of High Energy Physics, Beijing, China Institute of High Energy Physics, Beijing, China Institute of High Energy Physics, Beijing, China M. Ahmad, J.G. Bian, G.M. Chen, H.S. Chen, M. Chen, Y. Chen, C.H. Jiang, D. Leggat, M. Ahmad, J.G. Bian, G.M. Chen, H.S. Chen, M. Chen, Y. Chen, C.H. Jiang, D. Leggat, H. Liao, Z. Liu, F. Romeo, S.M. Shaheen, A. Spiezia, J. Tao, C. Wang, Z. Wang, E. Yazgan, H. Zhang, S. Zhang, J. Zhao State Key Laboratory of Nuclear Physics and Technology, Peking University, Beijing, China Y. Ban, G. Chen, Q. Li, S. Liu, Y. Mao, S.J. Qian, D. Wang, Z. Xu Universidad de Los Andes, Bogota, Colombia C. Avila, A. Cabrera, L.F. Chaparro Sierra, C. Florez, C.F. Gonz´alez Hern´andez, J.D. Ruiz Alvarez, M.A. Segura Delgado University of Split, Faculty of Electrical Engineering, Mechanical Engineering and Naval Architecture, Split, Croatia B. Courbon, N. Godinovic, D. Lelas, I. Puljak, P.M. Ribeiro Cipriano, T. Sculac University of Split, Faculty of Science, Split, Croatia Z. Antunovic, M. Kovac Institute Rudjer Boskovic, Zagreb, Croatia V. Brigljevic, D. Ferencek, K. Kadija, B. Mesic, A. Starodumov7, T. Susa University of Cyprus, Nicosia, Cyprus M.W. Ather, A. Attikis, G. Mavromanolakis, J. Mousa, C. Nicolaou, F. Ptochos, P.A. Razis, H. Rykaczewski – 16 – Charles University, Prague, Czech Republic M. Finger8, M. Finger Jr.8 Universidad San Francisco de Quito, Quito, Ecuador E. Carrera Jarrin Academy of Scientiﬁc Research and Technology of the Arab Republic of Egypt, Egyptian Network of High Energy Physics, Cairo, Egypt A.A. Abdelalim9,10, Y. Mohammed11, E. Salama12,13 National Institute of Chemical Physics and Biophysics, Tallinn, Estonia R.K. Dewanjee, M. Kadastik, L. Perrini, M. Raidal, A. Tiko, C. Veelken JHEP01(2018)045 Department of Physics, University of Helsinki, Helsinki, Finland P. Eerola, H. Kirschenmann, J. Pekkanen, M. Voutilainen Helsinki Institute of Physics, Helsinki, Finland Helsinki Institute of Physics, Helsinki, Finland J. Havukainen, J.K. Heikkil¨a, T. J¨arvinen, V. Karim¨aki, R. Kinnunen, T. Lamp´e J. Havukainen, J.K. Heikkila, T. Jarvinen, V. Karimaki, R. Kinnunen, T. Lampen, K. Lassila-Perini, S. Laurila, S. Lehti, T. Lind´en, P. Luukka, H. Siikonen, E. Tuominen, J. Tuominiemi Lappeenranta University of Technology, Lappeenranta, Finland J. Talvitie, T. Tuuva Lappeenranta University of Technology, Lappeenranta, Finland J. Talvitie, T. Tuuva IRFU, CEA, Universit´e Paris-Saclay, Gif-sur-Yvette, France M. Besancon, F. Couderc, M. Dejardin, D. Denegri, J.L. Faure, F. Ferri, S. Ganjour, S. Ghosh, A. Givernaud, P. Gras, G. Hamel de Monchenault, P. Jarry, I. Kucher, C. Leloup, E. Locci, M. Machet, J. Malcles, G. Negro, J. Rander, A. Rosowsky, M. ¨O. Sahin, M. Titov Laboratoire Leprince-Ringuet, Ecole polytechnique, CNRS/IN2P3, Univer- sit´e Paris-Saclay, Palaiseau, France Laboratoire Leprince-Ringuet, Ecole polytechnique, CNRS/IN2P3, Unive sit´e Paris-Saclay, Palaiseau, France A. Abdulsalam, C. Amendola, I. Antropov, S. Baﬃoni, F. Beaudette, P. Busson, L. Cadamuro, C. Charlot, R. Granier de Cassagnac, M. Jo, S. Lisniak, A. Lobanov, J. Martin Blanco, M. Nguyen, C. Ochando, G. Ortona, P. Paganini, P. Pigard, R. Salerno, J.B. Sauvan, Y. Sirois, A.G. Stahl Leiton, T. Strebler, Y. Yilmaz, A. Zabi, A. Zghiche Universit´e de Strasbourg, CNRS, IPHC UMR 7178, F-67000 Strasbourg, France J.-L. Agram14, J. Andrea, D. Bloch, J.-M. Brom, M. Buttignol, E.C. Chabert, N. Chanon, C. Collard, E. Conte14, X. Coubez, J.-C. Fontaine14, D. Gel´e, U. Goerlach, M. Jansov´a, A.-C. Le Bihan, N. Tonon, P. Van Hove Centre de Calcul de l’Institut National de Physique Nucleaire et de Physique des Particules, CNRS/IN2P3, Villeurbanne, France S. Gadrat – 17 – Universit´e de Lyon, Universit´e Claude Bernard Lyon 1, CNRS-IN2P3, Institut de Physique Nucl´eaire de Lyon, Villeurbanne, France S. Beauceron, C. Bernet, G. Boudoul, R. Chierici, D. Contardo, P. Depasse, H. El Mamouni, J. Fay, L. Finco, S. Gascon, M. Gouzevitch, G. Grenier, B. Ille, F. Lagarde, I.B. Laktineh, M. Lethuillier, L. Mirabito, A.L. Pequegnot, S. Perries, A. Popov15, V. Sordini, M. Vander Donckt, S. Viret Georgian Technical University, Tbilisi, Georgia T. Toriashvili16 Tbilisi State University, Tbilisi, Georgia Tbilisi State University, Tbilisi, Georgia Z. Tsamalaidze8 JHEP01(2018)045 Z. Tsamalaidze8 RWTH Aachen University, I. Physikalisches Institut, Aachen, Germany C. Autermann, L. Feld, M.K. Kiesel, K. Klein, M. Lipinski, M. Preuten, C. Schomakers, J. Schulz, V. Zhukov15 RWTH Aachen University, III. Physikalisches Institut A, Aachen, Germany RWTH Aachen University, III. Physikalisches Institut A, Aachen, Germany A. Albert, E. Dietz-Laursonn, D. Duchardt, M. Endres, M. Erdmann, S. Erdweg, T. Esch, R. Fischer, A. G¨uth, M. Hamer, T. Hebbeker, C. Heidemann, K. Hoepfner, S. Knutzen, M. Merschmeyer, A. Meyer, P. Millet, S. Mukherjee, T. Pook, M. Radziej, H. Reithler, M. Rieger, F. Scheuch, D. Teyssier, S. Th¨uer RWTH Aachen University, III. Physikalisches Institut B, Aachen, Germany G. Fl¨ugge, B. Kargoll, T. Kress, A. K¨unsken, T. M¨uller, A. Nehrkorn, A. Nowack, C. Pistone, O. Pooth, A. Stahl17 Deutsches Elektronen-Synchrotron, Hamburg, Germany M. Aldaya Martin, T. Arndt, C. Asawatangtrakuldee, K. Beernaert, O. Behnke, U. Behrens, A. Berm´udez Mart´ınez, A.A. Bin Anuar, K. Borras18, V. Botta, A. Camp- bell, P. Connor, C. Contreras-Campana, F. Costanza, C. Diez Pardos, G. Eckerlin, D. Eckstein, T. Eichhorn, E. Eren, E. Gallo19, J. Garay Garcia, A. Geiser, A. Gizhko, J.M. Grados Luyando, A. Grohsjean, P. Gunnellini, M. Guthoﬀ, A. Laboratoire Leprince-Ringuet, Ecole polytechnique, CNRS/IN2P3, Unive sit´e Paris-Saclay, Palaiseau, France Harb, J. Hauk, M. Hempel20, H. Jung, A. Kalogeropoulos, M. Kasemann, J. Keaveney, C. Kleinwort, I. Korol, D. Kr¨ucker, W. Lange, A. Lelek, T. Lenz, J. Leonard, K. Lipka, W. Lohmann20, R. Mankel, I.-A. Melzer-Pellmann, A.B. Meyer, G. Mittag, J. Mnich, A. Mussgiller, E. Ntomari, D. Pitzl, A. Raspereza, M. Savitskyi, P. Saxena, R. Shevchenko, S. Spannagel, N. Stefaniuk, G.P. Van Onsem, R. Walsh, Y. Wen, K. Wichmann, C. Wissing, O. Zenaiev University of Hamburg, Hamburg, Germany R. Aggleton, S. Bein, V. Blobel, M. Centis Vignali, T. Dreyer, E. Garutti, D. Gonzalez, J. Haller, A. Hinzmann, M. Hoﬀmann, A. Karavdina, R. Klanner, R. Kogler, N. Kovalchuk, S. Kurz, T. Lapsien, I. Marchesini, D. Marconi, M. Meyer, M. Niedziela, D. Nowatschin, F. Pantaleo17, T. Peiﬀer, A. Perieanu, C. Scharf, P. Schleper, A. Schmidt, S. Schumann, J. Schwandt, J. Sonneveld, H. Stadie, G. Steinbr¨uck, F.M. Stober, M. St¨over, H. Tholen, D. Troendle, E. Usai, A. Vanhoefer, B. Vormwald R. Aggleton, S. Bein, V. Blobel, M. Centis Vignali, T. Dreyer, E. Garutti, D. Gonzalez, J. Haller, A. Hinzmann, M. Hoﬀmann, A. Karavdina, R. Klanner, R. Kogler, N. Kovalchuk, S. Kurz, T. Lapsien, I. Marchesini, D. Marconi, M. Meyer, M. Niedziela, D. Nowatschin, F. Pantaleo17, T. Peiﬀer, A. Perieanu, C. Scharf, P. Schleper, A. Schmidt, S. Schumann, J. Schwandt, J. Sonneveld, H. Stadie, G. Steinbr¨uck, F.M. Stober, M. St¨over, H. Tholen, D. Troendle, E. Usai, A. Vanhoefer, B. Vormwald – 18 – Institut f¨ur Experimentelle Kernphysik, Karlsruhe, Germany M. Akbiyik, C. Barth, M. Baselga, S. Baur, E. Butz, R. Caspart, T. Chwalek, F. Colombo, W. De Boer, A. Dierlamm, N. Faltermann, B. Freund, R. Friese, M. Giﬀels, M.A. Har- rendorf, F. Hartmann17, S.M. Heindl, U. Husemann, F. Kassel17, S. Kudella, H. Mildner, M.U. Mozer, Th. M¨uller, M. Plagge, G. Quast, K. Rabbertz, M. Schr¨oder, I. Shvetsov, G. Sieber, H.J. Simonis, R. Ulrich, S. Wayand, M. Weber, T. Weiler, S. Williamson, C. W¨ohrmann, R. Wolf Institute of Nuclear and Particle Physics (INPP), NCSR Demokritos, Aghia Paraskevi, Greece JHEP01(2018)045 JHEP01(2018)045 G. Anagnostou, G. Daskalakis, T. Geralis, A. Kyriakis, D. Loukas, I. Topsis-Giotis National and Kapodistrian University of Athens, Athens, Greece G. Karathanasis, S. Kesisoglou, A. Panagiotou, N. Saoulidou National Technical University of Athens, Athens, Greece K. Kousouris University of Io´annina, Io´annina, Greece y , , I. Evangelou, C. Foudas, P. Kokkas, S. Mallios, N. Manthos, I. Papadopoulos, E. Paradas, J. Strologas, F.A. Triantis MTA-ELTE Lend¨ulet CMS Particle and Nuclear Physics Group, E¨otv¨os Lor´and University, Budapest, Hungary M. Csanad, N. Filipovic, G. Pasztor, O. Sur´anyi, G.I. Veres21 Wigner Research Centre for Physics, Budapest, Hungary G. Bencze, C. Hajdu, D. Horvath22, ´A. Hunyadi, F. Sikler, V. Veszpremi Institute of Nuclear Research ATOMKI, Debrecen, Hungary N. Beni, S. Czellar, J. Karancsi23, A. Makovec, J. Molnar, Z. Szillasi Institute of Physics, University of Debrecen, Debrecen, Hungary M. Bart´ok21, P. Raics, Z.L. Trocsanyi, B. University of Hamburg, Hamburg, Germany Ujvari Indian Institute of Science (IISc), Bangalore, India S. Choudhury, J.R. Komaragiri National Institute of Science Education and Research, Bhubaneswar, India S. Bahinipati24, S. Bhowmik, P. Mal, K. Mandal, A. Nayak25, D.K. Sahoo24, N. Sahoo, S.K. Swain Panjab University, Chandigarh, India Indian Institute of Technology Madras, Madras, India P.K. Behera Indian Institute of Technology Madras, Madras, India P.K. Behera Bhabha Atomic Research Centre, Mumbai, India Bhabha Atomic Research Centre, Mumbai, India R. Chudasama, D. Dutta, V. Jha, V. Kumar, A.K. Mohanty17, P.K. Netrakanti, L.M. Pant, P. Shukla, A. Topkar JHEP01(2018)045 Tata Institute of Fundamental Research-A, Mumbai, India T. Aziz, S. Dugad, B. Mahakud, S. Mitra, G.B. Mohanty, N. Sur, B. Sutar T. Aziz, S. Dugad, B. Mahakud, S. Mitra, G.B. Mohanty, N. Sur, B. Sutar Tata Institute of Fundamental Research-B, Mumbai, India S. Banerjee, S. Bhattacharya, S. Chatterjee, P. Das, M. Guchait, Sa. Jain, S. Kumar, M. Maity26, G. Majumder, K. Mazumdar, T. Sarkar26, N. Wickramage27 Indian Institute of Science Education and Research (IISER), Pune, India S. Chauhan, S. Dube, V. Hegde, A. Kapoor, K. Kothekar, S. Pandey, A. Rane, S. Sharma Institute for Research in Fundamental Sciences (IPM), Tehran, Iran S. Chenarani28, E. Eskandari Tadavani, S.M. Etesami28, M. Khakzad, M. Mohammadi Najafabadi, M. Naseri, S. Paktinat Mehdiabadi29, F. Rezaei Hosseinabadi, B. Safarzadeh30, M. Zeinali University College Dublin, Dublin, Ireland M. Felcini, M. Grunewald Panjab University, Chandigarh, India S. Bansal, S.B. Beri, V. Bhatnagar, R. Chawla, N. Dhingra, A.K. Kalsi, A. Kaur, M. Kau S. Bansal, S.B. Beri, V. Bhatnagar, R. Chawla, N. Dhingra, A.K. Kalsi, A. Kaur, M. Kaur, S. Kaur, R. Kumar, P. Kumari, A. Mehta, J.B. Singh, G. Walia S. Kaur, R. Kumar, P. Kumari, A. Mehta, J.B. Singh, G. Walia University of Delhi, Delhi, India Ashok Kumar, Aashaq Shah, A. Bhardwaj, S. Chauhan, B.C. Choudhary, R.B. Garg, S. Keshri, A. Kumar, S. Malhotra, M. Naimuddin, K. Ranjan, R. Sharma – 19 – Saha Institute of Nuclear Physics, HBNI, Kolkata, India Saha Institute of Nuclear Physics, HBNI, Kolkata, India R. Bhardwaj, R. Bhattacharya, S. Bhattacharya, U. Bhawandeep, S. Dey, S. Dutt, S. Dutta, S. Ghosh, N. Majumdar, A. Modak, K. Mondal, S. Mukhopadhyay, S. Nandan, A. Purohit, A. Roy, S. Roy Chowdhury, S. Sarkar, M. Sharan, S. Thakur R. Bhardwaj, R. Bhattacharya, S. Bhattacharya, U. Bhawandeep, S. Dey, S. Dutt, S. Dutt R. Bhardwaj, R. Bhattacharya, S. Bhattacharya, U. Bhawandeep, S. Dey, S. Dutt, S. Dutta, S. Ghosh, N. Majumdar, A. Modak, K. Mondal, S. Mukhopadhyay, S. Nandan, A. Purohit, A. Roy, S. Roy Chowdhury, S. Sarkar, M. Sharan, S. Thakur University College Dublin, Dublin, Ireland M. Felcini, M. Grunewald M. Felcini, M. Grunewald INFN Sezione di Bari a, Universit`a di Bari b, Politecnico di Bari c, Bari, Italy M. Abbresciaa,b, C. Calabriaa,b, A. Colaleoa, D. Creanzaa,c, L. Cristellaa,b, N. De Filippisa,c, M. De Palmaa,b, F. Erricoa,b, L. Fiorea, G. Iasellia,c, S. Lezkia,b, G. Maggia,c, M. Maggia, G. Minielloa,b, S. Mya,b, S. Nuzzoa,b, A. Pompilia,b, G. Pugliesea,c, R. Radognaa, A. Ranieria, G. Selvaggia,b, A. Sharmaa, L. Silvestrisa,17, R. Vendittia, P. Verwilligena P. Verwilligena INFN Sezione di Bologna a, Universit`a di Bologna b, Bologna, Italy G. Abbiendia, C. Battilanaa,b, D. Bonacorsia,b, L. Borgonovia,b, S. Braibant-Giacomellia,b, R. Campaninia,b, P. Capiluppia,b, A. Castroa,b, F.R. Cavalloa, S.S. Chhibraa, G. Codispotia,b, M. Cuﬃania,b, G.M. Dallavallea, F. Fabbria, A. Fanfania,b, D. Fasanellaa,b, P. Giacomellia, C. Grandia, L. Guiduccia,b, S. Marcellinia, G. Masettia, A. Montanaria, F.L. Navarriaa,b, A. Perrottaa, A.M. Rossia,b, T. Rovellia,b, G.P. Sirolia,b, N. Tosia INFN Sezione di Catania a, Universit`a di Catania b, Catania, Italy S. Albergoa,b, S. Costaa,b, A. Di Mattiaa, F. Giordanoa,b, R. Potenzaa,b, A. Tricomia,b, C. Tuvea,b INFN Sezione di Catania a, Universit`a di Catania b, Catania, Italy S. Albergoa,b, S. Costaa,b, A. Di Mattiaa, F. Giordanoa,b, R. Potenzaa,b, A. Tricomia C. Tuvea,b – 20 – INFN Sezione di Firenze a, Universit`a di Firenze b, Firenze, Italy G. Barbaglia, K. Chatterjeea,b, V. Ciullia,b, C. Civininia, R. D’Alessandroa,b, E. Focardia,b, P. Lenzia,b, M. Meschinia, S. Paolettia, L. Russoa,31, G. Sguazzonia, D. Stroma, b 17 INFN Sezione di Firenze a, Universit`a di Firenze b, Firenze, Italy G. Barbaglia, K. Chatterjeea,b, V. Ciullia,b, C. Civininia, R. D’Alessandroa,b, E. Focardia INFN Sezione di Firenze a, Universit`a di Firenze b, Firenze, Italy G. Barbaglia, K. Chatterjeea,b, V. Ciullia,b, C. Civininia, R. D’Alessandroa,b, E. Focardia,b, P. Lenzia,b, M. Meschinia, S. Paolettia, L. Russoa,31, G. Sguazzonia, D. Stroma, L. Viliania,b,17 P. Lenzia,b, M. Meschinia, S. Paolettia, L. Russoa,31, G. Sguazzonia, D. Stroma, L. Viliania,b,17 INFN Laboratori Nazionali di Frascati, Frascati, Italy L. Benussi, S. Bianco, F. Fabbri, D. Piccolo, F. Primavera17 INFN Laboratori Nazionali di Frascati, Frascati, Italy L. Benussi, S. Bianco, F. Fabbri, D. Piccolo, F. Primavera17 INFN Sezione di Genova a, Universit`a di Genova b, Genova, Italy V. Calvellia,b, F. Ferroa, E. Robuttia, S. Tosia,b JHEP01(2018)045 INFN Sezione di Milano-Bicocca a, Universit`a di Milano-Bicocca b, Milano, Italy A. Benagliaa, A. Beschi, L. Brianzaa,b, F. Brivioa,b, V. Cirioloa,b, M.E. Dinardoa,b, A. Benagliaa, A. Beschi, L. Brianzaa,b, F. Brivioa,b, V. Cirioloa,b, M.E. University College Dublin, Dublin, Ireland M. Felcini, M. Grunewald Dinardoa,b, S Fiorendia,b S Gennaia A Ghezzia,b P Govonia,b M Malbertia,b S Malvezzia g , , , , , , Fiorendia,b, S. Gennaia, A. Ghezzia,b, P. Govonia,b, M. Malbertia,b, S. Malvezzia, R.A. Manzonia,b, D. Menascea, L. Moronia, M. Paganonia,b, K. Pauwelsa,b, D. Pedrinia, S Pi i ia b 32 S R ia b N R d llia T T b lli d F ti a b R.A. Manzonia,b, D. Menascea, L. Moronia, M. Paganonia,b, K. Pauwelsa,b, D. Pedrinia, S. Pigazzinia,b,32, S. Ragazzia,b, N. Redaellia, T. Tabarelli de Fatisa,b INFN Sezione di Napoli a, Universit`a di Napoli ’Federico II’ b, Napoli, Italy, Universit`a della Basilicata c, Potenza, Italy, Universit`a G. Marconi d, Roma, Italy S. Buontempoa, N. Cavalloa,c, S. Di Guidaa,d,17, F. Fabozzia,c, F. Fiengaa S. Buontempoa, N. Cavalloa,c, S. Di Guidaa,d,17, F. Fabozzia,c, F. Fiengaa,b, A.O.M. Iorioa,b, W.A. Khana, L. Listaa, S. Meolaa,d,17, P. Paoluccia,17, C. Sciaccaa,b, F Thyssena S. Buontempoa, N. Cavalloa,c, S. Di Guidaa,d,17, F. Fabozzia,c, F. Fiengaa,b, A.O.M. Iorioa,b, W.A. Khana, L. Listaa, S. Meolaa,d,17, P. Paoluccia,17, C. Sciaccaa,b, S. Buontempoa, N. Cavalloa,c, S. Di Guidaa,d,17, F. Fabozzia,c, F. Fiengaa,b, A.O.M. Iorioa,b, W.A. Khana, L. Listaa, S. Meolaa,d,17, P. Paoluccia,17, C. Sciaccaa,b, F. Thyssena INFN Sezione di Padova a, Universit`a di Padova b, Padova, Italy, Universit`a di Trento c, Trento, Italy P. Azzia, N. Bacchettaa, L. Benatoa,b, D. Biselloa,b, A. Bolettia,b, R. Carlina,b, A. Car- valho Antunes De Oliveiraa,b, P. Checchiaa, P. De Castro Manzanoa, T. Dorigoa, U. Dossellia, F. Gasparinia,b, U. Gasparinia,b, A. Gozzelinoa, S. Lacapraraa, M. Margonia,b, A.T. Meneguzzoa,b, N. Pozzobona,b, P. Ronchesea,b, R. Rossina,b, F. Simonettoa,b, E. Torassaa, M. Zanettia,b, P. Zottoa,b, G. Zumerlea,b INFN Sezione di Pavia a, Universit`a di Pavia b, Pavia, Italy INFN Sezione di Pavia a, Universit`a di Pavia b, Pavia, Italy A. Braghieria, A. Magnania, P. Montagnaa,b, S.P. Rattia,b, V. Rea, M. Ressegottia,b, C. Riccardia,b, P. Salvinia, I. Vaia,b, P. Vituloa,b A. Braghieria, A. Magnania, P. Montagnaa,b, S.P. Rattia,b, V. Rea, M. Ressegottia C. Riccardia,b, P. Salvinia, I. Vaia,b, P. Vituloa,b INFN Sezione di Perugia a, Universit`a di Perugia b, Perugia, Italy L. Alunni Solestizia,b, M. Biasinia,b, G.M. Bileia, C. Cecchia,b, D. Ciangottinia,b, L. Fan`oa,b, P. Laricciaa,b, R. Leonardia,b, E. Manonia, G. Mantovania,b, V. Mariania,b, M. Menichellia, A. Rossia,b, A. Santocchiaa,b, D. Spigaa INFN Sezione di Pisa a, Universit`a di Pisa b, Scuola Normale Superiore di Pisa c, Pisa, Italy K. Androsova, P. Azzurria,17, G. Bagliesia, T. Boccalia, L. Borrello, R. Castaldia, K. Androsova, P. Azzurria,17, G. Bagliesia, T. Boccalia, L. Borrello, R. Castaldia, M.A. Cioccia,b, R. Dell’Orsoa, G. Fedia, L. Gianninia,c, A. Giassia, M.T. Grippoa,31, F. Ligabuea,c, T. Lomtadzea, E. Mancaa,c, G. Mandorlia,c, A. Messineoa,b, F. Pallaa, M.A. Cioccia,b, R. Dell’Orsoa, G. Fedia, L. Gianninia,c, A. Giassia, M.T. Grippoa,3 M.A. Ciocci , , R. Dell Orso , G. Fedi , L. Giannini , , A. Giassi , M.T. Grippo , , F. Ligabuea,c, T. Lomtadzea, E. Mancaa,c, G. Mandorlia,c, A. Messineoa,b, F. Pallaa, F. Ligabuea,c, T. Lomtadzea, E. Mancaa,c, G. Mandorlia,c, A. Messineoa,b, F. Pallaa, – 21 – A. Rizzia,b, A. Savoy-Navarroa,33, P. Spagnoloa, R. Tenchinia, G. Tonellia,b, A. Venturia, P.G. Verdinia INFN Sezione di Roma a, Sapienza Universit`a di Roma b, Rome, Italy L. Baronea,b, F. Cavallaria, M. Cipriania,b, N. Dacia, D. Del Rea,b,17, E. Di Marcoa,b, M. Diemoza, S. Gellia,b, E. Longoa,b, F. Margarolia,b, B. Marzocchia,b, P. Meridiania, G. Organtinia,b, R. Paramattia,b, F. Preiatoa,b, S. Rahatloua,b, C. Rovellia, F. Santanastasioa,b INFN Sezione di Torino a, Universit`a di Torino b, Torino, Italy, Universit`a del Piemonte Orientale c, Novara, Italy JHEP01(2018)045 JHEP01(2018)045 , , y N. Amapanea,b, R. Arcidiaconoa,c, S. Argiroa,b, M. Arneodoa,c, N. Bartosika, R. Bellana,b, C. Biinoa, N. Cartigliaa, F. Cennaa,b, M. Costaa,b, R. Covarellia,b, A. Deganoa,b, N. Demariaa, B. Kiania,b, C. Mariottia, S. Masellia, E. Migliorea,b, V. Monacoa,b, E. Monteila,b, M. Montenoa, M.M. Obertinoa,b, L. Pachera,b, N. Pastronea, M. Pelliccionia, G.L. Pinna Angionia,b, F. Raveraa,b, A. Romeroa,b, M. Ruspaa,c, R. Sacchia,b, K. Shchelinaa,b, V. Solaa, A. Solanoa,b, A. Staianoa, P. Traczyka,b G.L. Pinna Angionia,b, F. Raveraa,b, A. Romeroa,b, M. Ruspaa,c, R. Sacchia,b, K. Shchelinaa,b, V. Solaa, A. Solanoa,b, A. Staianoa, P. Korea University, Seoul, Korea S. Cho, S. Choi, Y. Go, D. Gyun, S. Ha, B. Hong, Y. Jo, Y. Kim, K. Lee, K.S. Lee, S. Lee, J. Lim, S.K. Park, Y. Roh INFN Sezione di Pavia a, Universit`a di Pavia b, Pavia, Italy Traczyka,b INFN Sezione di Trieste a, Universit`a di Trieste b, Trieste, Italy S. Belfortea, M. Casarsaa, F. Cossuttia, G. Della Riccaa,b, A. Zanettia Kyungpook National University, Daegu, Korea D.H. Kim, G.N. Kim, M.S. Kim, J. Lee, S. Lee, S.W. Lee, C.S. Moon, Y.D. Oh, S. Sekmen, D.C. Son, Y.C. Yang Chonbuk National University, Jeonju, Korea A. Lee Chonbuk National University, Jeonju, Korea A. Lee Chonnam National University, Institute for Universe and Elementary Particles, Kwangju, Korea Chonnam National University, Institute for Universe and Elementary Particles, K j K Kwangju, Korea Kwangju, Korea H. Kim, D.H. Moon, G. Oh Hanyang University, Seoul, Korea J.A. Brochero Cifuentes, J. Goh, T.J. Kim Korea University, Seoul, Korea Seoul National University, Seoul, Korea Seoul National University, Seoul, Korea J. Almond, J. Kim, J.S. Kim, H. Lee, K. Lee, K. Nam, S.B. Oh, B.C. Radburn-Smith, S.h. Seo, U.K. Yang, H.D. Yoo, G.B. Yu J. Almond, J. Kim, J.S. Kim, H. Lee, K. Lee, K. Nam, S.B. Oh, B.C. Radburn-Smith, S.h. Seo, U.K. Yang, H.D. Yoo, G.B. Yu University of Seoul, Seoul, Korea M. Choi, H. Kim, J.H. Kim, J.S.H. Lee, I.C. Park University of Seoul, Seoul, Korea M. Choi, H. Kim, J.H. Kim, J.S.H. Lee, I.C. Park M. Choi, H. Kim, J.H. Kim, J.S.H. Lee, I.C. Park M. Choi, H. Kim, J.H. Kim, J.S.H. Lee, I.C. Park Sungkyunkwan University, Suwon, Korea Y. Choi, C. Hwang, J. Lee, I. Yu Vilnius University, Vilnius, Lithuania V. Dudenas, A. Juodagalvis, J. Vaitkus V. Dudenas, A. Juodagalvis, J. Vaitkus National Centre for Particle Physics, Universiti Malaya, Kuala Lumpur, Malaysia I. Ahmed, Z.A. Ibrahim, M.A.B. Md Ali34, F. Mohamad Idris35, W.A.T. Wan Abdullah, M.N. Yusli, Z. Zolkapli Centro de Investigacion y de Estudios Avanzados del IPN, Mexico City, Mexico Reyes-Almanza, R, Ramirez-Sanchez, G., Duran-Osuna, M. C., H. Castilla-Valdez, E. De La Cruz-Burelo, I. Heredia-De La Cruz36, Rabadan-Trejo, R. I., R. Lopez-Fernandez, J. Mejia Guisao, A. Sanchez-Hernandez JHEP01(2018)045 Universidad Iberoamericana, Mexico City, Mexico S. Carrillo Moreno, C. Oropeza Barrera, F. Vazquez Valencia U ve s dad be oa e ca a, e co C ty, e co S. Carrillo Moreno, C. Oropeza Barrera, F. Vazquez Valencia Benemerita Universidad Autonoma de Puebla, Puebla, Mexico I. Pedraza, H.A. Salazar Ibarguen, C. Uribe Estrada Universidad Aut´onoma de San Luis Potos´ı, San Luis Potos´ı, Mexico A. Morelos Pineda University of Auckland, Auckland, New Zealand D. Krofcheck University of Canterbury, Christchurch, New Zealand P.H. Butler National Centre for Physics, Quaid-I-Azam University, Islamabad, Pakistan A. Ahmad, M. Ahmad, Q. Hassan, H.R. Hoorani, A. Saddique, M.A. Shah, M. Shoaib, M. Waqas Sungkyunkwan University, Suwon, Korea Y. Choi, C. Hwang, J. Lee, I. Yu Y. Choi, C. Hwang, J. Lee, I. Yu – 22 – Vilnius University, Vilnius, Lithuania Petersburg Nuclear Physics Institute, Gatchina (St. Petersburg), Russia Y. Ivanov, V. Kim40, E. Kuznetsova41, P. Levchenko, V. Murzin, V. Oreshkin, I. Smirnov, V. Sulimov, L. Uvarov, S. Vavilov, A. Vorobyev National Centre for Nuclear Research, Swierk, Poland H. Bialkowska, M. Bluj, B. Boimska, T. Frueboes, M. G´orski, M. Kazana, K. Nawrocki, M. Szleper, P. Zalewski Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Warsaw, Poland K. Bunkowski, A. Byszuk37, K. Doroba, A. Kalinowski, M. Konecki, J. Krolikowski, M. Misiura, M. Olszewski, A. Pyskir, M. Walczak Laborat´orio de Instrumenta¸c˜ao e F´ısica Experimental de Part´ıculas, Lisboa, Portugal P. Bargassa, C. Beir˜ao Da Cruz E Silva, A. Di Francesco, P. Faccioli, B. Galinhas, M G lli J H ll N L d L Ll t I l i M V N ll di J S i P. Bargassa, C. Beir˜ao Da Cruz E Silva, A. Di Francesco, P. Faccioli, B. Galinhas, M. Gallinaro, J. Hollar, N. Leonardo, L. Lloret Iglesias, M.V. Nemallapudi, J. Seixas, G St O T ld i D V d i J V l P. Bargassa, C. Beir˜ao Da Cruz E Silva, A. Di Francesco, P. Faccioli, B. Galinhas, M. Gallinaro, J. Hollar, N. Leonardo, L. Lloret Iglesias, M.V. Nemallapudi, J. Seixas, G. Strong, O. Toldaiev, D. Vadruccio, J. Varela M. Gallinaro, J. Hollar, N. Leonardo, L. Lloret Iglesias, M.V. Nemallapudi, J. Seixas, G. Strong, O. Toldaiev, D. Vadruccio, J. Varela , , , g , p , , G. Strong, O. Toldaiev, D. Vadruccio, J. Varela – 23 – Joint Institute for Nuclear Research, Dubna, Russia S. Afanasiev, P. Bunin, M. Gavrilenko, I. Golutvin, I. Gorbunov, A. Kamenev, V. Karjavi S. Afanasiev, P. Bunin, M. Gavrilenko, I. Golutvin, I. Gorbuno S. Afanasiev, P. Bunin, M. Gavrilenko, I. Golutvin, I. Gorbunov, A. Kamenev, V. Karjavin, A. Lanev, A. Malakhov, V. Matveev38,39, V. Palichik, V. Perelygin, S. Shmatov, S. Shulha, N. Skatchkov, V. Smirnov, N. Voytishin, A. Zarubin , , , , , , j , A. Lanev, A. Malakhov, V. Matveev38,39, V. Palichik, V. Perelygin, S. Shmatov, S. Shulha, N. Skatchkov, V. Smirnov, N. Voytishin, A. Zarubin Petersburg Nuclear Physics Institute, Gatchina (St. Petersburg), Russia Centro de Investigaciones Energ´eticas Medioambientales y Tec- nol´ogicas (CIEMAT), Madrid, Spain Centro de Investigaciones Energ´eticas Medioambientales y Tec- nol´ogicas (CIEMAT), Madrid, Spain J. Alcaraz Maestre, M. Barrio Luna, M. Cerrada, N. Colino, B. De La Cruz, A. Delgado Peris, A. Escalante Del Valle, C. Fernandez Bedoya, J.P. Fern´andez Ramos, J. Flix, M.C. Fouz, O. Gonzalez Lopez, S. Goy Lopez, J.M. Hernandez, M.I. Josa, D. Moran, A. P´erez-Calero Yzquierdo, J. Puerta Pelayo, A. Quintario Olmeda, I. Redondo, L. Romero, M.S. Soares, A. ´Alvarez Fern´andez Universidad Aut´onoma de Madrid, Madrid, Spain C. Albajar, J.F. de Troc´oniz, M. Missiroli JHEP01(2018)045 Universidad de Oviedo, Oviedo, Spain J. Cuevas, C. Erice, J. Fernandez Menendez, I. Gonzalez Caballero, J.R. Gonz´alez Fern´andez, E. Palencia Cortezon, S. Sanchez Cruz, P. Vischia, J.M. Vizan Garcia Instituto de F´ısica de Cantabria (IFCA), CSIC-Universidad de Cantabria, Santander, Spain I.J. Cabrillo, A. Calderon, B. Chazin Quero, E. Curras, J. Duarte Campderros, M. Fer- nandez, J. Garcia-Ferrero, G. Gomez, A. Lopez Virto, J. Marco, C. Martinez Rivero, P. Martinez Ruiz del Arbol, F. Matorras, J. Piedra Gomez, T. Rodrigo, A. Ruiz-Jimeno, L. Scodellaro, N. Trevisani, I. Vila, R. Vilar Cortabitarte CERN, European Organization for Nuclear Research, Geneva, Switzerland D. Abbaneo, B. Akgun, E. Auﬀray, P. Baillon, A.H. Ball, D. Barney, J. Bendavid, M. Bianco, P. Bloch, A. Bocci, C. Botta, T. Camporesi, R. Castello, M. Cepeda, G. Cerminara, E. Chapon, Y. Chen, D. d’Enterria, A. Dabrowski, V. Daponte, A. David, M. De Gruttola, A. De Roeck, N. Deelen, M. Dobson, T. du Pree, M. D¨unser, N. Dupont, A. Elliott-Peisert, P. Everaerts, F. Fallavollita, G. Franzoni, J. Fulcher, W. Funk, D. Gigi, A. Gilbert, K. Gill, F. Glege, D. Gulhan, P. Harris, J. Hegeman, V. Innocente, A. Jafari, P. Janot, O. Karacheban20, J. Kieseler, V. Kn¨unz, A. Kornmayer, M.J. Kortelainen, M. Krammer1, C. Lange, P. Lecoq, C. Louren¸co, M.T. Lucchini, L. Malgeri, M. Mannelli, A. Martelli, F. Meijers, J.A. Merlin, S. Mersi, E. Meschi, P. Milenovic45, F. Moortgat, M. Mulders, H. Neugebauer, J. Ngadiuba, S. Orfanelli, L. Orsini, L. Pape, E. Perez, M. Peruzzi, A. Petrilli, G. Petrucciani, A. Pfeiﬀer, M. Pierini, D. Rabady, A. Racz, T. Reis, G. Rolandi46, M. Rovere, H. Sakulin, C. Sch¨afer, C. Schwick, M. Seidel, M. Selvaggi, A. Sharma, P. Silva, P. Sphicas47, A. Stakia, J. Steggemann, M. Stoye, M. Tosi, D. Treille, A. Triossi, A. Tsirou, V. Veckalns48, M. Verweij, W.D. Zeuner Paul Scherrer Institut, Villigen, Switzerland W. Bertl†, L. Caminada49, K. Deiters, W. Erdmann, R. Horisberger, Q. Institute for Nuclear Research, Moscow, Russia Institute for Nuclear Research, Moscow, Russia JHEP01(2018)045 JHEP01(2018)045 Yu. Andreev, A. Dermenev, S. Gninenko, N. Golubev, A. Karneyeu, M. Kirsanov, N. Krasnikov, A. Pashenkov, D. Tlisov, A. Toropin Institute for Theoretical and Experimental Physics, Moscow, Russia V. Epshteyn, V. Gavrilov, N. Lychkovskaya, V. Popov, I. Pozdnyakov, G. Safronov, A. Spiridonov, A. Stepennov, M. Toms, E. Vlasov, A. Zhokin Moscow Institute of Physics and Technology, Moscow, Russia T. Aushev, A. Bylinkin39 National Research Nuclear University ’Moscow Engineering Physics Insti- tute’ (MEPhI), Moscow, Russia M. Chadeeva42, P. Parygin, D. Philippov, S. Polikarpov, E. Popova, V. Rusinov P.N. Lebedev Physical Institute, Moscow, Russia P.N. Lebedev Physical Institute, Moscow, Russia V Andreev M Azarkin39 I Dremin39 M Kirakosyan39 A Terkulov V. Andreev, M. Azarkin39, I. Dremin39, M. Kirakosyan39, A. Terkulov Skobeltsyn Institute of Nuclear Physics, Lomonosov Moscow State University, Moscow, Russia A. Baskakov, A. Belyaev, E. Boos, A. Demiyanov, A. Ershov, A. Gribushin, O. Kodolova, V. Korotkikh, I. Lokhtin, I. Miagkov, S. Obraztsov, S. Petrushanko, V. Savrin, A. Snigirev, I. Vardanyan Novosibirsk State University (NSU), Novosibirsk, Russia V. Blinov43, Y.Skovpen43, D. Shtol43 State Research Center of Russian Federation, Institute for High Energy Physics, Protvino, Russia I. Azhgirey, I. Bayshev, S. Bitioukov, D. Elumakhov, V. Kachanov, A. Kalinin, D. Kon- stantinov, P. Mandrik, V. Petrov, R. Ryutin, A. Sobol, S. Troshin, N. Tyurin, A. Uzunian, A. Volkov State Research Center of Russian Federation, Institute for High Energy Physics, Protvino, Russia I. Azhgirey, I. Bayshev, S. Bitioukov, D. Elumakhov, V. Kachanov, A. Kalinin, D. Kon- stantinov, P. Mandrik, V. Petrov, R. Ryutin, A. Sobol, S. Troshin, N. Tyurin, A. Uzunian, A. Volkov University of Belgrade, Faculty of Physics and Vinca Institute of Nuclear Sciences, Belgrade, Serbia P. Adzic44, P. Cirkovic, D. Devetak, M. Dordevic, J. Milosevic, V. Rekovic University of Belgrade, Faculty of Physics and Vinca Institute of Nuclear Sciences, Belgrade, Serbia Sciences, Belgrade, Serbia P. Adzic44, P. Cirkovic, D. Devetak, M. Dordevic, J. Milosevic, V. Rekovic P. Adzic44, P. Cirkovic, D. Devetak, M. Dordevic, J. Milosevic, V. Rekovic – 24 – Centro de Investigaciones Energ´eticas Medioambientales y Tec- nol´ogicas (CIEMAT), Madrid, Spain Ingram, H.C. Kaestli, D. Kotlinski, U. Langenegger, T. Rohe, S.A. Wiederkehr ETH Zurich - Institute for Particle Physics and Astrophysics (IPA), Zurich, Switzerland M. Backhaus, L. B¨ani, P. Berger, L. Bianchini, B. Casal, G. Dissertori, M. Dittma M. Backhaus, L. B¨ani, P. Berger, L. Bianchini, B. Casal, G. Dissertori, M. Dittmar, M. Doneg`a, C. Dorfer, C. Grab, C. Heidegger, D. Hits, J. Hoss, G. Kasieczka, T. Klijnsma, , , g , , , , , M. Doneg`a, C. Dorfer, C. Grab, C. Heidegger, D. Hits, J. Hoss, G. Kasieczka, T. Klijnsma, – 25 – W. Lustermann, B. Mangano, M. Marionneau, M.T. Meinhard, D. Meister, F. Micheli, P. Musella, F. Nessi-Tedaldi, F. Pandolﬁ, J. Pata, F. Pauss, G. Perrin, L. Perrozzi, M. Quit- tnat, M. Reichmann, D.A. Sanz Becerra, M. Sch¨onenberger, L. Shchutska, V.R. Tavolaro, K. Theoﬁlatos, M.L. Vesterbacka Olsson, R. Wallny, D.H. Zhu Universit¨at Z¨urich, Zurich, Switzerland T.K. Aarrestad, C. Amsler50, M.F. Canelli, A. De Cosa, R. Del Burgo, S. Donato, C. Galloni, T. Hreus, B. Kilminster, D. Pinna, G. Rauco, P. Robmann, D. Salerno, K. Schweiger, C. Seitz, Y. Takahashi, A. Zucchetta National Central University, Chung-Li, Taiwan V. Candelise, T.H. Doan, Sh. Jain, R. Khurana, C.M. Kuo, W. Lin, A. Pozdnyakov, S.S. Yu JHEP01(2018)045 National Taiwan University (NTU), Taipei, Taiwan Arun Kumar, P. Chang, Y. Chao, K.F. Chen, P.H. Chen, F. Fiori, W.-S. Hou, Y. Hsiung, Y.F. Liu, R.-S. Lu, E. Paganis, A. Psallidas, A. Steen, J.f. Tsai Chulalongkorn University, Faculty of Science, Department of Physics, Bangkok, Thailand Baylor University, Waco, U.S.A. Baylor University, Waco, U.S.A. A. Borzou, K. Call, J. Dittmann, K. Hatakeyama, H. Liu, N. Pastika, C. Smith Catholic University of America, Washington DC, U.S.A. R. Bartek, A. Dominguez The University of Alabama, Tuscaloosa, U.S.A. A. Buccilli, S.I. Cooper, C. Henderson, P. Rumerio, C. West The University of Alabama, Tuscaloosa, U.S.A. A. Buccilli, S.I. Cooper, C. Henderson, P. Rumerio, C. West Boston University, Boston, U.S.A. D. Arcaro, A. Avetisyan, T. Bose, D. Gastler, D. Rankin, C. Richardson, J. Rohlf, L. Sulak, D. Zou Imperial College, London, United Kingdom G. Auzinger, R. Bainbridge, J. Borg, S. Breeze, O. Buchmuller, A. Bundock, S. Casasso, M. Citron, D. Colling, L. Corpe, P. Dauncey, G. Davies, A. De Wit, M. Della Negra, R. Di Maria, A. Elwood, Y. Haddad, G. Hall, G. Iles, T. James, R. Lane, C. Laner, L. Lyons, A.-M. Magnan, S. Malik, L. Mastrolorenzo, T. Matsushita, J. Nash, A. Nikitenko7, V. Palladino, M. Pesaresi, D.M. Raymond, A. Richards, A. Rose, E. Scott, C. Seez, A. Shtipliyski, S. Summers, A. Tapper, K. Uchida, M. Vazquez Acosta64, T. Virdee17, N. Wardle, D. Winterbottom, J. Wright, S.C. Zenz JHEP01(2018)045 Thailand B. Asavapibhop, K. Kovitanggoon, G. Singh, N. Srimanobhas B. Asavapibhop, K. Kovitanggoon, G. Singh, N. Srimanobhas C¸ ukurova University, Physics Department, Science and Art Faculty, Adana, Turkey M.N. Bakirci51, A. Bat, F. Boran, S. Damarseckin, Z.S. Demiroglu, C. Dozen, S. Girgi M.N. Bakirci , A. Bat, F. Boran, S. Damarseckin, Z.S. Demiroglu, C. Dozen, S. Girgis, G. Gokbulut, Y. Guler, I. Hos52, E.E. Kangal53, O. Kara, U. Kiminsu, M. Oglakci, G. Onengut54, K. Ozdemir55, S. Ozturk51, A. Polatoz, B. Tali56, U.G. Tok, H. Topakli51, S. Turkcapar, I.S. Zorbakir, C. Zorbilmez G. Gokbulut, Y. Guler, I. Hos52, E.E. Kangal53, O. Kara, U. Kiminsu, M. Oglakci, G Onengut54 K O demir55 S O turk51 A Polato B Tali56 U G Tok H Topakli51 G. Onengut54, K. Ozdemir55, S. Ozturk51, A. Polatoz, B. Tali56, U.G. Tok, H. Topakli51, S. Turkcapar, I.S. Zorbakir, C. Zorbilmez S. Turkcapar, I.S. Zorbakir, C. Zorbilmez Middle East Technical University, Physics Department, Ankara, Turkey B. Bilin, G. Karapinar57, K. Ocalan58, M. Yalvac, M. Zeyrek Bogazici University, Istanbul, Turkey E. G¨ulmez, M. Kaya59, O. Kaya60, S. Tekten, E.A. Yetkin61 Bogazici University, Istanbul, Turkey Istanbul Technical University, Istanbul, Turkey M.N. Agaras, S. Atay, A. Cakir, K. Cankocak Institute for Scintillation Materials of National Academy of Science of Ukraine, Kharkov, Ukraine B. Grynyov National Scientiﬁc Center, Kharkov Institute of Physics and Technology, Kharkov, Ukraine L. Levchuk National Scientiﬁc Center, Kharkov Institute of Physics and Technology, Kharkov, Ukraine L. Levchuk University of Bristol, Bristol, United Kingdom F. Ball, L. Beck, J.J. Brooke, D. Burns, E. Clement, D. Cussans, O. Davignon, H. Flacher, J. Goldstein, G.P. Heath, H.F. Heath, L. Kreczko, D.M. Newbold62, S. Paramesvaran, T. Sakuma, S. Seif El Nasr-storey, D. Smith, V.J. Smith University of Bristol, Bristol, United Kingdom F. Ball, L. Beck, J.J. Brooke, D. Burns, E. Clement, D. Cussans, O. Davignon, H. Flacher, J. Goldstein, G.P. Heath, H.F. Heath, L. Kreczko, D.M. Newbold62, S. Paramesvaran, T. Sakuma, S. Seif El Nasr-storey, D. Smith, V.J. Smith – 26 – Rutherford Appleton Laboratory, Didcot, United Kingdom A. Belyaev63, C. Brew, R.M. Brown, L. Calligaris, D. Cieri, D.J.A. Cockerill, J.A. Cough- lan, K. Harder, S. Harper, E. Olaiya, D. Petyt, C.H. Shepherd-Themistocleous, A. Thea, I.R. Tomalin, T. Williams Imperial College, London, United Kingdom Brown University, Providence, U.S.A. G. Benelli, D. Cutts, A. Garabedian, M. Hadley, J. Hakala, U. Heintz, J.M. Hogan, K.H.M. Kwok, E. Laird, G. Landsberg, J. Lee, Z. Mao, M. Narain, J. Pazzini, S. Piperov, S. Sagir, R. Syarif, D. Yu University of California, Davis, Davis, U.S.A. University of California, Davis, Davis, U.S.A. R. Band, C. Brainerd, D. Burns, M. Calderon De La Barca Sanchez, M. Chertok, J. Conway, R. Conway, P.T. Cox, R. Erbacher, C. Flores, G. Funk, M. Gardner, W. Ko, R. Lander, C. Mclean, M. Mulhearn, D. Pellett, J. Pilot, S. Shalhout, M. Shi, J. Smith, D. Stolp, K. Tos, M. Tripathi, Z. Wang University of California, Los Angeles, U.S.A. Brunel University, Uxbridge, United Kingdom J.E. Cole, P.R. Hobson, A. Khan, P. Kyberd, I.D. Reid, P. Symonds, L. Teodorescu, M. Turner, S. Zahid University of California, San Diego, La Jolla, U.S.A. University of California, San Diego, La Jolla, U.S.A. J.G. Branson, S. Cittolin, M. Derdzinski, R. Gerosa, D. Gilbert, B. Hashemi, A. Holzner, D. Klein, G. Kole, V. Krutelyov, J. Letts, I. Macneill, M. Masciovecchio, D. Olivito, S. Padhi, M. Pieri, M. Sani, V. Sharma, S. Simon, M. Tadel, A. Vartak, S. Wasserbaech65, J. Wood, F. W¨urthwein, A. Yagil, G. Zevi Della Porta JHEP01(2018)045 University of California, Santa Barbara - Department of Physics, Santa Bar- bara, U.S.A. N. Amin, R. Bhandari, J. Bradmiller-Feld, C. Campagnari, A. Dishaw, V. Dutta, M. Franco Sevilla, C. George, F. Golf, L. Gouskos, J. Gran, R. Heller, J. Incandela, A. Ovcharova, H. Qu, J. Richman, D. Stuart, I. Suarez, J. Yoo California Institute of Technology, Pasadena, U.S.A. D. Anderson, A. Bornheim, J.M. Lawhorn, H.B. Newman, T. Nguyen, C. Pena, M. Spirop- ulu, J.R. Vlimant, S. Xie, Z. Zhang, R.Y. Zhu Carnegie Mellon University, Pittsburgh, U.S.A. M.B. Andrews, T. Ferguson, T. Mudholkar, M. Paulini, J. Russ, M. Sun, H. Vogel, I. Vorobiev, M. Weinberg Carnegie Mellon University, Pittsburgh, U.S.A. University of Colorado Boulder, Boulder, U.S.A. J.P. Cumalat, W.T. Ford, F. Jensen, A. Johnson, M. Krohn, S. Leontsinis, T. Mulholland, K. Stenson, S.R. Wagner Cornell University, Ithaca, U.S.A. J. Alexander, J. Chaves, J. Chu, S. Dittmer, K. Mcdermott, N. Mirman, J.R. Patterso J. Alexander, J. Chaves, J. Chu, S. Dittmer, K. Mcdermott, N. Mirman, J.R. Patterson, D. Quach, A. Rinkevicius, A. Ryd, L. Skinnari, L. Soﬃ, S.M. Tan, Z. Tao, J. Thom, J. Tucker, P. Wittich, M. Zientek Fermi National Accelerator Laboratory, Batavia, U.S.A. S. Abdullin, M. Albrow, M. Alyari, G. Apollinari, A. Apresyan, A. Apyan, S. Banerjee, L.A.T. Bauerdick, A. Beretvas, J. Berryhill, P.C. Bhat, G. Bolla†, K. Burkett, J.N. But- ler, A. Canepa, G.B. Cerati, H.W.K. Cheung, F. Chlebana, M. Cremonesi, J. Duarte, V.D. Elvira, J. Freeman, Z. Gecse, E. Gottschalk, L. Gray, D. Green, S. Gr¨unendahl, O. Gutsche, R.M. Harris, S. Hasegawa, J. Hirschauer, Z. Hu, B. Jayatilaka, S. Jindariani, M. Johnson, U. Joshi, B. Klima, B. Kreis, S. Lammel, D. Lincoln, R. Lipton, M. Liu, T. Liu, R. Lopes De S´a, J. Lykken, K. Maeshima, N. Magini, J.M. Marraﬃno, D. Ma- son, P. McBride, P. Merkel, S. Mrenna, S. Nahn, V. O’Dell, K. Pedro, O. Prokofyev, G. Rakness, L. Ristori, B. Schneider, E. Sexton-Kennedy, A. Soha, W.J. Spalding, L. Spiegel, S. Stoynev, J. Strait, N. Strobbe, L. Taylor, S. Tkaczyk, N.V. Tran, L. University of California, Los Angeles, U.S.A. M. Bachtis, C. Bravo, R. Cousins, A. Dasgupta, A. Florent, J. Hauser, M. Ignatenko, N. Mccoll, S. Regnard, D. Saltzberg, C. Schnaible, V. Valuev – 27 – University of California, Riverside, Riverside, U.S.A. E. Bouvier, K. Burt, R. Clare, J. Ellison, J.W. Gary, S.M.A. Ghiasi Shirazi, G. Hanso E. Bouvier, K. Burt, R. Clare, J. Ellison, J.W. Gary, S.M.A. Ghiasi Shirazi, G. Hanson, J. Heilman, E. Kennedy, F. Lacroix, O.R. Long, M. Olmedo Negrete, M.I. Paneva, W. Si, L. Wang, H. Wei, S. Wimpenny, B. R. Yates J. Heilman, E. Kennedy, F. Lacroix, O.R. Long, M. Olmedo Negrete, M.I. Paneva, W. Si, L. Wang, H. Wei, S. Wimpenny, B. R. Yates The University of Iowa, Iowa City, U.S.A. y , y, B. Bilki66, W. Clarida, K. Dilsiz67, S. Durgut, R.P. Gandrajula, M. Haytmyradov, V. Khristenko, J.-P. Merlo, H. Mermerkaya68, A. Mestvirishvili, A. Moeller, J. Nachtman, H. Ogul69, Y. Onel, F. Ozok70, A. Penzo, C. Snyder, E. Tiras, J. Wetzel, K. Yi Johns Hopkins University, Baltimore, U.S.A. University of California, San Diego, La Jolla, U.S.A. Uplegger, E.W. Vaandering, C. Vernieri, M. Verzocchi, R. Vidal, M. Wang, H.A. Weber, A. Whitbeck – 28 – University of Florida, Gainesville, U.S.A. D. Acosta, P. Avery, P. Bortignon, D. Bourilkov, A. Brinkerhoﬀ, A. Carnes, M. Carve D. Acosta, P. Avery, P. Bortignon, D. Bourilkov, A. Brinkerhoﬀ, A. Carnes, M. Carver, D. Curry, R.D. Field, I.K. Furic, S.V. Gleyzer, B.M. Joshi, J. Konigsberg, A. Korytov, K. Kotov, P. Ma, K. Matchev, H. Mei, G. Mitselmakher, D. Rank, K. Shi, D. Sperka, N. Terentyev, L. Thomas, J. Wang, S. Wang, J. Yelton Florida International University, Miami, U.S.A. Y.R. Joshi, S. Linn, P. Markowitz, J.L. Rodriguez Florida State University, Tallahassee, U.S.A. A. Ackert, T. Adams, A. Askew, S. Hagopian, V. Hagopian, K.F. Johnson, T. Kolberg, G. Martinez, T. Perry, H. Prosper, A. Saha, A. Santra, V. Sharma, R. Yohay JHEP01(2018)045 Johns Hopkins University, Baltimore, U.S.A. B. Blumenfeld, A. Cocoros, N. Eminizer, D. Fehling, L. Feng, A.V. Gritsan, P. Maksimovic, J. Roskes, U. Sarica, M. Swartz, M. Xiao, C. You The University of Kansas, Lawrence, U.S.A. A. Al-bataineh, P. Baringer, A. Bean, S. Boren, J. Bowen, J. Castle, S. Khalil, A. Kropivnit- skaya, D. Majumder, W. Mcbrayer, M. Murray, C. Royon, S. Sanders, E. Schmitz, J.D. Tapia Takaki, Q. Wang Kansas State University, Manhattan, U.S.A. A. Ivanov, K. Kaadze, Y. Maravin, A. Mohammadi, L.K. Saini, N. Skhirtladze, S. Toda Lawrence Livermore National Laboratory, Livermore, U.S.A. F. Rebassoo, D. Wright Florida Institute of Technology, Melbourne, U.S.A. Florida Institute of Technology, Melbourne, U.S.A. M.M. Baarmand, V. Bhopatkar, S. Colafranceschi, M. Hohlmann, D. Noonan, T. Roy, F. Yumiceva University of Illinois at Chicago (UIC), Chicago, U.S.A. M.R. Adams, L. Apanasevich, D. Berry, R.R. Betts, R. Cavanaugh, X. Chen, O. Evdoki- mov, C.E. Gerber, D.A. Hangal, D.J. Hofman, K. Jung, J. Kamin, I.D. Sandoval Gonzalez, M.B. Tonjes, H. Trauger, N. Varelas, H. Wang, Z. Wu, J. Zhang Massachusetts Institute of Technology, Cambridge, U.S.A. Massachusetts Institute of Technology, Cambridge, U.S.A. D. Abercrombie, B. Allen, V. Azzolini, R. Barbieri, A. Baty, R. Bi, S. Brandt, W. B D. Abercrombie, B. Allen, V. Azzolini, R. Barbieri, A. Baty, R. Bi, S. Brandt, W. Busza, I.A. Cali, M. D’Alfonso, Z. Demiragli, G. Gomez Ceballos, M. Goncharov, D. Hsu, M. Hu, Y. Iiyama, G.M. Innocenti, M. Klute, D. Kovalskyi, Y.S. Lai, Y.-J. Lee, A. Levin, P.D. Luckey, B. Maier, A.C. Marini, C. Mcginn, C. Mironov, S. Narayanan, X. Niu, C. Paus, C. Roland, G. Roland, J. Salfeld-Nebgen, G.S.F. Stephans, K. Tatar, D. Velicanu, J. Wang, T.W. Wang, B. Wyslouch University of Minnesota, Minneapolis, U.S.A. University of Minnesota, Minneapolis, U.S.A. A.C. Benvenuti, R.M. Chatterjee, A. Evans, P. Hansen, J. Hiltbrand, S. Kalafut, Y. Kubota, Z. Lesko, J. Mans, S. Nourbakhsh, N. Ruckstuhl, R. Rusack, J. Turkewitz, M.A. Wadud JHEP01(2018)045 University of Mississippi, Oxford, U.S.A. J.G. Acosta, S. Oliveros University of Nebraska-Lincoln, Lincoln, U.S.A. University of Nebraska-Lincoln, Lincoln, U.S.A. E. Avdeeva, K. Bloom, D.R. Claes, C. Fangmeier, R. Gonzalez Suarez, R. Kamalieddin, I. Kravchenko, J. Monroy, J.E. Siado, G.R. Snow, B. Stieger E. Avdeeva, K. Bloom, D.R. Claes, C. Fangmeier, R. Gonzalez Suarez, R. Kamalieddin, I. Kravchenko, J. Monroy, J.E. Siado, G.R. Snow, B. Stieger State University of New York at Buﬀalo, Buﬀalo, U.S.A. State University of New York at Buﬀalo, Buﬀalo, U.S.A. J. Dolen, A. Godshalk, C. Harrington, I. Iashvili, D. Nguyen, A. Parker, S. Rappoccio, B. Roozbahani Northeastern University, Boston, U.S.A. Northeastern University, Boston, U.S.A. G. Alverson, E. Barberis, A. Hortiangtham, A. Massironi, D.M. Morse, T. Orimoto, R. Teixeira De Lima, D. Trocino, D. Wood Northwestern University, Evanston, U.S.A. Northwestern University, Evanston, U.S.A. S. Bhattacharya, O. Charaf, K.A. Hahn, N. Mucia, N. Odell, B. Pollack, M.H. Schmitt, K. Sung, M. Trovato, M. Velasco University of Notre Dame, Notre Dame, U.S.A. N. Dev, M. Hildreth, K. Hurtado Anampa, C. Jessop, D.J. Karmgard, N. Kellams, K. Lannon, N. Loukas, N. Marinelli, F. Meng, C. Mueller, Y. Musienko38, M. Planer, A. Reinsvold, R. Ruchti, G. Smith, S. Taroni, M. Wayne, M. Wolf, A. Woodard The Ohio State University, Columbus, U.S.A. J. Alimena, L. Antonelli, B. Bylsma, L.S. Durkin, S. Flowers, B. Francis, A. Hart, C. Hill, W. Ji, B. Liu, W. Luo, B.L. Winer, H.W. Wulsin Princeton University, Princeton, U.S.A. University of Maryland, College Park, U.S.A. University of Maryland, College Park, U.S.A. C. Anelli, A. Baden, O. Baron, A. Belloni, B. Calvert, S.C. Eno, Y. Feng, C. Ferraioli, N.J. Hadley, S. Jabeen, G.Y. Jeng, R.G. Kellogg, J. Kunkle, A.C. Mignerey, F. Ricci-Tam, Y.H. Shin, A. Skuja, S.C. Tonwar y, , g, gg, , g y, , Y.H. Shin, A. Skuja, S.C. Tonwar Y.H. Shin, A. Skuja, S.C. Tonwar – 29 – Princeton University, Princeton, U.S.A. S. Cooperstein, O. Driga, P. Elmer, J. Hardenbrook, P. Hebda, S. Higginbotham, D. Lange, J. Luo, D. Marlow, K. Mei, I. Ojalvo, J. Olsen, C. Palmer, P. Pirou´e, D. Stickland, C. Tully University of Puerto Rico, Mayaguez, U.S.A. S. Cooperstein, O. Driga, P. Elmer, J. Hardenbrook, P. Hebda, S. Higginbotham, D. Lange, J. Luo, D. Marlow, K. Mei, I. Ojalvo, J. Olsen, C. Palmer, P. Pirou´e, D. Stickland, C. Tully University of Puerto Rico, Mayaguez, U.S.A. S. Malik, S. Norberg University of Puerto Rico, Mayaguez, U.S.A. S. Malik, S. Norberg – 30 – Purdue University, West Lafayette, U.S.A. A. Barker, V.E. Barnes, S. Das, S. Folgueras, L. Gutay, M.K. Jha, M. Jones, A.W. J A. Khatiwada, D.H. Miller, N. Neumeister, C.C. Peng, H. Qiu, J.F. Schulte, J. Sun, F. Wang, W. Xie Purdue University Northwest, Hammond, U.S.A. T. Cheng, N. Parashar, J. Stupak Rice University, Houston, U.S.A. Rice University, Houston, U.S.A. A. Adair, Z. Chen, K.M. Ecklund, S. Freed, F.J.M. Geurts, M. Guilbaud, M. Kilpatrick, W. Li, B. Michlin, M. Northup, B.P. Padley, J. Roberts, J. Rorie, W. Shi, Z. Tu, J. Zabel, A. Zhang JHEP01(2018)045 University of Rochester, Rochester, U.S.A. A. Bodek, P. de Barbaro, R. Demina, Y.t. Duh, T. Ferbel, M. Galanti, A. Garcia-Bellido, J. Han, O. Hindrichs, A. Khukhunaishvili, K.H. Lo, P. Tan, M. Verzetti The Rockefeller University, New York, U.S.A. R. Ciesielski, K. Goulianos, C. Mesropian Rutgers, The State University of New Jersey, Piscataway, U.S.A. A. Agapitos, J.P. Chou, Y. Gershtein, T.A. G´omez Espinosa, E. Halkiadakis, M. Heindl, E. Hughes, S. Kaplan, R. Kunnawalkam Elayavalli, S. Kyriacou, A. Lath, R. Montalvo, K. Nash, M. Osherson, H. Saka, S. Salur, S. Schnetzer, D. Sheﬃeld, S. Somalwar, R. Stone, S. Thomas, P. Thomassen, M. Walker S. Thomas, P. Thomassen, M. Walker University of Tennessee, Knoxville, U.S.A. A.G. Delannoy, M. Foerster, J. Heideman, G. Riley, K. Rose, S. Spanier, K. Thapa University of Tennessee, Knoxville, U.S.A. A.G. Delannoy, M. Foerster, J. Heideman, G. Riley, K. Rose, S. Spanier, K. Thapa Texas A&M University, College Station, U.S.A. O. Bouhali71, A. Castaneda Hernandez71, A. Celik, M. Dalchenko, M. De Mattia, A. Del- gado, S. Dildick, R. Eusebi, J. Gilmore, T. Huang, T. Kamon72, R. Mueller, Y. Pakhotin, R. Patel, A. Perloﬀ, L. Perni`e, D. Rathjens, A. Safonov, A. Tatarinov, K.A. Ulmer Texas Tech University, Lubbock, U.S.A. Texas Tech University, Lubbock, U.S.A. N. Akchurin, J. Damgov, F. De Guio, P.R. Dudero, J. Faulkner, E. Gurpinar, S. Kunori, K. Lamichhane, S.W. Lee, T. Libeiro, T. Mengke, S. Muthumuni, T. Peltola, S. Undleeb, N. Akchurin, J. Damgov, F. De Guio, P.R. Dudero, J. Faulkner, E. Gurpinar, S. Kunori, N. Akchurin, J. Damgov, F. De Guio, P.R. Dudero, J. Faulkner, E. Gurpinar, S. Kunori, K. Lamichhane, S.W. Lee, T. Libeiro, T. Mengke, S. Muthumuni, T. Peltola, S. Undleeb, I. Volobouev, Z. Wang I. Volobouev, Z. Wang Vanderbilt University, Nashville, U.S.A. Vanderbilt University, Nashville, U.S.A. S. Greene, A. Gurrola, R. Janjam, W. Johns, C. Maguire, A. Melo, H. Ni, K. Padeken, P. Sheldon, S. Tuo, J. Velkovska, Q. Xu y, , S. Greene, A. Gurrola, R. Janjam, W. Johns, C. Maguire, A. Melo, H. Ni, K. Padeken, P. Sheldon, S. Tuo, J. Velkovska, Q. Xu University of Virginia, Charlottesville, U.S.A. M.W. Arenton, P. Barria, B. Cox, R. Hirosky, M. Joyce, A. Ledovskoy, H. Li, C. Neu, T. Sinthuprasith, Y. Wang, E. Wolfe, F. Xia Wayne State University, Detroit, U.S.A. Wayne State University, Detroit, U.S.A. R. Harr, P.E. Karchin, N. Poudyal, J. Sturdy, P. Thapa, S. Zaleski R. Harr, P.E. Karchin, N. Poudyal, J. Sturdy, P. Thapa, S. University of Rochester, Rochester, U.S.A. Zaleski – 31 – University of Wisconsin — Madison, Madison, WI, U.S.A. University of Wisconsin — Madison, Madison, WI, U.S.A. M. Brodski, J. Buchanan, C. Caillol, S. Dasu, L. Dodd, S. Duric, B. Gomber, M. Groth , , , , , , , , M. Herndon, A. Herv´e, U. Hussain, P. Klabbers, A. Lanaro, A. Levine, K. Long, R. Loveless, G. Polese, T. Ruggles, A. Savin, N. Smith, W.H. Smith, D. Taylor, N. Woods M. Herndon, A. Herv´e, U. Hussain, P. Klabbers, A. Lanaro, A. Levine, K. Long, †: Deceased 1: Also at Vienna University of Technology, Vienna, Austria 2: Also at State Key Laboratory of Nuclear Physics and Technology, Peking University, Beijin China JHEP01(2018)045 3: Also at IRFU, CEA, Universit´e Paris-Saclay, Gif-sur-Yvette, France 4: Also at Universidade Estadual de Campinas, Campinas, Brazil 5: Also at Universidade Federal de Pelotas, Pelotas, Brazil 6: Also at Universit´e Libre de Bruxelles, Bruxelles, Belgium 7: Also at Institute for Theoretical and Experimental Physics, Moscow, Russia 8: Also at Joint Institute for Nuclear Research, Dubna, Russia 9: Also at Helwan University, Cairo, Egypt 10: Now at Zewail City of Science and Technology, Zewail, Egypt 10: Now at Zewail City of Science and Technology, Zewail, Egypt Now at Fayoum University, El-Fayoum, Egypt 12: Also at British University in Egypt, Cairo, Egypt 13: Now at Ain Shams University, Cairo, Egypt 14: Also at Universit´e de Haute Alsace, Mulhouse, France 14: Also at Universit´e de Haute Alsace, Mulhouse, France 15: Also at Skobeltsyn Institute of Nuclear Physics, Lomonosov Moscow State University, Moscow, Russia 16: Also at Tbilisi State University, Tbilisi, Georgia 17: Also at CERN, European Organization for Nuclear Research, Geneva, Switzerland Also at CERN, European Organization for Nuclear Research, Geneva, Switzerland 18: Also at RWTH Aachen University, III. Physikalisches Institut A, Aachen, Germany Also at RWTH Aachen University, III. Physikalisches Institut A, Aachen, Germany Also at RWTH Aachen University, III. Physika 19: Also at University of Hamburg, Hamburg, Germany 20: Also at Brandenburg University of Technology, Cottbus, Germany 20: Also at Brandenburg University of Technology, Cottbus, Germany 21: Also at MTA-ELTE Lend¨ulet CMS Particle and Nuclear Physics Group, E¨otv¨os Lor´and University, Budapest, Hungary 21: Also at MTA-ELTE Lend¨ulet CMS Particle and Nuclear Physics Group, E¨otv¨os Lor´and 21: Also at MTA-ELTE Lend¨ulet CMS Particle and Nuclear Physics Group, E¨otv¨os Lor´an 21: Also at MTA-ELTE Lendulet CMS Particle and Nuclear Physics Group, Eotvos Lorand University, Budapest, Hungary University, Budapest, Hungary 22: Also at Institute of Nuclear Research ATOMKI, Debrecen, Hungary 22: Also at Institute of Nuclear Research ATOMKI, Debrecen, Hungary 23: Also at Institute of Physics, University of Debrecen, Debrecen, Hungary 23: Also at Institute of Physics, University of Debrecen, Debrecen, Hungary 24: Also at Indian Institute of Technology Bhubaneswar, Bhub 25: Also at Institute of Physics, Bhubaneswar, India 26: Also at University of Visva-Bharati, Santiniketan, India 26: Also at University of Visva-Bharati, Santiniketan, India 27: Also at University of Ruhuna, Matara, Sri Lanka 27: Also at University of Ruhuna, Matara, Sri Lanka 28: Also at Isfahan University of Technology, Isfahan, Iran 28: Also at Isfahan University of Technology, Isfahan, Iran 29: Also at Yazd University, Yazd, Iran 30: Also at Plasma Physics Research Center, Science and Research Branch, Islamic Aza University, Tehran, Iran 30: Also at Plasma Physics Research Center, Science and Research Branch, Islamic Azad University, Tehran, Iran University, Tehran, Iran 31: Also at Universit`a degli Studi di Siena, Siena, Italy 31: Also at Universit`a degli Studi di Siena, Siena, Italy 32: Also at INFN Sezione di Milano-Bicocca; Universit`a di Milano-Bicocca, Milano, Italy 32: Also at INFN Sezione di Milano-Bicocca; Universit`a di Milano-Bicocca, Mila 33: Also at Purdue University, West Lafayette, U.S.A. 34: Also at International Islamic University of Malaysia, Kuala Lumpur, Malaysia 34: Also at International Islamic University of Malaysia, Kuala Lumpur, Malaysia 35: Also at Malaysian Nuclear Agency, MOSTI, Kajang, Malaysia 35: Also at Malaysian Nuclear Agency, MOSTI, Kajang, Malaysia 36: Also at Consejo Nacional de Ciencia y Tecnolog´ıa, Mexico city, Mexico 36: Also at Consejo Nacional de Ciencia y Tecnolog´ıa, Mexico city, Mexico 37: Also at Warsaw University of Technology, Institute of Electronic Systems, Warsaw, Poland – 32 – 38: Also at Institute for Nuclear Research, Moscow, Russia 39: Now at National Research Nuclear University ’Moscow Engineering Physics Insti- tute’ (MEPhI), Moscow, Russia 40: Also at St. Petersburg State Polytechnical University, St. Petersburg, Russia 41: Also at University of Florida, Gainesville, U.S.A. 42: Also at P.N. University, Tehran, Iran Lebedev Physical Institute, Moscow, Russia 43: Also at Budker Institute of Nuclear Physics, Novosibirsk, Russia 44: Also at Faculty of Physics, University of Belgrade, Belgrade, Serbia 45: Also at University of Belgrade, Faculty of Physics and Vinca Institute of Nuclear Sciences, Belgrade, Serbia 46: Also at Scuola Normale e Sezione dell’INFN, Pisa, Italy JHEP01(2018)045 47: Also at National and Kapodistrian University of Athens, Athens, Greece 47: Also at National and Kapodistrian University of Athens, Athens, Greece 48: Also at Riga Technical University, Riga, Latvia 49: Also at Universit¨at Z¨urich, Zurich, Switzerland 50: Also at Stefan Meyer Institute for Subatomic Physics (SMI), Vienna, Austria 50: Also at Stefan Meyer Institute for Subatomic Physics (SMI), Vienna, Austria 51: Also at Gaziosmanpasa University, Tokat, Turkey 52: Also at Istanbul Aydin University, Istanbul, Turkey 53: Also at Mersin University, Mersin, Turkey 54: Also at Cag University, Mersin, Turkey 55: Also at Piri Reis University, Istanbul, Turkey 56: Also at Adiyaman University, Adiyaman, Turkey 57: Also at Izmir Institute of Technology, Izmir, Turkey 58: Also at Necmettin Erbakan University, Konya, Turkey 59: Also at Marmara University, Istanbul, Turkey 60: Also at Kafkas University, Kars, Turkey 61: Also at Istanbul Bilgi University, Istanbul 62: Also at Rutherford Appleton Laboratory, Didcot, United Kingdom 62: Also at Rutherford Appleton Laboratory, Didcot, United Kingdom 63: Also at School of Physics and Astronomy, University of Southampton, Southampton, United Kingdom 64: Also at Instituto de Astrof´ısica de Canarias, La Laguna, Spain 64: Also at Instituto de Astrof´ısica de Canarias, La Laguna, Spain 65: Also at Utah Valley University, Orem, U.S.A. 66: Also at Beykent University, Istanbul, Turkey 67: Also at Bingol University, Bingol, Turkey 68: Also at Erzincan University, Erzincan, Turkey 69: Also at Sinop University, Sinop, Turkey 70: Also at Mimar Sinan University, Istanbul, Istanbul, Turkey 70: Also at Mimar Sinan University, Istanbul, Istanbul, Turkey 71: Also at Texas A&M University at Qatar, Doha, Qatar 71: Also at Texas A&M University at Qatar, Doha, Qatar 72: Also at Kyungpook National University, Daegu, Korea 72: Also at Kyungpook National University, Daegu, Korea – 33 –
https://openalex.org/W4388645399	https://ejournal.stmikbinapatria.ac.id/index.php/JT/article/download/347/218	Indonesian	null	Pengaruh Pengetahuan dasar dan Motivasi Belajar Terhadap Hasil belajar Algoritma Siswa SMK Negeri	Transformasi	2,023	cc-by-sa	3,901	1 “Prodi Teknologi Rekayasa Multimedia” Politeknik Bhakti Kartini Email : ninu.nanda@gmail.com 1 “Prodi Teknologi Rekayasa Multimedia” Politeknik Bhakti Kartini Email : ninu.nanda@gmail.com Abstrak Penelitian ini bertujuan untuk mengetahui pengaruh pengetahuan dasar dan motivasi belajar terhadap hasil belajar siswa. Penelitian yang berjenis cross sectional studi ini menggunakan sampel siswa dari sampel SMK Negeri di kota Bekasi. Survey disebar pada 120 responden dengan tingkat respon 50 persen. Sampel penelitian diperoleh melalui metode simple random sampling. Menggunakan 3 variabel yang diteliti yakni x1 = pengetahuan dasar, x2=motivasi belajar, dan y = hasil belajar. Analisis data menggunakan regresi liner berganda untuk mengetahui efek pengaruh pada variabelnya. Hasil penelitian menunjukkan analisis refresi linier untuk data ini adalah y= 0,354x1+0,283x2-0,448 ; r2 =0,813. Terdapat pengaruh yang signifikan antara pengetahuan dasar, motivasi belajar dengan hasil belajar Algoritma Pemrograman. Pengaruh variabel pengetahuan dasar dan motivasi belajar secara bersama-sama terhadap hasil belajar adalah sebesar 0,813, selebihnya dipengaruhi oleh variabel lain. Kata kunci : pengetahuan dasar komputer, motivasi, hasil belajar, algoritma pemrograman. Abstract This study aims to determine the effect of prior knowledge and learning motivation on student learning outcomes. This was a cross-sectional study used a sample of students from State Vocational High Schools in the city of Bekasi. The survey was distributed to 120 respondents with a response rate of 50 percent. The research sample was obtained through simple random sampling method. Using 3 variables studied, x1 = prior knowledge, x2 = learning motivation, and y = learning outcomes. Analyzing data used multiple linear regression to determine the effect on the variable. The results of this research show there is a linear reference analysis for this data . With regression equiation was y= 0.354x1+0.283x2-0.448 ; r2 = 0.813. From this research we could find that there was a significant influence between prior knowledge, learning motivation and learning outcomes of Programming Algorithms. The influence of prior knowledge and learning motivation simultaneously on learning outcomes is 0.813, the rest is influenced by other variables. Keywords : prior knowledge, motivation, learning outcomes, programming algorithms. PENGARUH PENGETAHUAN DASAR DAN MOTIVASI BELAJAR TERHADAP HASIL BELAJAR ALGORITMA SISWA SMK NEGERI Ibnu Adkha1 1.2. Rumusan Masalah Berdasarkan latar belakang dan identifikasi masalah serta batasan permasalahan di atas, maka rumusan masalah dalam penelitian ini adalah: Berdasarkan latar belakang dan identifikasi masalah serta batasan permasalahan di atas, maka rumusan masalah dalam penelitian ini adalah: 1. Apakah terdapat hubungan antara pengetahuan komputer dengan hasil belajar Algoritma Pemrograman? 2. Apakah terdapat hubungan antara motivasi dengan hasil belajar Algoritma Pemrograman? 3. Apakah terdapat hubungan antara pengetahuan komputer dan motivasi secara bersama-sama dengan hasil belajar Algoritma Pemrograman? “Jurnal TRANSFORMASI (Informasi & Pengembangan Iptek)” (STMIK BINA PATRIA) E-ISSN : 2827-8550 P- ISSN : 1978-5569 “Jurnal TRANSFORMASI (Informasi & Pengembangan Iptek)” (STMIK BINA PATRIA) E-ISSN : 2827-8550 P- ISSN : 1978-5569 tersebut merupakan prasyarat yang harus dipenuhi dalam belajar algoritma pemrograman. Pengetahuan komputer dalam proses belajar Algoritma Pemrograman sangat membantu siswa untuk menunjang keterampilan siswa dalam pembelajaran teori ataupun praktek membuat bahasa pemrograman. Motivasi adalah usaha-usaha yang menyebabkan seseorang atau kelompok bergerak melakukan sesuatu karena ingin mencapai tujuan yang dikehendaki. Namun pada intinya bahwa motivasi merupakan kondisi psikologis yang mendorong seseorang untuk melakukan sesuatu. Dalam kegiatan belajar, motivasi dapat dikatakan sebagai keseluruhan daya penggerak didalam diri siswa yang menjamin kelangsungan dan memberikan arah kegiatan belajar, sehingga diharapkan tujuan dapat tercapai. Dalam kegiatan belajar, motivasi sangat diperlukan, sebab seseorang yang tidak mempunyai motivasi dalam belajar, tidak akan mungkin melakukan aktivitas belajar. Merujuk pada uraian diatas, apakah terdapat hubungan pengetahuan komputer dan motivasi dengan hasil belajar Algoritma Pemrograman. Hal ini perlu dibuktikan secara empiris melalui data-data hasil penelitian di lapangan. 1.3. Tujuan Untuk mengetahui pengaruh variabel pengetahuan dasar komputer dan variabel motivasi terhadap hasil belajar Algoritma Pemrograman. 1.1. Latar Belakang Salah satu mata pelajaran kejuruan yang diberikan di SMK jurusan RPL adalah Algoritma Pemrograman. Mata pelajaran ini merupakan bidang studi yang menuntut agar siswa mempunyai kemampuan untuk menggunakan teknologi komputer dalam kehidupan sehari-hari dan mengaplikasikan komputer sesuai dengan standar kompetensi. Hasil belajar siswa dipengaruhi oleh faktor pengetahuan dasar dan motivasi (Ramaliza & Mutiara, 2021). Pengetahuan komputer dijadikan sebagai parameter bagi siswa untuk mendapat hasil belajar algoritma pemrograman yang baik, karena penguasaan pengetahuan komputer 61 Jurnal TRANSFORMASI, Vol. 19, No. 1, 2023 : 61 - 68 61 “Jurnal TRANSFORMASI (Informasi & Pengembangan Iptek)” (STMIK BINA PATRIA) E-ISSN : 2827-8550 P- ISSN : 1978-5569 “Jurnal TRANSFORMASI (Informasi & Pengembangan Iptek)” (STMIK BINA PATRIA) E-ISSN : 2827-8550 P- ISSN : 1978-5569 mengembangkannya lebih lanjut, misalnya melalui pembelajaran aktif atau melalui pembelajaran terbalik. Dalam metode pembelajaran terbalik, diuangkap bahwa siswa yang diminta balajar terlebih dahulu/ studi literature sebelum pembelajaran dimulai mendapatkan pengetahuan dasar yang lebih baik saat proses pembelajaran tatap muka dengan guru, sehingga hasil belajarnya lebih baik dibandingkan dengan kelompok kontrolnya (Van Alten, Phielix, Janssen, & Kester, 2019). Begitupun studi literatur oleh Hartikainen, Rintala, Pylväs, & Nokelainen, (2019), diungkap pembahasan mengenai pembelajaran aktif dimana siswa diberikan situasi berinteraksi yang lebih besar dengan guru, hal ini memerlukan pengetahuan dasar yang didapat dari sifat penasaran yang meningkat dan kebiasaan belajar yang positif. Pada akhirnya hal ini akan memperbaiki hasil belajar siswa. Pengetahuan dasar bukan merupakan satu-satunya faktor yang menentukan keberhasilan seseorang, karena ada faktor lain yang mempengaruhi. Motivasi belajar merupakan faktor lain yang mempengatuhi hasil belajar siswa. Motivasi belajar berkenaan dengan faktor-faktor yang mendorong tingkah laku dan memberikan arah kepada tingkah laku yakni untuk belajar. Motivasi belajar berpengaruh positif terhadap hasil belajar siswa, artinya semakin tinggi motivasi belajar maka hasil belajar siswa akan menjadi lebih baik (Rahman, 2022). Motivasi belajar dapat berwujud beberapa faktor, yaitu faktor internal dan faktor eksternal atau kombinasinya. Motivasi intrinsik adalah motivasi yang berasal dari dorongan mental dalam diri individu itu sendiri, terkait dengan kemauan, kemampuan, tujuan individu itu sendiri dan orang lain. Sedangkan motivasi ekstrinsik bersumber dari dorongan di luar dirinya atau lingkungannya. Perbedaan motivasi dalam dan luar ini pernah diteliti yang hasilnya adalah terdapat perbedaan hasil belajar pada siswa yang memiliki motivasi belajar intrinsik lebih tinggi daripada siswa dengan motivasi belajar ekstrinsik dengan menggunaan pendekatan metode tertentu (Hasan, Pomalato, & Uno, 2020). Hasil belajar mencerminkan sejauh mana tujuan proses pembelajaran telah tercapai atau tercapai. Hasil belajar dapat ditingkatkan dengan beberapa metode. Dalam penelitiian sebelumnya oleh Van Alten et al., (2019) dengan menggunakan metode terbalik, siswa lebih mampu mencapai hasil belajar yang lebih tinggi, karena ada lebih banyak waktu di kelas yang tersedia untuk kegiatan pembelajaran. Hal ini dapat menumbuhkan keaktiftifan, konstruktif, dan interaktif dari siswa. Sebaliknya, kelas konvensional memeberikan efek membosankan karena sebagian besar waktu kelas dikhususkan untuk kegiatan seperti tatap muka serta keterlibatan yang pasif. Dalam pelaksanaan pembelajaran perlu memperhatikan banyak aspek yang harus diperhatikan yakni diantaranya adalah aspek kognitif, afektif, dan psikologis yang saling sinergis. 2. Kajian Literatur Pembelajaran merupakan sebuah proses kompleks yang berbeda pada tiap individunya. Oleh karenanya diperlukan sebuah mekanisme tertentu untuk mengkaji dan memahami bagaimana cara untuk membuat proses pembelajaran menjadi lebih efektif dari waktu ke waktu. Belajar membutuhkan suasana belajar yang kondusif agar siswa tidak mudah jenuh,bosan selain itu juga dapat menumbuhkan minat motivasi belajar (Arianti, 2019). Evaluasi pembelajaran perlu dilakukan dari waktu ke waktu. Evaluasi ini bersifat menyeluruh dan terpadu. Evaluasi bersifat menyeluruh maknanya adalah penilaian berfokus pada semua pihak yang terlibat dalam proses pembelajaran, sedangkan terpadu merupakan proses pelibatan semua pihak dalam proses pembelajaran, terutama siswa. Kegiatan belajar mengajar perlu memberikan pengalaman belajar yang terhubung dengan pengetahuan dasar untuk membantu siswa menambah khasanah pengetahuan dasar siswa selanjutnya yang sinergis(Thoyib, Subandowo, & Wiyarno, 2021). j y y g g y y Pengetahuan dasar dapat menentukan hasil belajar bagi siswa dalam kegiatan pembelajaran (Francis, Wieth, Zabel, & Carr, 2020). Terdapat hubungan yang saling terkait antara efek pengetahuan dasar terhadap hasil belajar, yakni pengetahuan dasar seseorang dapat meningkatkan sifat penasaran untuk memperoleh lebih dan lebih dalam belajar. Sifat penasaran akan berpengaruh juga kepada kebiasaan belajar yang meningkat sehingga dapat membantu siswa dalam memperoleh hasil belajar yang lebih baik dari waktu ke waktu (Wade & Kidd, 2019). Guru perlu mengungkap pengetahuan dasar murid dan Jurnal TRANSFORMASI, Vol. 19, No. 1, 2023 : 61 - 68 62 62 4. Hasil dan Pembahasan 4.1.1. Deskripsi Analisis Data 4.1.1. Deskripsi Analisis Data Data yang disajikan kemudian diolah menggunakan teknik statistik dalam bentuk distribusi frekuensi, skor tertinggi, skor terendah, nilai rerata, dan simpangan baku. Deskripsi data hasil penelitian pengetahuan dasar, kecerdasan emosional, motivasi belajar, dan hasil belajar tiap perlakuan dapat dilihat pada Tabel 1. Tabel 1. Data Pengetahuan Dsar, Motivasi Belajar, dan Hasil Belajar Variabel jangkauan min maks rerata Pengetahuan Dasar 0,429 0,5 0,929 0,740 Motivasi Belajar 0,909 2,817 3,726 3,325 Hasil Belajar 0,464 0,5 0,964 0,757 Sumber : Data hasil penelitian yang diolah, 2023 Tabel 1. Data Pengetahuan Dsar, Motivasi Belajar, dan Hasil Belajar Sumber : Data hasil penelitian yang diolah, 2023 3. Metode Penelitian Penelitian ini merupakan penelitian deskriptif crossectional. Populasi dalam penelitian ini adalah seluruh siswa SMK Negeri 5 Kota Bekasi yang terdaftar pada tahun ajaran 2018/2019. Teknik pengambilan sampel dilakukan dengan simple random sampling. Variabel yang digunakan pada penelitian ini tediri dari dua variabel yaitu variabel bebas (X) dan variabel terikat (Y). Variabel bebas terdiri dari pengetahuan dasar (X1), motivasi belajar (X2), sedangkan variabel terikat yaitu hasil belajar Algoritma (Y). Jenis data dalam penelitian ini diperoleh dari: (1) data kuantitatif, yaitu data yang diperoleh dari hasil tes pengetahuan dasar siswa; (2) data kualitatif, yaitu angket kecerdasan emosional dan motivasi belajar yang diberikan kepada responden yang telah ditetapkan sebagai sampel. Pengumpulan data menggunakan 2 cara yaitu tes dalam bentuk pilihan ganda dan angket. Angket diberikan kepada siswa untuk memperoleh informasi mengenai motivasi belajar yang dimiliki oleh siswa dalam mempengaruhi hasil belajar algoritma. Jurnal TRANSFORMASI, Vol. 19, No. 1, 2023 : 61 - 68 63 “Jurnal TRANSFORMASI (Informasi & Pengembangan Iptek)” (STMIK BINA PATRIA) E-ISSN : 2827-8550 P- ISSN : 1978-5569 Aspek-aspek yang digunakan utuk mengukur seberapa besar motivasi belajar siswa ditinjau dari faktor intrinsik dan faktor ekstrinsik pada diri siswa. Angket kecerdasan emosional dan motivasi belajar masing-masing terdiri dari 28 butir pernyataan. Tes objektif/pilihan ganda diberikan guna mengukur pengetahuan dasar siswa berdasarkan kemampuan kognitif yang dimiliki. Proses analisis data pengaruh pengetahuan dasar dan motivasi belajar terhadap hasil belajar Algoritma siswa SMK Negeri 5 Kota Bekasi menggunakan analisis regresi linear berganda dengan bantuan program Minitab 21.3 64 bit. 4.1.2. Uji Normalitas j Uji normalitas penelitian menggunakan metode One-Sample Kolmogorov-Smirnov Test dengan taraf probabilitas Sig. α = 0,05. Hasil uji normalitas data dari variabel penelitian ini disajikan pada Tabel 2. Tabel 2. Hasil Uji Normalitas Data Variabel Penelitian Tabel 2. Hasil Uji Normalitas Data Variabel Penelitian Variabel Nilai K-Z Nilai signifikansi Pengetahuan Dasar 1,036 0,234 Motivasi Belajar 0,804 0,537 Hasil Belajar 0,880 0,421 Sumber : Data hasil penelitian yang diolah, 2023 Sumber : Data hasil penelitian yang diolah, 2023 Menggunakan metode one KS, maka keseluruhan data dianggap terdistribusi secara normal. Oleh karena itu maka analisis data menggunakan metode parametrik yakni analisis regresi linier berganda. 4.2 Pembahasan Perubahan kemampuan yang dimiliki oleh siswa dalam berbagai bidang dapat terjadi dalam proses pembelajaran, dan kemampuan itu diperoleh karena adanya belajar. Fenomena yang kini terjadi pada siswa di SMK Negeri 5 Kota Bekasi adalah rendahnya hasil belajar. Rendahnya hasil belajar tersebut tidak terlepas dari faktor yang berasal dari dalam diri siswa itu sendiri yang disebut sebagai faktor internal. Hasil penelitian menunjukkan bahwa pengetahuan dasar dan motivasi belajar mempunyai pengaruh positif terhadap hasil belajar pada mata pelajaran Algoritma. “Jurnal TRANSFORMASI (Informasi & Pengembangan Iptek)” (STMIK BINA PATRIA) E-ISSN : 2827-8550 P- ISSN : 1978-5569 Tabel 3. Analisis regresi linier berganda koefisien Nilai t Nilai sig R2 X1 0,354 5,133 0 0,813 X2 0,283 8,387 0 Konstanta -0,448 -4,983 0 Sumber : Data hasil penelitian yang diolah, 2023 Tabel 3. Analisis regresi linier berganda Sumber : Data hasil penelitian yang diolah, 2023 Berdasarkan hasil analisis regresi linier berganda pada Tabel 3. dapat dikonversikan kedalam model persamaan regresi linear berganda dalam penelitian ini, maka dapat dibuat suatu bentuk persamaan sebagai berikut : erganda dalam penelitian in 813 Koefisien ini bernilai positi i variabel-varibel x nya (yak ariabel (x1 & x2) terseb dap hasil belajar siswa. Peng kup besar (yakni mendekati ain. engaruh sinergis kedua var hasil nilai F hitung 123,725 d oleh siswa dalam berbagai mpuan itu diperoleh kare SMK Negeri 5 Kota Beka ebut tidak terlepas dari fakt but sebagai faktor interna n motivasi belajar mempun Algoritma. adap Hasil belajar Algoritm riabel pengetahuan dasar te at diketahui nilai t hitung se ai t hitung > t tabel (5,155 ifikan antara pengetahuan an dasar yang tinggi maka y= 0,354x1+0,283x2-0,448 ; R2 =0,813 (1) y= 0,354x1+0,283x2-0,448 ; R2 =0,813 (1) (1) Koefisien determinasi (R2) sebesar 0,813. Koefisien ini bernilai positif, maka variabel tak bebas (Y) dapat dijelaskan oleh variasi dari variabel-varibel x nya (yakni x1 dan x2). Nilai ini memberikan makna bahwa kedua variabel (x1 & x2) tersebut secara simultan mempunyai pengaruh sebesar 81,3% terhadap hasil belajar siswa. Pengaruh kedua variabel ini secara simultan pada penelitian ini cukup besar (yakni mendekati nilai 1). Selebihnya sebesar 18,7 % dipengaruhi oleh variabel lain. Selain regresi linier berganda juga diuji pengaruh sinergis kedua variabel X terhadap Y menggunakan pendekatan Anova, dengan hasil nilai F hitung 123,725 dan nilai Sig =0,000. 4.1.3. Analisis Regresi Linear Berganda g g Regresi linier berganda adalah model persamaan yang menggambarkan pengaruh dua variabel independen (x1 & x2 ) atau lebih terhadap variabel dependen (Y). Hasil analisis regresi linear berganda dari 60 responden dapat dilihat pada tabel 3 berikut : 64 Jurnal TRANSFORMASI, Vol. 19, No. 1, 2023 : 61 - 68 64 “Jurnal TRANSFORMASI (Informasi & Pengembangan Iptek)” (STMIK BINA PATRIA) E-ISSN : 2827-8550 P- ISSN : 1978-5569 karena penguasaan pengetahuan komputer tersebut merupakan prasyarat yang harus dipenuhi dalam belajar Algoritma Pemrograman, sehingga akan sangat membantu dalam menunjang keterampilan siswa dalam belajar teori ataupun praktek untuk menganalisa atau membuat bahasa pemrograman pada mapel tersebut. Selain itu juga pengetahuan komputer merupakan parameter keberhasilan bagi siswa dalam proses belajar mengajar, sehingga variabel ini dapat mempengaruhi secara signifikan hasil belajar siswa. karena penguasaan pengetahuan komputer tersebut merupakan prasyarat yang harus dipenuhi dalam belajar Algoritma Pemrograman, sehingga akan sangat membantu dalam menunjang keterampilan siswa dalam belajar teori ataupun praktek untuk menganalisa atau membuat bahasa pemrograman pada mapel tersebut. Selain itu juga pengetahuan komputer merupakan parameter keberhasilan bagi siswa dalam proses belajar mengajar, sehingga variabel ini dapat mempengaruhi secara signifikan hasil belajar siswa. 4.2.2. Pengaruh Motivasi Belajar Terhadap Hasil Belajar Pengaruh Pengetahuan dasar dan Motivasi Belajar Terhadap Hasil Belajar Dengan hipotesis bahwa : H0 : 1 = 2 = 0; (variable X1 dan X2 tidak berpengaruh terhadap Y) (2) H1 :1 ≠ 2 ≠ 0; (variabel X1 dan X2 berpengaruh terhadap Y) (3) Nilai taraf signifikansi (α) = 5% Tabel uji F untuk (α) = 5% dengan df pembilang = 59 dan df penyebut = 1 Dan F tabel = 3,06, maka hasil Uji Koefisien regresi secara bersama-sama (Uji F) output analisis regresi dapat diketahui nilai F sebesar 123,725 atau dari hasil analisis tersebut dapat diketahui nilai F hitung > F tabel (123,725 > 4,004), maka H0 ditolak, artinya ada pengaruh secara signifikan antara pengetahuan dasar dan motivasi belajar secara bersama-sama terhadap hasil belajar siswa. Hal ini diperkuat oleh nilai sig yakni 0,0 yang kurang dari 4.2.3. Pengaruh Pengetahuan dasar dan Motivasi Belajar Terhadap Hasil Belajar Dengan hipotesis bahwa : H0 : 1 = 2 = 0; (variable X1 dan X2 tidak berpengaruh terhadap Y) (2) H1 :1 ≠ 2 ≠ 0; (variabel X1 dan X2 berpengaruh terhadap Y) (3) Nilai taraf signifikansi (α) = 5% Tabel uji F untuk (α) = 5% dengan df pembilang = 59 dan df penyebut = 1 Dan F tabel = 3,06, maka hasil Uji Koefisien regresi secara bersama-sama (Uji F) output analisis regresi dapat diketahui nilai F sebesar 123,725 atau dari hasil analisis tersebut dapat diketahui nilai F hitung > F tabel (123,725 > 4,004), maka H0 ditolak, artinya ada pengaruh secara signifikan antara pengetahuan dasar dan motivasi belajar secara bersama-sama terhadap hasil belajar siswa. Hal ini diperkuat oleh nilai sig yakni 0,0 yang kurang dari 4.2.3. Pengaruh Pengetahuan dasar dan Motivasi Belajar Terhadap Hasil Belajar Dengan hipotesis bahwa : (2) (3) Nilai taraf signifikansi (α) = 5% g ( ) Tabel uji F untuk (α) = 5% dengan df pembilang = 59 dan df penyebut = 1 j ( ) g p g p y Dan F tabel = 3,06, maka hasil Uji Koefisien regresi secara bersama-sama (Uji F) output analisis regresi dapat diketahui nilai F sebesar 123,725 atau dari hasil analisis tersebut dapat diketahui nilai F hitung > F tabel (123,725 > 4,004), maka H0 ditolak, artinya ada pengaruh secara signifikan antara pengetahuan dasar dan motivasi belajar secara bersama-sama terhadap hasil belajar siswa. Hal ini diperkuat oleh nilai sig yakni 0,0 yang kurang dari Jurnal TRANSFORMASI, Vol. 19, No. 4.2.1. Pengaruh Pengetahuan Dasar Terhadap Hasil belajar Algoritma Berdasarkan hasil analisis statistik variabel pengetahuan dasar terhadap hasil belajar dengan menggunakan analisis regresi dapat diketahui nilai t hitung sebesar 5,155 dan sig 0,000. Hasil analisis regresi diperoleh nilai t hitung > t tabel (5,155 > 1.671) maka H0 ditolak, artinya ada pengaruh secara signifikan antara pengetahuan dasar dengan hasil belajar. Apabila siswa memiliki pengetahuan dasar yang tinggi, maka akan mempengaruhi dan meningkatkan hasil belajar Algoritma siswa di SMK Negeri 5 Kota Bekasi. Hal ini diperkuat dengan nilai sig 0,000 yang kurang dari 0,05. Sehingga variabel X1 berpengaruh terhadap variabel Y. Pengetahuan komputer merupakan pengetahuan dasar mengenai dasar-dasar komputer yang berhubungan dengan hardware, software dan brainware suatu komputer. Pengetahuan komputer yang tinggi sebaiknya dimiliki oleh siswa yang mengambil jurusan yang berhubungan dengan komputer khususnya disini pada jurusan RPL yang di dalamnya terdapat mata pelajaran Algoritma Pemrograman. Dalam proses pembelajaran Algoritma Pemrograman terdapat tujuan yang ingin dicapai salah satu tujuan pembelajaran yang utama adalah meningkatnya hasil belajar yang baik. Untuk mendapat hasil belajar Algoritma Pemrograman yang baik, maka siswa harus mempunyai pengetahuan komputer, Jurnal TRANSFORMASI, Vol. 19, No. 1, 2023 : 61 - 68 65 65 “Jurnal TRANSFORMASI (Informasi & Pengembangan Iptek)” (STMIK BINA PATRIA) E-ISSN : 2827-8550 P- ISSN : 1978-5569 4.2.2. Pengaruh Motivasi Belajar Terhadap Hasil Belajar Berdasarkan hasil analisis statistik variabel motivasi terhadap hasil belajar dengan menggunakan analisis regresi linear berganda dapat diketahui nilai t hitung sebesar 8,376. Hasil analisis regresi liner berganda tersebut diperoleh nilai t hitung > t tabel (8,376 > 1,66) maka H0 ditolak, artinya bahwa ada pengaruh secara signifikan antara motivasi belajar dengan hasil belajar. Dalam penelitian ini dapat disimpulkan bahwa motivasi belajar berpengaruh positif terhadap hasil belajar Algoritma pada siswa di SMK Negeri 5 Kota Bekasi. Hal ini diperkuat dengan nilai sig 0,000 yang kurang dari 0,05. Sehingga variabel X2 berpengaruh terhadap variabel Y. Motivasi merupakan segala sesuatu yang menjadi pendorong timbulnya suatu tingkah laku demi tercapainya tujuan ataupun cita-cita yang diharapkan. Siswa yang dalam proses belajar mempunyai motivasi yang kuat dan jelas pasti akan tekun dan berhasil belajarnya dalam artian motivasi itu bukan hanya berfungsi sebagai penentu terjadinya suatu perbuatan tetapi juga merupakan penentu hasil perbuatan. Siswa yang termotivasi secara intrinsik dapat terlihat dari kegiatannya yang tekun dalam mengerjakan tugas-tugas belajar karena butuh dan ingin mencapai tujuan belajar yang sebenarnya. Dengan kata lain, motivasi intrinsik dilihat dari segi tujuan kegiatan yang dilakukan adalah ingin mencapai tujuan yang terkandung di dalam perbuatan itu sendiri. Siswa yang memiliki motivasi intrinsik menunjukkan keterlibatan dan aktivitas yang tinggi dalam belajar. Motivasi ekstrinsik dapat membangkitkan motivasi intrinsik, sehingga motivasi ekstrinsik sangat diperlukan dalam pembelajaran. Siswa yang memiliki motivasi yang tinggi akan mempersiapkan diri dalam mengikuti proses pembelajaran dikelas meskipun menghadapi kesulitan-kesulitan materi yang ada. Siswa yang memiliki motivasi belajar rendah sesuai dengan kemampuan pengetahuan komputernya dimana memerlukan kemampuan untuk meningkatkan hasil belajarnya. Hasil belajar Algoritma Pemrograman dapat ditingkatkan dengan berbagai jenis usaha. Salah satunya untuk meningkatkan dengan cara diberikanya tambahan materi tentang pengetahuan komputer sebelum proses pembelajaran, sehingga motivasi belajar yang tinggi dapat membantu pencapaian hasil belajar. Hasil dari penelitian ini adalah motivasi dalam belajar mempunyai hubungan yang positif dan signifikan dengan hasil belajar Algoritma Pemrograman. 4.2.3. 5. Kesimpulan Berdasarkan hasil penelitian yang telah dilakukan, maka dapat disimpulkan bahwa terdapat pengaruh yang positif (R2=0,813) dan signifikan (sig=0,000) antara pengetahuan dasar komputer (variabel x1) dan motivasi belajar (x2) secara bersama-sama dengan hasil belajar (y) Algoritma Pemrograman pada siswa SMK 5 Kota Bekasi. Selain itu masing-masing variabel x (yakni pengetahuan dasar komputer (thitung=5,155) dan motivasi belajar (thitung=8,376)) secara individual juga memiliki pengaruh yang positif (>tteoritis=1,66)terhadap variabel y (hasil belajar). Perlu dilakukan analisis terpisah mengenai pengaruh variabel motivasi intrinsik dan ekstrinsik terhadap hasil belajar. 4.2.2. Pengaruh Motivasi Belajar Terhadap Hasil Belajar 1, 2023 : 61 - 68 66 66 0,05, sehingga secara serempak variabel bebas berpengaruh signifikan terhadap variable tak bebas untuk taraf signifikan 5 %. Dalam proses pembelajaran, penguasaan pengetahuan dasar komputer dikatakan mampu membangkitkan motivasi siswa mengenai performa kegiatan pembelajaran yang optimal. Pengetahuan komputer dasar tingkat tinggi dan pengetahuan komputer dasar tingkat rendah memiliki satu kesamaan, yaitu keduanya memiliki pengetahuan komputer dasar. Namun, siswa dengan tingkat pengetahuan dasar komputer yang tinggi cenderung lebih interaktif dibandingkan dengan siswa dengan tingkat pengetahuan komputer dasar yang rendah, dimana proses pembelajaran berlangsung pada waktu yang sama, guru yang sama tanpa adanya perbedaan perlakuan. Algoritma merupakan deretan langkah komputasi yang mentransformasikan masukan menjadi keluaran dalam waktu yang terbatas. Algoritma baru efektif jika dijalankan oleh sebuah pemroses (prosesor). Pemroses itu bisa manusia, robot, komputer, mesin, dan sebagainya. Pemroses membaca setiap instruksi di dalam algoritma lalu mengerjakannya. Hasil belajar Algoritma Pemrograman merupakan tingkat keberhasilan atau penguasaan seorang siswa terhadap mata pelelajaran Algoritma Pemrograman setelah menempuh proses belajar mengajar yang terlihat pada nilai yang diperoleh dari tes hasil belajarnya. Baik dalam menguasai pengetahuan dasar dan motivasi belajar yang ada pada diri siswa selama proses belajar mengajar, dimana hasil belajar Algoritma Pemrograman dapat diukur dengan menggunakan alat evaluasi yang biasanya disebut tes hasil belajar. Pada penelitian ini diketahui hasil bahwa variabel pengetahuan dasar, motivasi belajar pada siswa SMK 5 kota bekasi secara bersama-sama dapat mempengaruhi hasil belajar secara positif. Daftar Pustaka Arianti, A. (2019). Urgensi lingkungan belajar yang kondusif dalam mendorong siswa belajar aktif. Didaktika: Jurnal Kependidikan, 11(1), 41–62. Francis, A. P., Wieth, M. B., Zabel, K. L., & Carr, T. H. (2020). A classroom study on the role of prior knowledge and retrieval tool in the testing effect. Psychology Learning & Teaching, 19(3), 258–274. Hartikainen, S., Rintala, H., Pylväs, L., & Nokelainen, P. (2019). The Concept of Active Learning and the Measurement of Learning Outcomes: A Review of Research in Engineering Higher Education. Education Sciences, 9(4), 276. Hasan, F., Pomalato, S. W. D., & Uno, H. B. (2020). Pengaruh Pendekatan Realistic Mathematic Education (RME) terhadap Hasil Belajar Matematika Ditinjau dari Motivasi Belajar. Jambura Journal of Mathematics Education, 1(1), 13–20. Rahman, S. (2022). Pentingnya motivasi belajar dalam meningkatkan hasil belajar. In Prosiding Seminar Nasional Pendidikan Dasar Pasca Sarjana Universitas Gorontalo. Merdeka Belajar dalam Menyambut Era Masyarakat 5.0, November, 289–302 67 Jurnal TRANSFORMASI, Vol. 19, No. 1, 2023 : 61 - 68 67 Ramaliza, T., & Mutiara, E. (2021). Hubungan Pengetahuan Bahan Makanan Dan Motivasi Belajar Dengan Hasil Belajar Kue Indonesia Smk Negeri 1 Pantai Labu. GARNISH : Jurnal Pendidikan Tata Boga, 5(2), 79–89. Thoyib, M., Subandowo, M., & Wiyarno. (2021). Penerapan E-Learning dengan Analisis Pengetahuan Awal Terhadap Prestasi Belajar Bahasa Inggris Siswa SMK. Edcomtech: Jurnal Kajian Teknologi Pendidikan, 6(1), 13–25. Van Alten, D. C., Phielix, C., Janssen, J., & Kester, L. (2019). Effects of flipping the classroom on learning outcomes and satisfaction: A meta-analysis. Educational Research Review, 28, 100281. Wade, S., & Kidd, C. (2019). The role of prior knowledge and curiosity in learning. Psychonomic Bulletin and Review, 26(4), 1377–1387. https://doi.org/10.3758/s13423-019-01598-6 68 68 Jurnal TRANSFORMASI, Vol. 19, No. 1, 2023 : 61 - 68 68
https://openalex.org/W4392830062	https://conferencias.saludcyt.ar/index.php/sctconf/article/download/604/533	es		Descripción de la neumonía asociada a la ventilación mecánica	Salud, Ciencia y Tecnología - Serie de Conferencias	2,023	cc-by	5,114	Salud, Ciencia y Tecnología – Serie de Conferencias. 2023; 2:604 doi: 10.56294/sctconf2023604 Categoría: Seminario Científico Metodológico de la Universidad de Ciencias Médicas de Pinar del Río REVISIÓN Description of ventilator-associated pneumonia Descripción de la neumonía asociada a la ventilación mecánica Yanara González Baños1 , Osiris Oscar Terrado Almarales1 , Heidy Rego Avila2 1 Universidad de Ciencias Médicas de Pinar del Río. Facultad de Ciencias Médicas Ernesto Guevara de la Serna. Pinar del Río, Cuba. Universidad de Ciencias Médicas de Pinar del Río. Hospital General Docente “Abel Santamaría Cuadrado”. Pinar del Río, Cuba. 2 Citar como: González Baños Y, Terrado Almarales OO, Rego Avila H. Descripción de la neumonía asociada a la ventilación mecánica. Salud, Ciencia y Tecnología - Serie de Conferencias 2023; 2:604. https://doi.org/10.56294/sctconf2023604 Recibido: 26-06-2023 Revisado: 23-08-2023 Aceptado: 25-10-2023 Publicado: 26-10-2023 ABSTRACT Pneumonia associated with mechanical ventilation constitutes one of the main infections associated with health services and the main complication of mechanical ventilation. The present investigation was developed with the aim of describing ventilator-associated pneumonia. A literature review was carried out in the Scopus, SciELO and Redalyc databases, obtaining 37 references. This entity is an infection of the lower airway that produces an acute inflammatory lesion of the pulmonary parenchyma that occurs in response to the arrival of the microorganism to the distal airway, and which is acquired in the hospital 48 hours after endotracheal intubation. The symptoms and signs may manifest as the presentation of a series of symptoms and signs, either progressive or abrupt, such as dyspnea, fever, tachypnea, purulent expectoration, hemoptysis, rhonchi, crackles, hypoventilation and bronchospasm. Diagnosis is clinical, radiological and laboratory. Keywords: Ventilator Associated Pneumonia; Healthcare Associated Pneumonia; Bacterial Pneumonia; Intensive Care Units; Hospital Emergency Department. RESUMEN La neumonía asociada a la ventilación mecánica constituye una de las principales infecciones asociadas a los servicios de salud y la principal complicación de la ventilación mecánica. La presente investigación se desarrolló con el objetivo de describir la neumonía asociada a la ventilación mecánica. Se realizó una revisión de la literatura en las bases de datos Scopus, SciELO y Redalyc, obteniéndose 37 referencias. Esta entidad es una infección de la vía aérea inferior que produce una lesión inflamatoria aguda del parénquima pulmonar que se produce como respuesta a la llegada del microorganismo a la vía aérea distal, y que se adquiere en el hospital 48 horas después de la intubación endotraqueal. pueden manifestarse como la presentación de una serie de síntomas y signos, bien de manera progresiva o brusca, tales como disnea, fiebre, taquipnea expectoración purulenta, hemoptisis, roncus, crepitantes, hipoventilación y broncoespasmo. Su diagnóstico es clínico, radiológico y de laboratorio. Palabras clave: Neumonía Asociada Al Ventilador; Neumonía Asociada A La Atención Médica; Neumonía Bacteriana; Unidades De Cuidados Intensivos; Servicio De Urgencia En Hospital. © Autor(es); 2023. Este es un artículo en acceso abierto, distribuido bajo los términos de una licencia Creative Commons (https://creativecommons.org/licenses/by/4.0) que permite el uso, distribución y reproducción en cualquier medio siempre que la obra original sea correctamente citada. Salud, Ciencia y Tecnología – Serie de Conferencias. 2023; 2:604 2 INTRODUCCIÓN La Unidad de Cuidados Intensivos (UCI) es el área hospitalaria dedicada a la atención integral de los enfermos graves. Estos servicios altamente especializados en el cuidado de pacientes críticos juegan un papel fundamental en los hospitales de moderada y alta complejidad.(1,2) La supervivencia o la muerte de los pacientes en una UCI dependen del equilibrio dinámico entre la magnitud de la enfermedad y la suficiencia de las respuestas fisiológicas protectoras esenciales para conservar el aporte de sustratos y el medio interno, necesarios para el metabolismo y la función celular de todo el cuerpo. Los resultados de la asistencia ofrecida en las UCI han sido frecuentemente valorados por estudios de morbilidad y de mortalidad, y la mortalidad ha sido definida como el primer marcador asistencial en la práctica médica.(3) Esta unidad ocupa un lugar preponderante en la atención del paciente en estado crítico, que ingresa a los hospitales de segundo y tercer niveles de atención en Cuba y en los países desarrollados. (4) La infección asociada a los servicios de salud continúa siendo un evento adverso que afecta a gran número de pacientes graves que residen en las UCI. La naturaleza extremadamente vulnerable de estos pacientes, los múltiples procedimientos, el uso de dispositivos invasivos, el uso prolongado de regímenes antimicrobianos y los organismos resistentes a múltiples fármacos están convirtiendo a las UCI en epicentros de infección. Las infecciones adquiridas en el hospital asociadas a dispositivos (DAHAI, por sus siglas en inglés) son amenazas principales para la seguridad de los pacientes en UCI que son responsables del aumento de la morbilidad y mortalidad del paciente. En consecuencia, están asociados con estadías prolongadas en el hospital, discapacidades a largo plazo, mayor resistencia a los medicamentos y una carga financiera adicional para las familias. La neumonía asociada al ventilador (NAVM), la infección del torrente sanguíneo asociada a la línea central (ITSALC) y la infección del tracto urinario asociada al catéter (ITUAC) son las infecciones asociadas con el dispositivo común en la UCI. (5) La ventilación artificial mecánica (VM) es, sin dudas, la técnica de sustitución de funciones de sistemas más utilizada en las Unidades de Cuidados Intensivos (UCI). (6,7) La terapia con ventilación mecánica tuvo sus orígenes contemporáneos en el siglo 19; en ese momento usando presión subatmosférica justificada para reemplazar el trabajo aumentando de los músculos respiratorios. Empezando ésta con dispositivos tipo caja en donde el paciente podía estar sentado o acostado y la caja se colocaba del cuello para abajo usando un pistón o émbolo para aumentar o disminuir la presión facilitando la inspiración o espiración. (8,9) El primer pulmón de hierro funcional fue introducido en 1876, con todos los inconvenientes para tener acceso al cuerpo del paciente. Este fue sustituido por los cuartos de ventilación en donde el paciente tenía la cabeza fuera e incluso se podían tratar varios pacientes al mismo tiempo, con la misma mecánica de manipulación de la presión atmosférica con enormes pistones. Estos fueron populares en Boston (Estados Unidos de América) durante varias epidemias entre ellas la del Polio. ( 8,9) Nuevamente un anestesista entrenado en Boston de apellido Ibsen hizo observaciones con respecto a la ventilación con presión positiva en pacientes con traqueostomía y documentó un descenso importante en la mortalidad, desde un 80 % hasta un 40 % aproximadamente, con la complicación de que 3 no existían equipos para tal fin valiéndose de la ventilación manual por bolsa en manos de estudiantes de medicina. ( 8,9) En la década de los 60 se introduce los dispositivos cíclicos de presión con la idea de reemplazar el trabajo de los músculos respiratorios, disminuir el reflejo tusígeno, reducir el colapso basal pulmonar, y mejorar la terapéutica con aerosoles.( 8,9) Es habitual, en las UCI, el ingreso de pacientes postoperados de cirugía cardiovascular, torácica o abdominal, así como los que presentan procesos agudos como sepsis o fallo respiratorio, entre otros. Todos ellos pueden desarrollar alteración en la oxigenación y/o ventilación.(10) En la mayoría de las ocasiones la VM no cura las causas que producen una insuficiencia respiratoria, pero sí garantiza el funcionamiento de los pulmones y sus importantes efectos para el mantenimiento de https://doi.org/10.56294/sctconf2023604 3 González Baños et al. la vida, lo cual proporciona el tiempo necesario para poder curar o aliviar determinadas afecciones que perjudican de forma directa o indirecta la función pulmonar, la oxigenación e influir sinérgicamente en la fisiología de la mecánica pulmonar. Además, este proceder es una de las principales indicaciones de ingreso a las UCI, utilizada en 1 de cada 3 pacientes. (11) Entre las complicaciones mortales más importantes con el proceder se encuentran la lesión pulmonar generada o asociada a un desajuste en la regularización de la respuesta inflamatoria, que además de incrementar el daño en los pulmones, lleva los mediadores inflamatorios a la circulación general y produce insuficiencia multiorgánica; la neumonía asociada a la ventilación mecánica (NAVM), que resulta un problema epidemiológico en Cuidados Intensivos, la atelectasia, observada en alrededor de 80 % de las radiografías de tórax, que a más de agravar la hipoxemia existente, es causa contribuyente de NAVM; el sangrado digestivo alto, los trastornos hidroelectrolíticos, las arritmias cardíacas agudas y la trombosis venosa profunda.(11,12) La neumonía asociada a la ventilación mecánica tiene una alta incidencia en la provincia, en Cuba y el mundo, constituyendo una importante amenaza para la salud pública. Su evolución tórpida puede relacionarse con diferentes factores que condicionan la muerte. De ahí que la identificación de factores asociados a la mortalidad puede permitir trazar estrategias para mejorar la evolución del paciente, disminuyendo la mortalidad. La presente investigación se desarrolló con el objetivo de describir la neumonía asociada a la ventilación mecánica. MÉTODO Se realizó una búsqueda de información en las bases de datos Scopus, SciELO, Redalyc y PubMed/MedLine en abril de 2022. Para la obtención de la información se empleó una fórmula de búsqueda empleando los términos “Neumonía”, “Neumonía asociada al ventilador”, “Neumonía asociada a la atención médica” y los operadores booleanos para su interconexión. La estructura de la fórmula de búsqueda fue específica para cada base de datos. Se emplearon filtros idiomáticos, empleando aquellos publicados en español e inglés y se empleó como marco temporal el periodo 2017-2022, aunque se emplearon artículos externos al periodo por su importancia. Posterior a la obtención de los artículos se procedió a realizar una lectura de los títulos y resúmenes para comprobar la pertinencia de los mismos. En aquellos que generaron confusión se realizó la lectura del texto in extenso. Resultó de esta criba la selección de 37 artículos y libros, las cuales sustentaron el desarrollo de la presente. DESARROLLO Aún con los grandes avances en el conocimiento de la anatomía, de la fisiopatología pulmonar, el desarrollo científico y técnico de los instrumentos utilizados para la ventilación mecánica, ya sea invasiva o no invasiva, todavía es necesario buscar un mayor acercamiento a la dinámica pulmonar real que se adapte a las necesidades del paciente, precisamente porque la incidencia de eventos adversos, complicaciones y mortalidad por el proceder, se mantienen elevadas. (6) Se ha reportado una incidencia de 21 casos de NAVM por cada 1 000 hospitalizaciones en hospitales de Estados Unidos. (12) De acuerdo con el National Healthcare Safety Network, del 2006 al 2008 la tasa se registró en 3,7 por 1 000 días-ventilador en ese país.(13) En España se reportan que aproximadamente el 10 % de los pacientes ventilados desarrollan NAVM (14) y el reporta del Estudio Nacional de Vigilancia de Infección Nosocomial de España, del 2000 al 2009 la tasa de incidencia de NAV fue de 15 casos por 1.000 días-ventilador.(15) En un hospital de Lima, Perú se reporta una incidencia del 2,37 % y una mortalidad del 73,07 % en servicios de Medicina Interna.(16) Otiniano y colaboradores(17) reportaron una incidencia del 11 % en una https://doi.org/10.56294/sctconf2023604 Salud, Ciencia y Tecnología – Serie de Conferencias. 2023; 2:604 4 unidad de Cuidados Intensivos, mientras que Chincha y colaboradores (18) reportan una incidencia de 28,6 %. En México e la Red Hospitalaria de Vigilancia Epidemiológica (RHOVE) de la Dirección General de Epidemiología de la Secretaría de Salud, las neumonías ocupan el primer lugar de las IAAS notificadas en las UCIA, con un 40% de prevalencia, con una tasa de incidencia de 18,6 casos por 1 000 días-ventilador.(19) Datos recopilados en un informe elaborado por la international nosocomial infection control consortium (INICC) muestran una incidencia de 18,6/1 000 días de ventilación mecánica, esto en 78 unidades de cuidados intensivos de 13 naciones.(20) En Cuba, investigaciones sobre el tema informan que la NAVM es un tipo de infección nosocomial frecuente en los pacientes críticos y se asocia a altas tasas de morbilidad y mortalidad. Esta enfermedad depende de múltiples factores de riesgo causados por microorganismos multirresistentes, y se asocia a una mayor mortalidad.(21,22,23) En Pinar del Río, se ha reportado alta morbilidad y mortalidad por NAVM.(24,25) Neumonía La neumonía es una infección de la vía aérea inferior que produce una lesión inflamatoria aguda del parénquima pulmonar que se produce como respuesta a la llegada del microorganismo a la vía aérea distal. Esta inflamación es consistente en la radiografía de tórax u otras pruebas de imagen, con un nuevo infiltrado en el parénquima pulmonar. Se asocia con una alta morbimortalidad, siendo la primera causa de sepsis y shock séptico.(26) Se produce un efecto nocivo por parte de los microorganismos en los alvéolos (pequeños sacos) de los pulmones, que en vez de llenarse de aire (lo que sucede en condiciones de salud) lo hacen de líquido y pus, condición que genera que el infectado sienta dolor al respirar y limite su absorción de oxígeno.(27) Dependiendo de la zona afectada, se puede referir a neumonía lobular (cuando implica por completo un lóbulo pulmonar); bronconeumonía (cuando lo afectado es un segmento de lóbulo, a los alvéolos próximos a los bronquios) o neumonía intersticial (afección asociada al tejido intersticial).(28) Neumonía nosocomial La neumonía nosocomial es un proceso inflamatorio pulmonar de origen infeccioso, ausente en el momento del ingreso hospitalario, y que se desarrolla tras más de 48 h de haber ingresado en el hospital, es la segunda infección más frecuente en el hospital y la primera en las Unidades de Medicina Intensiva, la cual se puede incrementar hasta 20 veces en presencia de ventilación mecánica (VM), siendo considerada entonces como neumonía asociada a la VM (NAV). (29) Neumonía asociada a la ventilación mecánica La NAV es un tipo de neumonía adquirida en el hospital que ocurre más de 48 horas después de la intubación endotraqueal. Puede ser adicionalmente clasificada como de aparición temprana (dentro de las primeras 96 horas de VM) y de aparición tardía (más de 96 horas después de iniciada la VM), la cual es más comúnmente atribuible a patógenos resistentes a múltiples drogas. (30) Por otra parte, es importante definir de que se trata la ventilación mecánica, para se considera idóneo referir lo establecido por Sociedad Torácica Americana (ATS, por sus siglas en inglés), de quienes se ha comprendido que esto se trata de un tratamiento de soporte vital con una máquina que auxilia en la función respiratoria de aquellas personas cuando por sí mismas no pueden respirar lo suficiente. (31) Dinámica del microbioma pulmonar Durante los pasados 10 años se ha creído que un pulmón sano es un pulmón estéril, este paradigma ha venido cambiando. El pulmón aparentemente sano parece estar colonizado por múltiples bacterias residentes, las cuales migran desde la cavidad oral hasta las vías aéreas distales, Se asume que el https://doi.org/10.56294/sctconf2023604 5 González Baños et al. microbioma respiratorio representa una comunidad dinámica en donde el punto de equilibrio esta determinado por el balance entre la inmigración y la eliminación. (32) En la ventilación mecánica se produce un desbalance debido a varios factores entre los cuales está la abolición del reflejo de la tos, la disrupción de la limpieza mucociliar y la misma presencia del tubo endotraqueal disminuye la extinción bacteriana de las vías respiratorias inferiores. Patógenos potenciales pueden causar neumonía una vez que una adecuada carga bacteriana encuentre el medio adecuado para crecer en una fisiología pulmonar alterada. Estudios publicados en poblaciones sometidas a este tratamiento evidencian cambios en el microbioma tanto en pacientes que desarrollan neumonía como los que no. Sin embargo, los cambios vistos en los que la desarrollan parecen ser más profundos en términos de disbiósis del tracto respiratorio.(32) El espectro de especies detectadas y su diversidad parecen no estar afectadas por el uso de antibióticos tanto de moderado como de amplio espectro. Se especula que el microbioma detectado o al menos parte de este refleja el biofilm microbiano sobre la superficie del tubo endotraqueal. Es sabido que los tubos endotraqueales forman una barrera entre las bacterias y el sistema circulatorio del paciente y como consecuencia limitan el acceso de los antibióticos. Además, las bacterias colectadas del tubo muestran una resistencia antibiótica incrementada.(32) Sin embargo, no se puede rechazar la hipótesis de que los antibióticos no modifican el microbioma, pero sí se podría extrapolar que, si bien existe modificación, esta no es la esperada como se venía suponiendo hasta ahora.(32) Fisiopatología La fisiopatología de la neumonía asociada a la ventilación mecánica comprende varios fenómenos y está en gran parte mediada por la introducción de un cuerpo extraño en la vía aérea alta (el tubo endotraqueal), con la consecuente alteración de los mecanismos naturales que impiden el acceso de microorganismos al tracto respiratorio bajo.(8,9) El efecto mecánico sobre la tráquea que ejerce el tubo con su balón hace que se complique significativamente la evacuación de secreciones mucociliares. La formación de una bio-película o bio-film de bacterias sobre el polímero del tubo endotraqueal, la microaspiración y filtración de secreción orofaríngea alrededor del balón inflado del tubo endotraqueal. (8,9) Todos estos factores se exacerban en el contexto de la ventilación mecánica con presión positiva, que se complica con la mermada capacidad del paciente para movilizar secreciones y la ya alterada respuesta inmune innata y adaptativa del paciente críticamente enfermo.( 8,9) Aunque clásicamente se han venido distinguiendo 4 vías patogénicas para el desarrollo de NAVM (aspiración de secreciones colonizadas procedente de la orofaringe, por contigüidad, por vía hematógena, y a través de los circuitos o tubuladuras), la aspiración de secreciones procedentes de la orofaringe es la vía mayoritaria y casi única. La vía aérea inferior es una zona habitualmente estéril en personas sanas, la excepción se limita a pacientes con enfermedades crónicas pulmonares. En los pacientes bajo ventilación mecánica, la intubación endotraqueal, en cambio, rompe el aislamiento de la vía aérea inferior.(33) El neumotaponamiento del tubo endotraqueal es un sistema diseñado para aislar la vía aérea, evitando pérdidas aéreas y la entrada de material a los pulmones, pero no es completamente estanco. Por encima del neumotaponamiento se van acumulando secreciones que, provenientes de la cavidad oral, están contaminadas por los patógenos que colonizan la orofaringe. Estas secreciones contaminadas pasan alrededor del neumotaponamiento y alcanzan la vía aérea inferior. Esta cantidad o inóculo será escaso si existen pocas secreciones acumuladas, pero si la integridad del sistema está alterada, el inóculo que pueda llegar al parénquima pulmonar será mayor.(33) Cuando este inóculo supera la capacidad de defensa del huésped, se produce la reacción inflamatoria cuya expresión histológica es la aparición de infiltrado agudo con leucocitos polimorfonucleares. https://doi.org/10.56294/sctconf2023604 Salud, Ciencia y Tecnología – Serie de Conferencias. 2023; 2:604 6 Externamente, apreciaremos la existencia de secreciones respiratorias, que son aspiradas con sondas de aspiración por dentro del tubo endotraqueal. Se ha comprobado que una baja presión del neumotaponamiento, que permitiría un mayor paso de secreciones, se puede asociar al desarrollo de NAVM.(33) Manifestaciones clínicas La NAV pueden manifestarse como la presentación de una serie de síntomas y signos, bien de manera progresiva o brusca, tales como disnea, fiebre, taquipnea expectoración purulenta, hemoptisis, roncus, crepitantes, hipoventilación y broncoespasmo. También pueden desarrollarse datos de encefalopatía o sepsis. En pacientes sometidos a VM en los que se observa una disminución del volumen corriente (tidal) o un incremento en la presión inspiratoria habrá que sospechar la presencia de NAV. (14) En cuanto a parámetros de laboratorio, el empeoramiento de la hipoxemia, la leucocitosis o el aumento de reactantes de fase aguda deberán hacernos sospechar estas entidades clínicas. Las pruebas de imagen (radiografía, ecografía o tomografía computadorizada de tórax) mostrarán un nuevo infiltrado o progresión de uno previo.(14) Diagnóstico El diagnóstico clínico de estas neumonías es difícil, ya que los hallazgos no son específicos. La guía de la IDSA/ATS continúa recomendando el diagnóstico basado en la aparición de un nuevo infiltrado en una prueba de imagen, junto con la evidencia clínica de un origen infeccioso del mismo como la fiebre, purulencia del esputo, la leucocitosis o la diminución de la oxigenación. (34) A pesar de ello, la clínica es inespecífica en pacientes con VM, pudiendo confundirse con otras entidades como atelectasias, tromboembolismo pulmonar y sepsis de otro origen distinto al respiratorio.(34) Dado que el riesgo de que la NAV sea secundaria a gérmenes multirresistentes es alto, se recomienda obtener siempre muestras de secreciones respiratorias. Por otro lado, hasta un 15 % de los pacientes tienen hemocultivos positivos, por lo que debe realizarse su extracción. De todas formas, hay que tener en cuenta que hasta un 25 % de los hemocultivos positivos pueden tener un origen distinto al respiratorio. En el caso de la NN, los cultivos de las muestras respiratorias suelen ser menos rentables que en la NAV.ser menos rentables que en la NAV. La obtención de las muestras puede ser invasiva o no invasiva. Los métodos no invasivos hacen referencia sobre todo al aspirado traqueal. Esta es la forma más simple y la más utilizada en la práctica.(35,36) El método invasivo más extendido es el lavado broncoalveolar (BAL) obtenido mediante fibrobroncoscopio. No hay diferencias significativas entre el uso de aspirado traqueal y el BAL en cuanto a la mortalidad, el uso de antibióticos, la duración de la VM o la estancia en la UCI. Las pruebas de diagnóstico molecular desarrolladas en los últimos años para la detección de patógenos respiratorios ofrecen una ventaja para una identificación más rápida del agente causal de la NN o NAV. (35,36) Además, pueden aportar información sobre algunos de los patrones de resistencia, como por ejemplo la resistencia a meticilina en el caso del S. aureus o a los carbapenem en enterobacterias. Biomarcadores En estos años se han empleado diversos biomarcadores con la intención de mejorar la sensibilidad y la especificidad en el diagnóstico de NAV. Entre los más estudiados destaca la procalcitonina, la proteína C reactiva y el sTREM-1 (soluble triggering receptor expressed on myeloid cells). Actualmente, el uso de estos marcadores no parece ayudar en la toma de decisión a la hora de iniciar antibioterapia, siendo preferible basarse únicamente en la clínica y la radiología . (35,36,37) https://doi.org/10.56294/sctconf2023604 7 González Baños et al. CONCLUSIONES La neumonía asociada a la ventilación mecánica constituye una de las principales infecciones asociadas a los servicios de salud y la principal complicación de la ventilación mecánica. Es una infección de la vía aérea inferior que produce una lesión inflamatoria aguda del parénquima pulmonar que se produce como respuesta a la llegada del microorganismo a la vía aérea distal, y que se adquiere en el hospital 48 horas después de la intubación endotraqueal. pueden manifestarse como la presentación de una serie de síntomas y signos, bien de manera progresiva o brusca, tales como disnea, fiebre, taquipnea expectoración purulenta, hemoptisis, roncus, crepitantes, hipoventilación y broncoespasmo. Su diagnóstico es clínico, radiológico y de laboratorio. REFERENCIAS BIBLIOGRÁFICAS 1. Blanco-Gómez C, Delgado-Reyes A, Reyes-Rivadulla C, Gómez-Vázuquez M, Pérez-Martín L. Caracterización de pacientes con infecciones asociadas a la asistencia sanitaria. Revista de Ciencias Médicas de Pinar del Río 2023; 27:5755. https://revcmpinar.sld.cu/index.php/publicaciones/article/view/5755 2. Abdo-Cuza A, Jordan-Gonzalez J, Pi-Avila J, Espinosa-Nodarse N, Machado-Martinez R. Hallazgos tomográficos y ecográficos pulmonares en dos pacientes con neumonía por SARS-CoV-2. Universidad Médica Pinareña 2021; 18(1):804. https://revgaleno.sld.cu/index.php/ump/article/view/804 3. González Rodríguez R, García Acosta JA, Barcón Díaz L, Álvarez Dubé E. Variables asociadas a la mortalidad en pacientes ventilados de una unidad de terapia intermedia. Rev Ciencias Médicas [Internet]. 2018 Feb [citado 02/05/2022]; 22(1): 21-28. Disponible en: http://scielo.sld.cu/scielo.php?script=sci_arttext&pid=S1561-31942018000100005&lng=es 4. Álvarez-Alonso I, Lagar-Martínez R, Lagar-Álvarez R, Echevarría-Padrón D, Lagar-Álvarez J. Caracterización clínica-epidemiológica de la neumonía de la comunidad complicada en niños ingresados en la Unidad de Cuidados Intensivos del Hospital Pediátrico “Pepe Portilla”. Universidad Médica Pinareña 2022; 18(4):910. https://revgaleno.sld.cu/index.php/ump/article/view/910 5. Hernández-Suárez N, Ferro-Gonzalez B, Labrado-Alemán R, Tamayo-Batista I, Sandrino-Sánchez M, García-Miranda A. Regularidades de la superación profesional sobre Neumonía Adquirida en la Comunidad para docentes de Medicina Interna. Universidad Médica Pinareña 2022; 18(4):925. https://revgaleno.sld.cu/index.php/ump/article/view/925 6. Hernández Ruiz A, Delgado Fernández RI, Alcalde Mustelier GR, Collazo Ramos MI, Garcia Collazo CM. Mortalidad en pacientes con ventilación mecánica ingresados en una Unidad de Cuidados Intensivos. Rev haban cienc méd [Internet]. 2018 [citado 02/05/2022]; 17(6):885-895. Disponible en: http://www.revhabanera.sld.cu/index.php/rhab/article/view/2328 7. Palomino-Cabrera A, Cruz-González M, Potete-Morejón R, Soto-Bello Y, Moreira-Díaz L. Caracterización clínico – epidemiológica de pacientes politraumatizados atendidos en el Hospital General “Comandante Pinares”. Universidad Médica Pinareña 2021; 17(3):782. https://revgaleno.sld.cu/index.php/ump/article/view/782 8. Peñate-Brito JB, Rodríguez-Caballero CD, Guerra-Sánchez M. Medicina física y rehabilitación en pacientes con COVID-19. Rev Méd Electrón 2023; 45(4):4985. https://revmedicaelectronica.sld.cu/index.php/rme/article/view/4985 https://doi.org/10.56294/sctconf2023604 Salud, Ciencia y Tecnología – Serie de Conferencias. 2023; 2:604 8 9. Arredondo-Bruce AE, Arredondo-Rubido AE. Aspectos novedosos del diagnóstico del SARS-CoV-2 en la era poscovid. Rev Méd Electrón 2022; 44(4):4912. https://revmedicaelectronica.sld.cu/index.php/rme/article/view/4912 10. Goni-Viguria R, Yoldi-Arzoz E, Casajús-Sola L, Aquerreta-Larraya T, Fernández-Sangil P, GuzmánUnamuno E, et al. Fisioterapia respiratoria en la unidad de cuidados intensivos: Revisión bibliográfica. Enferm Intensiva [Internet]. 2018[citado 02/05/2022]; 29(4):168-181. Disponible en: https://www.sciencedirect.com/science/article/pii/S1130239918300580 11. Betancourt-Reyes GL, Betancourt-Betancourt Gd. El debate actual sobre el empleo de la ventilación mecánica no invasiva. Rev Méd Electrón 2022; 44(1):4418. https://revmedicaelectronica.sld.cu/index.php/rme/article/view/4418 12. Giuliano KK, Baker D, Quinn B. The epidemiology of nonventilator hospital-acquired pneumonia in the United States. Am J Infect Control [Internet]. 2017 [citado 02/05/2022];46(3):322-327. Disponible en: https://doi.org/10.1016/j.ajic.2017.09.005 13. Rufín-Gómez LÁ, Martínez-Morejón A, Rufín-Bergado AM, Méndez-Martínez J. Síndrome metabólico, un factor de riesgo en pacientes de COVID-19. Rev Méd Electrón 2022; 44(1):4367. https://revmedicaelectronica.sld.cu/index.php/rme/article/view/4367 14. Bravo Quiroga L, Sánchez Fraga S. Neumonías nosocomiales y asociadas a ventilación mecánica invasiva. Medicine 2018; 12(64):3763-9. Disponible en: https://www.sciencedirect.com/science/article/pii/S0304541218302300 15. Sociedad Española de Medicina Intensiva Crítica y Unidades Coronarias. Estudio Nacional de Vigilancia de Infección Nosocomial en UCI (ENVIN-UCI) 2001-2009 [Internet]; 2012 [citado 02/05/2022]. Disponible en: http://hws.vhebron.net/envin-helics 16. León-Chahua C, Oscanoa-Espinoza T, Chávez-Gutiérrez C, Chávez-Gutiérrez J. Características epidemiológicas de la neumonía intrahospitalaria en un servicio de medicina interna del Hospital Guillermo Almenara Irigoyen de Lima, Perú. Horiz Med [Internet]. 2016[citado 02/05/2022]; 16 (3): 4349. Disponible en: http://www.scielo.org.pe/scielo.php?pid=S1727558X2016000300007&script=sci_arttext&tlng=pt 17. Otiniano A, Gómez M. Factores de riesgo asociados a neumonía intrahospitalaria en pacientes de la unidad de cuidados intensivos. Rev Soc Peruana Med Interna [Internet]. 2021 [citado 02/05/2022];24(3):121-127. Disponible en: http://www.revistamedicinainterna.net/index.php/spmi/article/view/458 18. Castro Sánchez JA, Jiménez Hurtado WA. Nursing staff experiences for the prevention of ventilator associated pneumonia. Salud, Ciencia y Tecnología 2023; 3:380. https://revista.saludcyt.ar/ojs/index.php/sct/article/view/380 19. Vaca Moreno AP, Quinteros Portilla RE, Paredes Garcés MG, Acosta Nuñez JM. Prevention of pneumonia associated with invasive mechanical ventilation in an intensive care unit. Salud, Ciencia y Tecnología 2023; 3:326. https://revista.saludcyt.ar/ojs/index.php/sct/article/view/326 https://doi.org/10.56294/sctconf2023604 9 González Baños et al. 20. Betancourt-Reyes G, Betancourt-Betancourt G. Predictores de éxito en el empleo de la ventilación mecánica no invasiva. Revista de Ciencias Médicas de Pinar del Río [revista en Internet]. 2023; 27:5929. https://revcmpinar.sld.cu/index.php/publicaciones/article/view/5929 21. Vitón-Castillo A, Rego-Ávila H, Delgado-Rodríguez A. Consideraciones sobre el manejo de la vía aérea y la ventilación en el paciente crítico con la COVID-19. Revista de Ciencias Médicas de Pinar del Río 2020; 24(3):4520. https://revcmpinar.sld.cu/index.php/publicaciones/article/view/4520 22. Durán Rodríguez R, Rubio Méndez AM, Cobas Sánchez A, Rodríguez Paján N, Castillo Pérez Y. Comportamiento de neumonía asociada a ventilación mecánica en cuidados intensivos de adultos. Rev. Inf. Cient [internet]. 2017 [citado 02/05/2022];96(4):[aprox. 10 p.]. Disponible en: http://www.revinfcientifica.sld.cu/index.php/ric/article/view/1182 23. Bulacio S. Impact of the implementation of a combo of measures for the prevention of pneumonias associated with mechanical ventilation. Salud, Ciencia y Tecnología 2023; 3:548. https://revista.saludcyt.ar/ojs/index.php/sct/article/view/548 24. Miranda Pedroso R. Neumonía asociada a la ventilación mecánica artificial. Rev Cub Med Int Emerg [Internet]. 2019 [citado 02/05/2022];18(3):e592. Disponible en: http://www.revmie.sld.cu/index.php/mie/article/view/592 25. Rego-Avila H, Delgado-Rodríguez A, Vitón-Castillo A, Piñeiro-Izquierdo S, Machado-Mato O. Neumonía asociada a la ventilación mecánica en pacientes atendidos en una unidad de cuidados intensivos. Revista de Ciencias Médicas de Pinar del Río 2019; 24(1):4137https://revcmpinar.sld.cu/index.php/publicaciones/article/view/4137 26. Graziani Noriega D, Ampuero López A. Protocolo diagnóstico y tratamiento empírico en urgencias de las infecciones respiratorias. Medicine [Internet]. 2018 [citado 02/05/2022];12(64):3794-800. Disponible en: https://www.clinicalkey.es/service/content/pdf/watermarked/1-s2.0S030454121830235X.pdf?locale=es_ES&searchIndex 27. Organización Mundial de la Salud. Neumonía. OMS [Internet]. 2021 [Actualizado 2021 Ene 2; citado 02/05/2022]. Disponible en: https://www.who.int/es/news-room/fact-sheets/detail/pneumonia 28. Tapia Pilamonta EJ, Jaramillo Ruales EK. Carbapenem-resistant Pseudomonas aeruginosa before and during the covid-19 pandemic, a review in Latin America. Salud, Ciencia y Tecnología 2023; 3:477. https://revista.saludcyt.ar/ojs/index.php/sct/article/view/477 29. Cantón-Bulnes ML, Garnacho-Montero J. Antisepsia orofaríngea en el paciente crítico y en el paciente sometido a ventilación mecánica. Med Intensiva [Internet]. 2019 [citado 02/05/2022];43(S1):2330. Disponible en: https://www.sciencedirect.com/science/article/pii/S0210569118302559 30. Miller, F. Neumonía asociada al ventilador. Worl Federation of Societies of Anaesthesiologists. Disponible en: https://www.wfsahq.org/components/com_virtual_library/media/74d02bfd1d8ced1516fe305f960f1698382-Neumon--a-Asociada-a-Ventilador.pdf https://doi.org/10.56294/sctconf2023604 Salud, Ciencia y Tecnología – Serie de Conferencias. 2023; 2:604 10 31. Fan E, Del Sorbo L, Goligher EC, Hodson CL, Munshi L, Walkey AJ, et al. An Official American Thoracic Society/European Society of Intensive Care Medicine/Society of Critical Care Medicine Clinical Practice Guideline: Mechanical Ventilation in Adult Patients with Acute Respiratory Distress Syndrome. Am J Resp Crit Care Med [Internet]. 2018 [citado 02/05/2022]; 195(9):[aprox 45 pp.]. Disponible en: https://www.atsjournals.org/doi/full/10.1164/rccm.201703-0548ST 32. Zakharkina T,Martín-Loeches I, Matamoros S, Povoa P, Torres A, Kastelijn JB, et al. The dynamics of the pulmonary micro biome during mechanical ventilation in the intensive care unit and the association with occurrence of pneumonia.Thorax [Internet].2017 [citado 02/05/2022];72:803-810. Disponible en: https://thorax.bmj.com/content/72/9/803.short 33. Vásquez Gaibor AA; Reinoso Tapia SC, Lliguichuzca Calle MN, Cedeño Caballero JV. Neumonía asociada a ventilación mecánica. Revista Científica Mundo de la Investigación y el Conocimiento [Internet].2019 [citado 02/05/2022];3(3):1118-1139.: http://recimundo.com/index.php/es/article/view/562 34. Rochwerg B, Brochard L, Elliott MW, Hess D, Hill NS, Nava S, et al. Official ERS/ATS clinical practice guidelines: noninvasive for acute respiratory failure. Eur Respir J [Internet]. 2017 [citado 02/05/2022];50(2):1602426. Disponible en: https://www.thoracic.org/statements/resources/cc/nivguidelines.pdf 35. Kalil AC, Metersky ML, Klompas M, Muscedere J, Sweeney DA, Palmer LB, et al. Management of adults with hospital-acquired and ventilatorassociated pneumonia: 2016 Clinical Practice Guidelines by the Infectious Diseases Society of America and the American Thoracic Society. Clin Infect Dis [Internet]. 2016[citado 02/05/2022];63:e61. Disponible en: https://academic.oup.com/cid/article/63/5/e61/2237650 36. Kumar ST, Yassin A, Bhowmick T, Dixit D. Recommendations from the 2016 guidelines for the management of adults with hospital-acquired or ventilator-associated pneumonia. Pharmacy Therap [Internet]. 2017 [citado 02/05/2022];42(12):767-72. Disponible en: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5720490/ 37. Sierra Arguello JC, Montoya Torres C, Gonzalez M, Gil B, Echeverri JL, Molina F, et al. Perfil microbiológico y de resistencia en pacientes con neumonía adquirida en comunidad, ingresados a unidades de cuidados intensivos de la ciudad de Medellín, Colombia, en 2015. Acta Col. Cuid Intensiv [Internet]. 2018 [citado 02/05/2022];17(4):247-257. Disponible en: https://www.clinicalkey.es/#!/content/playContent/1-s2.0S0122726217300514?returnurl=null&referrer=null CONFLICTO DE INTERESES No existen. FINANCIACIÓN La autora no recibió financiación para el desarrollo de la presente investigación CONTRIBUCIÓN DE AUTORÍA Conceptualización: Yanara González Baños, Osiris Oscar Terrado Almarales, Heidy Rego Avila. Curación de datos: Yanara González Baños, Osiris Oscar Terrado Almarales, Heidy Rego Avila. https://doi.org/10.56294/sctconf2023604 11 González Baños et al. Análisis formal: Yanara González Baños, Osiris Oscar Terrado Almarales, Heidy Rego Avila. Investigación: Yanara González Baños, Osiris Oscar Terrado Almarales, Heidy Rego Avila. Metodología: Yanara González Baños, Osiris Oscar Terrado Almarales, Heidy Rego Avila. Administración del proyecto: Yanara González Baños, Osiris Oscar Terrado Almarales, Heidy Rego Avila. Supervisión: Yanara González Baños, Osiris Oscar Terrado Almarales, Heidy Rego Avila. Validación: Yanara González Baños, Osiris Oscar Terrado Almarales, Heidy Rego Avila. Visualización: Yanara González Baños, Osiris Oscar Terrado Almarales, Heidy Rego Avila. Redacción – borrador original: Yanara González Baños, Osiris Oscar Terrado Almarales, Heidy Rego Avila. Redacción – revisión y edición: Yanara González Baños, Osiris Oscar Terrado Almarales, Heidy Rego Avila. https://doi.org/10.56294/sctconf2023604
https://openalex.org/W2796030171	http://www.e-compos.org.br/e-compos/article/download/273/254	Portuguese	null	Um conto de três cidades: música e sensibilidades culturais urbanas	E- compós	2,008	cc-by	6,350	www.e-compos.org.br \| E-ISSN 1808-2599 \| www.e-compos.org.br \| E-ISSN 1808-2599 \| Resumo Revista da Associação Nacional dos Programas de Pós-Graduação em Comunicação \| E-compós, Brasília, v.11, n.1, jan./abr. 2008. Este artigo tem por objetivo discutir as complexas e profusas articulações entre música popular e a cidade. Quando pensamos nessas articulações, imediatamente são feitas associações mentais entre determinados gêneros e determinadas cidades: Nova Orleans e o jazz tradicional; Nova York e o hip-hop; Chicago e o blues; Detroit e o som de Motown; Nashville e o country; Belém do Pará e o tecnobrega; o Rio de Janeiro e o samba; São Luís e o reggae, entre outras. Aqui vamos apresentar de modo necessariamente panorâmico a análise de três cenas distintas (no tempo e no espaço) ligadas às culturas pop e rock. São três épocas, três estilos e três cidades diferentes: Manchester, na Inglaterra, do final dos anos 70 aos anos 90; Seattle no início dos anos 90 e Recife dos anos 90 aos 2000. A idéia é identificar certas recorrências e diferenças entre as cenas para compreender como o engendramento de sensibilidades culturais e a configuração de cenas musicais modelam e redesenham não apenas as próprias cidades, mas o modo como os sujeitos apreendem e circulam nesses espaços. Este artigo tem por objetivo discutir as complexas e profusas articulações entre música popular e a cidade. Para isso recortamos três épocas, três estilos e três cidades diferentes: Manchester do final dos anos 70 aos anos 90; Seattle no início dos anos 90; e Recife dos anos 90 aos 2000. O propósito é identificar certas recorrências e diferenças entre as cenas para compreender como o engendramento de “sensibilidades culturais” e a configuração de cenas musicais modelam e redesenham não apenas as próprias cidades, mas o modo como os sujeitos apreendem e circulam nesses espaços. Um conto de três cidades: música e sensibilidades culturais urbanas Angela Prysthon O conceito de “sensibilidade cultural” empregado aqui é tributário do trabalho de Celeste Olalquiaga sobre o pós-modernismo, no qual ela define sensibilidade “como uma predisposição coletiva para certas práticas culturais” (OLALQUIAGA, 1998, p. 16). E de fato há nos cenários observados aqui uma predisposição coletiva que construiu em torno da música um novo imaginário para as três cidades em questão. Evidentemente, esse novo imaginário não foi constituído apenas pelos produtos da indústria musical (discos, CDs, shows e presença midiática), mas por uma gama de articulações entre estes – que poderíamos classificar como “catalisadores” – e indivíduos, grupos e signos. de informação, da diferença e de novos espaços urbanos. Pois, além de um território conceitual necessariamente mais fluido, tem-se em vista uma nova materialidade. As world cities, cidades do mundo em constante processo de mutação, não são necessariamente as maiores cidades, mas lugares onde a diversidade se multiplica a cada instante, ora num movimento integrativo, ora na dissolução em partes isoladas. Revista da Associação Nacional dos Programas de Pós-Graduação em Comunicação \| E-compós, Brasília, v.11, n.1, jan./abr. 2008. As cidades mundiais são sítios nos quais en- contramos a justaposição de ricos e pobres, a nova classe média de profissionais liberais e os sem-teto, e uma variedade de outras identifi- cações étnicas, de classe e tradicionais, como também pessoas do centro e da periferia que são colocadas dentro de uma mesma localiza- ção especial. [tradução nossa] (FEATHERSTO- NE, 1995, p. 118) Desse modo, a própria configuração urbana contemporânea vai sendo permeada pelo imaginário cultural e conceitual do pós-moderno. Featherstone também fala de um entrelaçamento entre as esferas cultural, social e econômica dessa cidade pós-moderna: A cidade pós-moderna é portanto muito mais auto-consciente imagética e culturalmente; é um centro de consumo tanto cultural como geral, e assim como este último não pode ser desvincu- lado dos signos e imaginários culturais, os esti- los de vida urbanos, o cotidiano e as atividades de lazer estão todos em maior ou menor graus influenciados pelas tendências pós-moderna si- mulativas (FEATHERSTONE, 1991, p. 99) As transformações do cenário urbano mundial são quiçá lentas, graduais, mas certamente são bastante concretas. Angela Prysthon \| prysthon@gmail.com Doutora em Critical Theory And Hispanic Studies pela Universidade de Nottingham. Professora do Programa de Pós-Graduação em Comunicação da Universidade Federal de Pernambuco – UFPE. Doutora em Critical Theory And Hispanic Studies pela Universidade de Nottingham. Professora do Programa de Pós-Graduação em Comunicação da Universidade Federal de Pernambuco – UFPE. www.e-compos.org.br \| E-ISSN 1808-2599 \| Guerra, na qual Manchester teve grande parte do seu centro histórico destruído pelas bombas. A cidade também sofreu de modo particularmente intenso as reformas econômicas da era Thatcher: indústrias fechadas, altos índices de desemprego e o fechamento do porto em 1982. O que afetou, obviamente, as formas de produzir e consumir cultura na cidade. Guerra, na qual Manchester teve grande parte do seu centro histórico destruído pelas bombas. A cidade também sofreu de modo particularmente intenso as reformas econômicas da era Thatcher: indústrias fechadas, altos índices de desemprego e o fechamento do porto em 1982. O que afetou, obviamente, as formas de produzir e consumir cultura na cidade. hipótese aqui é que a música (e os processos sociais ligados a ela) vai ser essencial para o engendramento dessas transformações. A nossa principal 2/13 As cidades mundiais são sítios nos quais en- contramos a justaposição de ricos e pobres, a nova classe média de profissionais liberais e os sem-teto, e uma variedade de outras identifi- cações étnicas, de classe e tradicionais, como também pessoas do centro e da periferia que são colocadas dentro de uma mesma localiza- ção especial. [tradução nossa] (FEATHERSTO- NE, 1995, p. 118) É necessário compreender, portanto, como se desenhou essa gama de articulações nas três cenas a partir de uma noção diferente de cidade que deixa evidente a urgência de um constante deslocamento conceitual, vinculado ao marco teórico do pós-moderno. Pois, se na modernidade tínhamos, de certa maneira, algumas convicções em relação à natureza da cidade, seus componentes, suas articulações, a partir da pós- modernidade não apenas teremos que renegociar e retrabalhar todo esse elenco de noções, como também inserir uma série de novos paradigmas e termos. Claro que sem esquecer do flâneur, do cosmopolitismo e da modernidade – elementos constitutivos do urbano que entram em cena na cidade pós-moderna de maneira muito mais enfática que antes através da descentralização, dos meios de comunicação de massa, das redes Desse modo, a própria configuração urbana contemporânea vai sendo permeada pelo imaginário cultural e conceitual do pós-moderno Featherstone também fala de um entrelaçamen entre as esferas cultural, social e econômica dessa cidade pós-moderna: www.e-compos.org.br \| E-ISSN 1808-2599 \| www.e-compos.org.br \| E-ISSN 1808-2599 \| Já Reel around the fountain evoca uma representação mediatizada de Manchester ao citar diretamente frases do filme A Taste of Honey (Tony Richardson, 1962), ambientado em Manchester e uma constante referência para o grupo, em capas, vídeos e letras: noturno aberto em 1982), Tony Wilson (jornalista e empresário), Rob Gretton (empresário de bandas), Peter Saville (designer gráfico). Os fragmentos urbanos indubitavelmente permeiam a história musical da cidade. Por exemplo, nas letras de Morrissey, vocalista da banda extinta em 1986 The Smiths, as paisagens de Manchester estão sempre presentes. Desde a alusão a trens, pontes, fontes, cemitérios e escolas, até crimes, filmes e livros que tematizam a cidade. Como na canção Suffer Little Children (1983), que alude aos assassinatos em série de crianças perpetrados por Myra Hindley e Ian Brady nos anos 60 e na qual Morrissey vai mencionando os nomes das vítimas: Reel around the fountain Slap me on the patio I’ll take it now Oh ... Fifteen minutes with you Well, I wouldn’t say no People said that you were virtually dead And they were so wrong4 acional dos Programas de Pós-Graduação em Comunicação \| E-compós, Brasília, v.11, n.1, jan./abr. 2008 Entretanto, de todas as figuras individuais da cidade, a que talvez sintetize melhor esse período da história da música pop de Manchester seja Tony Wilson, o empresário e jornalista que impulsionou a “movida mancuniana”, desde os primeiros shows punk até a cristalização de Madchester (como Manchester começou a ser chamada a partir do final dos anos 80 e da configuração da cena acid e techno na cidade). Em meados dos 70, Wilson era um repórter de TV local com o que ele chamava de ‘excesso de orgulho cívico’ e grandes planos para a cultura do noroeste da Inglaterra (24 Hour Party People): ocupando o lugar central nesses planos estava a Factory Records, a gravadora que de certo modo Outro exemplo direto da presença de Manchester no cancioneiro dos Smiths é The Headmaster Ritual (1984), na qual é descrita a rotina numa escola da cidade: 2 “Lesley-Anne, com seu bonito colar de bolinhas brancas/ Oh, John, você nunca será um homem/ E você nunca verá sua casa novamente/ Oh, Manchester, tanto para dar conta”. [tradução nossa] 3 “ 4 “ 3 “Zumbis beligerantes/ Dirigem as escolas de Manchester/ Suínos invertebrados/ Mentes cimentadas”. 2 Manchester, so much to answer for Giacomo Bottà (2006), falando sobre a influência efetiva da música popular sobre a cidade, enumera algumas maneiras concretas através das quais é exercida essa influência: a da Associação Nacional dos Programas de Pós-Graduação em Comunicação \| E-compós, Brasília, v.11, n.1, jan./abr. 2008. A partir do final da década de 1970 surge uma cena musical vibrante na esteira da subcultura punk em ascensão em toda a Inglaterra (subcultura, aliás, deflagrada a partir de um cenário de decadência pós-industrial extremamente semelhante ao contexto particular mancuniano1). Podemos ver na trajetória dessa cena (que começa com o agrupamento de bandas em artistas a partir do impulso dos primeiros shows dos Sex Pistols na cidade em 1976 e, de certo modo, tem um fechamento simbólico a partir do final da era “Madchester” com o encerramento das atividades do clube Haçienda em 1997) um conjunto de exemplar de modos de articulação entre música e cidade. Vários grupos – em certa altura já classificados como pós-punk –, lugares e indivíduos podem ser mencionados como parte desse período histórico tão especial para a mitologia pop: Buzzcocks, The Fall, The Smiths e Morrissey, A certain ratio, Durutti Column, The Stone Roses, Joy Division e Ian Curtis, New Order, Happy Mondays, Factory Records (gravadora), Haçienda (clube 3/13 Este é o resultado de uma superposição em camadas: a música popular media lugares como paisagens textuais, sonoras e visu- ais. As letras de músicas que se referem a lugares se configuram como as paisagens textuais. O uso da tradição musical local, ver- nacular ou ruídos tipicamente urbanos cons- tituem as paisagens sonoras de uma banda. Finalmente as paisagens propriamente ditas consistem de todos os elementos visuais (por exemplo, as capas) que se referem à mesma localidade. Voltando para o nível da regeneração, parece importante notar que a música em si é etérea, mas sua produção, circulação e fruição dependem de fatores materiais localizados nas cidades. [tradução nossa] (p. 121) Manchester é uma cidade do noroeste da Inglaterra, mais conhecida como o berço da Revolução Industrial e como uma das maiores cidades da Grã-Bretanha (a zona metropolitana de Manchester é a segunda maior aglomeração urbana do Reino Unido depois de Londres). Além das marcas da revolução industrial e da sua subseqüente decadência, o imaginário da cidade foi profundamente marcado pela Segunda 1 Relativo a Manchester. www.e-compos.org.br \| E-ISSN 1808-2599 \| estabeleceu novos parâmetros para o lançamento e a circulação do rock e da música pop no mundo. Nem todos os artistas e grupos importantes da cena mancuniana faziam parte da Factory, mas a relação da gravadora com a cidade foi tão intensa que deixou marcas muito fortes mesmo após a falência em 1992. O selo, lançado em 1978, empregava um sistema de catalogação inusitado no qual não apenas os lançamentos musicais, mas também os trabalhos gráficos, edifícios e outros objetos recebiam um número. Até o caixão no qual foi enterrado Wilson em agosto de 2007 recebeu um número de catálogo: FAC 501. O caso da Factory sintetiza todas as possibilidades de relação entre música e cidade. Aliás, a Factory surge das entranhas de Manchester, é um projeto completamente desvinculável da cidade, em todos os seus aspectos (visuais, sonoros, líricos). talvez seja nos landscapes propriamente ditos que a conexão entre cidade e música feita pela Factory seja mais explícita ou bem sucedida: não somente o clube Haçienda se tornou uma espécie de símbolo cultural mor da cidade durante a sua existência, como no trabalho gráfico que marcou a gravadora (especialmente aquele empreendido pelo designer Peter Saville). [tradução nossa] 4 “Carretel em volta da fonte/Estapeia-me no pátio/ Tomarei agora/ Oh.../ Quinze minutos com você/ Eu não recusaria/ As pessoas disseram que você estava virtualmente morto/Mas eles estavam tão errados. [tradução nossa] www.e-compos.org.br \| E-ISSN 1808-2599 \| www.e-compos.org.br \| E-ISSN 1808-2599 \| dois grandes nomes do grunge para o sucesso mundial através daqueles que que talvez sejam os dois álbuns mais “clássicos” da cena: Nirvana, com Nevermind, e Pearl Jam, com Ten, ambos lançados no segundo semestre de 1991. Além do Nirvana e do Pearl Jam, outras bandas chegaram ao estrelato (nem todas vinculadas ao Sub Pop) do mesmo modo ambíguo e indeciso – um misto de atração e repulsa em relação à indústria fonográfica e ao showbiz: Alice in Chains, Soundgarden, Mudhoney, Green River, Melvins, entre outras de menor expressão. comportamento anti-establishment dos músicos) e associada ao grunge aos olhos do mundo. Para além de sua fama como um concentrado de empresas tecnológicas (especialmente aquelas ligadas à informática), como o berço da Starbucks e dos cafés de designer, mas de certo modo associado a tudo isso, Seattle foi se tornando sinônimo do grunge. Começou-se a prestar atenção naquela isolada e fria cidade do noroeste americano a partir de uma cena que pouco tinha de hedonista e afirmativa. Mas a interessante contradição é que tudo o que o movimento tinha de negação (rejeição do mainstream, do padrão, antiesteticismo, antiindústria) foi sendo capitalizado para a caracterização de Seattle como um dos pólos criativos mundiais de maior impacto e relevância na década de 1990. Outro aspecto que chama a atenção é como, de um modo até mais intenso que Manchester – que tinha algo de misoginia –, a cena de Seattle é emblemática da predominância masculina nos seus grupos musicais e na própria configuração das redes sociais ligadas a ela. Algo que pode ser vagamente relacionado ao boom da tecnologia na década de 90 com a chegada massiva de homens solteiros na região para trabalhar em empresas como Microsoft, por exemplo. A cena se configurou quase como uma catarse tanto para os jovens que faziam a música, como para aqueles que a consumiam. Curiosamente, o vigor do grunge só foi possível por causa do relativo isolamento cultural vivido pela cidade até então: De acordo com aqueles que estiveram lá, Se- attle era um lugar bem isolado culturalmente. As grandes bandas sequer incluíam Seattle nas turnês americanas da Costa Oeste, e a cena local estava repleta bandas derivativas que faziam o máximo para soar como outras. Não era um ambiente que anunciasse uma explo- são de vitalidade musical original. Ainda assim, o ambiente parece ser um conceito-chave para explicar a década de 1985-1995. 3 Smells like teen spirit Revista da Associação Nacional dos Programas de Pós-Graduação em Comunicação \| E-compós, Brasília, v.11, n.1, jan./abr. 2008. O caso de Seattle difere do de Manchester pela ausência de referências tão explícitas. Não é possível fazer a mesma taxonomia de letras, capas e sons do grunge que tenham rastros tão plásticos da cidade. Entretanto, é provável que o impacto da cena grunge em Seattle tenha sido ainda maior (até pensarmos nos termos da influência internacional que teve o movimento). O grunge é um estilo de rock alternativo surgido na segunda metade dos anos 80 no estado de Washington nos Estados Unidos, especialmente na área de Seattle. As influências do punk, do heavy metal e do hardcore aliadas a uma estética visual despojada, letras que versavam principalmente sobre a apatia e a angústia da chamada “geração X” (pessoas que estavam na casa dos vinte anos na década de 90) e uma rejeição do glamour e da performance estilizada que caracterizou o rock e o pop dos anos 80. Assim como em Manchester, um selo em particular vai ter preponderância na cena: a gravadora Sub Pop, que concentrou grande parte das bandas de Seattle e catapultou os Um dos exemplos mais notáveis dessa simbiose, especialmente relativa aos “soundscapes” urbanos são as gravações do grupo Joy Division, especialmente aquelas produzidas por Martin Hamnett, nas quais aparecem ruídos de trens, alarmes, maquinaria pesada, a acústica dos grandes armazéns, entre outros efeitos (OTT, 2003). Nas letras das canções de vários dos grupos da Factory, inclusive do Joy Division, também é possível vislumbrar algumas referências mais indiretas, especialmente a uma atmosfera lúgubre e sombria que remete a certa decadência pós- industrial. Assim como nas letras dos Happy Mondays, evocativas da psicodelia e do ecstasy massivamente consumidos em Madchester. Mas www.e-compos.org.br \| E-ISSN 1808-2599 \| www.e-compos.org.br \| E-ISSN 1808-2599 \| 4 A cidade não pára Em vários trabalhos sobre a cena Mangue foi apontada a ligação do movimento com a cidade, com a cultura urbana e com a emergência de novas identidades sociais na periferia. Emergindo da “periferia da periferia”, da lama, o mangue bit (como foi chamado no início pelos grupos que o constituíam) ou mangue beat (como ficou conhecido através da mídia nacional) vai transformar a cidade do Recife. Assim como Manchester e Seattle, a perspectiva de transformação urbana através da música, da cultura, é o motor das sensibilidades culturais. Em vários trabalhos sobre a cena Mangue foi apontada a ligação do movimento com a cidade, com a cultura urbana e com a emergência de novas identidades sociais na periferia. Revista da Associação Nacional dos Programas de Pós-Graduação em Comunicação \| E-compós, Brasília, v.11, n.1, jan./abr. 2008. justamente o “inchaço” do Recife, a sujeira e, simultaneamente, a música de suas ruas. O diálogo entre as dualidades tradição/ modernidade, centro/periferia, nacionalismo/ cosmopolitismo vai ser explorado nos trabalhos seguintes, por exemplo, no segundo disco, inclusive quando vai ser indiretamente assumida certa herança do Tropicalismo com a participação especial de Gilberto Gil na faixa Macô e a regravação de Maracatu Atômico de Jorge Mautner. Em Enquanto o mundo explode, Science afirma: um curupira já tem seu tênis importado não conseguimos acompanhar o motor da história mas somos batizados pelo batuque e apreciamos agricultura celeste. (1996) O outro grupo mais proeminente do mangue beat, o mundo livre s/a, embora ritmicamente mais convencional que o Nação Zumbi, reunindo algumas características do samba e do rock, procura explicitar a posição da periferia em relação ao mundo globalizado. Recife continua sendo referência importante como perspectiva periférica: 7/13 O diálogo entre as dualidades tradição/ modernidade, centro/periferia, nacionalism cosmopolitismo vai ser explorado nos trabal seguintes, por exemplo, no segundo disco, inclusive quando vai ser indiretamente assumida certa herança do Tropicalismo co participação especial de Gilberto Gil na faix Macô e a regravação de Maracatu Atômico Jorge Mautner. Em Enquanto o mundo exp Science afirma: Emergindo da “periferia da periferia”, da lama, o mangue bit (como foi chamado no início pelos grupos que o constituíam) ou mangue beat (como ficou conhecido através da mídia nacional) vai transformar a cidade do Recife. Assim como Manchester e Seattle, a perspectiva de transformação urbana através da música, da cultura, é o motor das sensibilidades culturais. hip hop e rock. As letras do Nação Zumbi freqüentemente tentam essa equação entre o local (as especificidades de viver numa cidade particularmente subdesenvolvida de um país subdesenvolvido, as gírias e os mitos recifenses) e o universal (as relações com a tecnologia, as imagens metropolitanas). As canções mais conhecidas do grupo tematizam justamente o “inchaço” do Recife, a sujeira e, simultaneamente, a música de suas ruas. hip hop e rock. As letras do Nação Zumbi freqüentemente tentam essa equação entre o local (as especificidades de viver numa cidade particularmente subdesenvolvida de um país subdesenvolvido, as gírias e os mitos recifenses) e o universal (as relações com a tecnologia, as imagens metropolitanas). As canções mais conhecidas do grupo tematizam justamente o “inchaço” do Recife, a sujeira e, simultaneamente, a música de suas ruas. francamente fechado e provinciano. A cidade se torna plataforma para a elaboração de estratégias de superação deste contexto, ela é a malha através das quais as predisposições coletivas são articuladas. (HOWITT) [do- cumento eletrônico] Contudo, o mais relevante da cena grunge em conexão com a cidade de Seattle é simultaneamente o modo como ela foi transformada pelos símbolos (ou poderíamos dizer anti-símbolos) associados à música (sobretudo a moda – as camisas de flanela, os cabelos sujos e desgrenhados, os coturnos – e o Ou seja, a cena surge exatamente de uma carência, aparece como resposta a um contexto www.e-compos.org.br \| E-ISSN 1808-2599 \| Mesmo podendo ser considerado mais culto que outros fenômenos mais populares (o hip-hop de São Paulo, o funk carioca e o pagode, por exemplo) por suas características ideológicas, discursivas e metalingüísticas, lança alguns dados importantes para o redimensionamento do papel do popular no contexto contemporâneo: se no início eram bandas urbanas lideradas por filhos da classe média (mesmo que em alguns grupos houvesse a presença de membros das classes mais baixas) a ganharem expressão nacional, nos últimos anos da década de 90 foram mercantilizados sob esse rótulo (às vezes até inadequadamente) artistas de origem indiscutivelmente “popular” e proletária, como Selma do Coco (uma senhora de idade “revelada” ao público num festival de rock em Recife em 1996), a cirandeira Lia de Itamaracá ou as bandas hardcore do subúrbio Alto José do Outro dado importante na conjuntura do manguebit é, indubitavelmente, como o discurso da identidade e da tradição ultrapassou as barreiras da cultura das classes médias e letradas para influenciar a produção musical mais popular. Vemos, assim, a emergência de artistas realmente periféricos (periféricos dentro da própria periferia) fazendo uso do discurso da identidade nacional e de suas relações com formas globais de expressão. Mesmo podendo ser considerado mais culto que outros fenômenos mais populares (o hip-hop de São Paulo, o funk carioca e o pagode, por exemplo) por suas características ideológicas, discursivas e metalingüísticas, lança alguns dados importantes para o redimensionamento do papel do popular no contexto contemporâneo: se no início eram bandas urbanas lideradas por filhos da classe média (mesmo que em alguns grupos houvesse a presença de membros das classes mais baixas) a ganharem expressão nacional, nos últimos anos da década de 90 foram mercantilizados sob esse rótulo (às vezes até inadequadamente) artistas de origem indiscutivelmente “popular” e proletária, como Selma do Coco (uma senhora de idade “revelada” ao público num festival de rock em Recife em 1996), a cirandeira Lia de Itamaracá ou as bandas hardcore do subúrbio Alto José do 8/13 Pinho, também de Recife. Todos conquistaram espaço na mídia, desde programas de auditório de grande audiência a documentários na MTV ou na TV Cultura. O mangue reanima, abastece Injeta, recarrega as baterias Da Veneza esclerosada Mangue, manguedown Cidade complexo, caos portuário Mangue, Manguetown. (1994) Pinho, também de Recife. Todos conquistaram espaço na mídia, desde programas de auditório de grande audiência a documentários na MTV ou na TV Cultura. onal dos Programas de Pós-Graduação em Comunicação \| E-compós, Brasília, v.11, n.1, jan./abr. 2008. Contudo, é provável que a grande contribuição do Mangue seja realmente a interferência na cultura da cidade. Um dos aspectos mais relevantes da história do movimento é precisamente essa relação com o Recife, ou, melhor ainda, a maneira como seus produtos, manifestações, modos e modas foram construindo ao longo da década de 90 uma nova relação com a cidade, uma nova cultura urbana. Em várias “genealogias” do Mangue se aponta a influência do Recife (e quase sempre a influência da pobreza, da miséria e das mazelas da cidade) nas letras, nas músicas, no visual e na atitude dos músicos, onde talvez o ponto mais interessante seja a forma como todos esses elementos acabaram por transformar o imaginário urbano recifense, a maneira como o Mangue construiu uma política de diferença cultural para a cidade, o modo como, através dos mais variados fenômenos culturais, o Recife se viu repentinamente inserido num contexto pós- moderno. O Recife foi, pois, reinventado a partir do movimento Mangue, ou melhor, da “cena Mangue”, como preferem seus “fundadores”. 8/1 Outro dado importante na conjuntura do manguebit é, indubitavelmente, como o discurso da identidade e da tradição ultrapassou as barreiras da cultura das classes médias e letradas para influenciar a produção musical mais popular. Vemos, assim, a emergência de artistas realmente periféricos (periféricos dentro da própria periferia) fazendo uso do discurso da identidade nacional e de suas relações com formas globais de expressão. 4 A cidade não pára Nos trabalhos das bandas manguebit (mesmo aquelas que rejeitam o rótulo, eventualmente) estão presentes tanto a rearticulação da tradição (através de ritmos populares de Pernambuco e alusões ao folclore da região), como a preocupação com as últimas tendências da cultura pop mundial. Recuperando o elo perdido (e certa independência vital e muitas vezes franca oposição) em relação ao tropicalismo, Chico Science & Nação Zumbi, por exemplo, em Da lama ao caos mistura ritmos brasileiros como o maracatu, a ciranda ou o coco com o samba, com música eletrônica, www.e-compos.org.br \| E-ISSN 1808-2599 \| Contudo, chama a atenção como recorrência o modo a partir do qual as sensibilidades culturais aparecem como constitutivas do tecido urbano, como tais articulações (tanto a música propriamente dita, como todo o seu entorno, seus acessórios – moda, audiovisual, códigos de comportamento, etc.) se tornam as bases para a inserção (ou reinserção) dessas cidades num contexto globalizado. Manchester, Seattle, Recife, em espaços-tempos distintos e cada uma de sua maneira particular, demonstram o funcionamento do que poderíamos chamar de cosmopolitismo pós-moderno ou cosmopolitismo periférico (PRYSTHON, 2002), processo sublinhado e condicionado por uma série de remapeamentos culturais implicados na globalização e numa reconfiguração pós- moderna do conceito de cidade. eixo” – seja Manchester, Seattle ou Recife – como encarnações urbanas do conceito de periferia). O cosmopolitismo vai-se reconfigurando através do percurso de autodescoberta feito pelas margens. Uma autodescoberta que pode levar ao estabelecimento das primeiras políticas da diferença e para a afirmação de um novo conceito de urbano. O cosmopolita periférico tenta se colocar, produzir e se autodefinir a partir de uma instância ambígua (ser e estar na periferia, desejar estar na metrópole, no centro) e aponta justamente os elementos que fazem da periferia um modelo de modernidade alternativa (problemática, incompleta, contraditória). Ele trabalha nos interstícios de uma realidade e tradições locais e de uma cultura urbana internacional, aspiracional e moderna. Temos assim outro cosmopolitismo que indubitavelmente transforma a própria noção de cidade, de experiência urbana na contemporaneidade. As sensibilidades culturais das três cenas apontam justamente para o momento de ruptura representado pelo pós-modernismo para a cultura das cidades. Elas são exemplos bastante concretos de como o pós-modernismo e a pós-modernidade têm relações, ou antes, podem ser conseqüências da política mundial contemporânea e de uma completamente nova configuração global de poder, “in which the old imperial maps have been lost” (na qual os velhos mapas imperiais se perderam) – como faz Robert Young (1990, p. 117). 5 Convergências periféricas As três cenas são evidentemente distintas, são espacialmente muito distantes umas das outras, mesmo que temporalmente haja coincidências. www.e-compos.org.br \| E-ISSN 1808-2599 \| Seattle e Recife demonstraram nas três últimas décadas, há uma clara propensão para que essas tendências apareçam de muitos outros lugares, difundam-se e dissolvam-se de forma muito mais rápida. A gradual superação desses esquemas oposicionais e a crescente descentralização cultural da contemporaneidade vão, assim, modificando profundamente a própria estrutura da teorização sobre a cidade e nossas próprias experiências. a emergência dessa sociedade pós-industrial, ou “sociedade de informação”– com todas as suas nuances, entre elas a valorização do periférico, do exótico, do excêntrico (refletidos no multiculturalismo) – desesestabiliza a força centralizadora das metrópoles modernas. O cosmopolitismo pós-moderno e periférico vai ser diferente, sobretudo porque ele não supõe necessariamente um ponto norteador (algo essencial no cosmopolitismo moderno com a Paris-mito dos modernos e os subseqüentes prolongamentos dessa Paris na periferia – São Paulo, Buenos Aires, etc.). Referências Bibliográficas BOTTÀ, Giacomo. Pop music, cultural sensibilities and places: Manchester 1976–1997. In: FORNÄS, Johan (ed). THE ESF-LIU CONFERENCE CITIES AND MEDIA: cultural perspectives on urban identities in a mediatized world, 2006, Vadstena, Suécia. Disponível em: <http://www.ep.liu.se/> Acesso em: 3 mar. 2008. Portanto, se o cosmopolitismo moderno é essencialmente centrípeto, a força centrífuga da pós-modernidade começa a relativizar a importância das grandes metrópoles mundiais em termos de disseminação das informações. O que antes era quase um sistema de oposições – campo/cidade; provinciano/cosmopolita; bárbarie/ civilização; caos/ordem –, torna-se uma rede de múltiplas interdependências, confluências e novos parâmetros. E é justamente a cidade que se torna o território intersticial onde se encadeiam, intercalam-se e se confrontam tais oposições. Ao invés de ser apenas mais um elemento do binarismo oposicional, a cidade passa a ser ela própria um processo dialético dos embates pós-modernos. Ou seja, poderíamos pensar no momento de ruptura do pós-moderno como o momento de autoconsciência cultural da periferia (e entendendo essas cidades “fora do Revista da Associação Nacional dos Programas de Pós-Graduação em Com As teorias pós-modernas e do pós-moderno, inevitavelmente, pois, lançam outras dimensões ao conceito de cosmopolitismo: a sua constante remissão ao crescente descentramento da vida urbana e da cultura pós-moderna, a evidente globalização em diversas esferas da sociedade – entre elas economia e cultura –, a insistência pelo relativismo cultural e o estabelecimento de um ciberespaço agora como realidade e não mais alucinação futurista são algumas das razões mais importantes para essa redefinição do cosmopolitismo. Basicamente, entretanto, www.e-compos.org.br \| E-ISSN 1808-2599 \| www.e-compos.org.br \| E-ISSN 1808-2599 \| Palabras clave Keywords City. Popular music. Sociability. Cultural sensibilities. www.e-compos.org.br \| E-ISSN 1808-2599 \| FEATHERSTONE, Mike. Consumer culture and postmodernism. Londres: Sage, 1991. FEATHERSTONE, Mike. Consumer culture and postmodernism. Londres: Sage, 1991. FEATHERSTONE, Mike. Consumer culture and postmodernism. Londres: Sage, 1991. ______. Undoing culture: globalization, postmodernism and identity. Londres: Sage, 1995. ______. Undoing culture: globalization, postmodernism and identity. Londres: Sage, 1995. Revista da Associação Nacional dos Programas de Pós-Graduação em Comunicação \| E- ______. Undoing culture: globalization, postmodernism and identity. Londres: Sage, 1995. HOWITT, Bernie. Popular culture- grunge. In: Society and culture Association. Disponível em: <http://www. ptc.nsw.edu.au/scansw/grunge.html>. Acesso em: 3 mar. 2008. KING, Emily (ed). Designed by Peter Saville. Nova York: Princeton Architectural Press, 2003. OLALQUIAGA, Celeste. Megalópolis: sensibilidades culturais contemporâneas. São Paulo: Studio Nobel, 1998. OTT, Chris. Joy Division’s unknown pleasures. Londres: Continuum, 2003. PRYSTHON, Angela. Cosmopolitismos periféricos: ensaios sobre modernidade, pós-modernidade e Estudos Culturais na América Latina. Recife: Bagaço, 2002. O que não significa, obviamente, que deixem de existir os grandes centros de onde emanam as tendências culturais. Porém como Manchester, REYNOLDS, Simon. Beijar o céu. São Paulo: Conrad, 2006. ROBERTSON, Matthew. Factory records: the complete graphic album. San Francisco: Chronicle Books, 2007. WILSON, Tony. Twenty four hour party people: what the sleeve notes never tell you. Londres: Channel 4 Books, 2002. YOUNG, Robert. White Mythologies. Writing History and the West. Londres: Routledge, 1990. REYNOLDS, Simon. Beijar o céu. São Paulo: Conrad, 2006. ROBERTSON, Matthew. Factory records: the complete graphic album. San Francisco: Chronicle Books, 2007. REYNOLDS, Simon. Beijar o céu. São Paulo: Conrad, 2006. ROBERTSON, Matthew. Factory records: the complete graphic album. San Francisco: Chronicle Books, 2007. WILSON, Tony. Twenty four hour party people: what the sleeve notes never tell you. Londres: Channel 4 Books, 2002. YOUNG, Robert. White Mythologies. Writing History and the West. Londres: Routledge, 1990. Revista da Associação Nacional dos Programas de Pós-Graduação em Comunicação \| E-compós, Brasília, v.11, n.1, jan./abr. 2008. 11/13 Resumen This article aims to discuss the complex and profuse articulations between popular music and the city. We chose as objects three different cities, styles and historical periods: Manchester from the end of the 1970s to the beginning of the 1990s; Seattle at the beginning of the 1990s and Recife from the 1990s to the 2000s. Our purpose is to identify certain recurrences and differences between these scenes to understand how cultural sensibilities are established and how musical movements model and redesign not only the actual cities, but also the way in which individuals apprehend and circulate in theses spaces. Este artículo tiene por objetivo discutir las complejas y profusas articulaciones entre la música popular y la ciudad. Para esto elegimos tres épocas, tres estilos y tres ciudades distintas: Manchester desde el final de los años 70 hasta los años 90; Seattle en principios de los 90 y Recife de los años 90 a los 2000. El propósito es identificar recurrencias y diferencias entre las escenas para comprender cómo las sensibilidades culturales son engendradas y cómo las escenas musicales modelan y rediseñan no sólo las propias ciudades, pero la manera cómo los sujetos aprehenden y circulan en esos espacios. A tale of three cities: music and urban cultural sensibilities A tale of three cities: music and urban cultural sensibilities Un cuento de tres ciudades: música y sensibilidades culturales urbanas Un cuento de tres ciudades: música y sensibilidades culturales urbanas Keywords Ciudad. Música popular. Sociabilidad. Sensibilidades culturales. Ciudad. Música popular. Sociabilidad. Sensibilidades culturales. City. Popular music. Sociability. Cultural sensibilities. City. Popular music. Sociability. Cultural sensibilities. Revista da Associação Nacional dos Programas de Pós-Graduação em Comunicação \| E-compós, Brasília, v.1 Recebido em: 24 de setembro de 2008 Aceito em: 1o de outubro de 2008 Recebido em: 24 de setembro de 2008 Aceito em: 1o de outubro de 2008 Aceito em: 1o de outubro de 2008 CONSELHO EDITORIAL Afonso Albuquerque Universidade Federal Fluminense, Brasil Alberto Carlos Augusto Klein Universidade Estadual de Londrina, Brasil Alex Fernando Teixeira Primo Universidade Federal do Rio Grande do Sul, Brasil Alfredo Vizeu Universidade Federal de Pernambuco, Brasil Ana Carolina Damboriarena Escosteguy Pontifícia Universidade Católica do Rio Grande do Sul, Brasil Ana Silvia Lopes Davi Médola Universidade Estadual Paulista, Brasil André Luiz Martins Lemos Universidade Federal da Bahia, Brasil Ângela Freire Prysthon Universidade Federal de Pernambuco, Brasil Antônio Fausto Neto Universidade do Vale do Rio dos Sinos, Brasil Antonio Carlos Hohlfeldt Pontifícia Universidade Católica do Rio Grande do Sul, Brasil Arlindo Ribeiro Machado Universidade de São Paulo, Brasil César Geraldo Guimarães Universidade Federal de Minas Gerais, Brasil Cristiane Freitas Gutfreind Pontifícia Universidade Católica do Rio Grande do Sul, Brasil Denilson Lopes Universidade Federal do Rio de Janeiro, Brasil Eduardo Peñuela Cañizal Universidade Paulista, Brasil Erick Felinto de Oliveira Universidade do Estado do Rio de Janeiro, Brasil Francisco Menezes Martins Universidade Tuiuti do Paraná, Brasil Gelson Santana Universidade Anhembi/Morumbi, Brasil Hector Ospina Universidad de Manizales, Colômbia Ieda Tucherman Universidade Federal do Rio de Janeiro, Brasil Itania Maria Mota Gomes Universidade Federal da Bahia, Brasil Janice Caiafa Universidade Federal do Rio de Janeiro, Brasil Jeder Silveira Janotti Junior Universidade Federal da Bahia, Brasil COMISSÃO EDITORIAL vista da Associação Nacional dos Programas COMPÓS \| www.compos.org.br Associação Nacional dos Programas de Pós-Graduação em Comunicação Presidente Erick Felinto de Oliveira Universidade do Estado do Rio de Janeiro, Brasil erickfelinto@uol.com.br Vice-presidente Ana Silvia Lopes Davi Médola Universidade Estadual Paulista, Brasil asilvia@faac.unesp.br Secretária-Geral Denize Correa Araújo Universidade Tuiuti do Paraná, Brasil denizearaujo@hotmail.com Ana Gruszynski \| Universidade Federal do Rio Grande do Sul, Brasil João Freire Filho \| Universidade Federal do Rio de Janeiro, Brasil Rose Melo Rocha \| Escola Superior de Propaganda e Marketing, Brasil Expediente 13/13 John DH Downing University of Texas at Austin, Estados Unidos José Luiz Aidar Prado Pontifícia Universidade Católica de São Paulo, Brasil José Luiz Warren Jardim Gomes Braga Universidade do Vale do Rio dos Sinos, Brasil Juremir Machado da Silva Pontifícia Universidade Católica do Rio Grande do Sul, Brasil Lorraine Leu University of Bristol, Grã-Bretanha Luiz Claudio Martino Universidade de Brasília, Brasil Maria Immacolata Vassallo de Lopes Universidade de São Paulo, Brasil Maria Lucia Santaella Pontifícia Universidade Católica de São Paulo, Brasil Mauro Pereira Porto Tulane University, Estados Unidos Muniz Sodre de Araujo Cabral Universidade Federal do Rio de Janeiro, Brasil Nilda Aparecida Jacks Universidade Federal do Rio Grande do Sul, Brasil Paulo Roberto Gibaldi Vaz Universidade Federal do Rio de Janeiro, Brasil Renato Cordeiro Gomes Pontifícia Universidade Católica do Rio de Janeiro, Brasil Ronaldo George Helal Universidade do Estado do Rio de Janeiro, Brasil Rosana de Lima Soares Universidade de São Paulo, Brasil Rossana Reguillo Instituto Tecnológico y de Estudios Superiores do Occidente, México Rousiley Celi Moreira Maia Universidade Federal de Minas Gerais, Brasil Sebastião Carlos de Morais Squirra Universidade Metodista de São Paulo, Brasil Simone Maria Andrade Pereira de Sá Universidade Federal Fluminense, Brasil Suzete Venturelli Universidade de Brasília, Brasil Valério Cruz Brittos Universidade do Vale do Rio dos Sinos, Brasil Veneza Mayora Ronsini Universidade Federal de Santa Maria, Brasil Vera Regina Veiga França Universidade Federal de Minas Gerais, Brasil COMPÓS \| www.compos.org.br Associação Nacional dos Programas de Pós-Graduação em Comunicação Presidente Erick Felinto de Oliveira Universidade do Estado do Rio de Janeiro, Brasil erickfelinto@uol.com.br Vice-presidente Ana Silvia Lopes Davi Médola Universidade Estadual Paulista, Brasil asilvia@faac.unesp.br Secretária-Geral Denize Correa Araújo Universidade Tuiuti do Paraná, Brasil denizearaujo@hotmail.com Expediente E-COMPÓS \| www.e-compos.org.br \| E-ISSN 1808-2599 Revista da Associação Nacional dos Programas de Pós-Graduação em Comunicação. Brasília, v.11, n.1, jan./abr. 2008. A identificação das edições, a partir de 2008, passa a ser volume anual com três números. Revista da Associação Nacional dos Programas de Pós-Graduação em Comunicação. Brasília, v.11, n.1, jan./abr. 2008. A identificação das edições, a partir de 2008, passa a ser volume anual com três números. A revista E-Compós é a publicação científica em formato eletrônico da Associação Nacional dos Programas de Pós-Graduação em Comunicação (Compós). Lançada em 2004, tem como principal finalidade difundir a produção acadêmica de pesquisadores da área de Comunicação, inseridos em instituições do Brasil e do exterior. Revista da Associação Nacional dos Programas de Pós-Graduação em Comunicação \| E-compós, Brasília, v.11, n.1, jan./abr. 2008. CONSULTORES AD HOC Bianca Freire-Medeiros \| Fundação Getulio Vargas, Brasil Josimey Costa da Silva \| Universidade Federal do Rio Grande do Norte, Brasil Maria Conceição Golobovante \| Pontifícia Universidade Católica de São Paulo, Brasil Marlyvan Moraes de Alencar \| Centro Universitário SENAC-SP, Brasil Miriam de Souza Rossini \| Universidade Federal do Rio Grande do Sul, Brasil Paulo Ribeiro \| Pontifícia Universidade Católica do Rio de Janeiro, Brasil Rita Alves de Oliveira \| Centro Universitário SENAC, Brasil REVISÃO DE TEXTO E TRADUÇÃO \| Everton Cardoso REVISÃO DE TEXTO E TRADUÇÃO \| Everton Cardoso ASSISTÊNCIA EDITORIAL E EDITORAÇÃO ELETRÔNICA \| Raquel Castedo
https://openalex.org/W4213082132	https://jurnal.polines.ac.id/index.php/keunis/article/download/3154/107744	Indonesian	null	ANALISIS PENCATATAN DAN PELAPORAN KEUANGAN UMKM	Keunis/Keunis	2,022	cc-by-sa	5,117	P-ISSN: 2302-9315 E-ISSN: 2714-7274 https://jurnal.polines.ac.id/index.php/keunis P-ISSN: 2302-9315 E-ISSN: 2714-7274 https://jurnal.polines.ac.id/index.php/keunis JURNAL KEUNIS (Keuangan dan Bisnis) Vol. 10, No. 1, Januari 2022, Hlm. 33-42 © Copyright: The Author(s) This is an open access article under the CC BY-SA license JURNAL KEUNIS (Keuangan dan Bisnis) Vol. 10, No. 1, Januari 2022, Hlm. 33-42 © Copyright: The Author(s) This is an open access article under the CC BY-SA license https://jurnal.polines.ac.id/index.php/keunis Keywords: MSMEs, Accounting Systems, Financial Statements, Financial Accounting Standards Abstrak: Penelitian ini bertujuan untuk menggambarkan sistem pencatatan dan pelaporan keuangan usaha mikro, kecil, dan menengah (UMKM). Populasi ini adalah sebanyak 19.197 pelaku UMKM dan sampel penelitian sebanyak 27 pelaku UMKM di Kota Palu. Data diperoleh melalui kuisioner dan wawancara kepada pelaku UMKM. Teknik analisis data menggunakan analisis deskriptif. Hasil penelitian menunjukkan bahwa mayoritas UMKM yang termasuk dalam kelompok usaha mikro belum melakukan pencatatan dan pelaporan keuangan. Akan tetapi, UMKM yang berskala kecil dan menengah sebagian besar telah melakukan pencatatan dan pelaporan keuangan baik secara manual dan terkomputerisasi. Selanjutnya, sebagian besar UMKM belum pernah mengikuti pelatihan pengelolaan keuangan, khususnya pencatatan dan pelaporan keuangan. Oleh karena itu, pelaku UMKM menginginkan dilakukannya pelatihan maupun pendampingan pencatatan dan pelaporan keuangan oleh pemerintah atau lembaga-lembaga lainnya. Informasi ini penting bagi pihak pemerintah daerah dan instansi terkait dalam rangka penyuluhan dan pendampingan pengelolaan usaha di masa mendatang. Kata kunci: UMKM, Sistem Akuntansi, Laporan Keuangan, Standar Akuntansi Keuangan ANALISIS PENCATATAN DAN PELAPORAN KEUANGAN UMKM DI KOTA PALU SYAMSUL STIE Panca Bhakti Palu syamsulsyahrir@gmail.com Abstract: This study aims to describe the financial recording and reporting system of micro, small and medium enterprises (MSMEs). This population is 19,197 MSMEs and the research sample is 27 MSMEs in Palu City. Data were obtained through questionnaires and interviews with MSMEs. The data analysis technique used analysis descriptive. The results showed that the majority of MSMEs included in the micro-enterprise group had not recorded and reported financial statements. However, most of the small and medium-sized MSMEs have carried out financial recording and reporting both manually and computerized. Furthermore, most MSMEs have never attended financial management training, particularly financial recording and reporting. Therefore, MSMEs want training and assistance in financial recording and reporting by the government or other institutions. This information is important for the local government and related agencies in the context of counseling and mentoring business management in the future. Keywords: MSMEs, Accounting Systems, Financial Statements, Financial Accounting Standards PENDAHULUAN Usaha mikro, Kecil, dan Menengah (UMKM), memegang peran strategis dalam peningkatan perekonomian dan kesejahteraan di Indonesia. Kontribusi UMKM sangat berarti terhadap produk domestk bruto (PDB), dan meningkatkan peluang kerja sehingga mereduksi tingkat pengangguran (Savitri & Saifudin, 2018). Oleh karena itu, eksistensi dan pengembangan UMKM perlu dipertahankan. Salah satunya melalui peningkatan keahlian pelaku UMKM dalam mengelola usahanya, khususnya dalam bidang keuangan. Siagian & Indra (2019) mengungkapkan bahwa pelaku UMKM tidak luput dari berbagai persoalan dan tantangan yang dihadapi, diantaranya dalam hal pengelolaan keuangan, utamanya dari segi pencatatan dan pelaporan keuangan usaha. Padahal, menurut Kurniwati et al. (2012) pencatatan dan pelaporan keuangan yang baik merupakan salah satu faktor utama penyebab keberhasilan UMKM. Pencatatan dan pelaporan keuangan sangat bermanfaat sebagai sumber informasi dalam pengambilan keputusan suatu bisnis dalam rangka pengembangan dan kesuksesan UMKM (Kurniwati 33 Jurnal Keunis, Vol. 10, No. 1 Januari 2022 et al., 2012). Akan tetapi, sampai saat ini sebagian besar pelaku UMKM belum menerapkan pencatatan dan pelaporan keuangan berdasarkan siklus akuntansi, yang dimulai dari penyediaan bukti transaksi sampai dengan terbitnya laporan keuangan, dan tidak sejalan dengan standar akuntansi yang telah ditetapkan. Argumen ini tercermin dari temuan penelitian terdahulu. Misalnya, penelitian Ramdani et al. (2018) yang bertujuan menelusuri permasalahan pencatatan laporan keuangan berbasis SAK ETAP pada UMKM Warkop di Kota Makassar. Temuan mereka mengindikasikan bahwa pencatatan dan penyusunan laporan keuangan yang disusun pengelola UMKM Warkop, hanya sebatas laporan bisnis sesuai dengan pemahaman dan kebutuhan mereka saja. Kemudian, Savitri & Saifudin (2018) meneliti tentang bagaimana praktik pencatatan akuntansi dan persepsi pemilik UMKM terhadap pencatatan akuntansi. Hasilnya, menunjukkan bahwa UMKM MR. Pelagi Semarang, yang menjadi subjek penelitian mereka ternyata belum maksimal dalam menerapkan sistem pencatatan akuntansi, karena pemilik berpendapat bahwa akuntansi merupakan masalah yang rumit. Selanjutnya, penelitian Siagian & Indra (2019) bertujuan mengetahui pengetahuan akuntansi pelaku UMKM. Hasil analisis mengindikasikan bahwa pelaku usaha mikro telah menyusun laporan keuangan atau catatan pembukuan meskipun terbilang sederhana. Selain itu, mereka juga menginformasikan bahwa pengelola UMKM masih kesulitan untuk membuat laporan keuangan sebagaimana mestinya. Terakhir, penelitian Suryani & Subardjo (2020) dengan desain penelitian kualitatif melalui pendekatan deskriptif dan interpretatif. Hasilnya, menunjukkan bahwa seluruh informan penelitian telah melakukan pencatatan transaksi dan penyusunan laporan keuangan, tetapi masih sangat sederhana dan didasarkan pada pemahaman tiap pelaku UMKM. Sejatinya, dengan pencatatan dan pelaporan keuangan yang memadai, UMKM dapat memenuhi persyaratan dalam pengajuan kredit yaitu laporan keuangan, menilai kinerja, menganalisis posisi keuangan, dan mengetahui jumlah pajak (Warsono & Darmawan, 2010). PENDAHULUAN p g g j p j ( ) Beberapa penelitian yang ada, lihat Salmiah et al. (2018), Savitri & Saifudin (2018), Ramdani et al. (2018) Siagian & Indra (2019), Suryani & Subardjo (2020) belum mengungkapkan secara utuh setiap tahapan pencatatan dan pelaporan keuangan, dengan kata lain penelitian tersebut dalam mengkaji pengelolaan keuangan UMKM belum sesuai dengan siklus akuntansi. Padahal, informasi tentang itu sangat bermanfaat dalam menyampaikan sampai sejauhmana praktik pencatatan dan pelaporan keuangan dilakukan oleh pelaku UMKM. Informasi ini penting untuk diketahui sebagai bahan evaluasi bagi pengambil kebijakan dalam rangka meningkatkan pengelolaan keuangan UMKM. Oleh karena itu, penelitian ini hadir dalam rangka memberikan gambaran secara utuh dari setiap tahapan pencatatan dan pelaporan keuangan UMKM berdasarkan siklus akuntansi, mulai dari bukti transaksi hingga penyusunan laporan keuangan. Secara khusus, pertanyaan penelitian ini adalah bagaimana pencatatan dan pelaporan UMKM di Kota Palu? Dengan mengetahui jawaban atas pertanyaan penelitian ini, diharapkan dapat dijadikan rujukan oleh Pemerintah Kota Palu dan dinas terkait, maupun lembaga lainnya dalam rangka peningkatan kualitas pengelolaan keuangan UMKM di Kota Palu, ditinjau dari segi pencatatan dan pelaporan keuangan. Merujuk pada Undang-Undang Nomor 20 Tahun 2008 tentang Usaha Mikro, Kecil, dan Menengah (UMKM), disebutkan usaha mikro adalah usaha produktif milik orang perorangan dan atau badan usaha perorangan yang memenuhi kriteria usaha mikro. Kriterianya yaitu jumlah kekayaan bersih yang dimiliki lebih kecil dari atau sama dengan Rp50.000.000,00 dengan total penjualan tahunan tidak lebih dari Rp300.000.000,00. Usaha kecil adalah usaha produktif yang berdiri sendiri, yang dilakukan oleh perseorangan atau badan usaha bukan merupakan anak cabang perusahaan yang dimiliki, dikuasai, atau menjadi bagian baik langsung maupun tidak langsung dari usaha menengah atau besar yang memenuhi kriteria usaha kecil. Kriterianya, yaitu mempunyai kekayaan bersih diatas dari Rp50.000.000,00 sampai batas paling banyak Rp500.000.000,00 tidak termasuk tanah dan bangunan tempat usaha, dan mendapatkan omzet penjualan tahunan lebih dari Rp300.000.000,00 sampai 34 E-ISSN: 2714-7274 P-ISSN: 2302-9315 Syamsul dengan paling banyak Rp2.500.000.000,00. Kemudian, usaha menengah adalah usaha ekonomi produktif yang berdiri sendiri, yang dilakukan oleh orang perorangan atau badan usaha bukan merupakan anak perusahaan atau cabang perusahaan yang dimiliki, dikuasai, atau menjadi bagian baik langsung maupun tidak langsung dengan usaha kecil atau usaha besar dengan jumlah kekayaan bersih atau hasil penjualan tahunan. Kriteria usaha menengah, yaitu kekayaan bersih lebih besar dari Rp500.000.000,00 sampai dengan paling banyak Rp10.000.000.000,00 tidak termasuk tanah dan bangunan tempat usaha, dan omzet penjualan tahunan lebih dari Rp2.500.000.000,00 sampai paling banyak Rp50.000.000.000,00. PENDAHULUAN Intinya, UMKM adalah aktivitas bisnis yang dibentuk oleh masyarakat, baik berupa usaha perorangan maupun badan usaha (Farida, 2016) yang memenuhi kriteria UMKM yang ditetapkan oleh pemerintah. y g p p Menurut Jusup (2016:4) akuntansi adalah sistem informasi yang mengukur aktivitas bisnis mengolah data menjadi laporan, dan mengomunikasikan hasilnya kepada para pengambil keputusan. Lebih lanjut, Jusup (2016:5) mendefinisikan akuntansi dari dua sudut pandang, yaitu dari sudut pemakai jasa akuntansi, dan dari sudut proses kegiatannya. Dilihat dari sudut pemakainya, akuntansi adalah sebagai suatu disiplin yang menyediakan informasi yang diperlukan untuk melaksanakan kegiatan secara efisien dan mengevaluasi kegiatan-kegiatan suatu entitas. Sementara itu, dari sudut kegiatannya, akuntansi adalah sebagai proses pencatatan, penggolongan, peringkasan, pelaporan, dan penganalisisan data keuangan suatu entitas. Intinya, akuntansi adalah sebuah proses mengidentifikasi, mengukur, mencatat, dan melaporkan aktivitas keuangan perusahan kepada pemangku kepentingan, baik dari pihak internal maupun pihak eksternal. Selanjutnya, siklus akuntansi adalah tahapan kegiatan dalam proses pencatatan dan pelaporan akuntansi, berawal dari terjadinya transaksi hingga terbitnya laporan keuangan. Menurut Soemarso (2004:110) siklus akuntansi dimulai dari transaksi dan bukti transaksi, jurnal, buku besar, daftar saldo, kertas kerja dan jurnal penyesuaian, laporan keuangan, jurnal penutup, daftar saldo penutup, jurnal pembalik. Secara umum terdapat lima laporan keuangan, yaitu laporan laba rugi, laporan perubahan modal, laporan neraca, laporan arus kas, dan catatan atas laporan keuangan (CaLK). Kelima laporan tersebut, masing-masing mempunyai fungsi menyediakan informasi yang berkaitan dengan posisi bisnis suatu usaha (Kurniwati et al., 2012). Standar Akuntansi Keuangan Entitas Tanpa Akuntabilitas Publik (SAK ETAP). Standar ini merupakan pedoman bagi entitas yang tidak memiliki akuntabilitas publik signifikan dan tidak menerbitkan laporan keuangan untuk tujuan umum bagi pengguna eksternal. Dengan adanya SAK ETAP, usaha kecil dan menengah dapat menyusun sendiri laporan keuangan dan laporan tersebut diaudit dan memperoleh opini audit. Sehingga, laporan tersebut dapat digunakan untuk mendapatkan dana (misalnya dari Bank) untuk pengembangan usaha (Martani et al., 2012). Pada intinya, SAK ETAP ini disusun, bertujuan untuk menciptakan fleksibilitas dalam implementasinya dan memberikan kemudahan akses ETAP kepada sumber pendanaan dari perbankan (Hetika & Mahmudah, 2017). Adapun jenis laporan keuangan menurut SAK ETAP terdiri dari Laporan Neraca, Laba Rugi, Perubahan Modal, Arus Kas, dan Catatan atas Laporan Keuangan (CaLK). Selanjutnya, standar Entitas Mikro, Kecil, dan Menengah (EMKM) merupakan pedoman untuk entitas yang lebih dikhususkan bagi pemilik usaha yang telah memenuhi kriteria sesuai dengan Undang Undang Nomor 20 Tahun 2008 tentang Usaha Mikro, Kecil, dan Menengah (UMKM). PENDAHULUAN Menurut Suryani & Subardjo (2020) standar EMKM ditetapkan sebagai bentuk penyederhanaan dari SAK ETAP, namun laporan keuangannya tetap memenuhi kebutuhan yang diharapkan pelaku bisnis. Meskipun, bentuknya penyederhanaan, standar ini diharapkan mengurangi anggapan negative pelaku usaha bahwa pencatatan dan pelaporan keuangan dalam akuntansi itu merupakan aktivitas yang sulit. Penyajian laporan keuangan berdasarkan pada SAK EMKM, hanya terdapat tiga laporan keuangan yang harus dipersiapkan oleh entitas, yaitu Laporan Laba Rugi, Laporan Posisi Keuangan dan Catatan atas Laporan Keuangan (CaLK). 35 Jurnal Keunis, Vol. 10, No. 1 Januari 2022 METODE PENELITIAN Jenis penelitian ini adalah deskriptif dengan pendekatan kuantitatif, dimaksudkan untuk menjelaskan tentang pencatatan dan pelaporan keuangan UMKM. Data penelitian dikumpulkan melalui wawancara dan kuisioner. Populasi penelitian berjumlah 19.197 Pelaku UMKM di Kota Palu. Data tersebut, di peroleh dari Dinas Koperasi dan UMKM Kota Palu Tahun 2020. Teknik penetapan sampel menggunakan rumus slovin dengan tingkat kesalahan 15 persen, sehingga sampel penelitian ini sebanyak 44 pelaku UMKM. Akan tetapi, terdapat beberapa kuisioner yang dibagikan tidak diisi lengkap, sehingga tersisa 27 kuisioner yang dapat diolah. Pada penelitian ini pencatatan dan pelaporan keuangan didefinisikan sebagai sebuah sistem pengelolaan keuangan UMKM dimulai dari bukti transaksi sampai dengan penyusunan laporan keuangan. Adapun dimensi dan indikatornya dapat dilihat pada tabel berikut ini. dilihat pada tabel berikut ini. Tabel 1. Dimensi dan Indikator Objek Penelitian Dimensi Indikator - Transaksi - Bukti Transaksi - Jurnal - Penjualan (Pendapatan) Kredit - Penjualan (Pendapatan) Tunai - Pembelian Kredit - Pembelian Tunai - Penerimaan Uang (Kas Masuk) - Pengeluaran Uang (Kas Keluar) - Buku Besar - Kas - Piutang - Persediaan - Pelengkapan - Aset Tetap - Akumulasi Penyusutan Aset Tetap - Utang - Modal - Prive - Penjualan (Pendapatan) - Pembelian - Beban-Beban - Neraca Saldo - Neraca Saldo - Jurnal Penyesuaian - Jurnal Penyesuaian - Kertas Kerja - Kertas Kerja - Laporan Keuangan - Laporan Laba Rugi - Laporan Perubahan Modal - Laporan Neraca - Laporan Arus Kas - Catatan Atas Laporan Keuangan - Jurnal Penutup - Jurnal Penutup - Neraca Saldo Setelah Penutupan - Neraca Saldo Setelah Penutupan Sumber: diolah dari berbagai literature (2021) Tabel 1. Dimensi dan Indikator Objek Penelitian 36 E-ISSN: 2714-7274 P-ISSN: 2302-9315 E-ISSN: 2714-7274 P-ISSN: 2302-9315 Syamsul Secara khusus, pengukuran unit analisis (objek penelitian) menggunakan skor dikotomi. Setiap item pertanyaan pada kuisioner yang dijawab “Ya” oleh responden diberi nilai 1, dan untuk item pertanyaan yang dijawab “Tidak” diberi nilai 0. Selanjutnya, dihitung jumlah responden yang menjawab “Ya” dan jumlah responden yang menjawab “Tidak” dari setiap item pertanyaan. Kemudian, data tersebut diolah dengan menggunakan analisis deskriptif dan diberikan penjelasan tambahan melalui hasil wawancara. HASIL DAN PEMBAHASAN Berdasarkan pada tabel 2, dapat dijelaskan bahwa pelaku UMKM yang terlibat dalam penelitian ini, mayoritas UMKM yang berbentuk perseorangan, yaitu sebanyak 81,48 persen. Kemudian, UMKM tersebut sebagian besar berjenis usaha dagang yaitu sebanyak 70,37 persen. Tabel 2. Deskripsi Bentuk, dan Jenis Usaha Responden Bentuk Usaha Jumlah % Jenis Usaha Jumlah % Perseorangan 22 81,48 Jasa 6 22,22 CV 3 11,11 Dagang 19 70,37 PT 2 7,41 Manufaktur 2 7,41 Total 27 100 Total 27 100 Sumber: Data primer diolah (2021) Kemudian, tabel 3 juga terlihat bahwa responden atau pengelola UMKM yang terlibat dalam survei ini adalah sebagian berjenis kelamin laki-laki sebesar 59,26 persen, dan memiliki tingkat pendidikan mayoritas sarjana yaitu 40,74 persen. j y Tabel 3. Jenis Kelamin dan Tingkat Pendidikan Jenis Kelamin Jumlah % Tingkat Pendidikan Jumlah % Laki-Laki 16 59,26 SD 1 3,70 Perempuan 11 40,74 SMP 5 18,52 SMA 10 37,04 Sarjana 11 40,74 Total 27 100 Total 27 100 Sumber: Data primer diolah (2021) Selanjutnya, pada tabel 4 dapat disampaikan bahwa transaksi yang paling sering dicatat ole Selanjutnya, pada tabel 4 dapat disampaikan bahwa transaksi yang paling sering dicatat oleh pelaku UMKM adalah transaksi penjualan saja, baik secara tunai maupun kredit. Pencatatan keuangan khususnya pada UMKM yang berskala mikro, pencatatan yang dilakukan sebatas mencatat jumlah barang yang dijual, itupun tidak semua transanski dicatat seperti itu, dengan istilah lain kadang-kadang mereka melakukan pencatatan. Kondisi ini, diperjelas dari hasil wawancara pelaku usaha, sebagai berikut. “.…usaha ini pencatatan tidak dilakukan secara detail, hanya seadanya saja. Untuk mengetahui berapa pemasukan dan pengeluaran setiap bulannya.…” (I Melda Candra Pemilik Joyful Bakery) “.…untuk mengetahui pengeluaran dan pemasukan.…” (Serni Dewangga Pemilik Warung Morut Sederhana) ) “.…kurangnya pengetahuan dalam pembuatan pencatatan jadi catat yang seperlunya seperti hasil- hasil penjualan per hari lalu diakumulasikan di akhir bulan ya begitu saja pencatatannya.…” (Mukli Pemilik Somai Mujur). ) “.…kurangnya pengetahuan dalam pembuatan pencatatan jadi catat yang seperlunya seperti hasil- hasil penjualan per hari lalu diakumulasikan di akhir bulan ya begitu saja pencatatannya.…” (Mukli Pemilik Somai Mujur). 37 Jurnal Keunis, Vol. 10, No. 1 Januari 2022 Tabel 4. Pencatatan Transaksi UMKM No. HASIL DAN PEMBAHASAN Transaksi yang Dicatat dalam Jurnal UMKM Jumlah Mencatat Tidak Mencatat UMKM Jumlah % Jumlah % Total % 1 Penjualan (Pendapatan) Kredit 12 44,44 15 55,56 27 100 2 Penjualan (Pendapatan) Tunai 12 44,44 15 55,56 27 100 3 Pembelian Kredit 6 22,22 21 77,78 27 100 4 Pembelian Tunai 6 22,22 21 77,78 27 100 5 Penerimaan Uang (Kas Masuk) 4 14,81 23 85,19 27 100 6 Pengeluaran Uang (Kas Keluar) 6 22,22 21 77,78 27 100 Sumber: Data primer diolah (2021) Tabel 4. Pencatatan Transaksi UMKM Selain itu, hasil survei ini menunjukkan bahwa pelaku UMKM melakukan pencatatan keuangan semata-mata hanya bertujuan mengetahui jumlah pemasukan dan pengeluaran. Hasil ini sesuai dengan temuan Ramdani et al. (2018) yang menginformasikan bahwa komponen yang dilaporkan oleh pengelola UMKM, lebih dominan hanya sebatas pencatatan kas masuk dan kas keluar. y Tabel 5. Penyusunan Buku Besar UMKM Tabel 5. Penyusunan Buku Besar UMKM No. Menyusun Buku Besar UMKM Jumlah Membuat Tidak Membuat UMKM Jumlah % Jumlah % Total % 1 Kas 11 40,74 16 59,26 27 100 2 Piutang 9 33,33 18 66,67 27 100 3 Persediaan 12 44,44 15 55,56 27 100 4 Pelengkapan 7 25,93 20 74,07 27 100 5 Aset Tetap 9 33,33 18 66,67 27 100 6 Ak. Penyusutan Aset Tetap 4 14,81 23 85,19 27 100 7 Utang 11 40,74 16 59,26 27 100 8 Modal 9 33,33 18 66,67 27 100 9 Prive 5 18,52 22 81,48 27 100 10 Penjualan (Pendapatan) 15 55,56 12 44,44 27 100 11 Pembelian 15 55,56 12 44,44 27 100 12 Beban-Beban 12 44,44 15 55,56 27 100 Sumber: Data primer diolah (2021) Pada tabel 5, dapat dijelaskan bahwa sebagian besar pelaku UMKM tidak menyusun buku besar dalam pengelolaan keuangan usaha mereka. Selain itu, terlihat bahwa pelaku UMKM yang menyusun buku besar penjualan dan pembelian masing-masing sebesar 55,56 persen. Setelah itu, pelaku UMKM yang menyusun buku besar beban-beban dan persediaan masing sebesar 44,44 persen, penyusunan buku besar kas dan utang hanya sebesar 40,74 persen dari seluruh pelaku UMKM yang diteliti. Sementara itu, juga terekam kurang dari 40 persen pelaku UMKM yang menyusun buku besar lainnya. Fakta ini, menunjukkan bahwa mayoritas pelaku UMKM hanya menyusun buku besar penjualan, pembelian, beban, persediaan, kas dan utang sebagai laporan dari pengelolaan keuangan usaha mereka. Hasil wawancara dengan pelaku usaha juga membuktikan kondisi tersebut. HASIL DAN PEMBAHASAN (Dodi Jhon Sampouw Pemilik Rumah Makan Seafood Banggai) “….tidak melakukan pencatatan keuangan, karena belum terlalu menguasai tentang pembukuan pencatatan keuangan….” (Asnawati Pemilik Anugrah Indah) Hasil ini sesuai dengan temuan Savitri & Saifudin (2018) mengemukakan bahwa pembukuan bagi pelaku UMKM merupakan sesuatu yang sulit dilaksanakan, disebabkan sempitnya pemahaman mereka tentang ilmu akuntansi, bahkan sebagian dari pelaku UMKM beranggapan bahwa laporan keuangan bukanlah hal yang penting untuk disusun. Akan tetapi, kondisi ini sangat berbeda dengan Pelaku UMKM yang masuk dalam kategori usaha kecil dan menengah mayoritas sudah mengoperasikan sistem kompeterisasi dalam pencatatan dan pelaporan keuangan usaha. Fakta ini tercermin dari kutipan wawancara berikut ini. “….kami tidak bisa melakukan atau membuat catatan keuangan sebagaimana mestinya… (Gladis Taga Pemilik Depot Lahiro) g “….kami tidak bisa melakukan atau membuat catatan keuangan sebagaimana mestinya….” (Gladis Taga Pemilik Depot Lahiro) “….kami tidak bisa melakukan atau membuat catatan keuangan sebagaimana mestinya….” (Gladis Taga Pemilik Depot Lahiro) “….kami tidak pencatatan keuangan karena kurangnya pengetahuan tentang ilmu akuntansi….”. (Dodi Jhon Sampouw Pemilik Rumah Makan Seafood Banggai) “….tidak melakukan pencatatan keuangan, karena belum terlalu menguasai tentang pembukuan pencatatan keuangan….” (Asnawati Pemilik Anugrah Indah) Hasil ini sesuai dengan temuan Savitri & Saifudin (2018) mengemukakan bahwa pembukuan bagi pelaku UMKM merupakan sesuatu yang sulit dilaksanakan, disebabkan sempitnya pemahaman mereka tentang ilmu akuntansi, bahkan sebagian dari pelaku UMKM beranggapan bahwa laporan keuangan bukanlah hal yang penting untuk disusun. Akan tetapi, kondisi ini sangat berbeda dengan Pelaku UMKM yang masuk dalam kategori usaha kecil dan menengah mayoritas sudah mengoperasikan sistem kompeterisasi dalam pencatatan dan pelaporan keuangan usaha. Fakta ini tercermin dari kutipan wawancara berikut ini. “….kami tidak pencatatan keuangan karena kurangnya pengetahuan tentang ilmu akuntansi… .kami tidak pencatatan keuangan karena kurangnya pengetahuan tentang ilmu akuntansi…. . di Jhon Sampouw Pemilik Rumah Makan Seafood Banggai) p g g y p g g (Dodi Jhon Sampouw Pemilik Rumah Makan Seafood Banggai) ( gg ) “….tidak melakukan pencatatan keuangan, karena belum terlalu menguasai tentang pembukua pencatatan keuangan….” (Asnawati Pemilik Anugrah Indah) Hasil ini sesuai dengan temuan Savitri & Saifudin (2018) mengemukakan bahwa pembukuan bagi pelaku UMKM merupakan sesuatu yang sulit dilaksanakan, disebabkan sempitnya pemahaman mereka tentang ilmu akuntansi, bahkan sebagian dari pelaku UMKM beranggapan bahwa laporan keuangan bukanlah hal yang penting untuk disusun. Akan tetapi, kondisi ini sangat berbeda dengan Pelaku UMKM yang masuk dalam kategori usaha kecil dan menengah mayoritas sudah mengoperasikan sistem kompeterisasi dalam pencatatan dan pelaporan keuangan usaha. HASIL DAN PEMBAHASAN g p j g “….secara rinci sampai pembuatan neraca dan buku besar saya tidak paham, apalagi bagi saya yang tidak mempunyai dasar dibidang itu….” (Patina Pemilik Bakery Kue Tradisonal) g p j g “….secara rinci sampai pembuatan neraca dan buku besar saya tidak paham, apalagi bagi saya yang tidak mempunyai dasar dibidang itu….” (Patina Pemilik Bakery Kue Tradisonal) 38 E-ISSN: 2714-7274 P-ISSN: 2302-9315 Syamsul “….pencatatan atau pembukuan yang perlu dilakukan hanyalah berupa pembelian bahan baku yang digunakan setiap harinya, seperti sabun bubuk dan sampo motor. Dan juga pencatatan keuangan yang masuk setiap harinya….” (Agung Hirawan Pemilik Cuci Motor Swaha). “….pencatatan atau pembukuan yang perlu dilakukan hanyalah berupa pembelian bahan baku yang digunakan setiap harinya, seperti sabun bubuk dan sampo motor. Dan juga pencatatan keuangan yang masuk setiap harinya….” (Agung Hirawan Pemilik Cuci Motor Swaha). y g p y ( g g ) Pada tabel 6, terlihat bahwa hanya beberapa pelaku UMKM yang menyusun laporan keuangan berupa laporan laba rugi dan perubahan modal, yaitu sekitar 25 persen dari seluruh UMKM yang ditelusuri. Sementara itu, pelaku UMKM yang menyusun laporan neraca, arus kas, dan catatan atas laporan keuangan kurang dari 25 persen. Angka ini, menunjukkan bahwa mayoritas pelaku UMKM belum menyusun laporan keuangan atas usaha yang mereka jalankan. Kemudian, berdasarkan penelusuran ditemukan bahwa pelaku UMKM yang menyusun laporan keuangan adalah UMKM yang tergolong dalam bentuk usaha CV dan PT dengan kategori usaha kecil dan menengah. g g g g Tabel 6. Penyusunan Laporan Keuangan UMKM Tabel 6. Penyusunan Laporan Keuangan UMKM Tabel 6. Penyusunan Laporan Keuangan UMKM No. Menyusun Laporan Keuangan UMKM Jumlah Menyusun Tidak Menyusun UMKM Jumlah % Jumlah % Total % 1 Laporan Laba Rugi 7 25,93 20 74,07 27 100 2 Laporan Perubahan Modal 7 25,93 20 74,07 27 100 3 Laporan Neraca 6 22,22 21 77,78 27 100 4 Laporan Arus Kas 6 22,22 21 77,78 27 100 5 Catatan Atas Laporan Keuangan 6 22,22 21 77,78 27 100 Sumber: Data primer diolah (2021) p ( ) Sederhananya, mayoritas pelaku UMKM belum menyusun laporan keuangan sebagaimana mestinya, utamanya UMKM yang masih masuk dalam kategori usaha kecil. Hal ini sesuai dengan ungkapan pelaku usaha berikut ini. g p p “….kami tidak bisa melakukan atau membuat catatan keuangan sebagaimana mestinya….” (Gladis Taga Pemilik Depot Lahiro) “….kami tidak pencatatan keuangan karena kurangnya pengetahuan tentang ilmu akuntansi….”. HASIL DAN PEMBAHASAN Fakta ini tercermin dari kutipan wawancara berikut ini. p “…. Sistem pencatatan keuangan perusahaan ini sudah berbasis komputer, makanya lebih mudah….” (Johny Limbunan Pemilik PT Patrako Abadi) p “…. Sistem pencatatan keuangan perusahaan ini sudah berbasis komputer, makanya lebih mudah….” (Johny Limbunan Pemilik PT Patrako Abadi) Sederhananya, pada tabel 7 terlihat bahwa mayoritas pelaku UMKM yang diteliti tidak menerapkan sistem akuntansi dalam pencatatan (pengelolaan) keuangan usaha mereka. Dapat dilihat pada tabel tersebut, bahwa lebih dari 50 persen pelaku usaha tidak menjalankan prosedur akuntansi dalam pengelolaan keuangan mereka, mulai dari penyediaan bukti transaksi, pembuatan jurnal, buku besar, neraca saldo, penyusunan jurnal penyesuaian, kertas kerja, jurnal penutup, dan neraca saldo setelah penutupan. Kondisi ini pun, tercermin dari hasil kutipan wawancara pelaku UMKM, sebagai berikut. 39 Jurnal Keunis, Vol. 10, No. 1 Januari 2022 “…. Memang pencatatan keuangan itu penting, tetapi karena usaha yang berjalan usaha kecil, makanya saya tidak menggunakan pencatatan keuangan….” (Moh. Fauzan Pemilik Fauzan Babershop). “.... saya enggan membuat catatan keuangan, dengan cara saya sendiri saya dapat mengetahui dengan mudah jumlah uang yang keluar dan masuk….” (Purnomo Pemilik Kartika Motor). Tabel 7 Penerapan Sistem Akuntansi UMKM “…. Memang pencatatan keuangan itu penting, tetapi karena usaha yang berjalan usaha kecil, makanya saya tidak menggunakan pencatatan keuangan….” (Moh. Fauzan Pemilik Fauzan Babershop). “.... saya enggan membuat catatan keuangan, dengan cara saya sendiri saya dapat mengetahui Babershop). “.... saya enggan membuat catatan keuangan, dengan cara saya sendiri saya dapat mengetahui dengan mudah jumlah uang yang keluar dan masuk….” (Purnomo Pemilik Kartika Motor). Tabel 7 Penerapan Sistem Ak ntansi UMKM p) “.... saya enggan membuat catatan keuangan, dengan cara saya sendiri saya dapat mengetahui dengan mudah jumlah uang yang keluar dan masuk….” (Purnomo Pemilik Kartika Motor). abe e e apa S ste u ta s U No. HASIL DAN PEMBAHASAN Penerapan Sistem Akuntansi UMKM Jumlah Membuat Tidak Membuat UMKM Jumlah % Jumlah % Total % 1 Bukti Transaksi 12 44,44 15 55,56 27 100 2 Jurnal 12 44,44 15 55,56 27 100 3 Buku Besar 6 22,22 21 77,78 27 100 4 Neraca Saldo 6 22,22 21 77,78 27 100 5 Jurnal Penyesuaian 4 14,81 23 85,19 27 100 6 Kertas Kerja 6 22,22 21 77,78 27 100 7 Laporan Keuangan 6 22,22 21 77,78 27 100 8 Jurnal Penutup 6 22,22 21 77,78 27 100 9 Neraca Saldo Setelah Penutupan 6 22,22 21 77,78 27 100 Sumber: Data primer diolah (2021) Merujuk pada hasil survei ini, dapat dijelaskan bahwa yang paling banyak disusun oleh pelaku UMKM dari setiap tahapan pencatatan, yaitu hanya pada penyediaan bukti transaksi dan pencatatan ke dalam jurnal. Mereka hanya membuat catatan sederhana, dimana catatan tersebut memperlihatkan jumlah pembelian (modal barang) dan penjualan (omzet penjualan). Sehingga, jika mereka ingin mengetahui apakah terjadi laba atau rugi, yaitu mereka tinggal membandingkan antara jumlah penjualan dengan jumlah pembelian. Ketika jumlah penjualan lebih tinggi daripada jumlah pembelian, selisihnya itulah yang disebut untung (laba). Sebaliknya, jika jumlah penjulan lebih kecil daripada jumlah pembelian, kelebihannya itu mereka sebut rugi. Sampai pada tahapan itu saja, itupun tidak seperti yang terdapat dalam literatur-literatur akuntansi maupun standar akuntansi yang telah ditetapkan. Oleh karena itu, sebagian besar para pelaku UMKM belum memiliki sistem pencatatan keuangan sebagaimana mestinya, artinya tidak sesuai dengan sistem akuntansi. Pelaku UMKM yang tidak membuat pencatatan dan pelaporan keuangan usaha, karena memang mereka sama sekali belum mengetahuinya. Meskipun, terdapat UMKM khususnya yang termasuk dalam kelompok mikro melakukan pencatatan dan pelaporan keuangan, namun belum sesuai dengan sistem akuntansi. Artinya, pencatatan keuangan yang diterapkan belum mengikuti tahapan-tahapan di dalam siklus akuntansi, dan pencatatan tersebut hanya dapat dipahami oleh pelaku UMKM itu sendiri. Hasil ini, sejalan dengan Salmiah et al. (2018) yang menemukan bahwa penerapan akuntansi bagi UMKM masih sangat sederhana atau tidak mengikuti tahapan-tahapan dalam siklus akuntansi. Begitupula, penelitian Ramdani et al. (2018) yang mengemukakan bahwa pencatatan dan penyusunan laporan keuangan yang dilakukan pengelola usaha hanya sebatas laporan bisnis yang dibuat sesuai dengan pemahaman dan kebutuhan masing-masing pengelola UMKM. Mestinya, hasil dari pencatatan dan pelaporan keuangan tersebut juga dapat memberikan informasi kepada pihak eksternal tentang kondisi keuangan perusahaan, sehingga memenuhi karakteristik informasi yaitu mudah dipahami, relevan dan akurat (Alfitri et al., 2014). HASIL DAN PEMBAHASAN ( ) Selanjutnya, pelaku UMKM juga mengatakan bahwa selama ini memang mereka belum pernah terlibat dalam pelatihan pengelolaan keuangan instansi pemerintah maupun lembaga-lembaga lainnya. 40 E-ISSN: 2714-7274 P-ISSN: 2302-9315 Syamsul “….saya tidak pernah mendapat pelajaran tentang keuangan….”. (Agung Hirawan Pemilik Cuci Motor Swaha) Kemudian, pelaku UMKM juga memiliki harapan untuk diberikan pelatihan khusus tentang pengelolaan keuangan yang sederhana dan mudah dipahami. Seperti yang disampaikan oleh pelaku UMKM pada kutipan wawancara berikut. “…. agar kedepannya bisa mengikuti pelatihan pengelolaan keuangan untuk usaha….” (Gladis Taga Pemilik Depot Lahiro) “…. agar kedepannya bisa mengikuti pelatihan pengelolaan keuangan untuk usaha….” (Gladis Taga Pemilik Depot Lahiro) “….kedepannya ada yang mengajari masalah keuangan yang benar….” (Agung Hirawan Pemilik Cuci Motor Swaha) “….kedepannya ada yang mengajari masalah keuangan yang benar….” (Agung Hirawan Pemilik Cuci Motor Swaha) “….dilakukan bimbingan berupa pelatihan pengelolaan usaha….” (Gisel Pemilik Kios Gisel) “….dilakukan bimbingan berupa pelatihan pengelolaan usaha….” (Gisel Pemilik Kios Gise “….harapannya pemerintah menyediakan pelatihan pencatatan keuangan khususnya pada “….harapannya pemerintah menyediakan pelatihan pencatatan keuangan khususnya pada yan mempunyai usaha….” (Asnawati Pemilik Anugrah Indah). mempunyai usaha….” (Asnawati Pemilik Anugrah Indah). Temuan penelitian ini menunjukkan bahwa pelaku UMKM yang masih berskala mikro belum melakukan pencatatan dan pelaporan keuangan sebagaimana mestinya, hal ini terjadi karena kurangnya pemahaman mereka dibidang tersebut. Hasil ini menguatkan argumentansi dari Savitri & Saifudin (2018) yang menjelaskan bahwa pelaku UMKM tidak mengetahui bagaimana pencatatan dan penyusunan laporan keuangan yang baik dan benar. Dengan demikian, diperlukan pelatihan dan pendampingan kepada pelaku UMKM, utamanya dibidang pencatatan dan pelaporan keuangan usaha. Sementara itu, pelaku UMKM yang tergolong dalam kelompok usaha kecil dan menengah sebagian besar telah menerapkan pencatatan dan pelaporan keuangan sesuai dengan tahapan dalam siklus akuntansi. Pencatatan dan pelaporan keuangan mereka pun, telah berbasis komputer (software). Selanjutnya, penerapan pencatatan dan pelaporan keuangan, pelaku UMKM dapat mengetahui kondisi usahanya, apakah terjadi perkembangan ataukah penurunan kinerja perusahaan dari tahun ke tahun. Bahkan, dapat memberikan informasi posisi keuangan perusahaan, besarnya pemasukan dan pengeluaran perusahaan, dan informasi perubahan modal dari periode ke periode, serta jumlah pajak yang harus dibayar. Selain itu, dengan adanya laporan keuangan yang handal dan dapat dipercaya dapat digunakan oleh pelaku UMKM dalam berbagai tujuan. Misalnya, memenuhi persyaratan untuk memperolah tambahan modal pengembangan usaha dari pihak kreditur. Dengan kata lain, apabila pelaku UMKM konsisten dalam melaksanakan sistem pencatatan dan pelaporan keuangan dengan baik dan memadai. HASIL DAN PEMBAHASAN Maka, berbagai macam manfaat yang diperoleh dari laporan keuangan tersebut untuk pengembangan usaha di masa mendatang, utamanya UMKM di Kota Palu. PENUTUP Penelitian ini menginformasikan bahwa mayoritas pelaku UMKM yang berskala mikro di Kota Palu tidak melakukan pencatatan keuangan dari semua jenis transaksi yang terjadi, tidak menyediakan buku besar, dan belum menyusun laporan keuangan. Artinya, pelaku UMKM di Kota Palu belum menerapkan sistem akuntansi dalam pencatatan (pengelolaan) keuangan usahanya. Namun demikian, untuk UMKM yang berskala kecil dan menengah sebagain besar telah menerapkan pencatatan dan pelaporan keuangan, baik secara manual dan terkomputerisasi. Penelitian ini terbatas dilakukan pada beberapa UMKM saja yang di Kota Palu, dan didominasi UMKM berskala mikro. Oleh karena itu, saran untuk penelitian selanjutnya dapat mengambil sampel penelitian yang lebih berimbang dari setiap kelompok UMKM, sehingga lebih kelihatan perbedaan antara pencatatan dan pelaporan keuangan UMKM berskala mikro, kecil, dan menengah. Penelitian lain, juga dapat mengidentifikasi faktor – faktor pendukung dan penghambat penerapan sistem pencatatan dan pelaporan keuangan pelaku UMKM. 41 Januari 2022 Jurnal Keunis, Vol. 10, No. 1 DAFTAR PUSTAKA Alfitri, A., Ngadiman, & Sohidin. (2014). Penerapan Standar Akuntansi Keuangan Entitas Tanpa Akuntabilitas Publik (SAK-ETAP) Pada Usaha Mikro Kecil Menengah (UMKM) Perajin Mebel Desa Gondangsari Kecamatan Juwiring Kabuaten Klaten. Jupe UNS, 2(2), 135–147. g g p ( ) Farida, U. I. (2016). Akuntansi Untuk Umkm (F. Nurhayani (ed.); 1st ed.). Penerbit CV Kekata Group. http://eprints.umpo.ac.id/2828/5/UMKM LAYFIX.pdf Hetika, H., & Mahmudah, N. (2017). Penerapan Akuntansi Dan Kesesuaiannya Dengan Sak Etap Pada Umkm Kota Tegal. JURNAL AKUNTANSI, EKONOMI Dan MANAJEMEN BISNIS, 5(2), 259. https://doi.org/10.30871/jaemb.v5i2.531 Jusup, A. H. (2016). Dasar-dasar Akuntansi Jilid 1 (IV). Bagian Penerbitan Sekolah Tinggi Ilmu Ekonomi YKPN. Kurniwati, E. P., Nugroho, P. I., & Arifin, C. (2012). Penerapan Pencatatan dan Laporan Akuntansi Pada Usaha Mikro Kecil Dan Menengah (UMKM). Informatics and Business Insitute Darmajaya, 2(2), 1– 10. https://doi.org/10.31294/jabdimas.v2i2.5818 Martani, D., Veronica, S., Wardhani, R., Farahmita, A., & Tanujaya, E. (2012). Akuntansi Keuangan Menengah Berbasis PSAK. Salemba Empat. Ramdani, M. R., Kamidin, M., & Ajmal, A. (2018). Implementasi SAK-ETAP Pada UMKM Warkop di Kota Makassar. Jurnal RAK (Riset Akuntansi Keuangan), 3(2), 0–19. Republik Indonesia. (2008). Undang-Undang Nomor 20 Tahun 2008 tentang Usaha Mikro, Kecil, dan Menengah. Sekretariat Negara. Salmiah, N., Indarti Siregar, & Fitri, I. (2018). Analisis Penerapan Akuntansi dan Kesesuaiannya Dengan Standar Akuntansi Keuangan Entitas Tanpa Akuntabilitas Publik (Pada UMKM di Kecamatan Sukajadi Binaan DisKop & UMKM Kota Pekanbaru). Jurnal Akuntansi, 3(2), 212–226. Savitri, R. V., & Saifudin, . . (2018). Pencatatan Akuntansi Pada Usaha Mikro Kecil Dan Menengah (Studi Pada Umkm Mr. Pelangi Semarang). JMBI UNSRAT (Jurnal Ilmiah Manajemen Bisnis Dan Inovasi Universitas Sam Ratulangi)., 5(2), 117–125. https://doi.org/10.35794/jmbi.v5i2.20808 Siagian, A. O., & Indra, N. (2019). Pengetahuan Akuntansi Pelaku Usaha Mikro Kecil dan Menengah (UMKM) Terhadap Laporan Keuangan. Syntax Literate: Jurnal Ilmiah Indonesia, 1(1), 41–57. S (2004) Ak i S P B k 1 S l b E Soemarso. (2004). Akuntansi Suatu Pengantar Buku 1. Salemba Empat. Suryani, N. H., & Subardjo, A. (2020). Penerapan Akuntansi Pelaku UMKM dan Kesesuaiannya Dengan SAK EMKM. Jurnal Ilmu Dan Riset Akuntansi, 9. Warsono, S., & Darmawan, A. (2010). Akuntansi UMKM Ternyata Mudah Dipahami dan Dipratikkan. Asgard Chapter. 42
https://openalex.org/W2947222441	https://europepmc.org/articles/pmc6538676?pdf=render	English	null	Publisher Correction: Nasal mucus glutathione transferase activity and impact on olfactory perception and neonatal behavior	Scientific reports	2,019	cc-by	394	www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Publisher Correction: Nasal mucus glutathione transferase activity and impact on olfactory perception and neonatal behavior xx OPEN Published: xx xx xxxx Aline Robert-Hazotte1, Philippe Faure1, Fabrice Neiers1, Catherine Potin1, Yves Artur1, Gérard Coureaud2 & Jean-Marie Heydel1 Correction to: Scientific Reports https://doi.org/10.1038/s41598-019-39495-6, published online 28 February 2019 The original version of this Article was published with an incorrect version of the Supplementary Information file due to a technical error. This has now been corrected in the HTML; the PDF version of the paper was correct from the time of publication. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. 1 (2019) 9 8111 \| htt //d i /10 1038/ 41598 019 44135 0 © The Author(s) 2019 1Centre des Sciences du Goût et de l′Alimentation, UMR 6265 CNRS/1324 INRA/Université de Bourgogne Franche- Comté, 9 boulevard Jeanne d’Arc, F-21000, Dijon, France. 2Centre de Recherche en Neurosciences de Lyon, INSERM U1028/CNRS UMR 5292/Université Lyon 1, CH Le Vinatier-Bâtiment 462 Neurocampus, 95 boulevard Pinel, F-69675, Bron, France. Aline Robert-Hazotte and Philippe Faure contributed equally. Correspondence and requests for materials should be addressed to G.C. (email: gerard.coureaud@cnrs.fr) or J.-M.H. (email: jean-marie.heydel@u- bourgogne.fr) © The Author(s) 2019 1Centre des Sciences du Goût et de l′Alimentation, UMR 6265 CNRS/1324 INRA/Université de Bourgogne Franche- Comté, 9 boulevard Jeanne d’Arc, F-21000, Dijon, France. 2Centre de Recherche en Neurosciences de Lyon, INSERM U1028/CNRS UMR 5292/Université Lyon 1, CH Le Vinatier-Bâtiment 462 Neurocampus, 95 boulevard Pinel, F-69675, Bron, France. Aline Robert-Hazotte and Philippe Faure contributed equally. Correspondence and requests for materials should be addressed to G.C. (email: gerard.coureaud@cnrs.fr) or J.-M.H. (email: jean-marie.heydel@u- bourgogne.fr) Scientific Reports \| (2019) 9:8111 \| https://doi.org/10.1038/s41598-019-44135-0
https://openalex.org/W2501334413	https://inria.hal.science/hal-01555338v1/file/LNCS6117DL.pdf	English	null	Formal Techniques for Distributed Systems	Lecture notes in computer science	2,011	cc-by	3,011	Lecture Notes in Computer Science Commenced Publication in 1973 Founding and Former Series Editors: Gerhard Goos, Juris Hartmanis, and Jan van Leeuwen Lecture Notes in Computer Science Commenced Publication in 1973 Founding and Former Series Editors: Gerhard Goos, Juris Hartmanis, and Jan van Leeuwen 6117 Editorial Board Friedemann Mattern ETH Zurich, Switzerland John C. Mitchell Stanford University, CA, USA John Hatcliff Elena Zucca (Eds.) Formal Techniques for Distributed Systems Joint 12th IFIP WG 6.1 International Conference, FMOODS 2010 and 30th IFIP WG 6.1 International Conference, FORTE 2010 Amsterdam, The Netherlands, June 7-9, 2010 Proceedings John Hatcliff Elena Zucca (Eds.) Formal Techniques for Distributed Systems Joint 12th IFIP WG 6.1 International Conference, FMOODS 2010 and 30th IFIP WG 6.1 International Conference, FORTE 2010 Amsterdam, The Netherlands, June 7-9, 2010 Proceedings 1 3 Moni Naor Weizmann Institute of Science, Rehovot, Israel Moni Naor Weizmann Institute of Science, Rehovot, Israel Oscar Nierstrasz University of Bern, Switzerland C. Pandu Rangan Indian Institute of Technology, Madras, India Bernhard Steffen TU Dortmund University, Germany Madhu Sudan Microsoft Research, Cambridge, MA, USA Demetri Terzopoulos University of California, Los Angeles, CA, USA Doug Tygar University of California, Berkeley, CA, USA Gerhard Weikum Max-Planck Institute of Computer Science, Saarbruecken, Germany CR Subject Classiﬁcation (1998): D.2, D.2.4, I.2.2, D.3, F.3, F.4, I.2.3 LNCS Sublibrary: SL 2 – Programming and Software Engineering 1 3 Volume Editors Volume Editors John Hatcliff Kansas State University, Computing and Information Sciences 234 Nichols Hall, Manhattan, KS 66502, USA E-mail: hatcliff@ksu.edu John Hatcliff Kansas State University, Computing and Information Sciences 234 Nichols Hall, Manhattan, KS 66502, USA E-mail: hatcliff@ksu.edu Elena Zucca DISI, University of Genova Via Dodecaneso 35, 16146 Genova, Italy E-mail: zucca@disi.unige.it Library of Congress Control Number: Applied for Typesetting: Camera-ready by author, data conversion by Scientiﬁc Publishing Services, Chennai, India Printed on acid-free paper 06/3180 Foreword In 2010 the international federated conferences on Distributed Computing Tech- niques (DisCoTec) took place in Amsterdam, during June 7-9. It was hosted and organized by the Centrum voor Wiskunde & Informatica. In 2010 the international federated conferences on Distributed Computing Tech- niques (DisCoTec) took place in Amsterdam, during June 7-9. It was hosted and organized by the Centrum voor Wiskunde & Informatica. g y DisCoTec conferences jointly cover the complete spectrum of distributed computing subjects ranging from theoretical foundations to formal speciﬁca- tion techniques to practical considerations. The 12th International Conference on Coordination Models and Languages (Coordination) focused on the design and implementation of models that allow compositional construction of large- scale concurrent and distributed systems, including both practical and founda- tional models, run-time systems, and related veriﬁcation and analysis techniques. The 10th IFIP International Conference on Distributed Applications and Inter- operable Systems in particular elicited contributions on architectures, models, technologies and platforms for large-scale and complex distributed applications and services that are related to the latest trends in bridging the physical/virtual worlds based on ﬂexible and versatile service architectures and platforms. The 12th Formal Methods for Open Object-Based Distributed Systems and 30th Formal Techniques for Networked and Distributed Systems together emphasized distributed computing models and formal speciﬁcation, testing and veriﬁcation methods. Each of the three days of the federated event began with a plenary speaker nominated by one of the conferences. The ﬁrst day Joe Armstrong (Ericsson Telecom AB) gave a keynote speech on Erlang-style concurrency, the second day Gerard Holzmann (Jet Propulsion Laboratory, USA) discussed the question “Formal Software Veriﬁcation: How Close Are We?”. The third and last day Joost Roelands (Director of Development Netlog) presented the problem area of distributed social data. In addition, there was a joint technical session consisting of one paper from each of the conferences and an industrial session with presen- tations by A. Stam (Almende B.V., Information Communication Technologies) and M. Verhoef (CHESS, Computer Hardware & System Software) followed by a panel discussion. LNCS Sublibrary: SL 2 – Programming and Software Engineering ISSN 0302-9743 ISBN-10 3-642-13463-7 Springer Berlin Heidelberg New York ISBN-13 978-3-642-13463-0 Springer Berlin Heidelberg New York p g g ISBN-13 978-3-642-13463-0 Springer Berlin Heidelberg New York This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, speciﬁcally the rights of translation, reprinting, re-use of illustrations, recitation, broadcasting, reproduction on microﬁlms or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer. Violations are liable to prosecution under the German Copyright Law. springer.com © IFIP International Federation for Information Processing 2010 Printed in Germany Typesetting: Camera-ready by author, data conversion by Scientiﬁc Publishing Services, Chennai, India Printed on acid-free paper 06/3180 Typesetting: Camera-ready by author, data conversion by Scientiﬁc Publishing Services, Chennai, India Printed on acid-free paper 06/3180 Foreword There were four satellite events: the Third DisCoTec Workshop on Context- Aware Adaptation Mechanisms for Pervasive and Ubiquitous Services (CAM- PUS), the First International Workshop on Interactions Between Computer Sci- ence and Biology (CS2BIO) with keynote lectures by Luca Cardelli (Microsoft Research - Cambridge, UK) and J´erˆome Feret (INRIA and ´Ecole Normale Su- prieure - Paris, France), the First Workshop on Decentralized Coordination of Distributed Processes (DCDP) with a keynote lecture by Tyler Close (Google), and the Third Interaction and Concurrency Experience Workshop with keynote lectures by T. Henzinger (IST, Austria) and J.-P. Katoen (RWTH Aachen Uni- versity, Germany). VI Preface This volume contains the proceedings of the IFIP International Conference on Formal Techniques for Distributed Systems. The conference was organized as the joint activity of two conferences: FMOODS (Formal Methods for Open Object- Based Distributed Systems) and FORTE (Formal Techniques for Networked and Distributed Systems). FMOODS/FORTE was part of the federated conference event DisCoTec (Distributed Computing Techniques) 2010, which also included the 12th International Conference on Coordination Models and Languages (CO- ORDINATION) and the 10th IFIP International Conference on Distributed Ap- plications and Interoperable Systems (DAIS). p p y ( ) The goal of FMOODS/FORTE is to provide a forum for fundamental research on theory and applications of distributed systems. The conference emphasizes the use of a variety of techniques for development of concurrent and distributed systems including model-based design, component and object technology, type systems, formal speciﬁcation and veriﬁcation, and formal approaches to testing. The conference encourages contributions that combine theory and practice in application areas of telecommunication services, Internet, embedded and real- time systems, networking and communication security and reliability, sensor networks, service-oriented architecture, and Web services. The FMOODS/FORTE 2010 program consisted of 13 regular and 6 short pa- pers. These papers were selected by a 30-member Program Committee (PC) from among 38 submissions. Each paper was assigned to at least four PC members for a detailed review. Additional expert reviews were solicited if the reviews of a paper had diversiﬁed assessments or the reviewers indicated low conﬁdence. The ﬁnal decision of acceptance was based on a 10-day online discussion of the PC. The selected papers constituted a strong program of stimulating and timely top- ics in the areas of formal veriﬁcation, algorithms and implementations, modeling and testing, process algebra and calculus, and analysis of distributed systems. FORTE traces its heritage back to the Protocol Speciﬁcation, Testing and Veriﬁcation (PSTV) conference ﬁrst held in 1981 in Teddington, UK. Since that ﬁrst meeting, PSTV has evolved into FORTE and now into FMOODS/FORTE and this year’s conference in Amsterdam was the 30th meeting in the conference series. To highlight this milestone, this year’s DisCoTec plans to include a spe- cial celebration reﬂecting on the progress of the community since 1981 and on challenges for the future. In the ﬁrst PSTV meeting in 1981, Gerard Holzmann published a paper containing his early views of what would become the SPIN model checker. Foreword I hope this rich program oﬀered every participant interesting and stimulating events. It was only possible thanks to the dedicated work of the Publicity Chair Gianluigi Zavattaro (University of Bologna, Italy), the Workshop Chair Marcello Bonsangue (University of Leiden, The Netherlands) and the members of the Organizing Committee, Susanne van Dam, Immo Grabe, Stephanie Kemper and Alexandra Silva. To conclude I want to thank the sponsorship of the International Federation for Information processing (IFIP), the Centrum voor Wiskunde & Informatica and the Netherlands Organization for Scientiﬁc Research (NWO). Frank S. de Boer June 2010 June 2010 Preface We were pleased to note this special work in the history of FORTE by featuring Dr. Holzmann as the keynote speaker of FMOODS/FORTE 2010 as part of this special celebration of PSTV - FORTE - FMOODS history. VIII We are deeply indebted to the PC members and external reviewers for their hard and conscientious work in preparing 154 reviews. We thank Frank de Boer, the DisCoTec General Chair, for his support, and the Steering Committees of FMOODS and FORTE for their guidance. Our gratitude goes to the authors for their support of the conference by submitting their high-quality research works. We thank the providers of the conference tool EasyChair that was a great help in organizing the submission and reviewing process. John Hatcliﬀ Elena Zucca June 2010 Organization Program Chairs ETH, Switzerland Technical University of Berlin, Germany Complutense University of Madrid, Spain Complutense University of Madrid, Spain University of Oslo, Norway Program Committee Program Committee University of Ottawa, Canada Federal University of Pernambuco, Brazil University of Bologna, Italy INT Evry, France University of Sheﬃeld, UK University of Turin, Italy Queens University, Kingston, Ontario, Canada University of G¨ottingen, Germany INRIA, France ENS Cachan - Bretagne, France University of Oslo, Norway Concordia University, Canada The Ohio State University, USA University of South Florida, USA University of Quebec - Outaouais, Canada University of Lisbon, Portugal ETH, Switzerland Technical University of Berlin, Germany Complutense University of Madrid, Spain University of Oslo, Norway CRIM Montreal, Canada Katholieke Universiteit Leuven, Belgium University of Kaiserslautern, Germany University of Oslo, Norway University of Stirling, UK LMU Munich, Germany Nara Institute of Science and Technology, Japan Imperial College London, UK Gregor v. Bochmann Paulo Borba Mario Bravetti Ana Cavalli John Derrick Mariangiola Dezani Juergen Dingel Dieter Hogrefe Valerie Issarny Claude Jard Einar Broch Johnsen Ferhat Khendek David Lee Jay Ligatti Luigi Logrippo Antonia Lopes Peter Mueller Uwe Nestmann Manuel N´u˜nez Olaf Owe Alexandre Petrenko Frank Piessens Arnd Poetzsch-Heﬀter Martin Steﬀen Ken Turner Martin Wirsing Keiichi Yasumoto Nobuko Yoshida Gregor v. Bochmann Paulo Borba Mario Bravetti Ana Cavalli John Derrick Mariangiola Dezani Juergen Dingel Dieter Hogrefe Valerie Issarny Claude Jard Einar Broch Johnsen Ferhat Khendek David Lee Jay Ligatti Luigi Logrippo Antonia Lopes Peter Mueller Uwe Nestmann Manuel N´u˜nez Olaf Owe Alexandre Petrenko Frank Piessens Arnd Poetzsch-Heﬀter Martin Steﬀen Ken Turner Martin Wirsing Keiichi Yasumoto Nobuko Yoshida The Ohio State University, USA University of South Florida, USA University of Quebec - Outaouais, Canada University of Lisbon, Portugal University of Lisbon, Portugal Program Chairs Kansas State University, USA University of Genoa, Italy Kansas State University, USA University of Genoa, Italy John Hatcliﬀ Elena Zucca CRIM Montreal, Canada CRIM Montreal, Canada Katholieke Universiteit Leuven, Belgium Katholieke Universiteit Leuven, Belgium University of Kaiserslautern, Germany University of Kaiserslautern, Germany University of Oslo, Norway University of Oslo, Norway University of Stirling, UK University of Stirling, UK LMU Munich, Germany Nara Institute of Science and Technology, Imperial College London, UK Organization X Table of Contents Invited Talk Formal Software Veriﬁcation: How Close Are We? (Abstract) . . . . . . . . . . 1 Gerard J. Holzmann Formal UML Modeling Exploiting the Hierarchical Structure of Rule-Based Speciﬁcations for Decision Planning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Artur Boronat, Roberto Bruni, Alberto Lluch Lafuente, Ugo Montanari, and Generoso Paolillo Reactive Semantics for Distributed UML Activities . . . . . . . . . . . . . . . . . . . 17 Frank Alexander Kraemer and Peter Herrmann Components and Architecture Statistical Abstraction and Model-Checking of Large Heterogeneous Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 Ananda Basu, Saddek Bensalem, Marius Bozga, Benoˆıt Caillaud, Benoˆıt Delahaye, and Axel Legay Formal Semantics and Analysis of Behavioral AADL Models in Real-Time Maude . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 Peter Csaba ¨Olveczky, Artur Boronat, and Jos´e Meseguer Testing Probabilistic Distributed Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 Robert M. Hierons and Manuel N´u˜nez Speciﬁcation and Testing of E-Commerce Agents Described by Using UIOLTSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78 Juan Jos´e Pardo, Manuel N´u˜nez, and M. Carmen Ruiz Testing Attribute-Based Transactions in SOC . . . Additional Referees Ernesto Posse Cristian Prisacariu Ismael Rodriguez Fernando Rosa-Velardo Jan Sch¨afer Rudolf Schlatte Sven Schneider Jens Sch¨onborn Adenilso Simao Carron Shankland Jan Smans Fausto Spoto Volker Stolz Dries Vanoverberghe Mahesh Viswanathan Frederic Vogels Neil Walkinshaw Yannick Welsch Hirozumi Yamaguchi Gianluigi Zavattaro Toqeer Israr Juliano Iyoda Bart Jacobs Willy Jimenez Ioannis Kassios Christian Kissig Ilham Kurnia Felipe Lalanne David Lee Ugo de’ Liguoro Luis Llana Savi Maharaj Thi Mai Thuong Tran Francisco Martins Tiago Massoni Alexandre Mota Akio Nakata Luca Padovani Miguel Palomino Andrew Phillips Suzana Andova Lucian Bentea Lorenzo Bettini Sergiy Boroday Florent Bouchy Roberto Bruni Marco Carbone Corina Cirstea Ferruccio Damiani Dominique Devriese Mustafa Emre Dincturk Jose Pablo Escobedo Pietro Ferrara David de Frutos-Escrig Simon Gay Nils Gesbert Rohit Gheyi Qiang Guo Yating Hsu Iksoon Hwang Timed Process Algebra Timed Process Algebra Forgetting the Time in Timed Process Algebra: Timeless Behaviour in a Timestamped World . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 Anton Wijs Theory and Implementation of a Real-Time Extension to the π-Calculus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 Ernesto Posse and Juergen Dingel Timed and Hybrid Automata Fuzzy-Timed Automata . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140 F. Javier Crespo, Alberto de la Encina, and Luis Llana Model Checking of Hybrid Systems Using Shallow Synchronization . . . . . 155 Lei Bu, Alessandro Cimatti, Xuandong Li, Sergio Mover, and Stefano Tonetta Program Logics and Analysis Heap-Dependent Expressions in Separation Logic . . . . . . . . . . . . . . . . . . . . 170 Jan Smans, Bart Jacobs, and Frank Piessens Static Type Analysis of Pattern Matching by Abstract Interpretation . . . 186 Pietro Ferrara Reasoning about Distributed Systems On-the-Fly Trace Generation and Textual Trace Analysis and Their Applications to the Analysis of Cryptographic Protocols . . . . . . . . . . . . . . 201 Yongyuth Permpoontanalarp On Eﬃcient Models for Model Checking Message-Passing Distributed Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216 P´eter Bokor, Marco Seraﬁni, and Neeraj Suri Logics for Contravariant Simulations . . . . . . . . . . . . . . . . . Table of Contents . . . . . . . . . . . . . . . . . . . . 87 Laura Bocchi and Emilio Tuosto Joint DisCoTec Session Grouping Nodes in Wireless Sensor Networks Using Coalitional Game Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 Fatemeh Kazemeyni, Einar Broch Johnsen, Olaf Owe, and Ilangko Balasingham Table of Contents XII Timed Process Algebra Timed Process Algebra . . . . . . . . . . . . . . 224 Ignacio F´abregas, David de Frutos Escrig, and Miguel Palomino Author Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
https://openalex.org/W4366548928	https://www.frontiersin.org/articles/10.3389/fpos.2023.1140084/pdf	English	null	Patching identity. How Russian language media in Estonia reconstitutes our understanding of citizenship	Frontiers in political science	2,023	cc-by	11,015	OPEN ACCESS EDITED BY Ian A. Morrison, American University in Cairo, Egypt REVIEWED BY Greg Nielsen, Concordia University, Canada Helga Kristin Hallgrimsdottir, University of Victoria, Canada CORRESPONDENCE Ivan Polynin ivan.polynin@tlu.ee SPECIALTY SECTION This article was submitted to Political Participation, a section of the journal Frontiers in Political Science RECEIVED 08 January 2023 ACCEPTED 27 March 2023 PUBLISHED 20 April 2023 CITATION Polynin I (2023) Patching identity. How Russian language media in Estonia reconstitutes our understanding of citizenship. Front. Polit. Sci. 5:1140084. doi: 10.3389/fpos.2023.1140084 EDITED BY Ian A. Morrison, American University in Cairo, Egypt REVIEWED BY Greg Nielsen, Concordia University, Canada Helga Kristin Hallgrimsdottir, University of Victoria, Canada CORRESPONDENCE Ivan Polynin ivan.polynin@tlu.ee SPECIALTY SECTION This article was submitted to Political Participation, a section of the journal Frontiers in Political Science RECEIVED 08 January 2023 ACCEPTED 27 March 2023 PUBLISHED 20 April 2023 CITATION Polynin I (2023) Patching identity. How Russian language media in Estonia reconstitutes our understanding of citizenship. Front. Polit. Sci. 5:1140084. doi: 10.3389/fpos.2023.1140084 EDITED BY Ian A. Morrison, American University in Cairo, Egypt Ivan Polynin* School of Governance, Law and Society, Tallinn University, Tallinn, Estonia The conceptual novelty of this article rests in seeing identity not as a nominal category, but as a complex sequence of relationships between groups and narratives. It ofers a deeper reading of Engin Isin’s “citizenship in practice” and an empirical interpretation of how Andersen’s imagined communities are brought to life through print media. Drawing from Raivo Vetik’s analysis of the Estonian ethnopolitical ﬁeld the author explores narratives of two major Russian language web-portals in Estonia: Rus.Postimees and Rus.Delﬁ. As a result, the reader may observe how the practice of citizenship simultaneously constitutes and is constituted by the minority’s identity and subject position. The content analysis conducted from the samples of the aforementioned media outlets shows that the lack of shared citizenship practices between the majority and the minority causes a voluntary grouping along the lines of legal status, language, space and ethnicity. Discussing what constitutes Isin’s act of citizenship the author concludes that acts are far more elusive than the ruptures they cause. A media analysis shows that aside from bearing long-term ruptures such as geographical, linguistic or formal, Estonian citizenship practice also received a new one, namely the symbolic rupture caused by the war in Ukraine. TYPE Hypothesis and Theory PUBLISHED 20 April 2023 DOI 10.3389/fpos.2023.1140084 TYPE Hypothesis and Theory PUBLISHED 20 April 2023 DOI 10.3389/fpos.2023.1140084 TYPE Hypothesis and Theory PUBLISHED 20 April 2023 DOI 10.3389/fpos.2023.1140084 COPYRIGHT COPYRIGHT © 2023 Polynin. 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. KEYWORDS social identity, Estonia, minority identity, media analysis, nation building, relational approach, discourse, Russian minority Frontiers in Political Science OPEN ACCESS By breaking the previous status-quo, it pushes forward securitization and forces the minority to contest, redeﬁne, and reestablish its allegiance and perceptions of its place in Estonian society. Introduction 68). However, if researchers excessively rely on the self- perception of their respondents, they tend to repeat the same mistake as the majority of those respondents: to treat features forming identity as real. But what problems does it actually entail and how can they be avoided? The second section presents a broad exposition of the Estonian case using Isin’s notions of citizenship practice, acts, and ruptures. These notions allow us to gain a deeper understanding of how citizenship practically acquires its meaning, content and purpose. Moreover, it shows how, thanks to acts, ruptures become part of citizenship practices. Also, this section strongly draws from Vetik’s insight into the asymmetry of the Estonian ethnopolitical ﬁeld and empirical data of Integration monitoring 2020. The third part, delves deeper into Benedict’s Anderson’s vision of imagined communities, print-capitalism, media and language domination. Using the Estonian example, I claim that identity is not simply narrated by print-capitalism, but rather engaged in a mutually constitutive relationship with it. In the ﬁnal section, I am analyzing the role of largest Russian language news portals in forming our understanding of Estonian citizenship, particularly its vocabulary, practices and ruptures. Thus, before looking at the broader picture of interethnic relations in Estonia and the fragmentary nature of a citizenship practice, we need to ask what exactly is this identity that being imagined? Before giving a comprehensive answer to the question asked in the previous paragraph let us clarify the most serious contradiction between the micro and macro level understandings of citizenship and identity. The micro level understanding of citizenship perceives it as a membership or a position of an individual in a certain group. This level is arguably the closest to the everyday understanding of identity by a person without familiarity of the intricacies of existing debates in modern social science. By thinking about citizenship in terms of membership, an individual deﬁnes one’s own place in a certain group (I am ethnic Estonian, ethnic Russian) and can formulate relatively clear criteria by which people are ascribed to an inner or an outer group (Estonian/Russian is my mother tongue, I can hear your accent and tell to which group you belong). Tajfel and Turner (1986), standing at the roots of Social Identity Theory called this process: internalization of identity (p. 17). Introduction Rogers Brubaker’s relational attack on the mainstream usage of categories such as nations, groups and diasporas has left a certain vacuum at the junction of nationalism, migration and citizenship studies. Calling for a “groupless language”, Brubaker focused on studying ethnic group mobilization in relational terms, while overlooking how his fellow- researchers construct those theoretical categories (Csergo, 2008, p. 393). The following article aims to overcome this shortcoming by appealing to Engin Isin’s reading of citizenship within the Bourdieusian ﬁeld theory. I analyze the discourse of the Russian speaking media in Estonia as an example of Isin’s citizenship in practice. Speciﬁcally, I claim that the practice of constitution of media narrative is the actual mechanism behind the creation of what Benedict Anderson called an “imagined community” (Anderson, 1983). Anderson stated that the “written word” plays a key role in forming a community, alluding to the invention of the printing press and multiple local communities transforming into a single nation by reading the same news, and imagining that it happens to the virtual “them”. Frontiers in Political Science 01 frontiersin.org Polynin Polynin 10.3389/fpos.2023.1140084 10.3389/fpos.2023.1140084 but must be unpacked. It is in this context that I claim that Anderson’s imagined community and consumption of media help to demonstrate an ethnopolitical context in which claims of identity are standardized, negotiated, and contested. Further, Tajfel’s insight relating complex micro-level subjectivities to macro- scale dynamics of identity can help to explore the nexus of relationships that constitute media discourses in Estonia. The structure of the article is as follows: A relational orientation to identities is undertaken by an explicit unpacking of Vetik’s recent work in the Estonian ethnopolitical ﬁeld. The need to explore the institutional, discursive and symbolic realms in tandem is explored through making a distinction between micro and macro identity partially covered in Tajfel’s works. In this context, I explicitly bring Vetik in dialogue with Tajfel and other scholars in the ﬁeld of ethnic identity construction to demonstrate how the basis of identity necessarily draws from self-identiﬁcation of a group and external categorizations by out-groups. With this theoretical unpacking I aim to clarify what role identity has in the relational concept of citizenship. According to Tajfel the social identity is “that part of an individual’s self-concept which derives from his knowledge of his membership in a social group (or groups) together with the value and emotional signiﬁcance attached to that membership” (Tajfel, 1978, p. Introduction In other words, it is not enough for you to call yourself Russian, the members ascribing themselves to the group of ethnic Russians should also agree with you. This process also scales to a higher level: individuals need to internalize group membership in a way that should be recognized by the out-group (Tajfel and Turner, 1986, p. 17), or within the scope of this article, by ethnic Estonians. At this point Tajfel and Turner express a notable shift from nominal to more relational language. They emphasize that the attributes for the intergroup comparison must have a comparative value. Not all of the diﬀerences/similarities are valid when trying to tell one group from the other, but only those charged with the aim to achieve a superiority of one group vis-à-vis the other (Tajfel and Turner, 1986, pp. 17–18). Frontiers in Political Science frontiersin.org Micro and macro level identity Instead, he provides the following relational deﬁnition: In constructing an identity based on both internal and external identiﬁcations of the other, the ethnic-minority in Estonia is simultaneously viewed by the Estonian state lens and by the lens of Estonian history as an external other in close proximity to the Russian federation (Pettai, 2006; Makarychev, 2019; Vetik, 2019). Thus, minorities in Estonia occupy the unique position of being considered a potential source of threat and a group requiring the focus of Estonian politicians, the ministry of internal aﬀairs, and security agencies. For example, in February 2020, Riigikogu (2020) (Estonian national legislature) passed a statement “On Historical Memory and Falsiﬁcation of History”, in which it condemned the Molotov–Ribbentrop Pact, laid equal blame on the USSR and Nazi Germany for the instigation of the Second World War, and accused modern Russia of weaponizing history. Later, on May 8th all three Baltic countries issued similar statements. The objectivity of these allegations notwithstanding, these interpretations of history provide a contentious backdrop where the wedge between the Russian minority and ethnic majorities can be played out. In this context, Russians in Estonia are led to reorient their identities and strategies of belonging, either in favoring the Riigikogu perspective or aligning themselves with the view oﬀered by the Kremlin in which Russians are celebrated as the primary liberators of Europe from Nazism. Drawing from the idea that “collective memory is the keystone of national identity” (David and Bar-Tal, 2009, p. 168), I argue that, recasting of history is neither abstract nor passive, but is instead played out in active politics and used for solidifying identities as much as rupturing them. “Citizenship is a dynamic (political, legal, social and cultural but perhaps also sexual, aesthetic and ethical) institution of domination and empowerment that governs who citizens (insiders), subjects (strangers, outsiders) and abjects (aliens) are and how these actors are to govern themselves and each other in a given body politic” (Isin, 2009, p. 371). This deﬁnition allows us to look at citizenship as a macrolevel phenomenon including the membership function of identity, but not limited by it. I emphasize that such approach does not discard the theoretical concepts of Tajfel’s and Turner’s intergroup behavior, but instead utilizes them while looking beyond the microlevel internalization of identity. As we can see from the deﬁnition, the relational approach brings power and institutional structure into the picture. Micro and macro level identity If the ﬁrst two fall under the umbrella of citizenship as status, the third category is much closer to “citizenship as a practice” associated with the issue of belonging, deﬁning one’s membership and place in the society. Even though Isin warns against either objectivism or subjectivism, it is impossible to avoid them entirely. We cannot deny the fact that properties of social identity constantly manifest themselves in reality. Neither can we dismiss the subjective understandings of individuals about themselves and the out-group, because the sum of those subject position constitutes the relational ﬁeld of a nation. The ﬁrst thing we can do to solve this conundrum is to look within the ﬁeld of psychology. For example, Ehala (2018) suggests stopping treatment of signs of identity as indices having a causal connection between the sign and its deﬁnition like a name or ﬁngertips. Instead, he proposed perceiving the signs of identity as actual symbols deﬁned in arbitrary terms, but requiring the agreement of the absolute majority (Ehala, 2018, p. 48). What are the most important factors contextualizing minority citizenship practice in Estonia? Russians are the largest minority in Estonia. According to data from 2022, out of the total population of 1, 331, 796, there are 315, 242 Russian minority members, who comprise about 23.6% of the aforementioned total (Statistics Estonia, 2022). Most of the current minority members are ﬁrst/second generation immigrants, or descendants of those, who came to Estonia after the Second World War. According to the demographic statistics from 1934, during the time of the ﬁrst Estonian republic, Russians were a rather small minority, representing only 8.2% of the population, while by the end of the Soviet reign they amounted to 30.3% (Eesti Entsükloopedia, 2012). Such a rapid migration along with a diﬀerent attitude toward the fall of the Soviet Union predetermined a number of long-term problems in the Estonian interethnic relations. Identity is but a part of citizenship, which is hardly possible to deﬁne without being political. Taking into consideration the complex process of internalization discussed at the beginning of the section, I claim that social identity is the closest thing individuals have to membership. However, Isin speciﬁcally points out that citizenship should not be considered as membership (Isin, 2009, p. 371). Micro and macro level identity Even by ascribing oneself a certain identity an individual or a group claims dominance or resistance/adaptation to this dominance. At the same time, citizenship is not limited to the traditional static legal form, it also includes a practical everchanging aspect forming the positioning strategies of the engaged actors and the body politic itself. Nonetheless, the question which still remains is how does one operationalize Isin’s version of citizenship? Frontiers in Political Science Micro and macro level identity At this point we should distinguish the relational approach to group identity and citizenship from other essentialized versions. Vetik (2012, 2019) explores the Estonian ethnopolitical ﬁeld in a relational language past the binaries of nominal essentialized groups. Similar to Isin, Vetik’s insights provide an empirical unpacking of diﬀerences in subject positions (combination of opinions and perceptions deﬁning a place of an individual within a social ﬁeld) between competing majority and minority groups. Instead of seeing the categories of Russian or Russian Estonian, as nominal, Vetik invites reﬂection on the constitution of these subject positions. Following Bourdieu (1985), Vetik contends that identity and inter-ethnic dynamics are constituted in asymmetrical power relations. He further demonstrates how this ﬁeld of ethnic identity is a relational and complex sphere where subjects, orientations and positions compete to establish and maintain an objective and subjective existence. In this context, the struggles for identiﬁcation in ethnopolitics are not simple matters of the institutional vs. the symbolic, but draw in various scales from the macro and micro- level. Kruusvall et al. (2009) further argue that the space/ﬁeld/arena in which these identity claims are articulated and challenged requires active engagement and strategic adaption from all subjects including minority and majority groups. Thus, the examination of Estonians or Russians is not simply a case of them existing, Isin warns, that when studying such a complex set of relationships, there is always the danger of falling into either objectivism or subjectivism (Isin, 2002, p. 26). Excessive objectivism is one of the varieties of essentialism, when a parameter pertaining to an analytical construct, such as social identity is perceived as a feature deﬁning a group in reality. For example, when you frame the Russian minority in Estonia exclusively through ethnic, racial, labor market and language terms and expect the representatives of this group to behave in accordance to those properties. Subjectivism focuses on the self-perception of a person and assumes that self-deﬁnition is key to classiﬁcation, while in fact it is also not. A member of a group might lack knowledge for such identiﬁcation, never articulate it 02 frontiersin.org 10.3389/fpos.2023.1140084 Polynin explicitly, or not be accepted by the other members of the group (Isin, 2002, p. 26). So, how can these issues be addressed? beneﬁtting an actor. The third one lies in the aﬀective/symbolic dimension and signiﬁes the feeling of attachment to an imagined community (Fein and Straughn, 2014, p. 694). frontiersin.org Acts and practices of citizenship in Estonia Since the start of the Ukrainian war in February 2022, dilemmas for active association in history can be felt more tangibly in the Estonian ethnopolitical ﬁeld. A particularly insightful moment where competing narratives of belonging come to light is the Narva tank incident. In August 2022, the Estonian government moved a soviet T-34 tank from the outskirts of Narva to the military history museum in Viimsi. In addition, it also forcibly removed and utilized some of the old Soviet sculptures and monuments devoted to the Second World War. This action was preceded by several weeks of In Isin’s vocabulary citizenship may manifest itself in two diﬀerent ways: as a status and as a practice. Fein and Straughn (2014) suggest looking at citizenship from three basic understandings. The ﬁrst one is normative/territorial, traditionally associated with a legal status pertaining to a certain territory, reinforcing an individual’s human rights (reﬂected in the constitution and international law) and imposing obligations. The second is pragmatic/utilitarian, picturing citizenship as a choice 03 frontiersin.org 10.3389/fpos.2023.1140084 Polynin background, based on civic nationalist ideology” (Vetik, 2019, p. 413). protests attended by a notable number of local residents who stood vigil around the tank and surrounded it with ﬂowers and candles, making it look more like a place of religious worship than a military monument. The celebration and active call for preservation of these monuments demonstrates how, for many Russians in Estonia, monuments are not passive objects of a forgotten era, but rock-solid proof of the role Russians played in history of Estonia and the whole of Europe. However, from the dominating Estonian point of view, the Soviet monuments are a living reminder of one oppression after another and that freedom did not come until the very fall of the USSR. The last time when these two irreconcilable narratives clashed was in 2007 (Ehala, 2009), when in Tallinn the relocation of “Bronze soldier” (a monument commemorating the soldiers who died in the Second World War) caused violent riots and looting by the Russian community, which felt its identity threatened. Almost every recent security action taken by the government in the year 2022 causes a further asymmetry in the ﬁeld of interethnic relations. For example, the decision to conﬁscate ﬁrearms from permanent residents with Russian citizenship has branded them as potentially dangerous for Estonian security (RUS.ERR, 2022c). Acts and practices of citizenship in Estonia What is evident in the example above is the contested nature of historical memory and its association with Estonianness today. In navigating the complex discursive and historical domains of Russians in Estonia, the minority ﬁnds itself having to negotiate, contextualize and challenge the state position on its loyalty and contributions against fascism. Before revealing the next layer of Estonian interethnic ﬁeld, we need to take a step back to Isin and discuss another theoretical concept involving citizenship. So far, we have already encountered citizenship as a status (bearing a passport of certain country) and citizenship as practice (or actions, which take place regularly and originate from various subject positions). What unites them both is relative stability of conﬁguration: a legal status is visible, seldom changed, and easy to use for mobilizing identities, but may be misleading, as we can see from the previous paragraph. The practice of citizenship also presupposes repetitive patterns of behavior, which may be reﬂected in laws, customs, or other social institutions. In opposition to status and practice there stands an act of citizenship. According to Isin: The key to the Narva tank removal lies in the symbolic dimension. The government justiﬁed its actions by claiming that the Russian weaponry, which is currently deployed in Ukraine and has been used for Estonian oppression, should not be displayed as a monument and belongs in the past, as it provokes tensions (Õhtuleht, 2022). However, for a large share of the Russian minority community, these issues are not directly connected. As something used for liberation of Estonia from Nazism, the tank was a part of a positive identity. Nonetheless, it would be a mistake to present the entire problem as a dualistic conﬂict between the Soviet and the Estonian mainstream narratives. The entire ﬁeld of subject positions within the local community has multiple discourses concerning historical and present issues, where a pro- Soviet stance is far from dominant. Despite the forceful approach of the government, the monument removal did not cause the Russian community to unite against this decision, because the hybrid Russian and Soviet identity is strongly marginalized and has no recourse for producing a viable political force (Makarychev, 2019). However, the problem is that securitization of state politics inevitably polarizes society and deepens the divide along ethnic lines (Bigo, 2006). Acts and practices of citizenship in Estonia The decision of the new coalition, which equated the presence of Russian citizenship with a lack of loyalty to the Estonian state, was motivated by the current war in Ukraine. Therefore, such political actions polarize and simplify the ﬁeld, causing minority members to contest the disloyalty alleged on the basis of formal citizenship and appeal to the equal rights narrative. This became even more salient when the coalition started debating the removal of voting rights from the same minority group over similar security concerns (RUS.ERR, 2022a) or decided to drive all international students with a Russian passport out of the country by forbidding them to apply for new resident permits (RUS.ERR, 2022b). Under these conditions, formal citizenship status becomes a key criterion, equating citizenship with loyalty. This negatively aﬀects the unity of the nation as long term-minority members may feel their rights diminished. At the same time, recent migrants are outright rejected, as they are judged not on the basis of their actual political positions, skills and loyalty; but their citizenship, which they cannot easily change. This causes a notable shift in opinion on the issue among more mainstream-aligned members of the minority. Even if one does not exercise the right to bear arms and has zero regrets over Soviet symbols, it is still largely concerning to be treated as a threat and deprived of rights available to majority. Thus, due to this crisis, Estonian citizenship is being renegotiated in both of its aspects: as a status and as a practice, causing me to keep the focus on both. In contrast with 2007, the 2022 protests were peaceful and subsequently followed by negotiations between the prime minister Kaja Kallas and the Narva city council, which started productively, but ended in an ultimatum, in which the national government demanded the local authorities remove the tank, or it would do so itself (Õhtuleht, 2022). The Narva city council made a counteroﬀer of moving the monument from its previous location, but in the end, refused to take responsibility, as voting on the issue ended inconclusively. As a result, the central government delivered on its promise and using considerable police force and building equipment, moved the tank and destroyed the other monuments. Despite the preceding protests, in contrast with 2007, the whole action was carried out without any clashes with the local community. Frontiers in Political Science frontiersin.org Acts and practices of citizenship in Estonia That leaves both of the Estonian ethnic groups struggling for, as Vetik put it, the “moral high ground”, where the majority demands unity around ethnic nationalism and the minority group “calls for equal opportunities regardless of ethnic “How do we understand ‘acts of citizenship’? The term immediately evokes such acts as voting, taxpaying and enlisting. But these are routinized social actions that are already instituted. By contrast, acts make a diﬀerence. We make a diﬀerence when we actualize acts with actions. We make a diﬀerence when we break routines, understandings and practices” (Isin, 2009, p. 379). Isin argues that acts introduce ruptures into practices. In terms of citizenship practices a rupture is a break, which changes the conditions in which actors operate. Following Isin’s logic: if practice is following or reacting to a script, then an act 04 frontiersin.org 10.3389/fpos.2023.1140084 Polynin citizens of the ﬁrst Estonian republic prior to the 1940s. Many failed to produce such evidence, resulting in a large share of minority members in 1990s and later on opting for for an Estonian alien passport or Russian citizenship. This factor is quite well reﬂected in multiple works (Vetik, 2012; Fein and Straughn, 2014; Jašina- Schäfer and Cheskin, 2020), which try to distance themselves from a traditional state-centered view of citizenship and instead build their arguments around the logic of belonging and narratives behind the citizenship choice of the Russian speaking residents of Estonia. Balancing normative considerations, the actual utility of your passport, and the emotional connections to the Estonian state culture and nation, all of the aforementioned researchers touched upon the complexity of citizenship practices of the Russian minority of Estonia. means changing the script entirely, where rupture is the new beginning (Isin, 2009, p. 379–380). I ﬁnd the notion of an act of citizenship fascinating but rather problematic to approach empirically. An act is always purposeful, but is not necessarily conscious, and there is no way to tell if it is “inherently exclusive or inclusive, homogenizing or diversifying, or positive or negative”, as qualities appear after an act has already taken place (Isin, 2009, p. 381). Acts and practices of citizenship in Estonia This retrospectivity and the diﬃculty with deﬁning the level on which an act actually takes place gives a researcher immense freedom, where almost anything can be considered as an act of citizenship, given that it has transformed modes of being political and divided a nation into “citizens, strangers, outsiders and aliens” (Isin, 2009, p. 383). Arguing whether a particular set of actions is an act or not, goes beyond the scope of this study, and might not be as productive as focusing on ruptures instead. I claim that some ruptures in the Estonian notion of citizenship are much larger than a single act and might be caused by multiple acts over time. Yet, it is not easy to identify a time horizon of any one particular act resulting in rupture. Rather, these ruptures can widely be seen across the linguistic, spatial, legal (formal) and more recently, symbolic domains that collectively constitute/frame citizenship. Hence, these ruptures require a deeper embedding in the acts, memories and symbolism that frame them. For instance, problems with Estonian language proﬁciency in Ida- Virumaa (the most Eastern part of Estonia), cannot be understood solely in relation to the consequences of post-Second World War migration (discussed at the beginning of this chapter) but extend to current events in independent Estonia. According to the most recent Estonian Integration Monitoring, only 21% of Ida-Virumaa residents demonstrate active language proﬁciency (EIM, 2020, p. 35). By explaining this with the high concentration of minority members in the northeastern region of Estonia, where there is no Estonian language environment, we can identify two ruptures. The analysis of both existing linguistic and formal citizenship ruptures between the communities is now further complicated by newer symbolic ruptures resulting from the ongoing war in Ukraine. The ﬁrst one is related to the radical polarization within the Russophone communities in Estonia (more on this in the empirical section below) internally polarized the Russian speaking community in Estonia (Rus.Delﬁ, 2022). The attitude toward Ukraine and support of Ukrainian people ﬁghting for freedom, liberty, and survival of their nation has become a watershed, excluding a pro-Kremlin stance in any form from being a part of Russian Estonian identity or a minority citizenship practice. Acts and practices of citizenship in Estonia The most recent manifestation of this rupture is the Narva tank removal, which outlines the contested boundaries of what is considered to be an appropriate display of a Russian Estonian identity and what cannot be a part thereof. During the debates around the meaning of the Narva tank and the actual necessity of its removal, most of the engaged individuals one way or the other based their line of argument on symbolic allusion to the current war in Ukraine. Thus, what we observe across these ruptures, is a struggle for the moral high ground between the minority and majority members, where the majority’s citizenship strategy is centered around the core of Estonian ethnic identity, which demands ancestral descent or proof of loyalty from potential citizens. From the dominating point of view of the majority, the primary threat to Estonian national unity is the uneven demographic composition left as an aftermath of the Soviet Union (Vetik, 2019, p. 408). Four years ago Vetik claimed that the demographic situation and Russian expansionism have been used as a justiﬁcation by multiple Estonian politicians for pushing through security policies. They have tended to present those policies as countermeasures defending Estonian language and culture (Vetik, 2019, p. 409). Today we once again may witness the truth of this observation, as the war in Ukraine has been used in every instance when the minority rights or loyalty have been questioned (RUS.ERR, 2022a,b). First, there is an obvious linguistic rupture, which frames multiple practices from administrative (the language of government services available to the minority population) and political (language of political canvassing, communication with politicians) to education and media consumption. Second, the spatial rupture strongly inﬂuences language contact. Nation-wide data from Integration Monitoring indicates that most (51%) minority members interact with Estonians mostly at work or school and quite less often during their free time (32%) (EIM, 2020, p. 36). The lack of language proﬁciency and geographical distribution is constituted by and constitutes the format of contact between communities. Therefore, the nature of this contact reinforces the formality of majority-minority relationships, where the Estonian language gains a pragmatic role, rather than being socially or culturally uniting. In case of formal citizenship, loyalty is measured by the wish to learn the Estonian language, pass a constitution exam and undergo the naturalization procedure. Frontiers in Political Science Acts and practices of citizenship in Estonia From the view of majority, this is a logical continuation of a “language-centered model” of integration of the Estonian society, where ethnic diversity is recognized alongside the common language denominator and implies hierarchal inequalities (EIM, 2020, p. 103). From the view of minority, this is the issue of equal rights. For many members Second, about 6% (Statistics Estonia, 2022) of the current Estonian population still has a status of aliens or non-citizens. Those are Soviet Union citizens and their descendants, who by the decision of the authorities, did not qualify for Estonian citizenship right after Estonia regained its independence. According to the 1992 citizenship law, in order to be considered as Estonian national, these people should have provided proof, that their ancestors were 05 frontiersin.org Polynin 10.3389/fpos.2023.1140084 has already substituted Russian as a language of power and administration, which Russian used to be in the times of the Soviet Union. Nevertheless, an even more serious problem is in the Russian language being perceived as contender, which results in segregation of the Estonian education system. On the one hand, a large share of the Russian minority parents defend a persistent narrative of minority children being entitled to education in their own language. According to data from the most recent Estonian Integration Monitoring, about 44% of minority parents would prefer their children to study at fully Russian-language schools or Russian-language schools where some subjects are taught in Estonian (EIM, 2020, p. 20). In contrast to that ﬁrst group, is the 46% of minority parents, who would prefer fully Estonian schools, language immersion, or other Estonian medium schools with an in-depth focus on alternative languages (EIM, 2020, p. 20). of the Russian minority, being born in Estonia and living in it for dozens of years automatically means that one should qualify for Estonian citizenship and should not be treated as a migrant. Both narratives constantly compete with each other, and even though the majority’s narrative is clearly dominant, many minority members contest it with their resistance to applying for Estonian citizenship on these conditions (EIM, 2020, p. 46). Nonetheless, this practice of citizenship does not acquire a performative aspect. Isin tells us that for an act of citizenship to happen, a group of people needs to claim their rights and enact political subjectivity (Isin, 2009, p. 368). Imagining community Undoubtedly, Benedict Anderson’s “Imagined Communities” (Anderson, 1983) is one of the most well-known important works in the sphere of nation building. At the same time, it is one of the most misinterpreted studies in the ﬁeld. As Enric Castelló emphasized in his review of the Anderson’s work: many researchers tend to jump from the ﬁrst few introductory pages to an automatic conclusion that nations are imagined through media, while ignoring the rest of the book (Castelló, 2016, p. 61). At the same time Anderson hardly uses the word media throughout the whole text and is more focused on print-capitalism or the way industrial productions of written texts shift the way individuals perceive the group to which they belong. Anderson argued that printed capitalism aﬀected the creation of national consciousness in at least three ways. First, it allowed multiple people within a nation to understand each other better thanks to standardization of language. Second, the same process granted people a certain level of temporal ﬁxity and lowered the bar to accessing information, as instead of understanding the dialect or writing tradition of a speciﬁc monastery, people could rely on a certain standard. Third, printed media has inevitably empowered speciﬁc varieties of language while weakening others, which has made some of the ethnic groups dominant over the others, as their language became primary and all others turned into regional dialects (Anderson, 1983, p. 42–45). In an analysis of print media, both content and language become equally important in shaping and framing practices. Let us take a look at how a language may manifest its power in technocratic choice as much as in politicized and contested vocabularies. Being in a power position, the Estonian media writing in the Russian language gradually and, not always consciously, updates even the language itself. There is a considerable number of words reﬂecting a speciﬁc Estonian institutional reality, which are not present in the Russian language: “Bolnichnaya kassa” (Больничная касса—Estonian Health Insurance Fund), “kassa po bezrabotse” (касса по безработице—Estonian Unemployment Insurance Fund), “digitalnyi” (дигитальный—digital, in opposition to the oﬃcial Russian “electric” or “электронный”), etc. In these and many other cases, the power of Estonian language is not contested but even driven by the minority. Due to the institutional reality being accepted as a given, the dominance of the Estonian language is reinforced without a conscious understanding of the political nature of these repetitive actions. Frontiers in Political Science Acts and practices of citizenship in Estonia Despite the belligerent lexis of this deﬁnition, the act of claiming does not necessarily mean an aggressive mobilization of Russian minority’s ethnic identity, similar to the one which happened in the year 2007 when a mob outraged by the Bronze Soldier monument dismantling clashed with Police in Tallinn. Instead, it means the activation of an imagined identity trying to ﬁnd its place, alterity, and belonging. On the other hand, there is a certain resistance on the behalf of the Estonian schools who are trying to protect the Estonian language environment in the areas highly populated by the minority. Due to school classes being formed on the basis of language of instruction, it is frequent for Estonian and Russian speaking children to rarely meet in classes or sometimes even in a single school. A relatively recent problem in the Estonian language medium schools in Ida-Virumaa is an inﬂux of Russian speaking pupils (RUS.ERR, 2019). With a large number of children speaking Russian as a native tongue, these schools have insuﬃcient resources to guarantee the quality of education and the Estonian language environment. This problem concerns not only the schools of Ida- Virumaa, but also Russian schools in all major cities such as Tartu and Tallinn because few Estonian parents are keen on sending their children even to a language immersion school where their children learn both Russian and Estonian. Therefore, there is no consensus regarding the power position of the Russian language, but it gains the role of a contender in any case, notwithstanding the subject positions of those entering the school system. frontiersin.org Media, citizenship, and the war in Ukraine The Russian speaking Internet media in Estonia is represented by multiple sources, however the two largest of them act as the primary agents of print capitalism inﬂuencing the minority community. First is the oldest web portal’s name is Rus.Delﬁ(2022), which is in fact a multilingual network of portals. The Estonian language version of the portal was launched in 1999 along with the Latvian language one. The number of browser contacts (views from a single opened browser) for Rus.Delﬁﬂuctuates around 300–400 thousand views per week. Second, another portal, which constantly contests the title of the most popular Russian speaking news source in Estonia, is Rus.Postimees (2022). This portal is a Russian language version of the largest and the most popular Estonian newspaper established in 1886. During the 1990s, Postimees once again became a national Estonian paper and, between 2005 to 2016 issued a Russian language paper version. Since 2016, it has a news portal only. At the moment, the number of browser contacts ﬂuctuates weekly, with the winner of most visited website changing frequently. Anderson’s approach masterfully describes processes of identity formation, but at the same time he treats it as an empty box having almost no real continuity. He concentrates on his macrolevel model of analysis, where nationalism is always a product of language competition (Latin vs. vernacular, imperial European languages vs. colonial Creole languages) advanced through print capitalism and reﬂected in administration, bureaucracy, and education. Print media, for him, is always the means for achieving the ends in the form of an imagined community. He argues that nations are similar to children in the respect that their identity cannot be remembered, but only narrated to them by the continuing ﬂows of information produced by print capitalism (Anderson, 1983, p. 204). Both portals are an integral part of Estonian print-capitalism, which in contrast, with Rus.ERR (Russian version news outlet of the Estonian national TV and Radio company), are commercial institutions having private owners whose task and vision go beyond simply informing the population. They are both independent, compete for the media market, and serve as natural counterweights to the Russian news portals from Russia by propelling the Estonian mainstream narrative and acting as tools for internalizing the identity of Russian minority in Estonia. However, as we can see from the Estonian Integration Monitoring data, minority residents tend to be much more inclined toward local identities than toward the national one. Imagining community For example, the oﬃcial Estonian regulations prescribe writing the word of the capital Tallinn with two “n”-s, while transcribing it into Russian (Таллинн). At the same time, in the oﬃcial Russian language it must be written with a single “n” (Таллин). At some point the Russian authorities even argued against this to no avail, by claiming that laws of Estonia do In case of the major western powers, which acquired their independence a long time ago, the foundational part of the nation building has already ended. From Anderson’s point of view, European nations cement the status-quo when a language becomes a language of power and linguistic nationalism reaches the point where status is no longer contested by a serious competitor (Anderson, 1983, p. 42–45). This does not necessarily mean a dominance of a single language, but instead may result in an equilibrium achieved by several languages like in the case Switzerland, where several monolingual nations are united by a bilingual or trilingual political class (Anderson, 1983, p. 138). For Estonia this process is still ongoing and gradually happening even today. In a similar manner as vernacular languages overcame Latin as a lingua franca, the Estonian language 06 frontiersin.org Polynin 10.3389/fpos.2023.1140084 single force, but a ﬁeld of struggle, where multiple actors such as political parties, NGOs, various majority and minority interest groups, representatives of government institutions, private media editorials, journalists, bloggers, and even individuals compete for an actual narrative of identity delivered to recipients. not regulate the rules of the Russian language. Therefore, even the normalization of writing Tallinn with two n-s at the end is also an important part of symbolic power manifestation delivered through the Russian language print or internet media in Estonia. Due to the undertaken timeframe and a speciﬁc focus on the power of words, Benedict Anderson did not concern himself with TV or radio as much as with the print media. Building parallels between diﬀerent theoretical frameworks of this study, I claim that Anderson strongly focused on the standardization and the symbolic aspects of citizenship practice. He considered the action of reading the same news as a ritual, which produced the feeling of unity with thousands of people one had never met. Imagining community It created an illusion of belonging or at the very least existing in the same community with similar interests and discourses: Second, print capitalism and identity have always been engaged in a mutually constitutive relationship. Even though print capitalism facilitated modern nationalism (or the form of it that we know from the 19th century), the content of the actual identity largely depended on the speciﬁc circumstances of the nations where it arose. This is not to say that print capitalism in itself is a sole “basis” through which identity comes to be constituted, but rather, that it is embedded in a larger relational schema of politics, history, discourses, and ideas—which facilitate the rise and fall of certain dialects/languages and attitudes in the media. “The signiﬁcance of this mass ceremony—Hegel observed that newspapers serve modern man as a substitute for morning prayers—is paradoxical. It is performed in silent privacy, in the lair of the skull. Yet each communicant is well aware that the ceremony he performs is being replicated simultaneously by thousands (or millions) of others of whose existence he is conﬁdent, yet of whose identity he has not the slightest notion. Furthermore, this ceremony is incessantly repeated at daily or half-daily intervals throughout the calendar. What more vivid ﬁgure for the secular, historically clocked, imagined community can be envisioned?” (Anderson, 1983, p. 35). Frontiers in Political Science Media, citizenship, and the war in Ukraine Therefore, I argue that studying the inﬂuence of selected media outlets allows us to perfectly capture the dominant political discourses of the Russian minority community in Estonia. The war in Ukraine has drastically changed the discourse of the analyzed media outlets. From Figure 1 we may observe that 3 months before the war, even as the trouble that was brewing in Ukraine held much of the country’s attention, the discourse of both portals was mostly focused on Estonia and its internal aﬀairs: there were 5,387 mentions of Estonia for Postimees and 5,363 for Delﬁ. The overlapping themes were also focused on prices, stability of the Euro and coronavirus. We may also observe that Rus.Delﬁalso prioritized children in its coverage even before the war. As we can see from Figure 2, after the start of the war, the top 6 words become identical in both of the portals: Ukraine, Russian, Estonia, Military, Ukrainian, and Putin. The shock of the horrifying Russian assault on Ukraine was so strong that it did not just capture the whole of media discourse, but to a certain extent, it even synchronized the vocabulary of both media outlets. Let us take a look at the empirical evidence supporting this statement (from now on Rus. Postimees will be referred to as PM and Rus.Delﬁas D). Approaching the end of the most violent phase of the Russian oﬀensive, both portals had similar mentions of the word March (PM: 1,637 and D: 1,669) and focused on the actions of the word leaders, explain why the word President is frequently mentioned (PM: 1,471 and D: 1,387). Rus.Postimees has also started to actively cover the issues of refugees (1,497) and children (1,454). Rus.Delﬁ kept the focus on children (1,762), but despite devoting signiﬁcant attention to refugees (1,264 mentions), it was also more focused on residents (1,514): Ukrainian and local. For the purpose of this work, I analyze the quantitative sample, which includes 9 986 articles from Rus.Delﬁand 9 935 articles from Rus.Postimees for the period from 13.11.2021 to 18.04.2022, capturing the time before and after the war in Ukraine. The raw data was collected using four Python scripts scanning and processing information from the search pages of Rus.Delﬁand Rus.Postimees. The ﬁrst script scanned the websites for speciﬁc dates and extracted all the links, which were then stored in a table. Media, citizenship, and the war in Ukraine In other words, the acceptance or rejection of identities occurs in the terms formulated within the ruptures. Moreover, faced with the situation where the interests of a considerable part of the Russian minority conﬂict with the mainstream political agenda, we may observe how the media outlets function not only as separate actors informing the population but also as respondents, activists, or mutual translators, interpreting not only the actions of government to the general public but also vice-versa. distributed by Rus.Delﬁand Rus.Postimees are constantly being shared, contested, integrated into, or rejected from identities on a personal level. After multiple rounds of internalization (as described in the section on micro and macro identities) and standardization (as in the section on the imagining communities) these narratives are rooted in the ethnopolitical ﬁeld as subject positions (as described by Vetik). Even if a reader’s subject position is entirely diﬀerent from the mainstream narrative, it is still situated within the ﬁeld. In most of the cases even the vocabulary used by the competing narrative is not contested in terms of language or content. For example, when covering the issue of Narva tank, both the Estonian mainstream news and their Russian counter- parts used the same construction of “moving the Narva tank,” in Estonian “Narva tanki teisaldamine” and in Russian “Перенос Нарвского танка” Even the opponents of this measure did not use more assertive terms such as “sacrilege”, “destruction”, “vandalizing”, or even “removal.” Therefore, even the competing narratives were united by the sheer fact that the tank was simply “moved.” I argue that this strongly reﬂects the symbolic aspect described by Anderson. The practice of repetitive reading of the same news from the same portals and sharing or arguing over the discourse distributed by familiar media sources creates a strong sense of belonging. However, for this sense to arise the print-media ﬁeld should have a limited number of options and community members need to trust them. Internet media is much more accessible than a print newspaper and easily provides nation-wide coverage while generating a high level of trust. Russophones in Estonia clearly illustrate how this speciﬁcally works. According to EIM (2020) 72% of minority respondents named the Estonian Russian language news portals as a third important source of information outranked only by information from friends and relatives (89%) and colleagues/fellow students (75%) (EIM, 2020, p. 74). Media, citizenship, and the war in Ukraine For example, when asked to rate being informed about the events of their hometown during the last 5 years, minority respondents proved to be more informed about local news than about national news, while for majority members the picture is the other way around (EIM, 2020, p. 72). As much as I like this metaphor, I do not completely agree with it and consider it an overstatement. First, even if it is impossible to reproduce one’s exact identity or state of consciousness in childhood, this does not mean that there’s no continuity between diﬀerent life stages and that people are completely oblivious of how they thought when they were younger. Identity and its historical continuity being an active form of engagement draws in passive subjectivities of experience, memory, longing, and sense of belonging and association. Besides, if it was so easy to narrate an identity, then being the dominant actors driving and regulating print capitalism, governments would have no problem integrating large minorities into their societies, who themselves challenge/resist/reinterpret or reevaluate claims of belonging. That would probably also result in the Russian minority sharing the same citizenship practice as the Estonian majority and this article never being written. Thus, identity is indeed narrated by print capitalism but only to an extent limited by collective memory. Besides, it would be more reasonable to consider print capitalism not as a In the previous chapter I argued that rather than being simple identity narrators, print-capitalist media sources are inﬂuenced by their readers, as much as the other way around. The narratives 07 frontiersin.org 10.3389/fpos.2023.1140084 Polynin analysis employs Isin’s concept of ruptures and reveals them across diﬀerent dimensions, such as space, time, formal citizenship, language, ethnicity, and major events such as the war in Ukraine. Therefore, coding and grouping were conducted along those theoretical lines. Drawing from Anderson, I demonstrate how the mutually constituted media citizenship vocabulary shapes the readers’ perception of these ruptures, deﬁning a shared experience for them to imagine their identities. On the one hand, this approach does not allow for a detailed examination of the actual reactions to the media discourse per se. On the other hand, to outline the citizenship practices, the analysis of their vocabulary is more important, as these words serve as a container for multiple pro and contra subject positions. Frontiers in Political Science frontiersin.org Media, citizenship, and the war in Ukraine The second script retrieved the texts of articles saving them and the dates when they were written. The third script performed tokenization of the saved articles, meaning breaking the saved texts into individual words, and then lemmatization, or the process of converting each word to the base form and then saving the resulting data in a separate table. Finally, the fourth script searched for identical words, counted their occurrences, and recorded the results in a separate table. This large sample size provides a comprehensive and representative view of the citizenship vocabulary of the Russian-speaking media in Estonia, enabling the identiﬁcation of primary themes. From both sides of the theoretical framework of this study, the war in Ukraine has remarkably changed the citizenship practices of the Russian minority in Estonia. Aside from introducing the rupture between followers of the Kremlin’s narrative and the independent news, the detailed analysis of which goes beyond the scope of this article, the war in Ukraine has subtly become a component of citizenship itself. Tracking the news about the war has become as much of a practice for readers, as writing about it for journalists and bloggers (Netpeak Checker, 2022). It is certainly not the ﬁrst time that a subject position toward a war becomes a part of identity, but what makes this particular war diﬀerent for Estonia is For processing this data, I apply theory-driven content analysis to examine the most frequently used words in the articles. The 08 frontiersin.org Polynin 10.3389/fpos.2023.1140084 FIGURE 1 The top 6-word mentions before the start of the war in Ukraine (13.11.2021–23.02.2022). FIGURE 1 The top 6-word mentions before the start of the war in Ukraine (13.11.2021–23.02.2022). FIGURE 2 The top 10-word mentions after the start of the war in Ukraine (24.02.2022–18.04.2022). FIGURE 2 The top 10-word mentions after the start of the war in Ukraine (24.02.2022–18.04.2022). Frontiers in Political Science frontiersin.org Mapping fragmented citizenship practices on the hand, the absence of an active involvement in combat, and on the other hand, a continuous and circulating information ﬂow about the war, which challenges existing memories and identities and constantly raises the question of loyalty. No matter how we look at it: as Anderson’s mass ceremony of simultaneous media consumption or Isin’s collective act of citizenship, this results in the reimagining of the existing identities of Estonian Russians. As this process is currently continuing, it is too early to say how these identities will eventually look. The vocabulary of Estonian minority citizenship practices would not be complete, if we did not try to describe the four primary components it consists of: space and location, formal (legal) citizenship, language and ethnicity. Contrary to the Estonian language-centered integration model, media discourse is emphatically regional. As we can see from Frontiers in Political Science 09 frontiersin.org Polynin 10.3389/fpos.2023.1140084 FIGURE 3 Word frequencies characterizing the space and location aspect of Russian minority identity. FIGURE 4 Word frequencies characterizing the legal aspect of Russian minority identity. the Figure 3, the word combinations of “local” (PM: 1,359, D: 1,379) and “resident” (PM: 2,930, D: 2,397) are widely used in characterizing identities. The geographical component is also visible in an extensive thematic focus on the minority populated “Estonozemelets” in Russian or “Eestimaalane” in the Estonian language (the one living on the Estonian land). Due to the large presence of minority members having foreign or no citizenship, the Estonian political elite tried to propose a land-based identity, FIGURE 3 Word frequencies characterizing the space and location aspect of Russian minority identity. FIGURE 4 Word frequencies characterizing the legal aspect of Russian minority identity. “Estonozemelets” in Russian or “Eestimaalane” in the Estonian language (the one living on the Estonian land). Due to the large presence of minority members having foreign or no citizenship, the Estonian political elite tried to propose a land-based identity, which proved to be a rather contested substitute to legal or ethnic types of identities. Devoid of the imagining act, the term of “Estonozemelets” does not serve its primary purpose and instead of uniting the minority and majority, it stresses the existing status-quo the Figure 3, the word combinations of “local” (PM: 1,359, D: 1,379) and “resident” (PM: 2,930, D: 2,397) are widely used in characterizing identities. Frontiers in Political Science Mapping fragmented citizenship practices The geographical component is also visible in an extensive thematic focus on the minority populated areas, such as the region of Ida-Virumaa (PM: 315, D: 358, counting adjective forms) or the Lasnamäe district in Tallinn (PM: 373, D: 657). Another visible trend is a rather modest usage of a politically correct word form aiming to unite all Estonian residents 10 frontiersin.org Polynin 10.3389/fpos.2023.1140084 FIGURE 5 Word frequencies characterizing the legal aspect of Russian minority identity. FIGURE 5 Word frequencies characterizing the legal aspect of Russian minority identity. “Russian-speaking-one,” is an attempt to further diﬀerentiate the language and ethnic aspects of the word “Russian.” Using this model of perception, the Russian-speaking resident of Estonia is much more integrated into society than the “Estonian Russian,” which brings in an ethnic aspect and more political subjectivity, similar to “African American.” of living in the same space. That is why even the mainstream internet portals do not use this term often (PM: 68, D: 24) in comparison with the regional denominators. Legal status has become one of the primary stepping stones in identity diﬀerentiation. From Figure 4 we can see the following trends. First, we may observe the increased importance of the standard legal form of identity, which is rooted in words such as citizen and civic (PM: 1,843, D: 1,666). Also, due to historical developments, a popular manner of referral includes the word passport (PM: 312, D: 277) and adding an adjective red, blue or gray. Even though Estonian passports changed their color to red long time ago, in colloquial speech a red passport may often mean a Russian one, and a gray passport is way to refer to a person without citizenship. Second, as the Estonian authorities keep introducing new ways to sanction Russian passport holders, it has become of higher importance for both the mainstream media and ordinary Estonian minority readers to draw an additional line between themselves and “rossijane” (Russian nationals) (PM: 649, D: 845), meaning Russian nationals, but also having a connotation of those hailing from or residing in Russia. The frequent emphasis on the actions of “rossijane” emphasizes an attempt to further separate this identity from that of the local community. This stands in contrast to use of the word “russkiy”, which means an ethnic Russian. Finally, we see that attention on Estonian non-citizens has plummeted (PM: 11, D: 32). Mapping fragmented citizenship practices Considering that multiple researchers investigating the issue of minority citizenship in Estonia often focus on gray-passport holders, this trend signiﬁes a large diﬀerence between a mainstream research and an insider perspective on the issue. Adding the ﬁnal strokes to this picture of the citizenship vocabulary of the Russian language web-portals of Estonia, I would like to highlight two more features. First, the theme of “discrimination” is present, but far from persistent (PM: 47, D: 78). Despite clear problems with the securitization of the Russian identity, this has not yet fully aﬀected the minority community, which prefers the opposition strategy of diﬀerentiation, or distancing itself further from Russia and Russian nationals not just via legal status, but on the level of the discourse as well. Second, the dominating media discourse shows that the Russian minority of Estonia has accepted a one directional narrative of integration. In this narrative the non-majority members constitute an element that needs to be integrated (PM: 38, D: 40) rather than integrate itself (PM: 9, D:12). All of this shows that the Russian minority identity is currently in transition, the ﬁnal result of which is yet to be seen. Frontiers in Political Science References Anderson, B. (1983). Imagined Communities. Reﬂections on the Origin and Spread of Nationalism. London: Verso. Isin, E. (2002). Being Political: Genealogies of Citizenship. Minnesota: University of Minnesota Press. Balti Uuringute Instituut Tallinna Ülikool SA Poliitikauuringute Keskus Praxis (2020). Eesti ühiskonna integratsiooni monitoring [The integration monitoring of the Estonian society]. Tallinn: Kultuuriministeerium. Available online at: https://www.kul. ee/en/estonian-integration-monitoring-2020 (accessed June 10, 2022). Balti Uuringute Instituut Tallinna Ülikool SA Poliitikauuringute Keskus Praxis (2020). Eesti ühiskonna integratsiooni monitoring [The integration monitoring of the Estonian society]. Tallinn: Kultuuriministeerium. Available online at: https://www.kul. ee/en/estonian-integration-monitoring-2020 (accessed June 10, 2022). Isin, E. (2009). Citizenship in ﬂux: The ﬁgure of the activist citizen. Subjectivity 29, 367–388. doi: 10.1057/sub.2009.25 Isin, E. (2009). Citizenship in ﬂux: The ﬁgure of the activist citizen. Subjectivity 29, 367–388. doi: 10.1057/sub.2009.25 Jašina-Schäfer, A., and Cheskin, A. (2020). Horizontal citizenship in Estonia: Russian speakers in the borderland city of Narva. Citizenship Stud. 24, 93–110. doi: 10.1080/13621025.2019. 1691150 Bigo, D. (2006). “Globalized (in)security: The ﬁeld and the ban-opticon,” in Illiberal practices of liberal regimes: The (in)security games, eds. D. Bigo and A. Tsoukala (Paris: L’harmattan) 5–49. Kruusvall, J., Vetik, R., and Berry, J. W. (2009). The strategies of inter-ethnic adaptation of Estonian Russians. Stud. Transit. States Soc. 1, 3–24. Available online at: http://htk.tlu.ee/stss/wp-content/uploads/2009/12/STSS_Kruusvall_Vetik_Berry.pdf Bourdieu, P. (1985). The social space and the genesis of groups. Theory Soc. 14, 723–744. doi: 10.1007/BF00174048 Castelló, E. (2016). Anderson and the Media. The strength of “imagined communities”. Debats. J. Cult. Power Soc. 1, 59–63. doi: 10.28939/iam.debats-en .2016-5 Makarychev (2019). Estonia’s Russophones Tumble Between Two Populisms. PONARS Eurasia. Available online at: https://www.etis.ee/Portal/Publications/Display/ 12f51974-3d7c-48f8-bccc-9cf24c1c81d4 (accessed July 3, 2022). Csergo, Z. (2008). Review essay: Do we need a language shift in the study of nationalism and ethnicity? Reﬂections on Rogers Brubaker’s critical scholarly agenda. Nations Nation. 14, 393–398. doi: 10.1111/j.1469-8129.2008.00 346.x Netpeak Checker (2022). SEO website analytics. Available online at: https:// netpeaksoftware.com (accessed September 28, 2022). Õhtuleht (2022). Peaminister Kallas Narvas: tank tuleb teisaldada kiiremini kui kahe nädalaga. Prime-minister Kallas in Narva: The tank should be relocated quicker than in two weeks. Available online at: https://www.ohtuleht.ee/1067721/peaminister-kallas- narvas-tank-tuleb-teisaldada-kiiremini-kui-kahe-nadalaga (accessed September 16, 2022). David, O., and Bar-Tal, D. (2009). A sociopsychological conception of collective identity: The case of national identity as an example. Pers. Soc. Psychol. Rev. 13, 354–379. doi: 10.1177/1088868309344412 narvas-tank-tuleb-teisaldada-kiiremini-kui-kahe-nadalaga (accessed September 16, 2022). Eesti Entsükloopedia (2012). Rahvuskoosseis Eestis. [National composition of Estonia]. Available online at: http://entsyklopeedia.ee/artikkel/rahvuskoosseis_eestis (accessed May 30, 2022). Pettai, V. (2006). 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. Author contributions The author conﬁrms being the sole contributor of this work and has approved it for publication. Conclusions The relational framework provides a researcher with multiple theoretical tools serving to engage with rich empirical material, such as that provided by the Estonian ethnopolitical ﬁeld. In this sense the following article is an attempt to think along not only with Isin, but also Anderson, Vetik, Tajfel, and many other scholars. By employing relational language, I strive as much as possible to ﬁnd the balance between objectivism and subjectivism and transcend nominal binaries and essentialism. Instead of treating groups as the real entities, I consider the subject positions constituting the ethnopolitical ﬁeld of Estonia. I trace how citizenship becomes a ﬂuid nexus between a status and practice for minorities. I observe how language gains power through print media and becomes involved in a constitutive relationship with identity, becoming a narrator as much as a listener. In this light, the act of citizenship fades into the background, exposing the ruptures of citizenship practice in Estonia. Integration monitoring asserts that the Estonian model of integration prioritizes the Estonian language as a primary tool for integration (EIM, 2020, p. 102). Looking at the data from the analysis, we may ﬁnd additional support for this argument. Even discarding the contaminated data, in which it is impossible to diﬀerentiate between ethnicity and language, we can see from Figure 5 that linguistic indicators play an important part in identity construction. The “Russian-language-one” is a quite popular form designating a resident (PM: 312, D: 388). However, even though the Estonian-language-one (PM: 52, D: 39) is not as frequent, it demonstrates the dominating position of the Estonian language in society, as it reﬂects that people prioritize the word “Estonian” instead (PM: 3,002, D: 2,940). The intricate form of an adjective, 11 frontiersin.org Polynin Polynin 10.3389/fpos.2023.1140084 Data availability statement The linguistic, geographical and formal status ruptures ﬁnd their reﬂection in media narratives, forming patches of identities. Space and location become salient in the regional vocabulary, while the uniting mainstream identity based on land ﬁnds little support. Language rupture serves as an identity tool, where the Estonian language aﬃrms its dominating position in the narrative. The weakness of shared citizenship practice encourages grouping along the lines of legal status, where Russian nationals become ostracized in the narrative, while non-citizens almost disappear from the discourse. In turn, the minority’s subject position normalizes the language of integration narrative becoming primarily one- directional and inclining toward being integrated, rather than integrating itself. The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author. Conﬂict of interest The author declares that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. The war in Ukraine has inﬂicted a new rupture upon citizenship practice, causing securitization and polarizing identities. In the new conditions, the Russian minority is driven into ﬁnding ways to prove its loyalty and rethink its historical contributions and its current role in the Estonian society. Even though the act of the Narva tank relocation and comparison of its signiﬁcance with the Bronze soldier removal deserves a separate paper, in light of the rest of this analysis, it gives us an exact idea of how the war in Ukraine may manifest itself as a rupture in Estonian citizenship practices. As for now, the identity of the Russian minority remains fragmented and ﬁnding common citizenship practices with the Estonian majority is a task yet to be undertaken. References Explaining ethnic politics in the Baltic States: Reviewing the triadic nexus model. J. Baltic Stud. 37, 124–136. doi: 10.1080/01629770500 000291 Ehala, M. (2009). The bronze soldier: Identity threat and maintenance in Estonia. J. Baltic Stud. 40, 139–158. doi: 10.1080/016297709027 22294 Riigikogu (2020). The Riigikogu passed the Statement “On Historical Memory and Falsiﬁcation of History”. Press Releases. Available online at: https://www.riigikogu.ee/ en/press-releases/plenary-assembly/riigikogu-passed-statement-historical-memory- falsiﬁcation-history/ (accessed July 4, 2022). falsiﬁcation-history/ (accessed July 4, 2022). Ehala, M. (2018). Signs of Identity: The Anatomy of Belonging. NY: Routledge. doi: 10.4324/9781315271439 Rus.Delﬁ(2022). The oﬃcial website of the Russian version of Delﬁ. Available online at: https://rus.delﬁ.ee/ (accessed August 8, 2022). Fein, L. C., and Straughn, J. B. (2014). How citizenship matters: narratives of stateless and citizenship choice in Estonia. Citizenship Stud. 18, 690–706. doi: 10.1080/13621025.2014.944774 RUS.ERR (2019). Estonian schools cannot handle the inﬂux of Russian pupils (in Russian). Frontiers in Political Science 12 frontiersin.org 10.3389/fpos.2023.1140084 Polynin stat/rahvastik__rahvastikunaitajad-ja-koosseis__rahvaarv-ja-rahvastiku-koosseis/ RV0222U (accessed September 16, 2022). RUS.ERR (2022a). Reform gets behind stripping Russian citizens of right to vote. Available online at: https://news.err.ee/1608721012/reform-gets-behind-stripping- russian-citizens-of-right-to-vote (accessed September 19, 2022). Tajfel, H. (1978). “Social categorization, social identity, and social comparison,” in Diﬀerentiation between social groups: Studies in the social psychology of intergroup relations, ed. H. Tajfel (London: Academic Press) 61–76. RUS.ERR (2022b). Russian students: ’We can’t go back to Russia because we support Ukraine’. Available online at: https://news.err.ee/1608683119/russian-students-we- can-t-go-back-to-russia-because-we-support-ukraine (accessed August 13, 2022). Tajfel, H., and Turner, J. C. (1986). “The social identity theory of intergroup behavior,” in Psychology of Intergroup Relation, eds. S. Worchel, and W. G. Austin (Chicago: Hall Publishers) 7–24. RUS.ERR (2022c). Russian, Belarusian citizens will not be allowed to own ﬁrearms in Estonia. Available online at: https://news.err.ee/1608658276/russian-belarusian- citizens-will-not-be-allowed-to-own-ﬁrearms-in-estonia (accessed August 3, 2022). Vetik, R. (2012). Nation-Building in the Context of Post-Communist Transformation and Globalization. New York, NY: Peter Lang Press. doi: 10.3726/978-3-653- 01615-4 Rus.Postimees (2022). The oﬃcial website of the Russian version of Delﬁ. Available online at: https://rus.postimees.ee/ (accessed August 8, 2022). Vetik, R. (2019). National identity as interethnic (de) mobilization: a relational approach. Ethnopolitics 18, 406–422. doi: 10.1080/17449057.2019. 1613065 Statistics Estonia (2022). Population by ethnic nationality (RV0222U: Rahvastik soo, rahvuse ja maakonna järgi). Available online at: https://andmed.stat.ee/et/ 13 13 Frontiers in Political Science frontiersin.org Frontiers in Political Science
https://openalex.org/W4287279874	https://zenodo.org/records/4589108/files/Safety%20of%20Laparoscopy%20in%20Patients%20with%20Ventriculoperitoneal%20Shunts_Mohamed.pdf	English	null	Safety of Laparoscopy in Patients with Ventriculoperitoneal Shunts	DOAJ (DOAJ: Directory of Open Access Journals)	2,021	cc-by	5,793	IBEROAMERICAN JOURNAL OF MEDICINE 02 (2021) 130-137 IBEROAMERICAN JOURNAL OF MEDICINE 02 (2021) 130-137 ABSTRACT Article history: Received 01 February 2021 Received in revised form 20 February 2021 Accepted 05 March 2021 Keywords: Laparoscopy Surgery Ventriculoperitoneal shunts Safety Article history: Received 01 February 2021 Received in revised form 20 February 2021 Accepted 05 March 2021 Keywords: Laparoscopy Surgery Ventriculoperitoneal shunts Safety Article history: Received 01 February 2021 Received in revised form 20 February 2021 Accepted 05 March 2021 The relationship between the intra-abdominal pressure (IAP) and intracranial pressure (ICP) has been suspected for more than 100 years and was subsequently confirmed by numerous studies in both animals and humans which demonstrate the link and the positive correlation between IAP and ICP. There are mounting concerns that the pneumoperitoneum created during laparoscopic surgery to create space for instrument placement and to allow safe tissue dissection may result in an increase in the ICP secondary to the increase in the IAP which may result in serious consequences in patients with Ventriculoperitoneal (VP) shunts. There is uncertainty about the safety of laparoscopic surgery in VP shunt patients. The aim of this article is to review the literature to answer the question [Is laparoscopic surgery safe in VP shunt patients with and without intraoperative monitoring of ICP]? © 2021 The Authors. Published by Iberoamerican Journal of Medicine. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4.0/). HOW TO CITE THIS ARTICLE: Mohamed SA, Mohamed AA. Safety of Laparoscopy in Patients with Ventriculoperitoneal Shunts. Iberoam J Med. 2021;3(2):130-137. doi: 10.5281/zenodo.4589108. present, the VP shunt is the standard treatment of hydrocephalus. * Corresponding author. E-mail address: sarahabbas_90@hotmail.com ISSN: 2695-5075 / © 2021 The Authors. Published by Iberoamerican Journal of Medicine. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4.0/). http://doi.org/10.5281/zenodo.4589108 Sarah A. Mohamed a,*, Abbas AR Mohamed b a Department of Surgery, University of Wales, Cardiff, Wales,m United Kingdom b Department of General Surgery, National Guard Hospital, Al Madinah, Saudi Arabia Journal homepage: www.iberoamericanjm.tk HOW TO CITE THIS ARTICLE: Mohamed SA, Mohamed AA. Safety of Laparoscopy in Patients with Ventriculoperitoneal Shunts. Iberoam J Med. 2021;3(2):130-137. doi: 10.5281/zenodo.4589108. 2. HISTORICAL BACKGROUND OF VP SHUNT Trepanning was recommended by Hippocrates for the treatment of various diseases, including hydrocephalus, and was widely practiced since 400 BC. It was not until around the 17th century those physicians recognized its definitive role of surgical treatment for hydrocephalus [5]. Many types of VC catheters have been used for VP shunts including silicone (PDMS) catheters, the expanded polytetrafluoroethylene catheters (e-PTFE), VC coated with polyvinylpyrrolidone (PVP). At the turn of the millennium, new advances in VCs were the introduction of catheters (branded Bactiseal), which featured impregnation of 2 antibiotics, Rifampicin and Clindamycin HCL, into the silicone matrix [14]. Historically surprisingly, until the beginning of the present century, the pathology of hydrocephalus remained obscure, with no definitive recognized rational methods of therapy or successful surgical treatment [6]. Many practitioners continued to practice primitive methods in attempts to treat hydrocephalus, including repeated percutaneous punctures, head wrapping, and bloodletting, all with consistently fatal results [7]. Today, most VCs are made of silicone polymer tubes and are available in straight configurations that can be tailed to an appropriate length and angled configurations, which have a set length. Inner diameters of the tubing range between 1.0 mm and 1.6 mm and outer diameters between 2.1 mm and 3.2 mm [15]. During the early 20th century, significant progress was made in both understanding the etiology of hydrocephalus and the development of successful surgical treatments for it. This advancement was led by the pioneers of neurosurgery Sir Victor Horsley in England, and Harvey Cushing, Walter Dandy, and others at Johns Hopkins in the US [8]. No doubt the advances in the fields of biomaterials and biomedical engineering made significant contributions to the quality and ability of implanted CSF shunts to allow many patients with VP shunts to live relatively ordinary lives [13]. Walter Dandy was among the first to describe the basic mechanism and classification of hydrocephalus as obstructive or non-obstructive in 1913. He was the first to establish the principles of treatment of hydrocephalus by either reducing cerebrospinal fluids CSF formation, relieving the obstruction, or diverting the fluid to a part of the body in which it can be readily absorbed [9]. 1. INTRODUCTION Similarly, surgeons resisted and ignored laparoscopy and didn’t attempt to test its suitability for surgical applications since 1901, when Georg Kelling performed the first diagnostic laparoscopy on the peritoneal cavity of a dog using a cystoscope [3]. It was until the late eighties of the last century when the advent of computer chip-based television cameras resulted in its revival [4]. At present, laparoscopic surgery became the standard treatment of much surgical pathology. Although the first attempt of rerouting cerebrospinal (CSF) from the ventricles to the peritoneal cavity (VP shunt) was performed in 1905, the procedure of VP shunt was abandoned for more than the next 30 years [1]. Surgeons resisted the VP shunt techniques because of frequent occlusion of the tubes and recurrent infections. The introduction of silicone rubber tubes that prevented shunts from occlusion and the development of anti-microbial agents resulted in the revival of the producer [1, 2]. At There is an increased survival of patients with VP shunts There is an increased survival of patients with VP shunts IBEROAMERICAN JOURNAL OF MEDICINE 02 (2021) 130-137 131 ‘fell into disrepute’ and was virtually abandoned for more than the next 30 years [1]. Surgeons resisted the VP shunt techniques because of frequent occlusion of the tubes and recurrent infections. due to the advances in the techniques of cerebral shunts and improved medical therapies. At the same time, there is an increasing trend in laparoscopic surgery. Patients with hydrocephalus are living longer and may present with unrelated medical or surgical problems. The introduction of silicone rubber tubes that prevented shunts from occlusion and the development of anti- microbial agents resulted in the revival of the producer of VP shunts. [1, 2] Since the introduction of silicone catheters, obstruction by adhesion to proteins and cells of CSF and infections has been a primary focus of ventricular catheters (VC) researches [13]. 3. HISTORICAL BACKGROUND OF LAPAROSCOPY SURGERY The history of laparoscopy dates back to 1901, when Georg Kelling, a German surgeon, performed diagnostic laparoscopy on the peritoneal cavity of a dog using a cystoscope inserted through a trocar with the creation of pneumoperitoneum with filtered air [3]. Around the same time, the Swedish surgeon Dr. Jacobaeous published reports on laparoscopy on humans in the peritoneal, thoracic, and pericardial cavities and was credited with coining the term “laparoscopy” (“laparothorakoskopie”) [16]. The first sterile ventricular puncture and external ventricular drain insertion was performed by Carl Wernicke in 1881 [10]. External drainage by different devices like silk and catgut wicks became quite popular during the late 19th century [11]. However, due to the risks of open drainage, attempts were made at the beginning of the 20th century to introduce mechanisms for internal CSF diversion. The Polish-Austrian surgeon Jan Mikulicz-Radecki was the first to attempt rerouting of CSF from the ventricles to the subdural space by inserting a mass of glass wool in the shape of a nail into the ventricles of a child in 1893 [12]. During the early 20th century, it astonishes the degree to which the surgeons have ignored laparoscopy and didn’t attempt to test its suitability for surgical applications. However, gastroenterologists, internists, and gynecologists recognized its inherent value [17]. The first attempt of rerouting CSF from the ventricles to the peritoneal cavity VP shunt was performed by Kausch, a German neurosurgeon in 1905. The procedure of VP shunt Since the trials of Georg Kelling and Jacobaeous, no IBEROAMERICAN JOURNAL OF MEDICINE 02 (2021) 130-137 132 surgeon who was the second to perform video laparoscopic cholecystectomy in 1988 and was the first to publish his early experience [23]. The American College of Surgeons introduced the new technology to the general surgery world during the annual meeting of the college in October 1989 [20]. remarkable progress was made until 1929 when Heinz Kalk, a German gastroenterologist, later called Father of Modern Laparoscopy, developed a superior first forward- viewing scope with improved lenses [18]. Heinz's introduction of forward-viewing scope paved the way for the beginning of the era of operative laparoscopy. In 1933 the gynecologist Karl Fervers performed the first laparoscopic operative procedure of lysis of adhesions using cautery [19]. Fevers were followed a few years later by the Swiss gynecologist, Boesch, who performed the first laparoscopic ligation of the Fallopian tubes by electrocoagulation in 1936 [18, 19]. 3. HISTORICAL BACKGROUND OF LAPAROSCOPY SURGERY It didn't take long for the laparoscopic cholecystectomy to replace open cholecystectomy. Many papers were published documenting the advantages of laparoscopic surgery over open surgery in terms of less postoperative pain, early postoperative recovery, and early return to work. Development continued, and still going on, in the field of laparoscopic surgery in terms of improving laparoscopic camera resolutions, the introduction of more refined laparoscopic instruments, and improving surgeon's training to enable surgeons to perform more complex laparoscopic surgical procedures. At present, laparoscopic surgery almost replaced most open surgical procedures. The therapeutic modality of Fervers and Boesch was a definite breakthrough in the field of laparoscopic surgery that laid the foundations for operative laparoscopic surgery; although it took almost one-third of a century since Georg Kelling performed the first diagnostic laparoscopy. The progress continued to be slow during the next 40 years, and operative laparoscopy was limited only to tubal ligations. By the year 1971, 35 years after Fervers and Boesch’s breakthrough, only 1% of tubal ligations were performed laparoscopically in the United States and by 1976, the figure mounted up to reach 60% [19]. 4. THE RELATION BETWEEN INTRA- ABDOMINAL PRESSURE AND INTRACRANIAL PRESSURE The gradual and slow evolution of laparoscopic surgery at its early stages was related to limitations of technology and the skepticism of the medical and surgical communities [20]. The intraabdominal pressure (IAP) is a physiological parameter defined as the steady-state pressure concealed within the abdominal cavity [24]. The intravesicular pressure measurement provides a simple, convenient, and accurate measurement of IAP [25]. Values of up to 5mm of Hg are considered normal in adults under normal physiological conditions. [26]. It was not surprising that the pioneers of laparoscopic surgery, Kurt Semm, a German gynecologist, who was the first to perform laparoscopic appendectomy in 1983, and the German surgeon Erich Muhe who was also the first to perform laparoscopic cholecystectomy by a 3-cm, direct- vision laparoscope of his own design, in 1986, were both suffered skepticism, criticism and experienced many examples of repression by the old guard of traditional surgery [20, 21]. Intracranial pressure is the pressure exerted by fluids such as CSF and blood inside the skull and on the brain tissue. Changes in intracranial pressure (ICP) attributed to changes in the volume of one or more of these constituents. The Monro-Kellie hypothesis states that the cranium is a rigid vault that contains brain tissue, CSF, and blood [27]. If one of the three components increases in size the volume of the other two has to decrease to maintain equilibrium and to prevent a rise of ICP. The most important technological advancement in laparoscopic surgery is the advent of a video laparoscope. Video technology was developing in the 1960s and was being touted for teaching purposes and documentation, as the resolution was not sufficient for operative laparoscopy [22]. The ICP is one of the determinants of the cerebral perfusion pressure (CPP) as the CPP calculated by subtracting the ICP from the mean arterial pressure (MAP): CPP = MAP − ICP [28]. The CPP is normally fairly constant due to autoregulation, but it can be affected to a great extent by sustained changes in the mean arterial pressure and ICP. The advent of computer chip-based television cameras that project a magnified, view of the filmed object onto monitors and TV screens was considered a revolution in the field of video laparoscopy and laparoscopic surgery. 4. THE RELATION BETWEEN INTRA- ABDOMINAL PRESSURE AND INTRACRANIAL PRESSURE The advent of computer chip-based television cameras that project a magnified, view of the filmed object onto monitors and TV screens was considered a revolution in the field of video laparoscopy and laparoscopic surgery. The French surgeon Phillip Mouret performed the first video laparoscopic cholecystectomy in 1987. He was followed shortly by Francois Dubois, another French The French surgeon Phillip Mouret performed the first video laparoscopic cholecystectomy in 1987. He was followed shortly by Francois Dubois, another French The ICP can be measured and monitored by invasive and non-invasive techniques. The invasive techniques include external ventricular drainage through a catheter inserted in IBEROAMERICAN JOURNAL OF MEDICINE 02 (2021) 130-137 133 the lateral ventricles and micro transducer ICP monitoring devices. The non-invasive techniques include optic nerve sheath diameter, CT scan, MRI, transcranial Doppler, tympanic membrane displacement, and funduscopy [29]. demonstrated a significant and linear increase in ICP with increased IAP and Trendelenburg positions. The combination of the Trendelenburg position and increased IAP of 16 mmHg results in an increase in the ICP of 150% over control levels. They concluded that surgeons should take into consideration the IAP and Patient positioning when performing laparoscopy on patients with head trauma, cerebral aneurysms, and conditions associated with increased ICP. External ventricular drainage is the gold standard in terms of accuracy of measurement of ICP pressure, although micro transducers generally are just as accurate. The non- invasive techniques provide reliable alternatives to the invasive techniques and associated with minor risks of complications such as hemorrhage and infection. ICP is usually measured in millimeters of mercury (mmHg) and, at rest, ranges between 7and 15 mmHg for a supine adult [24]. A similar study conducted by Halverson et al. [35] by insufflating carbon dioxide at 1.5 l/min in the abdomen of nine 30-35-kg domestic pigs while recording the ICP, lumbar spinal pressure (LP), central venous pressure (CVP), and some others vital parameters. They recorded the different values of ICP at IAP of 0, 5, 10, and 15 mmHg with animals in supine, Trendelenburg, and Reverse Trendelenburg positions. They reported that the animals showed a significant increase in ICP (mmHg) with each 5- mmHg increase in IAP with a further increase occurred with Trendelenburg's position, without a reduction in reverse Trendelenburg positions. The increase in the IAP correlated with the increases in ICP and LP without significant change in CVP. 4. THE RELATION BETWEEN INTRA- ABDOMINAL PRESSURE AND INTRACRANIAL PRESSURE They concluded that care should be taken with laparoscopy in patients at risk for increased ICP. They also suggested that the mechanism of increased ICP associated with insufflations is most likely the impairment of the drainage of the lumbar venous plexus at an increased IAP. The relationship between the IAP and the ICP has been suspected for more than 100 years, but it was not clearly identified until Breschet demonstrated the multiple anastomoses and connections between the intracranial venous system and the vertebral venous system in the period between 1828 and 1832 [24]. Batson illustrated in 1957 the extent of connections of the multiple veins of the valveless spinal epidural venous network which was named after him. Batson's work explained the spread of metastasis and infections through these vertebral veins, into the spine and the central nervous system while bypassing both the liver and the lungs [30, 31]. The current evidence strongly supports that the IAP is transmitted to the central nervous system by two pathways. One pathway is retrograde flow through the venous plexus of the spinal canal and the intracranial veins. The second pathway is direct as elevations in the IAP transferred into the thoracic compartment, which in turn results in back pressure on the jugular veins and decreases the drainage of the CSF and blood, leading to an increased ICP [32]. Similarly, Josephs et al. [36] investigated the effect of pneumoperitoneum on ICP and CPP in an animal model of five 30-kg pigs. They monitored ICP, MAP, arterial blood gases, and IAP for 30 minutes before, during, and after the creation of pneumoperitoneum. They demonstrated a positive correlation between IAP and ICP that was independent of changes in arterial PCO2 or arterial PH. They advised that laparoscopy for evaluation of abdominal trauma victims must be used cautiously in patients with severe head injuries. The link and positive correlation between intra-abdominal pressure and intracranial pressure were confirmed by numerous studies in animals [33-36]. Bloomfield G.et al. [33] studied the effects of elevated IAP upon ICP and CPP in an animal model of five anesthetized swine. They increased the IAP to 25 mm Hg above baseline by inflating a balloon inserted inside the peritoneal cavity of the swine, measuring at the same time changes in ICP. They demonstrated a significant and linear increase in ICP with increased IAP and concluded that elevated IAP increases ICP and decreases CPP. 5. LAPAROSCOPIC SURGERY IN PATIENTS WITH VP SHUNTS There is an increased survival of patients with VP shunts due to the advances in the techniques of cerebral shunts and improved medical therapies. At the same time, there is an increasing trend in laparoscopic surgery. Patients with hydrocephalus are living longer and may present with unrelated medical or surgical problems [37]. It estimated that the number of patients with CSF shunts in the United States to be greater than 125,000 in 1995 [38]. Many In another animal study, Rosenthal et al. [34] studied the effect of pneumoperitoneum on the ICP in a large animal model of five pigs by recording arterial blood gases, mean arterial blood pressure, and ICP at different measures of IAP both in the supine and Trendelenburg positions. They IBEROAMERICAN JOURNAL OF MEDICINE 02 (2021) 130-137 134 patients with VP shunt may present with an indication for laparoscopic surgery. tubes subjected to increased backpressure, and none of them showed signs of valve failure. The risk of valve failure is noticed to be minimal even with IAP as high as 80 mm of Hg [45]. There are no absolute contraindications for laparoscopy in patients with VP shunts, although there are always concerns about the risks of an increase in the ICP secondary to an increase in IAP during laparoscopic surgery. The abdomen is usually insufflated during laparoscopic procedures with carbon dioxide to intraabdominal pressure of 12 to15 mmHg to create space for instrument placement and to allow safe tissue dissection during laparoscopic procedures. Similarly, Matsumoto et al. [46] studied five different valves simulating a closed system in Japan in 2010. There was no reflux of the CO2 for any of the valves with a pressure of less than 25 mm Hg.8 Surgeons have always been concerned about protecting shunts from potential reflux during laparoscopic surgery on patients with VP shunts. Different methods were advised for temporal protection of the VP shunts during laparoscopic procedures including clamping the shunt catheter intra-abdominally or through a skin incision, and externalizations of the shunt before carbon dioxide insufflation [46]. Some authors advised neurosurgery consultation before surgery to verify the proper function of the VP shunt [37]. The effect of increased intra-abdominal pressures in patients with VP shunts was extensively studied. Raised IAP that occur in individuals with ileus, small-bowel obstruction constipation, has been reported to play a role in malfunctioning VP shunts in patients with hydrocephalus [39]. 5. LAPAROSCOPIC SURGERY IN PATIENTS WITH VP SHUNTS Many factors have been thought to be the cause of obstruction or malfunction of VP shunts during laparoscopic procedures. Uzzo et al. [40] suggested in addition to the increases in ICP secondary to increases in the IAP, the pneumoperitoneum may increase the resistance to outflow through the distal peritoneal catheter, causing a partial or complete shunt obstruction. The safety of laparoscopic surgery in patients with VP shunts has always been controversial because of the potential risk of an increase in ICP, shunt malfunction, and infection. There is also the question of the need for routine monitoring of ICP intra-operatively [47]. Unfortunately, there is little published data on the issue due to the small number of reported cases and the lack of well-designed studies that include a large number of patients. Cobianchi et al. [41] suggested that obstruction of the antegrade flow of the cerebrospinal fluid as a result of increased IAP together with the retrograde passage of carbon dioxide through the shunt catheter result in a sudden increase in ICP during laparoscopic procedures. The hypercapnia-induced cerebral arterial dilatation and venous pressure elevation cause increased intracranial blood volume and increased ICP in the fixed volume of the cranium. Many authors reported the safety of laparoscopic surgery in patients with VP shunt without any precautions apart from routine anesthetic monitoring. On the other hand, some authors reported potential dangerous complications of laparoscopy in patients with VP shunts. A series of 4 patients with VP shunts who had laparoscopic cholecystectomy without intraoperative ICP monitoring was published by Collure et al. [42]. All patients didn’t show central nervous system sequelae postoperatively and the shunts remained intact and functioning. The authors concluded that laparoscopic cholecystectomy in patients with VP is safe without the need for invasive intraoperative monitoring of ICP or manipulation of the shunt. The risk of retrograde passage of carbon dioxide from the abdomen to the brain is minimal with advances in the fields of biomaterials and biomedical engineering and the advent of one-way valve VP shunt catheters that can withstand significantly high IAP pressures before allowing such reflux [42]. The shunt valve's hydrodynamic profile, as derived by catheter manufacturers, is a standard parameter that indicates the pressure that the valve can tolerate before allowing retrograde flow to occur. Most shunts have a one- way valve that can withstand a pressure of 300 mmHg before allowing retrograde flow. Collude et al. 5. LAPAROSCOPIC SURGERY IN PATIENTS WITH VP SHUNTS [43] suggested that pressure of 12-15 mmHg which is used to insufflate the abdomen during laparoscopic surgical procedures is unlikely to produce pneumocephalus. A retrospective study conducted by Jackman et al. [48] reviewing the anesthesia records of 18 patients with VP shunt who underwent 19 consecutive laparoscopic operations, looking for signs of increased ICP. They didn't document any evidence of clinically increased ICP and concluded that invasive methods for shunt monitoring are usually not required as routine anesthetic monitoring should remain the standard of care. In another retrospective study conducted by Fraser et al. [49] reviewing all pediatric patients with VP shunts who underwent laparoscopic and open abdominal operations in The risk of valve failure of shunt valves (in vitro model) was studied by Neale et al. [44] in nine different shunt IBEROAMERICAN JOURNAL OF MEDICINE 02 (2021) 130-137 135 monitoring of ICP intra-operatively. monitoring of ICP intra-operatively. monitoring of ICP intra-operatively. their institute in the period from 1998 to 2008. A total of 51 patients were operated laparoscopically out of 99 patients. They reported that there was no air embolism into the shunt in the laparoscopic group. Shunt infection occurred in one patient in the laparoscopic group in comparison to 3 patients in the open group. g p y The safety of laparoscopic surgery in patients with VP shunts has always been controversial. At present, there is no strong evidence to establish a solid consensus on the safety of laparoscopic surgery in patients with VP shunts. However, surgeons are advised to consider certain precautions that may reduce the risk of laparoscopy in this group of patients. As there is only little published data regarding the safety of laparoscopic surgery in patients with VP shunts due to the small number of reported cases and lacks of well-designed studies include a large number of patients, we recommend all cases of laparoscopic surgery in patients with VP shunts be reported to build up base data for futures studies. Yoshihara et al. [50] were performed laparoscopic cholecystectomy in four patients with shunts (two with ventriculoperitoneal shunts, and two with lumboperitoneal shunts). The shunt catheters were clamped during the pneumoperitoneum in three patients and the intraabdominal pressure was kept at 8 mmHg. They reported that all cases experienced an uneventful postoperative course, with no shunt-associated complications. 5. LAPAROSCOPIC SURGERY IN PATIENTS WITH VP SHUNTS A retrospectively from japan reported safe laparoscopic colorectal surgery in four patients with VP shunt who were operated with the pneumoperitoneum pressure set at 10 mmHg under routine anesthetic monitoring and without any manipulations such as clamping or externalization of the VP catheters [51]. One case of VP shunt failure in a patient with shunt- dependent hydrocephalus after laparoscopic placement of feeding jejunostomy was reported by Baskin et al. [52] postoperatively; the patient developed clinical and radiographic evidence of shunt failure and underwent emergent shunt revision that revealed an isolated distal shunt obstruction. They concluded that laparoscopic surgery represents a potential danger in patients with pre- existing CSF shunts. Schwed et al. [53] reported a case of a 73-year-old woman who had laparoscopic cholecystectomy 10 days after having insertion of a VP shunt. The patient suffered subcutaneous emphysema and impaired respiration immediately after surgery. The patient recovered uneventfully with no evidence of postoperative infection. 7. REFERENCES 1. Jackson IJ. A review of the surgical treatment of internal hydrocephalus. J Pediatr. 1951;38(2):251-8. doi: 10.1016/s0022-3476(51)80117-3. 1. Jackson IJ. A review of the surgical treatment of internal hydrocephalus. J Pediatr. 1951;38(2):251-8. doi: 10.1016/s0022-3476(51)80117-3. 26. De Keulenaer BL, De Waele JJ, Powell B, Malbrain ML. What is normal intra-abdominal pressure and how is it affected by positioning, body mass and positive end-expiratory pressure? Intensive Care Med. 2009;35(6):969-76. doi 10.1007/s00134-009-1445-0. 2. Alexander E Jr. Archaeology: the big neurosurgical dig. Clin Neurosurg.;28:323-73. doi: 10.1093/neurosurgery/28.cn_suppl_1.323. 27. Wilson MH. Monro-Kellie 2.0: The dynamic vascular and venous pathophysiological components of intracranial pressure. J Cereb Blood Flow Metab. 2016;36(8):1338-50. doi: 10.1177/0271678X16648711. 3. Lau WY, Leow CK, Li AK. History of endoscopic and laparoscopic surgery. World J Surg. 1997;21(4):444-53. doi: 10.1007/pl00012268. 4. Stellato TA. History of laparoscopic surgery. Surg Clin North Am. 1992;72(5):997-1002. doi: 10.1016/s0039-6109(16)45826-3. 28. Steiner LA, Andrews PJ. Monitoring the injured brain: ICP and CBF. Br J Anaesth. 2006;97(1):26-38. doi: 10.1093/bja/ael110. 5. Goodrich JT. History of Hydrocephalus and Its Surgical Treatment. In: Cinalli G., Ozek M., Sainte-Rose C, editors. Pediatric Hydrocephalus. Springer. 2018:1-45. doi: 10.1007/978-3-319-31889-9_34-1. 29. Raboel PH, Bartek J Jr, Andresen M, Bellander BM, Romner B. Intracranial Pressure Monitoring: Invasive versus Non-Invasive Methods-A Review. Crit Care Res Pract. 2012;2012:950393. doi: 10.1155/2012/950393. 6. Scarff JE. Treatment of hydrocephalus: an historical and critical review of methods and results. J Neurol Neurosurg Psychiatry. 1963;26(1):1-26. doi: 10.1136/jnnp.26.1.1. 30. Batson OV. The function of the vertebral veins and their role in the spread of metastases. Ann Surg. 1940;112(1):138-49. doi: 10.1097/00000658- 194007000-00016. 7. Rachel RA. Surgical treatment of hydrocephalus: a historical perspective. Pediatr Neurosurg. 1999;30(6):296-304. doi: 10.1159/000028814. 31. Doepp F, Schreiber SJ, Wandinger KP, Trendelenburg G, Valdueza JM. Multiple brain abscesses following surgical treatment of a perianal abscess. Clin Neurol Neurosurg. 2006;108(2):187-90. doi: 10.1016/j.clineuro.2004.11.024. 8. Corsello A, Di Dalmazi G, Pani F, Chalan P, Salvatori R, Caturegli P. Walter E. Dandy: his contributions to pituitary surgery in the context of the overall Johns Hopkins Hospital experience. Pituitary. 2017;20(6):683-91. doi: 10.1007/s11102-017-0834-6. 32. Scalea TM, Bochicchio GV, Habashi N, McCunn M, Shih D, McQuillan K, et al. Increased intra-abdominal, intrathoracic, and intracranial pressure after severe brain injury: multiple compartment syndrome. J Trauma. 2007;62(3):647-56; discussion 656. doi: 10.1097/TA.0b013e31802ee542. 9. Dandy WE, Blackfan KD. Internal hydrocephalus: A clinical and pathological study. Am J Dis Child. 1914;VIII(6):406-82. doi:10.1001/archpedi.1914.02180010416002. 33. Bloomfield GL, Ridings PC, Blocher CR, Marmarou A, Sugerman HJ. 7. REFERENCES Effects of increased intra-abdominal pressure upon intracranial and cerebral perfusion pressure before and after volume expansion. J Trauma. 1996;40(6):936-41; discussion 941-3. doi: 10.1097/00005373-199606000- 00012. 10. Keen WW. Surgery of the lateral ventricles of the brain. Lancet. 1890;136(3498):553-5. doi: 10.1016/S0140-6736(00)48676-9. 11. Srinivasan VM, O'Neill BR, Jho D, Whiting DM, Oh MY. The history of external ventricular drainage. J Neurosurg. 2014;120(1):228-36. doi: 10.3171/2013.6.JNS121577. 34. Rosenthal RJ, Hiatt JR, Phillips EH, Hewitt W, Demetriou AA, Grode M. Intracranial pressure. Effects of pneumoperitoneum in a large-animal model. Surg Endosc. 1997;11(4):376-80. doi: 10.1007/s004649900367. 12. Davidoff LM. Treatment of hydrocephalus: Historical review and description of a new method. Arch Surg. 1929;18(4):1737-62. doi:10.1001/archsurg.1929.01140130837055. 35. Halverson A, Buchanan R, Jacobs L, Shayani V, Hunt T, Riedel C, et al. Evaluation of mechanism of increased intracranial pressure with insufflation. Surg Endosc. 1998;12(3):266-9. doi: 10.1007/s004649900648. 13. Weisenberg SH, TerMaath SC, Seaver CE, Killeffer JA. Ventricular catheter development: past, present, and future. J Neurosurg. 2016;125(6):1504-12. doi: 10.3171/2015.12.JNS151181. 36. Josephs LG, Este-McDonald JR, Birkett DH, Hirsch EF. Diagnostic laparoscopy increases intracranial pressure. J Trauma. 1994;36(6):815-8; discussion 818-9. doi: 10.1097/00005373-199406000-00011. 14. Sylvia K. Sylvia K: AdvaMed Combination Products Workshop Case Study–Antibacterial Orthopedic and Neurological Devices. Available from: http://www.fda.gov.tw/upload/189/Content/2013011112025251410.pdf (accessed Feb 2021). 37. Hammill CW, Au T, Wong LL. Laparoscopic cholecystectomy in a patient with a ventriculoperitoneal shunt. Hawaii Med J. 2010;69(4):103-4. 38. Bondurant CP, Jimenez DF. Epidemiology of cerebrospinal fluid shunting. Pediatr Neurosurg. 1995;23(5):254-8; discussion 259. doi: 10.1159/000120968. 15. Drake JM, Rose CS. The Shunt Book: New York:Wiley; 1995. 16. Vecchio R, MacFayden BV, Palazzo F. History of laparoscopic surgery. Panminerva Med. 2000;42(1):87-90. 39. Martínez-Lage JF, Martos-Tello JM, Ros-de-San Pedro J, Almagro MJ. Severe constipation: an under-appreciated cause of VP shunt malfunction: a case-based update. Childs Nerv Syst. 2008;24(4):431-5. doi: 10.1007/s00381- 007-0514-3. 17. Litynski GS. Highlights in the History of Laparoscopy: the Development of Laparoscopic Techniques-a Cumulative. Effort of Internists, Gynecologists and Surgeons. Frankfurt: Barbara Bern-ert Verlag; 1996. 18. Nezhat C, Page B. Let There Be Light: a Historical Analysis of Endoscopy's Ascension since Antiquity. Available from: http://sls.org/nezhats- history-of-endoscopy/ (accessed Feb 2021). 40. Uzzo RG, Bilsky M, Mininberg DT, Poppas DP. Laparoscopic surgery in children with ventriculoperitoneal shunts: effect of pneumoperitoneum on intracranial pressure--preliminary experience. Urology. 1997;49(5):753-7. doi: 10.1016/S0090-4295(97)00233-1. 19. Kaiser AM, Corman ML. History of laparoscopy. Surg Oncol Clin N Am. 2001;10(3):483-92. 41. Cobianchi L, Dominioni T, Filisetti C, Zonta S, Maestri M, Dionigi P, et al. 6. CONCLUSIONS VP shunt is the standard treatment of hydrocephalus throughout the world. There has been increasing use of laparoscopic in daily surgical practices as well as an increasing number of patients with VP shunts due to the advances in the techniques of cerebral shunts and improved medical therapies. Surgeons may be faced with patients of VP shunts presenting with an indication for laparoscopic surgery. Surgeons have always been concerned about the risks of an increase in the intracranial pressures secondary to increases in the IAP during laparoscopic surgery on patients with VP shunts. There is also the question of the need for routine IBEROAMERICAN JOURNAL OF MEDICINE 02 (2021) 130-137 136 25. Sugrue M, De Waele JJ, De Keulenaer BL, Roberts DJ, Malbrain ML. A user's guide to intra-abdominal pressure measurement. Anaesthesiol Intensive Ther. 2015;47(3):241-51. doi: 10.5603/AIT.a2015.0025. 46. Matsumoto T, Endo Y, Uchida H, Kusumoto T, Muto Y, Kitano S. An examination of safety on laparoscopic surgery in patients with ventriculoperitoneal shunt by a CO2 reflux experiment. J Laparoendosc Adv Surg Tech A. 2010;20(3):231-4. doi: 10.1089/lap.2010.0038. 49. Fraser JD, Aguayo P, Sharp SW, Holcomb III GW, Ostlie DJ, St Peter SD. The safety of laparoscopy in pediatric patients with ventriculoperitoneal shunts. 45. Al-Mufarrej F, Nolan C, Sookhai S, Broe P. Laparoscopic procedures in adults with ventriculoperitoneal shunts. Surg Laparosc Endosc Percutan Tech. 2005;15(1):28-9. doi: 10.1097/01.sle.0000153733.78227.8f. 47. Martínez Ramos D, Gibert Gerez J, Salvador Sanchís JL. [Laparoscopic surgery in patients with a ventriculoperitoneal shunt]. Rev Esp Enferm Dig. 2006;98(10):795-6. doi: 10.4321/s1130-01082006001000015. 53. Schwed DA, Edoga JK, McDonnell TE. Ventilatory impairment during laparoscopic cholecystectomy in a patient with a ventriculoperitoneal shunt. J Laparoendosc Surg. 1992;2(1):57-9. doi: 10.1089/lps.1992.2.57. 48. Jackman SV, Weingart JD, Kinsman SL, Docimo SG. Laparoscopic surgery in patients with ventriculoperitoneal shunts: safety and monitoring. J Urol. 2000;164(4):1352-4. 45. Al-Mufarrej F, Nolan C, Sookhai S, Broe P. Laparoscopic procedures in adults with ventriculoperitoneal shunts. Surg Laparosc Endosc Percutan Tech. 2005;15(1):28-9. doi: 10.1097/01.sle.0000153733.78227.8f. 46. Matsumoto T, Endo Y, Uchida H, Kusumoto T, Muto Y, Kitano S. An examination of safety on laparoscopic surgery in patients with ventriculoperitoneal shunt by a CO2 reflux experiment. J Laparoendosc Adv Surg Tech A. 2010;20(3):231-4. doi: 10.1089/lap.2010.0038. 47. Martínez Ramos D, Gibert Gerez J, Salvador Sanchís JL. [Laparoscopic surgery in patients with a ventriculoperitoneal shunt]. Rev Esp Enferm Dig. 2006;98(10):795-6. doi: 10.4321/s1130-01082006001000015. 48. Jackman SV, Weingart JD, Kinsman SL, Docimo SG. Laparoscopic surgery in patients with ventriculoperitoneal shunts: safety and monitoring. J Urol. 2000;164(4):1352-4. 49 Fraser JD Aguayo P Sharp SW Holcomb III GW Ostlie DJ St Peter SD 50. Yoshihara T, Tomimaru Y, Noguchi K, Nagase H, Hamabe A, Hirota M, et al. Feasibility of laparoscopic cholecystectomy in patients with cerebrospinal fluid shunt. Asian J Endosc Surg. 2017;10(4):394-8. doi: 10.1111/ases.12380. 52. Baskin JJ, Vishteh AG, Wesche DE, Rekate HL, Carrion CA. Ventriculoperitoneal shunt failure as a complication of laparoscopic surgery. JSLS. 1998;2(2):177-80. 7. REFERENCES Ventriculoperitoneal shunt and the need to remove a gallbladder: Time to definitely overcome the feeling that laparoscopic surgery is contraindicated. Ann Med Surg (Lond). 2014;3(3):65-7. doi: 10.1016/j.amsu.2014.03.005. 20. Kelley WE Jr. The evolution of laparoscopy and the revolution in surgery in the decade of the 1990s. JSLS. 2008;12(4):351-7. 21. Périssat J, Collet D, Monguillon N. Advances in laparoscopic surgery. Digestion. 1998;59(5):606-18. doi: 10.1159/000007535. 42. Sankpal R, Chandavarkar A, Chandavarkar M. Safety of Laparoscopy in Ventriculoperitoneal Shunt Patients. J Gynecol Endosc Surg. 2011;2(2):91-3. doi: 10.4103/0974-1216.114082. 22. Berci G, Davids J. Endoscopy and television. Br Med J. 1962;1(5292):1610-3. doi: 10.1136/bmj.1.5292.1610. 43. Collure DW, Bumpers HL, Luchette FA, Weaver WL, Hoover EL. Laparoscopic cholecystectomy in patients with ventriculoperitoneal (VP) shunts. Surg Endosc. 1995;9(4):409-10. doi: 10.1007/BF00187161. 23. Dubois F, Icard P, Berthelot G, Levard H. Coelioscopic cholecystectomy. Preliminary report of 36 cases. Ann Surg. 1990;211(1):60-2. doi: 10.1097/00000658-199001000-00010. 44. Neale ML, Falk GL. In vitro assessment of back pressure on ventriculoperitoneal shunt valves. Is laparoscopy safe? Surg Endosc. 1999;13(5):512-5. doi: 10.1007/s004649901024. 24. Depauw PRAM, Groen RJM, Van Loon J, Peul WC, Malbrain MLNG, De Waele JJ. The significance of intra-abdominal pressure in neurosurgery and neurological diseases: a narrative review and a conceptual proposal. Acta Neurochir (Wien). 2019;161(5):855-64. doi: 10.1007/s00701-019-03868-7. IBEROAMERICAN JOURNAL OF MEDICINE 02 (2021) 130-137 137 J Laparoendosc Adv Surg Tech A. 2009;19(5):675-8. doi: 10.1089/lap.2009.0116. J Laparoendosc Adv Surg Tech A. 2009;19(5):675-8. doi: 10.1089/lap.2009.0116. 50. Yoshihara T, Tomimaru Y, Noguchi K, Nagase H, Hamabe A, Hirota M, et al. Feasibility of laparoscopic cholecystectomy in patients with cerebrospinal fluid shunt. Asian J Endosc Surg. 2017;10(4):394-8. doi: 10.1111/ases.12380. 51. Ichikawa Y, Matsuda C, Mizushima T, Takahashi H, Miyoshi N, Haraguchi N, et al. Safety of laparoscopic colorectal surgery in patients with ventriculoperitoneal shunt. Asian J Endosc Surg. 2019;12(3):264-8. doi: 10.1111/ases.12640. 52. Baskin JJ, Vishteh AG, Wesche DE, Rekate HL, Carrion CA. Ventriculoperitoneal shunt failure as a complication of laparoscopic surgery. JSLS. 1998;2(2):177-80. 53. Schwed DA, Edoga JK, McDonnell TE. Ventilatory impairment during laparoscopic cholecystectomy in a patient with a ventriculoperitoneal shunt. J Laparoendosc Surg. 1992;2(1):57-9. doi: 10.1089/lps.1992.2.57.
https://openalex.org/W2891484191	https://journals.uct.ac.za/index.php/JARER/article/download/553/541	English	null	A Performance Assessment of Local Authorities in Managing Public Housing in Ghana	Journal of African real estate research	2,018	cc-by	9,872	Journal of African Real Estate Research Volume 3, Issue 1 Journal of African Real Estate Research Volume 3, Issue 1 www.journals.uct.ac.za/index.php/JARER/index Abstract The level of maintenance and the condition of public housing has been a significant concern for tenants and housing researchers alike. The state of public housing is often a reflection of its local authority managers and the policy that surrounds it. This paper assesses the performance of local authorities in the management of public housing in Ghana. Data was collected through interviews with local authority housing officers and tenants as well as a small sample survey of tenants. The performance of the authorities mentioned above was measured by three factors; adequacy (house types and quantity), decency (maintenance and satisfaction with maintenance), and affordability (rent levels). The paper concludes that the current state of Ghanaian public housing, when measured against its mandate to provide adequate, decent and affordable housing to beneficiary government workers is substandard due to underperforming local authorities (LAs). The paper suggests that rent policy should be reviewed to empower LAs to self-determine and collect rent. Rents in the public housing sector should be reviewed to realistic levels so that they may generate funds for maintenance. Housing policy should institute internal and external mechanisms to monitor the performance of LAs in the management of public housing. Keywords: Performance Assessment; Local Authorities; Management; Public Housing; Ghana Keywords: Performance Assessment; Local Authorities; Management; Public Housing; Ghana Keywords: Performance Assessment; Local Authorities; Management; Public H Ghana A Performance Assessment of Local Authorities in Managing Public Housing in Ghana Samson Aziabah1,2 1 Department of Real Estate and Land Management, Faculty of Planning and Land Management, University for Development Studies, Ghana 2 Department of Management in the Built Environment, Faculty of Architecture and the Built Environment, Delft University of Technology, Netherlands To cite this article: Aziabah, S. (2018). A Performance Assessment of Local Authorities in Managing Public Housing in Ghana. Journal of African Real Estate Research, 3(1), pp.39-60. DOI: 10.15641/jarer.v1i1.553. 1. Introduction Global policy on housing has shifted from direct state-led housing production to the enablement approach whereby the state assumes the role of facilitator. These changes were initiated in a bid to provide adequate and decent housing 1 +233244732781; akanvose@gmail.com 2 +31685010725; a.b.aziabahakanvose@tudelft.nl 1 +233244732781; akanvose@gmail.com 2 +31685010725; a.b.aziabahakanvose@tudelft.nl Journal of African Real Estate Research Volume 3(1) 2018 39-60 to an increasing global population. Furthermore, these changes were influenced by the United Nations’ (1948) declaration which states that the right to decent housing is a human right (United Nations, 1948). In line with these global trends, Ghana has had a history of significant investment in housing production by the state. For instance, the colonial government in Ghana built houses for civil servants (Yankson & Gough, 2014); and veteran soldiers of the Second World War (Arku, 2006; Arku, 2009). In addition, the first and subsequent governments since Ghana’s independence in 1957 have built houses through state agencies such as the State Housing Corporation (SHC) and the Tema Development Corporation (TDC). However, after a change to the enablement approach, most of the houses were sold to individuals and institutions. The remainder were transferred to local authorities (LAs) to manage. Nevertheless, public housing remains significant as it facilitates labour mobility, especially for the civil servants it targets. It also serves as a form of social support in an economy where wages are said to be low (Arku, 2009). Local Authority Managing Public Houses in Ghana Local authority (LA) housing, that is; public housing managed by the district or municipal assemblies, can be found across the country in varied forms and scale (Ginsburg, 2005). The number of housing units in an estate range from 25 to 100 or more. The housing stock in a district can vary between 100 and 500 or more units. They are mostly located on prime land in an urban area. The housing types include single or two-bedroom detached or semi-detached houses, terraced houses, two-or-more-bedroom multi-storey apartments, and detached bungalows (Tipple et al., 2004). However, it should be noted that not all the types of houses can be found in all municipalities. Following the expansion of the responsibilities of LAs as part of the decentralisation of governance, and pursuant to the Local Government Act 1993, Act 462 (replaced by Local Governance Act 2016, Act 936), the Local Government Instrument 2009, Legislative Instrument (LI) 1961 establishes the Department of Works with a responsibility to: • Encourage and facilitate maintenance of public buildings and facilities in the district; • assist in the maintenance of public buildings made up of offices, residential accommodations, and ancillary structures; and • facilitate the registration and maintenance of data on public buildings. In practice, an officer is responsible for managing publicly owned houses, and an allocation committee assists him. Despite the clear responsibility of LAs, many authors (Tipple, 1999; Tufuor, 2004; Asabere, 2007) have been concerned about the quality of public housing because of their poor maintenance. These buildings are not well maintained, and consequently, their state causes agitation for some tenants (Benson, 2014). Poor public housing conditions have implications on the economy and society, including loss of investment return, shortening of the economic lifespan of the houses, and negative impact on the productivity of occupants (Tipple, 1999; Asabere, 2007). The problem of poor maintenance and the consequences on the quality 40 Journal of African Real Estate Research Volume 3(1) 2018 39-60 of housing in Ghana have often been blamed on poor management (Obeng- Odoom, 2011b). However, many of the studies (Konadu-Agyemang, 2001; Tufuor, 2004; Obeng-Odoom, 2011b, Obeng-Odoom & Amedzro, 2011) that have considered housing in Ghana have not assessed the performance of LAs in the management of public housing. Local Authority Managing Public Houses in Ghana Furthermore, studies that have assessed the performance of housing management have primarily done so from a tenant-satisfaction assessment perspective (Baiden et al., 2010; Ilesanmi, 2010; Olawore et al., 2011) or in relation to housing features or project location (Eyiah-Botwe, 2015). Not many authors have focused on assessing housing management performance from the viewpoint of both tenants and managers in relation to identified goals of management. Therefore, this paper aims to assess the performance of LAs as managers of public housing in Ghana. By assessing the performance of local authority management against the goals of public housing, targeted interventions can be suggested for improvement. Also, the outcome of performance assessment may give legitimacy and affirm the mandate of LAs to manage and maintain public housing (De Bruijn, 2002a). This paper presents a performance assessment of housing management primarily based on qualitative data derived from the perspective of managers and tenants. To achieve its aim, the paper answers the following questions: What is the goal of public housing in Ghana? To what extent is the goal of public housing being achieved? Moreover, what can be done to improve the performance of LAs as housing managers? The first section of this paper introduces local authority managed public housing in Ghana. The second section reviews the literature on performance assessment and concludes with the performance indicators used in this paper. The methods and approach to the study are presented in section three. The findings are presented in section four, while section five discusses the implications of the findings. The conclusion of the paper is presented in section six. 2.1 Performance Assessment in Public Housing Performance assessment is a widely used method of assessing the performance of public organisations against set objectives (Walker, 1994). Performance information may be compared with “price” and “quality” information in the private sector (Johnsen, 2005). Performance assessment is a New Public Management (NPM) tool introduced for clarifying the output of non-monetary services such as housing in the public sector (Kemp, 1995; Walker & van der Zon, 2000; Askim, 2009). Public housing provision is driven by social goals (Boyne, 2002). Therefore, performance assessment in public housing enables us to assess whether public housing managers are achieving social goals. 2. Literature Review Research into public housing is ubiquitous in academic literature. Various researchers have evaluated conditions and tenant satisfaction in public housing (Kaitilla, 1993; Komu, 2010; Ibem & Aduwo, 2013; Ibem & Amole, 2013); or have focused on transformations in public housing (Tipple, 1999; Tipple et al., 2004). Others have examined policy in public housing (Arku, 2006; Arku, 2009; Huchzermeyer, 2014), or discussed housing management (Obeng-Odoom, 2011b). Some authors have evaluated performance in public housing (Walker & Murie, 2004). While adequate research has been conducted in various aspects of housing, very little research has been conducted on the performance of housing management in a developing country context such as Ghana. Available literature on performance assessment largely relates to a developed country context (Boyne, 1997; Arimah, 2000; Boyne, 2002; Koopman et al., 2008). For instance, Walker and Boyne (2006) have assessed the impact of public sector reform on the performance of LAs in the United Kingdom. They collected information on 41 Journal of African Real Estate Research Volume 3(1) 2018 39-60 outputs, efficiency, effectiveness, value for money and consumer satisfaction. The systems approach was used by (Straub et al., 2010) to measure performance in Dutch housing associations by identifying indicators related to inputs, throughputs, outputs and outcomes. These studies underscore the importance of performance assessment in housing and show varied purposes, subjects and indicators of performance assessment. (a) What to Measure in Assessing Public Housing Management The widely used criteria that have dominated the literature on public sector performance measurement are famously known as the four “Es” –efficiency, effectiveness, economy and equity (Smith, 1995; Kendall & Knapp, 2000; Bouckaert & van Dooren, 2009). However, it has been argued that performance measurement should not be limited to these measurable aspects but should include quality of service (Jacobs & Manzi, 2000). Some argue that it should incorporate social, economic and environmental inputs and impacts (van Bortel & Gruis, 2011). Indeed, some authors (Kemp, 1995; Walker & Murie, 2004; Bouckaert & van Dooren, 2009) have argued that easy to collect indicators may not necessarily present an accurate reflection of housing management. Even though data about the quality of service indicators may be difficult to collect, they are critical in communicating the correct level of performance. Therefore, the question “what to measure” seems to be answered by a combination of measurable indicators where available (outputs), with other quality of life information (outcomes) to assess public housing performance. (b) Indicators of Performance Assessment in Public Housing Indicators are facts which help assess the achievement of targets and objectives (Smith & Walker, 1994). They give information that describes non-financial inputs, throughputs, outputs and outcomes (Askim, 2009). Therefore, indicators must be “smart measures” and should match the mission, goals and objectives of the organisation (De Bruijn, 2002b; Anheier, 2005; Terence, 2008). In public housing, the objectives may be derived from policy documents or legislation (Bouckaert & van Dooren, 2009). For example, the goal of public housing in Ghana may be discerned from policy documents regarding the accommodation of civil servants (UN-HABITAT, 2011). Therefore, indicators to assess housing performance must reflect the 42 Journal of African Real Estate Research Volume 3(1) 2018 39-60 interest of policymakers, and importantly, the concerns of its beneficiaries (Symon & Walker, 1995; Walker, 2001; Walker & Murie, 2004; Terence, 2008). The alternative is to seek the views of beneficiaries about housing management services directly. Finally, in choosing indicators of performance, one must be guided by the availability and ease of obtaining information. In light of the above, it is necessary to consider what measures and indicators are appropriate to measure public housing performance in Ghana. 2.2 Indicators of Performance Assessment in Public Housing in Ghana The starting point for performance assessment of local authority managed public housing in Ghana is to identify the goal of public housing. Ghana has no specific policy on the matter as the National Housing Policy (2015) does not outline specific goals for public housing. However, it may be inferred from National Policy. The National Housing Policy of Ghana (2015) outlines four primary goals: • To provide adequate, decent and affordable housing that is accessible to satisfy the needs of all people living in Ghana • To ensure that housing is designed and built to sustainable building principles leading to the creation of green communities • To ensure that there is the participation of all stakeholders in decision- making on housing development and allocation in their localities; and • To ensure adequate and sustainable funding for the supply of diverse mix of housing in all localities. (Government of Ghana, 2015) For the purpose of this paper, the first goal of the National Housing Policy is adapted for public housing. Thus, the goal of public housing in Ghana may be said to be: to provide adequate, decent and affordable housing that satisfies the need of beneficiary groups [government employees]. Adequacy describes housing availability in sufficient number and type that allows beneficiaries to choose, thereby enhancing mobility (Olawore et al., 2011). Decent describes dwellings meeting acceptable physical standards (Stone, 2006; Olawore et al., 2011). That is freedom from repair and maintenance (Stone & Hartman, 1983; van Mossel, 2008). Affordability defined for this study is the ability of a household to spend no more than 30% of its income on housing (Gabriel et al., 2005; Baker et al., 2015; Cai & Lu, 2015). This definition of affordability is based on the “ratio approach”, which is more appropriate for the context of this study (Cai & Lu, 2015). The literature (Smith, 1995; Walker & van der Zon, 2000; Straub et al., 2010) is replete with indicators relating to these measures. Table 1 presents selected indicators of the measures used in this paper. 3. Methods A mixed methods approach within the context of the definition by Creswell and Plano Clark (2011) was applied in the study to triangulate and complement the responses (Bryman, 2006). It involved collecting both numeric and textual data represented in both quantitative and qualitative forms. Nonetheless, the study was predominantly qualitative, employing structured interviews to collect data from district housing officers and tenants, and complemented by a quantitative aspect consisting of a small sample survey of tenants in three districts in two regions. The survey assessed tenants’ perception of the condition of the houses and measured their satisfaction with housing conditions. 2.2 Indicators of Performance Assessment in Public Housing in Ghana 43 Journal of African Real Estate Research Volume 3(1) 2018 39-60 Table 1: Measures and Indicators of Public Housing Assessment Measure Indicators Adequacy • Number of houses constructed per annum • Number of allocations (lettings) per annum • Type of houses • Satisfaction with the type of house Decency • Physical condition of dwellings (main components) • Number of dwellings maintained per annum • Response to requests for repairs • Satisfaction with housing condition • Satisfaction with maintenance Affordability • Amount of monthly income spent on rent Table 1: Measures and Indicators of Public Housing Assessment Local Authorities (Districts) and Tenants Three LAs, Tamale, Bolgatanga and Kassena-Nankana (see Table 2) were purposefully selected to represent the three categories of districts in Ghana. LAs are categorised into metropolitan and municipal districts, depending on the size and population of the area (Parliament of Ghana, 2016). The Tamale metropolitan district is one of four metropolises in the country. It hosts the Northern regional capital and is cosmopolitan in character. The metropolis hosts the headquarters of many private and non-governmental organisations. The only tertiary hospital that serves the three northern regions of Ghana is found in Tamale. Furthermore, Tamale has a relatively large public housing sector. Like Tamale, Bolgatanga municipality houses the capital of the Upper East region. It is similarly cosmopolitan given that the regional offices of many state agencies and private organisations are located there. Even though exact figures for the housing stock were not available, the housing officer estimated the stock size to be in the medium range. Therefore, Bolgatanga was chosen to represent that category of housing. The Kassena-Nankana municipality is relatively less cosmopolitan and has a small population of public houses. It was selected because of the public housing stock size. Other factors that influenced the choice of the three districts were the convenience in terms of time, availability and access to housing officers. Because of the qualitative nature of the study, and the objective of achieving depth rather than breadth in the assessment, the sample of LAs were purposefully selected. 44 Journal of African Real Estate Research Volume 3(1) 2018 39-60 The study interviewed three housing officers in each district and the coordinating director for Kassena-Nankana municipal. Housing Officers Housing officers loosely refer to the officers responsible for managing the housing stock in the districts; they are not necessarily trained estate officers. For instance, two of the officers are administrative assistants who have assumed additional responsibility for managing the houses. Their primary role is to coordinate allocations and repairs. The interviews with housing officers related to the goal of public housing, number and composition of the stock, quality of housing, and repair and maintenance. Tenants The number of tenants interviewed from each house type is presented in Table 2. Based on the focus of the study, which relates to depth rather than scale, the study sampled 20 tenants from each district to interview. However, only 4 tenants participated in the Kassena-Nankana district. Most tenants declined to participate either because they could not make time or because they are not happy with their conditions. The interviews with tenants related to their perception of the goal of public housing, housing affordability, repair and maintenance. The interviews with both housing officers and tenants were semi-structured and face-to-face. The same sample of tenants formed the sample for the survey. The small sample of 44 tenants was chosen due to the resistance of most tenants to participate. Notwithstanding the limitation of the sample size, the study considered it adequate to present an overview of tenants’ perception of the condition of housing and their satisfaction. Moreover, the main purpose of the survey was to complement the responses from the interviews. For these reasons, the survey did not seek to achieve statistical representativeness in its sample selection but rather variety in its respondents’ background including department and house type. It should be noted that some districts do not have some house types. Table 2: Basic Information About Local Authorities and Sample Region/ District Type of houses Total Sample Single Unit detached Semi-detached Flat Total Sample Total Sample Total Sample Northern region Tamale 246 4 251 12 17 4 514 20 Upper East region Bolgatanga - 2 - 13 - 5 - 20 Kassena- Nankana 9 - 60 4 - - 69 4 Total 255 6 311 29 17 9 583 44 Source: Tamale metropolitan, Bolgatanga, and Kassena-Nankana municipal districts, 2015. Table 2: Basic Information About Local Authorities and Sample 45 Journal of African Real Estate Research Volume 3(1) 2018 39-60 A convenience sampling method was used to select tenants. This was based on their willingness to be interviewed. It must be emphasised most tenants declined to participate in the study largely because they were upset with the conditions of the houses. The respondent tenants who participated in the study and their respective departments are presented in Table 3. The gender split was 27 males and 17 females. The range was 2-8 persons per household, and the average household size of the 37 tenants who responded was 5 people. Tenants This is higher than the national average of 4.4 but lower than the averages of the two regions – 5.5 and 7.7 for Upper East and Northern regions respectively (GSS, 2013). The modal household size was 4, and the duration of stay ranged from 3 months to 20 years; the average duration of stay was 1.8 years. The relatively short duration of stay may be because of the transfer of government employees across districts and regions. Nearly half (21) of the tenants had not lived in the houses for more than 5 years. Table 3: Departments of Tenant Respondents S. NO Department/agency Frequency 1 Ghana Education Service 11 2 Regional Coordinating Council 5 3 Ghana Health Service 4 18 State Transport Company 4 5 Lands Commission 2 7 National Youth Council 2 10 Ghana Police Service 2 11 Ghana Fire Service 2 12 Town and Country Planning 2 15 Local Government Service 2 4 National Health Insurance Scheme 1 6 Department of Agriculture 1 8 Commission for Human Rights and Administrative Justice 1 9 National Service Scheme 1 13 Environmental Sanitation Department 1 14 Audit Service 1 16 Centre for National Culture 1 17 Ghana Revenue Authority 1 Total 44 Table 3: Departments of Tenant Respondents Table 3: Departments of Tenant Respondents S. NO Department/agency Frequency 1 Ghana Education Service 11 2 Regional Coordinating Council 5 3 Ghana Health Service 4 18 State Transport Company 4 5 Lands Commission 2 7 National Youth Council 2 10 Ghana Police Service 2 11 Ghana Fire Service 2 12 Town and Country Planning 2 15 Local Government Service 2 4 National Health Insurance Scheme 1 6 Department of Agriculture 1 8 Commission for Human Rights and Administrative Justice 1 9 National Service Scheme 1 13 Environmental Sanitation Department 1 14 Audit Service 1 16 Centre for National Culture 1 17 Ghana Revenue Authority 1 Total 44 Condition and Satisfaction Assessments The survey was conducted to measure tenants' perceptions of the condition of houses, and their satisfaction with housing conditions. To measure the perception of the condition of houses, tenants were required to indicate their assessment (good, fair, poor) of the conditions of 10 physical elements of a house. These include internal walls, external walls, windows and frames, doors, floor, roof (cover), paintwork, plumbing, and electrical installations (van Mossel and Jansen, 2010; Ibem et al., 2013). As stated earlier, this is an assessment by non-professionals which is meant to complement interview responses. To assess the satisfaction with housing conditions, they were required to indicate their satisfaction (satisfied, indifferent, and dissatisfied) with the same ten elements of the house, the type of house, and level or 46 Journal of African Real Estate Research Volume 3(1) 2018 39-60 repairs. The outcome of the survey was then presented to supplement the qualitative responses concerning housing conditions and maintenance. Abductive methods were used to analyse the data. Themes were developed based on the goals of public housing to guide the data collection. The audio- recorded interviews were transcribed and coded according to themes with the aid of the Atlas.ti software. The data was then analysed for issues according to the themes. Where applicable, the issues in a theme were compared with the survey data and inferences were drawn. The survey data was presented in simple descriptive statistics such as frequencies and percentages to correlate with the qualitative responses of tenants. 4.1 Perception of the Goal of Public Housing As a starting point, the study wanted to know the respondents’ view of the goal of public housing in comparison with the goal adopted for the study. Three main goals emerged from tenant respondents and housing officers. First, public housing is meant to provide adequate, affordable housing for government workers. “Adequacy”, according to the respondents refers to both “adequacy” and “decency” as defined in this study. The second relates to productivity; by housing workers close to their workplaces, access to work is easy and convenient and may enhance productivity. The third goal of public housing according to respondents is to provide comfort and security to public sector workers. Some answers of respondents to the question include: "…most of those living there are public sector workers. My opinion is that it is to help public servants especially those who face challenges because they are not natives" (Tenant interview, October 2015). “…to be able to accommodate government workers for convenience, because it can affect productivity” (Tenant interview, October 2015). "…sometimes some of them [tenants] cannot meet the advance payment requested by private landlords, or they will like to have their privacy, so they fall on the RCC [local authority] …to do them a favour on the part of giving them decent accommodation, whereby they will have sound mind to be efficient with their officially assigned duties.” (Housing officer, Tamale, October 2015). This understanding of the goal of public housing is in line with that adopted for this study. 4.2 Performance of Local Authorities (a) Adequacy 47 47 Journal of African Real Estate Research Volume 3(1) 2018 39-60 Four indicators of adequacy were examined: number of houses constructed per annum, number of allocations per annum, type of houses and satisfaction with the type of houses. According to the housing officers, there have not been large-scale new constructions or conversions of buildings into residential units. They estimate that in a year only one or two residential properties may be constructed or none (Housing officers, Tamale Bolgatanga and Kassena-Nankana, October 2015). Therefore, the total stock has remained the same or barely increased. With regards to housing allocations, all the housing officers stated that the number of allocations in a year is low. A housing officer said: Four indicators of adequacy were examined: number of houses constructed per annum, number of allocations per annum, type of houses and satisfaction with the type of houses. According to the housing officers, there have not been large-scale new constructions or conversions of buildings into residential units. They estimate that in a year only one or two residential properties may be constructed or none (Housing officers, Tamale Bolgatanga and Kassena-Nankana, October 2015). Therefore, the total stock has remained the same or barely increased. With regards to housing allocations, all the housing officers stated that the number of allocations in a year is low. A housing officer said: “…I can say that about 10 houses become available for allocation to new tenants…in a year, we can receive about 15 or 20 [applications]. In a week, I have received about 8 applications” (Housing officer, Tamale, October 2015). The main types of houses and relative numbers in the study districts are presented in Table 2. Variation in house type should allow for housing applicants to choose their preferred living arrangement. However, according to all three housing officers, tenants do not have the opportunity to choose their preferred house type. A housing officer said, “Tenants do not have a choice. Allocations are according to rank. So, junior staff applies for junior staff quarters [mostly semi- detached dwellings], and senior staff apply for bungalows [mostly detached single unit houses]” (Housing officer, Bolgatanga, October 2015). Even though applicants may specify their preferred house type, practically, it does not affect the allocation process. The dominant criterion is the rank of the applicant, which entitles one to a single room, two bedroom or multiple room house. 4.2 Performance of Local Authorities The responses of tenants supported this situation about the choice of house type. “The rooms are not enough for the tenants”; “the houses are not ok for us in number for government workers”; “… but it may not be achieving much because of the limited number of dwellings” (Tenant interviews, October 2015). “The rooms are not enough for the tenants”; “the houses are not ok for us in number for government workers”; “… but it may not be achieving much because of the limited number of dwellings” (Tenant interviews, October 2015). On the question of tenants' satisfaction with house type, the responses show that they are generally satisfied. 55% of respondents indicated that they were satisfied with the type of houses available. (b) Decency To assess decency in public housing, the study examined the physical conditions of the houses, the number of dwellings maintained per annum, responses of housing officers to requests for repairs and maintenance, satisfaction with maintenance, and satisfaction with the condition of dwellings. Condition of Houses 48 Journal of African Real Estate Research Volume 3(1) 2018 39-60 Tenants indicated their assessment of 10 physical components of their dwellings. It may be argued that the responses may not be objective. However, it is hoped that by aggregating the responses, the effect of individual bias will be reduced. Even though it is an assessment by non- professionals, the responses suffice to paint a general picture of the condition of the houses. The condition assessment by tenants is presented in Table 4 below. Table 4: Condition Assessment of Components of Dwellings by Tenants (N=44) Component Good Fair Poor Non- response Total No % No % No % No % Internal walls 15 34 15 34 14 32 - - 44 External walls 11 25 11 25 20 45 2 5 44 Windows and frames 6 1 16 36 20 45 2 5 44 Doors 5 11 16 36 21 48 2 5 44 Floor 15 34 12 27 16 36 1 2 44 Ceiling 8 18 9 20 26 59 1 2 44 Roof (cover) 8 18 16 36 16 36 4 9 44 Paintwork 4 9 19 43 20 45 1 2 44 Plumbing 22 50 5 11 13 30 4 9 44 Electrical Installations 20 45 10 23 10 23 4 9 44 Overall assessment 6 14 15 34 12 27 11 25 44 Source: Field study, October 2015. Table 4: Condition Assessment of Components of Dwellings by Tenants (N=44) The assessment results show that where the majority assess the condition of a component to be poor, many more tenants are likely to share the same view as compared to where a majority assess the condition of a component to be good. Some remarks that support the assessment by respondents include: “…most of the various facets of the entire structure are completely failing”; “the building needs more attention on its structural elements”; “some parts of the wood is rotten”; “most sockets do not function well” “No, we don’t do any maintenance because they [government] should have been sending us funds or materials for the maintenance to be taken care of” (Housing officer, Tamale, October 2015). Maintenance by Local Authorities To gain an understanding of the maintenance practices in the three districts, housing officers were asked about responsive repairs, that is, repair requests made by tenants, and the number of dwellings maintained per annum. All the housing officers said the districts do not carry out responsive repairs. “No, we don’t do any maintenance because they [government] should have been sending us funds or materials for the maintenance to be taken care of” (Housing officer, Tamale, October 2015). 49 Journal of African Real Estate Research Volume 3(1) 2018 39-60 "We do not do any maintenance. We do it on our [referring to himself a tenant] own, I had to paint my place myself. The money [rent] is paid into the consolidated fund…we do not receive funds for maintenance. It used to be PWD that would receive the money and who was in charge of maintaining the dwellings, but that has ceased” (Housing officer, Bolgatanga, October 2015). According to the officers, in the past materials were procured by central government and maintenance was carried out by the Public Works Department (PWD). However, housing officers could not provide data on houses maintained per year. This practice of government-led maintenance has since stopped due to a lack of funding to LAs. Thus, tenants have to fund repairs by themselves, including significant works, and the cost may be set off against rent payable over time. Housing officers are quoted as saying: "…when we are allocating; we ask them to do the maintenance and submit receipts to be offset with the rent" (Housing officer, Bolgantanga, October 2015). "… you have to write to us, and if we don't have money to do it [the repairs] we will let our engineer make the estimates, and if you are in the position, you do it, and we use it to offset your rent” (Housing officer, Kassena-Nankana, October 2015). Consequently, tenants were asked where they make requests for repairs, what the responses were to their requests, and the kind of repairs tenants funded themselves. Most tenants said they make requests for repairs to the maintenance unit (Works Department) of the district. However, their requests are often not addressed. They said; "I don't request for repairs because they won't do it"; "I do not make a request to anyone because they never mind us"; "I have not made any request. Maintenance by Local Authorities I had to renovate it [the house] myself" I have reported to the authorities, but to no avail, so then, I pay eventually for all the damages ever since I lived in this residence" (Tenant interviews, October 2015). According to some tenant respondents, the cost of tenant self-financed repairs may be set off against rents, confirming the statements of various housing officers. “You write a letter and make estimates to them; if you decide to do it yourself, and they will give you the go-ahead to do it, and it is set off against your rent" (Tenant interview, October 2015). “You write a letter and make estimates to them; if you decide to do it yourself, and they will give you the go-ahead to do it, and it is set off against your rent" (Tenant interview, October 2015). The kind of repairs reportedly carried out by tenants themselves include painting and mending cracks on internal and external walls and repairing windows and frames. Furthermore, tenants undertook the changing and repair of locks, doors, ceiling, roofs as well as minor works on plumbing, electrical fitting and installations (Table 5, column 6). Table 5, columns 4 and 5, presents the frequency of reported repairs of the house components and ranking. 50 Journal of African Real Estate Research Volume 3(1) 2018 39-60 Table 5: Comparing Condition Assessment and Reported Repairs by Tenants Component Condition assessment Reported repairs Reported repairs funded by tenants Fair Poor Freq. Rank Paintwork X 20 1st Painting inside and outside walls Windows and frames X 18 2nd Changing frames, insect and burglar proofing, and louver blades Plumbing X 12 3rd Changing old water closets, showers, pipes Internal walls X 12 3rd Mending cracks, rendering and painting Ceiling X 10 4th Replaced ceiling External walls X 8 5th Mending cracks and painting Electrical Installations X 8 5th Changing wiring and electrical sockets Doors X 8 5th Changing door locks Roof (cover) X 5 6th Mending leaks in roof Floor X 4 7th Mending cracks in walls, laying tiles Source: Field data, October 2015. Table 5: Comparing Condition Assessment and Reported Repairs by Tenants The study compared the condition assessment by tenants and reported repairs conducted by tenants on individual house components (Table 5). It can be observed that, generally, where respondents assessed the condition of components to be fair, the same components recorded fewer instances of reported repairs, whereas components assessed to be in poor condition also recorded most repairs by tenants. Satisfaction with housing conditions and maintenance Tenants were asked to indicate their satisfaction with the condition of; the overall condition of their dwellings, the dwelling type, and the number of rooms (Table 6). The maintenance issues that tenants indicated their satisfaction on included; the medium of contact with LAs, the response of LAs to maintenance requests, and quality of repairs (Table 6). Table 6: Satisfaction with House Components, Characteristics, and Maintenance Component Level of satisfaction No response Total Satisfied Indifferent Dissatisfied Freq. (%) Freq. (%) Freq. (%) Freq. (%) Paintwork 9 20 17 39 17 39 1 2 44 Windows and frames 9 20 15 34 17 39 3 7 44 Plumbing 17 39 8 18 14 32 5 11 44 Internal walls 23 52 7 16 14 32 - - 44 Ceiling 9 20 9 20 24 55 2 5 44 External walls 15 34 7 16 22 50 - - 44 Electrical Installations 22 50 12 27 10 23 - - 44 Doors 10 23 15 34 19 43 - - 44 Roof (cover) 9 20 9 20 22 50 4 9 44 Floor 13 30 13 30 15 34 3 7 44 Overall condition 11 25 10 22 21 48 2 5 44 Table 6: Satisfaction with House Components, Characteristics, and Maintenance 51 Journal of African Real Estate Research Volume 3(1) 2018 39-60 Dwelling characteristics Type 24 55 7 16 9 20 4 9 44 Number of rooms 20 45 10 23 13 30 1 2 44 Maintenance Medium of contact with landlord 3 7 13 30 13 30 15 34 44 Response to maintenance request 1 2 9 20 18 41 16 64 44 Quality of repairs - - 8 18 20 45 16 36 44 Source: Tenant survey, October 2015. Aside from internal walls and electrical installations, there was a greater degree of consensus among respondents regarding components that caused dissatisfaction (see “Roof (cover)” in Table 6). There was a lesser degree of consensus regarding components that the majority of respondents deemed to be satisfactory (see “Plumbing” in Table 6). This observation is in line with the satisfaction of the overall condition of dwellings. The satisfaction scores for maintenance services show that the majority were dissatisfied with the quality of repairs and response to repairs requests. As stated earlier by some tenants, LAs do not carry out maintenance. Satisfaction with housing conditions and maintenance Therefore, tenants’ responses to the quality of repairs may have been construed to refer to tenant self-financed repairs. Furthermore, nearly half of tenants did not indicate their satisfaction in this category. Considered on the whole, it may be argued that the scores reinforce the absence of maintenance as found earlier. Comparing the condition and satisfaction assessments, it is observed that where a majority of tenants assess a component to be poor, a similar number were dissatisfied with the condition of the component. Again, this suggests that there is less consensus among respondents regarding satisfactory components. Furthermore, while a number of respondents assessed the overall condition of dwellings to be fair, an even greater majority were not satisfied with the overall conditions. It may be concluded that both, at the component level, and considered holistically, the condition and satisfaction assessments reinforce each other. That is, generally, the conditions of dwellings are poor, and consequently, tenants are not satisfied. (c) Affordability The leading indicator of affordability is the cost of housing or housing expenditure in relation to income (Cai & Lu, 2015). The study could not independently obtain data on incomes and rents paid by tenants as this is not available to LAs. Furthermore, as some respondents declined to disclose their incomes, estimates were determined by reference to their departments, house type (an indication of rank) and the average income in the department. Tenants pay 10% (determined by the central government) of basic salary as rent. Therefore, the stated salary and percentage of rent were used to compute average rent payable for instances where respondents did not provide data. The noted limitation with this approach is that, while the study relied on the income of one member of a household, there could be more than one income 52 Journal of African Real Estate Research Volume 3(1) 2018 39-60 earner in a household or income streams of a respondent. The average monthly income of tenants in the sample was GH¢1,321.591. This compares closely with the national average monthly income of GH¢1,387.00 (Ghana Statistical Service, 2014). Therefore, the computed housing affordability index for the three districts is 9.9%. It may be said that public housing is affordable because average monthly housing expenditure (rent) is less than the 30% of average income. In addition to the computed index, tenants were asked if they consider the rents low, and whether they would be willing to pay higher rents for maintenance. All the respondents said that rents are low. However, 86% of the respondents were willing to pay higher rents for better maintenance while 14% did not support higher rents. Their objection to rent increases was premised on the condition of the houses. "No, because nothing like repairs and maintenance has been done; there is no better service delivery; there has not been maintenance since the occupation, yet monthly rent is paid" (Tenants interview, October 2015). The study also asked the opinions of housing officers about the affordability of rent to compare with the computed finding. As managers, housing officers will have a fair idea of how rents in public housing compare with private sector rents. Basing their conclusions on a comparison of rent levels in public and private housing, and additional facilities (such as private toilets, baths, kitchen, and storeroom) available in public housing, the housing officers were unanimous that rents are affordable. g g , y ¢ 2. Some compound houses do not have kitchens. So tenants use parts of their lobbies as cooking areas UN-HABITAT 2011. Ghana Housing Profile, Nairobi, United Nations Human Settlements Programme. 1 Average exchange rate, May 2016: €1.00 = GH¢4.302 1 Average exchange rate, May 2016: €1.00 = GH¢4.302 5. Discussion For the goals of public housing to remain relevant, it is imperative to assess the performance of housing management regularly. For example, Dutch housing associations have a system of both internal and external assessment to ensure that they remain focused on their core goals (van Overmeeren et al., 2010; Aziabah, 2018). There is adequate evidence (Awotona, 1990; Asabere, 2007; Komu, 2010) to show that inadequate attention to public housing has led to undesirable conditions that have often triggered the sale of public housing. Regular performance assessment and monitoring is necessary to ensure adequate housing (Ouwehand & van Daalen, 2002; Newton & Tunstall, 2012; Aziabah, 2018). Going forward, tenants should be involved in performance assessment and in management to emphasise the collective responsibility of stakeholders to achieve adequate maintenance (Lee et al., 1998; Yusof et al., 2014). Tenants generally seem satisfied with the house types they were assigned however there remains an apparent situation of inadequate quantity from which applicants may choose. Furthermore, public housing has stagnated - either grown marginally or remained relatively the same. This is not surprising as changes in Ghana’s housing policy have favoured an enablement strategy and thus the government has not built large-scale public housing (see Tipple, 1999; Arku, 2009). However, in light of the failure of the private sector to meet housing needs, there have been recent arguments for the state to be involved in providing rental housing (Field & Ofori, 1989; Obeng-Odoom, 2011a; Acquah, 2015). The inability of the state to increase the quantity of housing can also be blamed on low rents, which some (for example Konadu-Agyemang, 2001) have argued is not adequate to carry out effective maintenance. Perhaps developing countries like Ghana need to re- examine public sector rents within the context of the broader housing sector and the realities of maintenance, especially if the government is to heed the call to participate in rental housing provision. In addition to the fact that rents are low, LAs are unable to carry out maintenance because they do not collect rents. In Ghana, a central government agency, the Controller and Account General’s Department (CAGD), collects rent as per policy agreement. This arrangement may have been introduced because the central government used to finance state agencies (for example the State Housing Corporation) to maintain houses. The current situation could be likened to what Kadiri Kabir (2004) refers to as lack of decentralisation of power. (c) Affordability In some private rented housing, tenants share facilities such as kitchens2, bathrooms, and toilets, whereas this is not the case in public housing. "Well with the private, why this [public housing] is cheaper is that one, you have privacy, and second, where you are, there is everything [referring to kitchens, tap water, storage areas, etc.]. You have, for instance, two bedrooms. For instance, in the private houses, people rent single rooms for GH¢50, so two rooms is GH¢100. In the public house, you may have two rooms, hall kitchen and bath and toilet to yourself. However, in a privately rented house, you have to share some of the facilities such as toilet and bath and kitchen. So, single room rented at GH¢50 and three rooms being rented at GHc100, you see that the public houses are cheaper. That's where the affordability comes." (Housing officer, October 2015). The findings show that generally, rents in public housing are affordable. However, the houses are not being maintained because LAs do not receive rents which are collected by central government. As a result, tenants largely finance repairs, especially those that are necessary. The impact of the lack of 53 Journal of African Real Estate Research Volume 3(1) 2018 39-60 maintenance is that respondents are not satisfied with the condition of the houses. Juxtaposing these findings against the goal of public housing – to provide affordable accommodation to government employees to make them comfortable, so that it may lead to increase in productivity - it may be said that LAs are not performing well in public housing management. 5. Discussion This rent collection arrangement needs to change to allow LAs to determine and collect rents. The central government could exercise oversight in housing management by formulating guidelines for districts, rather than directly assuming some management 54 Journal of African Real Estate Research Volume 3(1) 2018 39-60 responsibility. For example, housing associations in the Netherlands determine rents within a government rent policy which is reviewed annually (AEDES, 2016; Aziabah, 2018). The study also found that rents in Ghanaian public housing are affordable. However, some tenants argued against rent increases because they bear the cost of some repairs that would typically be the landlord’s responsibility. Therefore, one may argue that the real rents could be higher. That being said, it is still unlikely that rents will be closer to 30% of tenant households' income. This situation highlights the need for LAs to maintain the houses to an acceptable standard to justify rent increases. What remains to be seen is whether to increase rents to maintain the houses, or maintain the houses to justify any rent increases. If the latter option were to be taken, the question would be how to fund major repairs required. It is also vital for LAs to define their maintenance responsibilities and those of the tenants in rental agreements. By far the primary indicator of the performance of LAs is the condition of the dwellings. After all, the construction of a house is legitimated when adequate maintenance of its condition continues (van Wyk, 2006). Unfortunately, the findings of this study confirms those of other authors who have written about public housing in Ghana (UN-HABITAT, 2011; Yankson & Gough, 2014), and other African countries (fTipple et al., 2004, Komu, 2010, Otieno, 2014). For instance, Asabere (2007), states that the rents of public housing in Ghana was inadequate to cover the maintenance, and therefore it was one of the reasons that triggered the sale of the public houses to existing tenants. Also, Tipple et al. (2004), have found that most Sub-Saharan African government- built houses are in poor physical conditions or do not meet the expectations of occupants. The suggestion earlier to change the current policy of rent determination and collection in Ghana may go a long way to make funds available for maintenance. However, as Field and Ofori (1989) have pointed out in the case of Singapore, the commitment of central government and LAs is necessary in this regard. 5. Discussion This paper has combined qualitative and quantitative data to assess the performance of public housing management in Ghana. This technique offers the first step in benchmarking standards. Housing researchers may apply or adapt this approach to assess performance in similar contexts; in doing so, models of performance assessment in housing management may be developed for developing country context. 6. Conclusion This paper assesses the performance of LAs in the management of public housing in Ghana. This is done to contextualise the problem of poor maintenance and conditions in its public housing sector. The paper collected mainly qualitative and quantitative data through interviews with housing professionals and tenants in the Tamale metropolitan, and Bolgatanga and Kassena-Nankana municipalities. The paper assessed the adequacy, decency and affordability of public housing. It found that there is adequate variety in 55 Journal of African Real Estate Research Volume 3(1) 2018 39-60 house types but limited quantity mainly because there has not been an expansion of the stock. Regarding decency, the paper found that the conditions of housing are poor due mainly to inadequate maintenance by LAs. As a result, tenants are not satisfied with their houses. Furthermore, public housing is affordable as rents averagely account for only about 9% of household incomes. The paper, therefore, concludes that housing management is not achieving the aim of providing adequate, and decent housing to government workers. Based on the conclusions of this paper, LAs may consider a number of actions to improve performance. First, rent determination and collection should be wholly carried out by LAs to make rents available for maintenance. District assemblies must consider reviewing rents upwards to raise funds for maintenance, and in the long-term expand the housing stock. Finally, there is a need for policy to clearly define the goals of public housing, define performance indicators to measure progress towards achieving these goals, and institute both internal and external monitoring and evaluation mechanisms to track progress towards achieving the goals of public housing. References Acquah, P. K. (2015). Affordable Housing: Nothing Affordable. Modern Ghana [Online]. Available: Https://Www.Modernghana.Com/News/590609/Affordable- Housing-Nothing-Affordable.Html [Accessed 14/03/2016]. AEDES. (2016). Dutch Social Housing In A Nutshell: Examples of Social Innovation For People And Communities. In: Aedes (Ed.). Anheier, H. K. (2005). Nonprofit Organisations: Theory, Management, Policy, London, Routledge Taylor & Francis Group. Arimah, C. B. (2000). Housing-Sector Performance In Global Perspective: A Cross-City Investigation. Urban Studies, 37, pp.2551-2579. Arku, G. (2006). Housing And Development Strategies In Ghana, 1945-2000. International Development Planning Review, 28, pp.333-358. Arku, G. (2009). Housing Policy Changes In Ghana In The 1990s. Housing Studies, 24, pp.261-272. Asabere, P. (2007). The Demise Of The Rent-Controlled Public Housing Programmes Of Ghana: The Story Of The Tema Development Corporation. Urban Studies, 44, pp.1919-1935. Askim, J. (2009). The Demand Side Of Performance Measurement: Explaining Councillors' Utilization Of Performance Information in Policymaking. International Public Management Journal, 12 , pp.24- 47. Awotona, A. (1990). Nigerian Government Participation In Housing: 1970– 1980. Habitat International, 14, pp.17-40. Aziabah, S. (2018). Better Public Housing Management in Ghana. An Approach to Improve Maintenance and Housing Quality. Doctor Of Philosophy, Delft University Of Technology. Baiden, P., Arku, G., Luginaah, I. & Asiedu, A. (2010). An Assessment Of Residents’ Housing Satisfaction And Coping In Accra, Ghana. 56 Journal of African Real Estate Research Volume 3(1) 2018 39-60 Baker, E., Mason, K. & Bentley, R. (2015). Measuring Housing Affordability: A Longitudinal Approach. Urban Policy And Research, pp.1-16. Benson, I. (2014) . Nurses' Quarters Uninhabitable - B/A Regional Hospital Health Personnel. Joyonline News [Online]. Bouckaert, G. & Van Dooren, W. (2009). Performance Measurement And Management In Public Sector Organisations. Public Management And Governance. Third Ed. London: Routledge. Boyne, G. (1997). Comparing The Performance Of Local Authorities: An Evaluation Of The Audit Commission Indicators. Local Government Studies, 23, pp.17-43. Boyne, G. A. (2002). Public And Private Management: What’s The Difference? Journal Of Management Studies, 39, pp.97-122. Bryman, A. (2006). Integrating Quantitative And Qualitative Research: How Is It Done? Qualitative Research, 6, pp.97-113. Cai, W. & Lu, X. 2015. Housing Affordability: Beyond The Income And Price Terms, Using China As A Case Study. Habitat International, 47, pp.169-175. Creswell, W. J. & Plano Clark, L. V. (2011). Designing And Conducting Mixed Methods Research, Los Angeles, Sage Publications, Inc. De Bruijn, H.(2002a). Managing Performance In The Public Sector, London, Routledge. De Bruijn, H. (2002b). References Performance Measurement In The Public Sector: Strategies To Cope With The Risks Of Performance Measurement. International Journal Of Public Sector Management, 15, pp.578-594. Eyiah-Botwe, E. (2015). Assessing Housing Project End-Users Satisfaction In Ghana: A Case Study Of Ssnit Housing Flats In Asuoyeboa- Kumasi. Civil And Environmental Research, 7, pp.13-22. Field, B. & Ofori, G. (1989). Housing Stress And The Role Of The State. Habitat International, 13, pp.125-138. Gabriel, M., Jacobs, K., Arthurson, K., Burke, T. & Yates, J. (2005). Conceptualising And Measuring The Housing Affordability Problem. National Research Venture 3: Housing Affordability For Lower Income Australians. Research Paper 1. Australian Housing And Urban Research Institute. Ginsburg, N. (2005). The privatisation of council housing. Critical social policy, 25(1), pp.115-135 Ghana Statistical Service. (2014). Ghana Living Standards Survey Round 6 (Glss 6). In: Service, G. S. (Ed.). Accra: Ghana Statistical Service Government Of Ghana. (2015). National Housing Policy. In: Ministry Of Water Resources, W. A. H. (Ed.). Accra. GSS. (2013) . 2010 Population And Housing Census: National Analytical Report. In: Service, S. (Ed.). Accra: Ghana Statistical Service. Huchzermeyer, M. (2014). Changing Housing Policy In South Africa. In: Jan, B., Paul, V. L. & Peer, S. (Eds.) Affordable Housing In The Urban Global South. Hoboken: Taylor And Francis. Ibem, E. O. & Aduwo, E. B. (2013). Assessment of Residential Satisfaction in Public Housing in Ogun State, Nigeria. Habitat International, 40, pp.163-175. 57 Journal of African Real Estate Research Volume 3(1) 2018 39-60 Ibem, E. O., Opoko, A. P., Adeboye, A. B. & Amole, D. (2013). Performance Evaluation Of Residential Buildings in Public Housing Estates in Ogun State, Nigeria: Users' Satisfaction Perspective. Frontiers Of Architectural Research, 2, pp.178-190. Ibem, O. E. & Amole, D. (2013). Residential Satisfaction in Public Core Housing in Abeokuta, Ogun State, Nigeria. Social Indicators Research, 113, pp.563-581. Ilesanmi, A. O. (2010). Post-Occupancy Evaluation And Residents/' Satisfaction With Public Housing In Lagos, Nigeria. J Build Apprais, 6, pp.153-169. Jacobs, K. & Manzi, T. (2000). Performance Indicators And Social Constructivism: Conflict And Control In Housing Management. Critical Social Policy, 20, pp.85-103. Johnsen, Å. (2005). What Does 25 Years Of Experience Tell Us About The State Of Performance Measurement In Public Policy And Management? Public Money & Management, 25, pp.9-17. Kadiri Kabir, O. (2004). Low-Cost Technology And Mass Housing System In The Nigerian Housing. Jounrnal of Applied Sciences, 4(4), pp.565- 567. Kaitilla, S. (1993). References Satisfaction With Public Housing In Papua New Guinea: The Case Of West Taraka Housing Scheme. Environment And Behavior, 25, pp.514-545. Kemp, P. A. (1995). Researching Housing Management Performance. Urban Studies, 32, pp.779-790. Kendall, J. & Knapp, M. (2000). Measuring The Performance Of Voluntary Organizations. Public Management Review, 2, pp.105-132. Komu, F. (2010). Housing Decay And Maintenance: The Case Of Public Housing In Tanzania. PhD Dissertation, Royal Institute Of Technology. Konadu-Agyemang, K. (2001). A Survey Of Housing Conditions And Characteristics in Accra, An African City. Habitat International, 25, pp.15-34. Koopman, M., Van Mossel, H. J. & Straub, A. (2008). Performance Measurement in The Dutch Social Rented Sector, Amsterdam, Ios Press. Lee, L., Petrova, E., Shapiro, M. & Struyk, R. (1998). Housing Maintenance And Management In Russia During The Reforms. Housing Studies, 13, pp.679-696. Newton, R. & Tunstall, R. (2012). Lessons For Localism: Tenant Self- Management. Urban Forum. Obeng-Odoom, F. (2011a). Private Rental Housing In Ghana: Reform Or Renounce? Journal Of International Real Estate And Construction Studies,1, pp.71. Obeng-Odoom, F. (2011b). Reforming Housing Management In Ghana: The Role Of Education. International Public Management Review, 12 (1), pp.112-123. Obeng-Odoom, F. & Amedzro, L. (2011). Inadequate Housing In Ghana. Urbani Izziv, 22, pp.127-137. 58 58 Journal of African Real Estate Research Volume 3(1) 2018 39-60 Olawore, A., Ibem, E. O. & Amole, O. O. (2011). Assessment of The Qualitative Adequacy Of Newly Constructed Public Housing in Ogun State, Nigeria. Property Management, 29, pp.285-304. Otieno, O. S. 2014. Constraints In Housing Policy Towards Provision Of Low-Cost Housing To The Urban Poor In Kisumu City, Kenya. International Journal Of Humanities And Social Science Invention, 3, pp.31-42. Ouwehand, A. & Van Daalen, G.(2002). Dutch Housing Associations: A Model For Social Housing, Delft University Press. g y Parliament of Ghana. (2016). Act 936 : Local Goverenace Act. Ghana. Smith, P. (1995). Performance Indicators And Outcome In The Public Sector. Public Money & Management, 15, pp.13-16. Smith, R. & Walker, R. (1994). The Role Of Performance Indicators in Housing Management: A Critique. Environment And Planning A, 26, pp.609-621. pp Stone, M. E. (2006). What Is Housing Affordability? The Case For The Residual Income Approach. Housing Policy Debate, 17, pp.151-184. Stone, M. E. & Hartman, C. (1983). America's Housing Crisis: What Is To Be Done? New York: Routledge. Straub, A., Koopman, M. & Van Mossel, H.-J. (2010). Systems Approach And Performance Measurement By Social Enterprises. Facilities, 28, pp.321-331. Symon, P. References & Walker, R. M. (1995). A Consumer Perspective On Performance Indicators: The Local Housing Authority Reports To Tenants Regimes In England And Wales. Environment And Planning C: Government and Policy, 13, pp.195-216. Terence, Y. M. L. (2008). Optimisation Of Performance Management For Housing Services. Journal Of Facilities Management, 6, pp.226-240. Tipple, A. G. (1999). Transforming Government-Built Housing: Lessons From Developing Countries. Journal Of Urban Technology, 6, pp.17- 35. Tipple, A. G., Owusu, E. S. & Pritchard, C. (2004). User-Initiated Extensions in Government-Built Estates in Ghana and Zimbabwe: Unconventional But Effective Housing Supply. Africa Today, 51, pp.79-105. Tufuor, T. (2004). Reforming The Public Rental Sector In Accra. Master Of Science, Institute For Housing And Urban Development Studies (Ihs) Rotterdam, The Netherlands, And Lund University, Sweden (Hdm). y Un-Habitat. (2011). Ghana Housing Profile, Nairobi, United Nations Human Settlements Programme. g United Nations. (1948). Universal Declaration on Human Rights. Van Bortel, G. & Gruis, V. (2011). Performance Measurement In V., G. & Zijlstra, S. (Eds.) Housing Management: Basics Of Organisation, Network And Asset Management In Dutch Housing Associations. Tudelft. Van Mossel, H. J. (2008). Securing High-Performance Maintenance Service Delivery. In: Koopman, M., Van Mossel, H. J. & Straub, A. (Eds.) Performance Measurement In The Dutch Social Rented Sector. 19 Ed. Amsterdam: Ios Press Bv. 59 Journal of African Real Estate Research Volume 3(1) 2018 39-60 Van Mossel, H. J. & Jansen, S. J. T. (2010). Maintenance Services in Social Housing: What Do Residents Find Important? Structural Survey, 28, pp.215-229. Van Overmeeren, A., Gruis, V. & Haffner, M. (2010). Performance Assessment Of Housing Associations. Journal Of Housing And The Built Environment, 25, pp.139-151. Van Wyk, J. J. (2006). A Housing Management Model For Developing Countries. International Journal For Housing Science And Its Applications, 30, pp.105-122. pp pp Walker, R. (1994). Putting performance measurement into context: classifying social housing organisations. Policy and politics, 22 (3), pp.191-202. Walker, B. & Murie, A. 2004. The Performance of Social Landlords in Great Britain: What Do We Know And What Does It Show? Housing Studies, 19, pp.245-267. Walker, M. R. & Van Der Zon, M. J. F. (2000). Measuring The Performance of Social Housing Organisations in England and the Netherlands: A Policy Review And Research Agenda. Journal Of Housing And The Built Environment, 15, pp.183-194. Walker, R. M. (2001). How to Abolish Public Housing: Implications and Lessons from Public Management Reform. Housing Studies, 16, pp.675-696. Walker, R. M. References & Boyne, G. A. (2006). Public Management Reform And Organisational Performance: An Empirical Assessment Of The U.K. Labour Government's Public Service Improvement Strategy. Journal Of Policy Analysis And Management, 25, pp.371-393. Yankson, W. K. P. & Gough, V. K. (2014). Urban Low-Income Housing in Ghana. In: Bredenoord, J., Van Lindert, P. & Smets, P. (Eds.) Affordable Housing In The Urban Global South: Seeking Sustainable Solutions. New York: Routledge. Yusof, N. A., Abdullah, S. & Najib, N. U. M. (2014). How Does Communication Influence The Perceived Performance Of Maintenance Services In Multi-Storey Public Housing? International Journal Of Strategic Property Management, 18, pp.380-392. 60
https://openalex.org/W1082253719	https://www.nrel.gov/docs/fy15osti/63911.pdf	English	null	Low surface recombination velocity in solution-grown CH3NH3PbBr3 perovskite single crystal	Nature communications	2,015	cc-by	7,077	OPEN Received 22 Mar 2015 \| Accepted 1 Jul 2015 \| Published 6 Aug 2015 Results Si l Single crystal characterization. The solution-grown CH3NH3PbBr3 single crystals studied here have dimensions of B1.4 1.4 0.7 mm3 (inset of Fig. 1a). We measured the X-ray diffraction using a Bruker D8 Discover diffractometer with two-dimensional area detector. The X-ray beam is located at the front face of the crystal (Fig. 1a). A diffraction pattern of isolated spots, rather than con- tinuous arcs (Debye ring), is observed (Fig. 1b) and it is caused by X-ray diffraction from oriented lattice planes, consistent with high quality single crystals. To obtain the Miller index of the front face, the diffraction intensity is integrated for the region where the crystal front face and the plane determined by the incident and diffracted X-ray beams are perpendicular (inside the black lines in Fig. 1b, where w¼ 90±5). Such an integration produces intensity peaks that correspond to diffraction from the lattice planes that are parallel to the front surface of the single crystal (Fig. 1c). These diffraction peaks can be assigned to the family of (001) planes of a cubic phase perovskite crystal, suggesting that the front face belongs to this family of planes. The optical measurements in this work were all conducted with incident light on the front surface. We carried out similar X-ray diffraction measurements on the side face of the crystal and ﬁnd that it also belongs to the family of (001) planes (Supplementary Fig. 1). The integration of the whole X-ray diffraction pattern (w integrated from 0 to 180) produces the diffraction peaks corresponding to other lattice planes (Supplementary Fig. 1). (Supplementary Fig. 2), shows a prominent absorption band at 2.35 eV attributed to excitonic absorption17,18. We simulate the spectrum near the bandedge according to Elliott’s formula19,20: aðoÞ ¼A y ‘o Eg pepx sinh px ð Þ þ A Rex X 1 nex¼1 4p n3 ex d ‘o Eg þ Rex=n2 ex ð1Þ ð1Þ þ A Rex X 1 nex¼1 4p n3 ex d ‘o Eg þ Rex=n2 ex where A is a constant related to the transition matrix element, o is the frequency of light, y is the step function, Eg is the bandgap, x is deﬁned as R1=2 ex = ‘o Eg 1=2 where Rex is the exciton binding energy, nex is the principal quantum number and d denotes a delta function. ARTICLE ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms8961 X-ray diffraction intensity (a.u.) 80 70 60 50 40 30 20 10 2θ (degree) (001) (002) (003) (004) a b c d 1.0 0.8 0.6 0.4 0.2 0.0 (105 cm–1) 2.8 2.6 2.4 2.2 2.0 1.8 Energy (eV) Excitonic Continuum Absorption coefﬁcient Figure 1 \| Structure characterization and optical absorption spectrum. (a) The microscopic image of the single crystal. The cross-hair indicates that the X-ray beam is located at the front face. Inset is the photograph of the single crystal. (b) X-ray diffraction pattern measured by two- dimensional area detector. The right and left frame are corresponding to 2y C arrier recombination at semiconductor surfaces has a detrimental impact on solar cell performance because it decreases carrier lifetimes reducing the attainable short-circuit current and open-circuit voltage1. Therefore, surface passivation is a necessary component in achieving high efﬁciency solar energy conversion2. The knowledge of surface recombination can also provide guidance for optimizing the grain size in polycrystalline solar cells3. Solution processed lead halide based solar cells are undergoing impressive improvements in power conversion efﬁciency4–8. While thorough characterization in transport carrier parameters such as mobility and bulk lifetime shows sufﬁcient characteristics9–16, surface recombination has not been studied. X-ray diffraction intensity (a.u.) 80 70 60 50 40 30 20 10 2θ (degree) (001) (002) (003) (004) a b c b b a Here we directly probed the carrier dynamics at the surface of CH3NH3PbBr3 perovskite single crystals using broadband transient reﬂectance (TR) spectroscopy. The lead bromide perovskite single crystals are chosen because their sizes are sufﬁciently large for optical measurements and their native surfaces do not require further polishing or treatments. In the TR measurement, optical excitation of the perovskite single crystal can modulate the reﬂectance, R, near the bandgap and the relative reﬂectance change (DR(‘o)/R) is recorded by a white-light continuum. We quantitatively reproduce the spectrum using Kramers–Kronig integration of excitonic absorption bleach and ﬁnd a linear relationship between DR(‘o)/R and the total photoexcited carrier density, N, which includes both free carriers and excitons. Pumping at different photon energies with various intensities, we ﬁnd that kinetics of DR(‘o)/R is only sensitive to N near the surface, and is modulated by surface recombination and carrier diffusion that transports carriers away from the surface. ARTICLE The surface recombination velocity (SRV) and carrier diffusion coefﬁcient (D) are found to be 3.4±0.1 103 cm s 1 and 0.27±0.01 cm2 s 1, respectively, by global ﬁtting of a diffusion model to DR(‘o)/R kinetics collected at different excitation energies. d d 1.0 0.8 0.6 0.4 0.2 0.0 (105 cm–1) 2.8 2.6 2.4 2.2 2.0 1.8 Energy (eV) Excitonic Continuum Absorption coefﬁcient Energy (eV) Energy (eV) Figure 1 \| Structure characterization and optical absorption spectrum. Figure 1 \| Structure characterization and optical absorption spectrum. (a) The microscopic image of the single crystal. The cross-hair indicates that the X-ray beam is located at the front face. Inset is the photograph of the single crystal. (b) X-ray diffraction pattern measured by two- dimensional area detector. The right and left frame are corresponding to 2y centred at 30 and 60, respectively. (c) Integration of the X-ray diffraction pattern. The diffraction intensity is integrated in the region of w ¼ 90±5, indicated by the black lines in panel b. w is the angle between the plane determined by the incident and diffracted beams and the plane of the front face. (d) The absorption coefﬁcient (red circles) of the CH3NH3PbBr3 perovskite single crystal obtained from ellipsometry measurement. The black-line is the modelled absorption coefﬁcient with excitonic (green-dash line) and continuum (blue-dash line) components. NATURE COMMUNICATIONS \| 6:7961 \| DOI: 10.1038/ncomms8961 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. Low surface recombination velocity in solution-grown CH3NH3PbBr3 perovskite single crystal Ye Yang1, Yong Yan1, Mengjin Yang1, Sukgeun Choi1, Kai Zhu1, Joseph M. Luther1 & Matthew C. Beard1 Organic-inorganic hybrid perovskites are attracting intense research effort due to their impressive performance in solar cells. While the carrier transport parameters such as mobility and bulk carrier lifetime shows sufﬁcient characteristics, the surface recombination, which can have major impact on the solar cell performance, has not been studied. Here we measure surface recombination dynamics in CH3NH3PbBr3 perovskite single crystals using broadband transient reﬂectance spectroscopy. The surface recombination velocity is found to be 3.4±0.1 103 cm s 1, B2–3 orders of magnitude lower than that in many important unpassivated semiconductors employed in solar cells. Our result suggests that the planar grain size for the perovskite thin ﬁlms should be larger than B30 mm to avoid the inﬂuence of surface recombination on the effective carrier lifetime. 1 National Renewable Energy Laboratory (NREL), Golden, Colorado 80401, USA. Correspondence and requests for materials should be addressed to Y.Y. (email: Ye.Yang@nrel.gov) or to M.C.B. (email: Matt.Beard@nrel.gov). 1 National Renewable Energy Laboratory (NREL), Golden, Colorado 80401, USA. Correspondence and requests for mat (email: Ye.Yang@nrel.gov) or to M.C.B. (email: Matt.Beard@nrel.gov). NATURE COMMUNICATIONS \| 6:7961 \| DOI: 10.1038/ncomms8961 \| www.nature.com/naturecommunications 1 & 2015 Macmillan Publishers Limited. All rights reserved. & 2015 Macmillan Publishers Limited. All rights reserved. Results Si l 1d), N is the total carrier density, equal to the excitation density, and Ns is a saturation density (deﬁned to be the carrier density for which 50% of exciton absorption is bleached) and is related to the exciton Bohr radius (note, equation 4 is only valid when NooNS (ref. 25)). We are able to closely simulate DR(‘o)/R (blue-dash line, Fig. 2b) by substituting equations (4) and (3) into equation (2) with ﬁtting parameter, NS. The simulation reveals Da (green-dash line, Fig. 2b) and the best-ﬁt value of NS (6.0 1018 cm 3). These equations imply that Da and DR(‘o)/R are proportional to the excitation density, N, for NooNS. DR R ‘o ð Þ ¼ 4 n ‘o ð Þ2 1 Dn ‘o ð Þ ð2Þ ð2Þ where Dn is the frequency-dependent photon induced change in refractive index associated with a change of absorption coefﬁcient, Da, and is calculated using the Kramers–Kronig transformation22: The TR measurements were conducted under various pump intensities and the kinetics (probe photon energy of 2.38 eV) are shown in Fig. 3a. The kinetic traces do not display single- exponential behaviour. The decay is faster at early delay and slower at later delay. The initial magnitude shows a linear relationship with excitation density (inset Fig. 3a). At higher excitation levels, the bleach of the continuum band absorption due to the band ﬁlling begins to contribute and the exciton contribution saturates, resulting in a Da (as well as TR response) that is no longer linear with the excitation intensity14. In this work, excitation intensities are all controlled to be in the linear region (NB0.02NS) so the photo-induced exciton absorption bleach dominates the TR response. Dn ‘o ð Þ ¼ c p P Z1 0 Da ‘o ð Þ o02 o2 do0 ð3Þ ð3Þ 0.1 1 10 100 1,000 Delay (ps) 2.8 2.6 2.4 2.2 2.0 1.8 Energy (eV) –8 –4 0 4 8 Pump scattering b a –10 –5 0 5 10 2.8 2.6 2.4 2.2 2.0 1.8 Energy (eV) –4 –2 0 2 4 TR(2 ps) TR simulation Δ (calculation) Δ (102 cm–1) ΔR/R (10–3) ΔR/R (10–3) Figure 2 \| TR spectra and simulations. (a) Pseudocolor representation of the transient reﬂectance spectra. (b) The transient reﬂection spectrum at delay time of 2 ps (dotted line in panel a) and the corresponding simulation (blue-dash line). Results Si l The ﬁrst term describes the continuum state absorption and the second term is for excitonic states. We modelled the absorption (black line, Fig. 1d) accounting for inhomogeneous broadening by convolving with a Gaussian function (see Supplementary Table 1 for ﬁtting parameters). We neglect the excitonic transitions with nex larger than 6 because the oscillator strength decreases as 1/nex 3 . We ﬁnd the best-ﬁt Simulation of optical absorption near bandedge. The absorption coefﬁcient (Fig. 1d), determined from ellipsometry 2 ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms8961 where c is the speed of light, and P means Cauchy principal value of the integral. The photo-induced Da includes both bleach of the exciton absorption and continuum band absorption. However, at low excitation levels, the bleach of continuum band absorption, caused by band ﬁlling, is not important because of the low occupation probability. Thus, Da is dominated by the bleach of exciton absorption resulting from photo-induced carriers (free carriers and excitons) due to the phase-space ﬁlling effect, and can be expressed as23,24: values of Rex and Eg to be 41.6 meV and 2.394 eV with o0.1 meV ﬁtting uncertainty. The exciton binding energy found here is about half of the calculated value18. For photon energies greater than B2.55 eV, the absorption differs from the model because higher energy bands are not included. Transient reﬂectance spectroscopy. For the pump-probe TR measurements, the pump is monowavelength with photon energy of 2.48 eV and the probe is broadband with the spectrum ranging from 2.95 to 1.50 eV. The pump penetration depth is calculated as 190 nm from the absorption coefﬁcient while the effective detecting depth is Bl/4pn (n is refractive index)21 and is B18 nm for probe photon energies near bandgap. The pseudocolor image of the TR spectra is shown in Fig. 2a with a representative spectrum at 2 ps (Fig. 2b). There are two anti- symmetric peaks centred at 2.35 eV. At longer delays, the magnitude decreases while the spectral shape persists. Since DR(‘o)/R is small (B0.005) and the refractive index n(‘o) is much larger than the extinction coefﬁcient k(‘o) in the spectral region of interest (Supplementary Fig. 2), DR(‘o)/R can be approximated as: Da ¼ a0 N Ns ð4Þ ð4Þ where a0 is the steady state exciton absorption coefﬁcient (green-dash line, Fig. & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms8961 The normalized surface carrier density dynamics shows the same decay trend as the TR kinetics. The black dashed lines represent the global ﬁtting based on the carrier diffusion model. (b) Normalized carrier density distribution proﬁles for 2.48 eV pump at indicated delays in the single crystal. Inset shows the distributions within 100 nm from the surface, and the red shade represents the probe (at 2.38 eV) detection depth. Figure 4 \| Excitation energy dependent TR kinetics and carrier density Figure 4 \| Excitation energy dependent TR kinetics and carrier density distribution proﬁles. (a) The normalized TR kinetics recorded at 2.38 eV for three different pump energies. The normalized surface carrier density dynamics shows the same decay trend as the TR kinetics. The black dashed lines represent the global ﬁtting based on the carrier diffusion model. (b) Normalized carrier density distribution proﬁles for 2.48 eV pump at indicated delays in the single crystal. Inset shows the distributions within 100 nm from the surface, and the red shade represents the probe (at 2.38 eV) detection depth. Figure 3 \| Excitation intensity dependent TR kinetics. (a) The kinetics of transient reﬂectance recorded at 2.38 eV for different excitation intensities. The pump energy is 2.48 eV. Inset is the plot of maximum amplitude as function of excitation intensity. (b) The normalized kinetics shown in panel a. dynamics shows the same decay trend as the TR kinetics. The black dashed lines represent the global ﬁtting based on the carrier diffusion model. (b) Normalized carrier density distribution proﬁles for 2.48 eV pump at indicated delays in the single crystal. Inset shows the distributions within 100 nm from the surface, and the red shade represents the probe (at 2.38 eV) detection depth. above, the TR kinetics follow the total carrier dynamics in the effective detecting region. To understand the relationship between carrier density and surface recombination, we use a one- dimensional diffusion model that includes surface recombination to reproduce the TR kinetics. The analytical expression for the normalized carrier density distribution as function of time is26,27: 40 30 20 10 0 eff (ns) 30 25 20 15 10 5 0 Grain size (μm) S=10 cm s–1 S=100 cm s–1 S=1,000 cm s–1 S=10,000 cm s–1 S=3.4×103 cm s–1 (measured) b = 31 ns (measured) Figure 5 \| Effective lifetime (seff) in polycrystalline ﬁlms. teff are plotted as function of the grain size for various surface recombination velocity. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms8961 The black dashed line represents the measured bulk lifetime. The red-dash line is effective lifetime as function of grain size calculated according to the SRV determined in the perovskite single crystals. 40 30 20 10 0 eff (ns) 30 25 20 15 10 5 0 Grain size (μm) S=10 cm s–1 S=100 cm s–1 S=1,000 cm s–1 S=10,000 cm s–1 S=3.4×103 cm s–1 (measured) b = 31 ns (measured) N t; x ð Þ ¼ 1 2 exp x2 4Dt w a ﬃﬃﬃﬃﬃ Dt p x 2 ﬃﬃﬃﬃﬃ Dt p S þ aD S aD w a ﬃﬃﬃﬃﬃ Dt p þ x 2 ﬃﬃﬃﬃﬃ Dt p þ 2S S aD w S ﬃﬃﬃﬃ t D r þ 2x 2 ﬃﬃﬃﬃﬃ Dt p !# ð5Þ ð5Þ where t is delay time, x is distance from surface, D is the ambipolar diffusion coefﬁcient, a is absorption coefﬁcient at the excitation energy, S is surface recombination velocity, and w(z) ¼ exp(z2)[1 erf(z)]. Since the pump penetration depths are always larger than effective detecting depth, the carrier density in detecting region can be approximated as the carrier density evaluated at the surface, N(t,0)28,29 (the validity of this approximation is explored below). A non-linear global ﬁtting routine is used to model the three kinetic traces simultaneously by calculating N(t,0). In the ﬁtting procedure, the absorption where t is delay time, x is distance from surface, D is the ambipolar diffusion coefﬁcient, a is absorption coefﬁcient at the excitation energy, S is surface recombination velocity, and w(z) ¼ exp(z2)[1 erf(z)]. Since the pump penetration depths are always larger than effective detecting depth, the carrier density in detecting region can be approximated as the carrier density evaluated at the surface, N(t,0)28,29 (the validity of this approximation is explored below). A non-linear global ﬁtting routine is used to model the three kinetic traces simultaneously by calculating N(t,0). In the ﬁtting procedure, the absorption Figure 5 \| Effective lifetime (seff) in polycrystalline ﬁlms. teff are plotted Figure 5 \| Effective lifetime (seff) in polycrystalline ﬁlms. teff are plotted as function of the grain size for various surface recombination velocity. The black dashed line represents the measured bulk lifetime. The red-dash line is effective lifetime as function of grain size calculated according to the SRV determined in the perovskite single crystals. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms8961 the s of ies. as 1.0 0.8 0.6 0.4 0.2 0.0 Normalized ΔR/R 5,000 4,000 3,000 2,000 1,000 0 Delay (ps) 1.0 0.8 0.6 0.4 0.2 0.0 Normalized N (t,0) Normalized N (t,x) Pump 2.28 eV Pump 2.48 eV Pump 3.10 eV b a 1.0 0.8 0.6 0.4 0.2 0.0 2.0 1.5 1.0 0.5 0.0 Distance (μm) 10 ps 100 ps 500 ps 5,000 ps 1,000 ps 1.0 0.8 0.6 0.4 0.2 0.0 100 80 60 40 20 0 Distance (nm) 10 ps 100 ps 500 ps 5,000 ps 1,000 ps N (t,x) Figure 4 \| Excitation energy dependent TR kinetics and carrier density distribution proﬁles. (a) The normalized TR kinetics recorded at 2.38 eV for three different pump energies. The normalized surface carrier density dynamics shows the same decay trend as the TR kinetics. The black dashed lines represent the global ﬁtting based on the carrier diffusion model. (b) Normalized carrier density distribution proﬁles for 2.48 eV pump at indicated delays in the single crystal. Inset shows the distributions within 100 nm from the surface, and the red shade represents the probe (at 2.38 eV) detection depth. b a 1.0 0.8 0.6 0.4 0.2 0.0 5,000 4,000 3,000 2,000 1,000 0 Delay (ps) Excitation density: 0.49–4.99×1017 cm–3 15 10 5 0 ΔR/R (10–3) ΔR/R (a.u) ΔR/R (10–3) 5,000 4,000 3,000 2,000 1,000 0 Delay (ps) Excitation density (1017 cm–3) 0.49 0.92 1.68 2.97 4.99 15 10 5 0 5 4 3 2 1 0 TRmax N (1017 cm–3) Figure 3 \| Excitation intensity dependent TR kinetics. (a) The kinetics of transient reﬂectance recorded at 2.38 eV for different excitation intensities. The pump energy is 2.48 eV. Inset is the plot of maximum amplitude as function of excitation intensity. (b) The normalized kinetics shown in panel a. 1.0 0.8 0.6 0.4 0.2 0.0 Normalized ΔR/R 5,000 4,000 3,000 2,000 1,000 0 Delay (ps) 1.0 0.8 0.6 0.4 0.2 0.0 Normalized N (t,0) Normalized N (t,x) Pump 2.28 eV Pump 2.48 eV Pump 3.10 eV b a 1.0 0.8 0.6 0.4 0.2 0.0 2.0 1.5 1.0 0.5 0.0 Distance (μm) 10 ps 100 ps 500 ps 5,000 ps 1,000 ps 1.0 0.8 0.6 0.4 0.2 0.0 100 80 60 40 20 0 Distance (nm) 10 ps 100 ps 500 ps 5,000 ps 1,000 ps N (t,x) Figure 4 \| Excitation energy dependent TR kinetics and carrier density distribution proﬁles. NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms8961 (a) The normalized TR kinetics recorded at 2.38 eV for three different pump energies. The normalized surface carrier density dynamics shows the same decay trend as the TR kinetics. The black dashed lines represent the global ﬁtting based on the carrier diffusion model. (b) Normalized carrier density distribution proﬁles for 2.48 eV pump at indicated delays in the single crystal. Inset shows the distributions within 100 nm from the surface, and the red shade represents the probe (at 2 38 eV) detection depth b a 1.0 0.8 0.6 0.4 0.2 0.0 5,000 4,000 3,000 2,000 1,000 0 Delay (ps) Excitation density: 0.49–4.99×1017 cm–3 15 10 5 0 ΔR/R (10–3) ΔR/R (a.u) ΔR/R (10–3) 5,000 4,000 3,000 2,000 1,000 0 Delay (ps) Excitation density (1017 cm–3) 0.49 0.92 1.68 2.97 4.99 15 10 5 0 5 4 3 2 1 0 TRmax N (1017 cm–3) Figure 3 \| Excitation intensity dependent TR kinetics. (a) The kinetics transient reﬂectance recorded at 2.38 eV for different excitation intensitie The pump energy is 2.48 eV. Inset is the plot of maximum amplitude as function of excitation intensity. (b) The normalized kinetics shown in panel a. 1.0 0.8 0.6 0.4 0.2 0.0 Normalized ΔR/R 5,000 4,000 3,000 2,000 1,000 0 Delay (ps) 1.0 0.8 0.6 0.4 0.2 0.0 Normalized N (t,0) Pump 2.28 eV Pump 2.48 eV Pump 3.10 eV a a 15 10 5 0 ΔR/R (10–3) ΔR/R (10–3) 5,000 4,000 3,000 2,000 1,000 0 Delay (ps) Excitation density (1017 cm–3) 0.49 0.92 1.68 2.97 4.99 15 10 5 0 5 4 3 2 1 0 TRmax N (1017 cm–3) a a Normalized N (t,x) b 1.0 0.8 0.6 0.4 0.2 0.0 2.0 1.5 1.0 0.5 0.0 Distance (μm) 10 ps 100 ps 500 ps 5,000 ps 1,000 ps 1.0 0.8 0.6 0.4 0.2 0.0 100 80 60 40 20 0 Distance (nm) 10 ps 100 ps 500 ps 5,000 ps 1,000 ps N (t,x) b b 1.0 0.8 0.6 0.4 0.2 0.0 5,000 4,000 3,000 2,000 1,000 0 Delay (ps) Excitation density: 0.49–4.99×1017 cm–3 ΔR/R (a.u) Figure 4 \| Excitation energy dependent TR kinetics and carrier density distribution proﬁles. (a) The normalized TR kinetics recorded at 2.38 eV Figure 4 \| Excitation energy dependent TR kinetics and carrier density distribution proﬁles. (a) The normalized TR kinetics recorded at 2.38 eV for three different pump energies. Results Si l The calculated change in absorption coefﬁcient is shown as the green-dash line. 0.1 1 10 100 1,000 Delay (ps) 2.8 2.6 2.4 2.2 2.0 1.8 Energy (eV) –8 –4 0 4 8 Pump scattering a ΔR/R (10–3) p Figure 3b contains the normalized data from Fig. 3a, showing that TR kinetics for a given excitation energy are independent of excitation density, over 1 order of magnitude. The decay of the TR kinetics suggests the carrier depopulation and/or diffusion out of the probing region. In the bulk, the depopulation from ﬁrst-order radiative and/or Shockley–Read–Hall (nonradiative) recombination is independent of the excitation density. However, they are excluded because the ﬁrst-order bulk recombination occurs on a much longer time scale (B31 ns, measured by time- resolved photoluminescence, Supplementary Fig. 3). The decay of TR kinetics is therefore attributed to the surface recombination and the carrier diffusion from the surface into the bulk. Since no electric ﬁled is applied, the carriers are expected to diffuse together as either uncorrelated electron-hole pairs or excitons to maintain the charge neutrality. b b –10 –5 0 5 10 2.8 2.6 2.4 2.2 2.0 1.8 Energy (eV) –4 –2 0 2 4 TR(2 ps) TR simulation Δ (calculation) Δ (102 cm–1) ΔR/R (10–3) Δ (102 cm–1) Diffusion and surface recombination model. To explore DR/R recovery kinetics, we varied the excitation photon energy. In addition to 2.48 eV the sample was excited at 3.10 and 2.28 eV with corresponding penetration depths of 80 nm and 3.7 mm. We conﬁrmed for each photon energy that TR kinetics are independent of excitation density (Supplementary Figs 4 and 5). However, the DR/R dynamics exhibits a clear dependence on the excitation energy (Fig. 4a). In Fig. 4a the data for different excitation energies are normalized for comparison and we ﬁnd that the kinetics decays faster for larger excitation energies corresponding to shorter penetration depths. Because of the linear relationship between DR/R and excitation density discussed Figure 2 \| TR spectra and simulations. (a) Pseudocolor representation of the transient reﬂectance spectra. (b) The transient reﬂection spectrum at delay time of 2 ps (dotted line in panel a) and the corresponding simulation (blue-dash line). The calculated change in absorption coefﬁcient is shown as the green-dash line. 3 & 2015 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms8961 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms8961 Figure 5 \| Effective lifetime (seff) in polycrystalline ﬁlms. teff are plotted as function of the grain size for various surface recombination velocity. The black dashed line represents the measured bulk lifetime. The red-dash line is effective lifetime as function of grain size calculated according to the SRV determined in the perovskite single crystals. NATURE COMMUNICATIONS \| 6:7961 \| DOI: 10.1038/ncomms8961 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS \| 6:7961 \| DOI: 10.1038/ncomms8961 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. NATURE COMMUNICATIONS \| 6:7961 \| DOI: 10.1038/ncomms8961 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. 4 NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms8961 coefﬁcient, a, at each pump-photon energy is determined from the ellipsometry (Supplementary Fig. 2), and S and D are free ﬁtting parameters. The best-ﬁt results are shown in Fig. 4a with best-ﬁt parameters of S ¼ 3.4±0.1 103 cm s 1 and D ¼ 0.27±0.01 cm2 s 1. To directly compare with reported value, we calculated the ambipolar mobility as 10.8 cm2 V 1 s 1 from D via the Einstein relation. Our value is consistent with that determined from Hall effect measurement of CH3NH3PbBr3 single crystals9, B1–10 times larger than that obtained for CH3NH3PbX3 (X ¼ Br and I) polycrystalline ﬁlms11,12,15–17, and B10 times smaller than that obtained from time-of-ﬂight measurement of the perovskite single crystals9,10. The SRV determined here is B2–3 orders of magnitude smaller than those in other important semiconductors (before surface passivation) such as GaAs (SB8.5 105cms 1)30, GaP (SB2 106cms 1)31, InP (SB1.5 105 cms 1)26, p-Si (SB2.4 105cms 1)32, n-Si (SB1.2 106 cms 1)32, and CdTe (SB5 105cms 1)33. In solar cells, charge recombination across interfaces between active layers and charge transporting layers can also impact the performance. Recently, Tress et al. demonstrated that interfacial charge recombination could be suppressed by optimizing the thickness of the hole transporting layer in perovskite solar cells39. In this case, the open-circuit voltage is limited by the nonradiative bulk and surface recombination40. Considering the long bulk lifetime, surface recombination likely limits the open-circuit voltage for perovskite solar cells. Thus, lowering surface recombination should enhance the open-circuit voltage. It should be noted that the surface recombination measured here is from a single crystal surface without post treatment. However, in working solar cells, the surfaces could be unintentionally passivated by contacting with charge transport layers41 or forming a lead halide over layer42, which may lead to a reduction of surface recombination. The passivation dependent surface recombination is beyond the scope of this paper, and will be the subject of the future work. In Fig. 4b, we display the calculated carrier distributions at various delay times according to equation (5) for 2.48 eV excitation. The carrier density decreases near the surface and increases in the bulk due to diffusion. ð6Þ Sample preparation. To a 5 ml dimethylformaide solvent, PbBr2 (183 mg, 0.5 mmol) and CH3NH3Br (56 mg, 0.5 mmol) were dissolved. The mixture solution was heated slightly to obtain a transparent solution. This solution was further ﬁltered through a compacted celite column. The ﬁltrate was collected. Two millilitre of this solution was transferred into an inner vial (5 ml in total vial volume) that was placed in a larger outer vial (25 ml in total volume) with 5 ml of toluene inside. Finally the outer vial was carefully sealed. The diffusion of toluene from outer vial into the inner vial was slow and the crystallization process was maintained in dark and undisturbed environment for at least three days. The orange block-shaped single crystals were obtained and characterized by X-ray diffraction. where tb is the bulk carrier lifetime measured above, d is planar grain size. The effective lifetimes, teff, are calculated with D ¼ 0.27 cm2 s 1 plotted in Fig. 5 for various SRV. We ﬁnd that, ultra low SRV (SB10–100 cm s 1) is required to make teff approach tb when do5 mm, suggesting that surface passivation will be necessary for the perovskite cells with grain size smaller than 5 mm. For unpassivated perovskites (SB103–104 cm s 1), the surface recombination inﬂuence on the teff can be eliminated when the grain size is larger than B30 mm. The effective lifetime curve calculated with the measured S and D (Fig. 5, red-dash curve) shows close correspondence with the reported trend of the perovksite solar cell efﬁciency versus apparent grain size, in which the efﬁciency was boosted by 10 times as the grain size increased from 1 to B20 mm while efﬁciency growth slope became much slower when the grain size was larger than B20 mm (ref. 4). Discussion We measured TR kinetics for several different single crystals obtained from the same growth method and for different faces of the same crystal (Supplementary Fig. 6) and ﬁnd a high degree of reproducibility of our measurement. For single crystal semiconductors, midgap states created by surface defects are primarily responsible for surface recombination. Theoretical calculations suggest that defects at grain boundary or surfaces for CH3NH3PbX3 (X ¼ Br and I) perovskite in either cubic or tetragonal phase cause very few midgap states34–37, which may explain the low SRV determined here. Compared with the bromide perovskite, the iodide perovskite likely has less midgap states due to the narrower bandgap, and thus we expect an even lower SRV (ongoing measurements). NATURE COMMUNICATIONS \| 6:7961 \| DOI: 10.1038/ncomms8961 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. The inset shows N(t,x) close to surface, where the red shaded region represents the effective detecting depth (B18 nm). The carrier distribution is nearly uniform in this regime, conﬁrming that the carrier density that affects DR/R can be safely approximated by the surface density. In summary, we conducted TR measurements for perovskite single crystals. The TR spectra are quantitatively described by excitonic absorption bleach, and carrier dynamics is attributed to surface recombination and carrier diffusion from the surface into the bulk. By simultaneously ﬁtting the carrier decay for different excitation energies, SRV and diffusion coefﬁcient are obtained. The measured SRV is B2–3 order of magnitude smaller than that in many semiconductors used for solar cells. Our result also suggested that the grain size in polycrystalline perovskite ﬁlm should be larger than B30 mm or grain boundaries should be further passivated to eliminate the surface recombination inﬂuence on effective lifetime. 1. Gray, J. L. in Handbook of Photovoltaic Science and Engineering 61–112 (John Wiley & Sons, Ltd, 2005). 2. Green, M. A. The path to 25% silicon solar cell efﬁciency: history of silicon cell evolution. Prog. Photovolt: Res. Appl 17, 183–189 (2009). 3. Sopori, B. in Handbook of Photovoltaic Science and Engineering 307–357 (John Wiley & Sons, Ltd, 2005). 4. Nie, W. et al. High-efﬁciency solution-processed perovskite solar cells with millimeter-scale grains. Science 347, 522–525 (2015). NATURE COMMUNICATIONS \| 6:7961 \| DOI: 10.1038/ncomms8961 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved. Methods Transient re Transient reﬂectance spectroscopy. Femtosecond pump-probe TR experiments were performed based on a regeneratively ampliﬁed Ti:sapphire laser system that produces B4 mJ laser pulses at 800 nm with 1 KHz repetition rate. The pumps for TR are generated by an optical parametric ampliﬁer pumped by 800 nm fundamental pulses (B1.5 mJ per pulse), which is chopped at a rate of 500 Hz and attenuated by neutral density ﬁlter wheels. The broadband probe pulses (420–830 nm) are generated by focusing 800 nm light into a sapphire crystal. The probe pulses are delayed in time with respect to the pump pulses using a motorized translation stage mounted with a retroreﬂecting mirror. The pump and probe are spatially overlapped on the surface of the sample, and the probe pulses are directed to the multichannel complementary metal–oxide–semiconductor sensor. The size of the focused spot at the sample position for probe and pump beams are 180 and 560 mm, respectively. The total pump-photon ﬂux is determined by measuring the pump power after a pinhole with radius of 200 mm at the sample position. The input photon ﬂux is obtained by subtracting the reﬂected photon ﬂux from the total photon ﬂux. The excitation density is calculated as the ratio of input photon ﬂux to the effective depth that is 1 over absorption coefﬁcient at pumping energy. For polycrystalline thin ﬁlms, the surface recombination at grain boundaries can determine the effective carrier lifetime. To minimize the impact from surface recombination, the grain size cannot be too small. The effective lifetime as function of grain size with a given surface recombination velocity (S) is approximated by38: 1 teff ¼ 1 tb þ 2 d 2S þ 1 D d p 2 ð6Þ ð6Þ ARTICLE NATURE COMMUNICATIONS \| DOI: 10.1038/ncomms8961 5. Liu, D. & Kelly, T. L. Perovskite solar cells with a planar heterojunction structure prepared using room-temperature solution processing techniques. Nat. Photonics 8, 133–138 (2014). 32. Sabbah, A. J. & Riffe, D. M. Measurement of silicon surface recombination velocity using ultrafast pump–probe reﬂectivity in the near infrared. J. Appl. Phys. 88, 6954–6956 (2000). y 33. Cohen, R., Lyahovitskaya, V., Poles, E., Liu, A. & Rosenwaks, Y. Unusually low surface recombination and long bulk lifetime in n-CdTe single crystals. Appl. Phys. Lett. 73, 1400–1402 (1998). 6. Liu, M., Johnston, M. B. & Snaith, H. J. Efﬁcient planar heterojunction perovskite solar cells by vapour deposition. Nature 501, 395–398 (2013). y 7. Burschka, J. et al. Sequential deposition as a route to high-performance perovskite-sensitized solar cells. Nature 499, 316–319 (2013). 34. Yin, W.-J., Shi, T. & Yan, Y. Unique properties of halide perovskites as possible origins of the superior solar cell performance. Adv. Mater. 26, 4653–4658 (2014). perovskite-sensitized solar cells. Nature 499, 316–319 (2013). 8. Zhou, H. et al. Interface engineering of highly efﬁcient perovskite solar cells. Science 345, 542–546 (2014). 35. Shi, T., Yin, W.-J., Hong, F., Zhu, K. & Yan, Y. Unipolar self-doping behavior in perovskite CH3NH3PbBr3. Appl. Phys. Lett. 106, 103902 (2015). 9. Shi, D. et al. Low trap-state density and long carrier diffusion in organolead trihalide perovskite single crystals. Science 347, 519–522 (2015). p pp y 36. Yin, W. J., Shi, T. T. & Yan, Y. F. Unusual defect physics in CH3NH3PbI3 perovskite solar cell absorber. Appl. Phys. Lett. 104, 063903 (2014). p g y 10. Dong, Q. et al. Solar cells. Electron-hole diffusion lengths 4175 mum in solution-grown CH3NH3PbI3 single crystals. Science 347, 967–970 (2015). 37. Kim, J., Lee, S.-H., Lee, J. H. & Hong, K.-H. The role of intrinsic defects in methylammonium lead iodide perovskite. J. Phys. Chem. Lett. 5, 1312–1317 (2014). 11. Wehrenfennig, C., Liu, M., Snaith, H. J., Johnston, M. B. & Herz, L. M. Charge-carrier dynamics in vapour-deposited ﬁlms of the organolead halide perovskite CH3NH3PbI3-xClx. Energy Environ. Sci. 7, 2269 (2014). 38. Sproul, A. B. Dimensionless solution of the equation describing the effect of surface recombination on carrier decay in semiconductors. J. Appl. Phys. 76, 2851–2854 (1994). gy 12. Wehrenfennig, C., Eperon, G. E., Johnston, M. B., Snaith, H. J. & Herz, L. M. High charge carrier mobilities and lifetimes in organolead trihalide perovskites. Adv. Mater. ARTICLE 26, 1584–1589 (2014). 39. Marinova, N. et al. Light harvesting and charge recombination in CH3NH3PbI3 perovskite solar cells studied by hole transport layer thickness variation. ACS Nano. 9, 4200–4209 (2015). 13. Marchioro, A. et al. Unravelling the mechanism of photoinduced charge transfer processes in lead iodide perovskite solar cells. Nat. Photonics 8, 250–255 (2014). 40. Tress, W. et al. Predicting the open-circuit voltage of CH3NH3PbI3 perovskite solar cells using electroluminescence and photovoltaic quantum efﬁciency spectra: the role of radiative and non-radiative recombination. Adv. Energy Mater. 5, 1400812 (2015). 14. Manser, J. S. & Kamat, P. V. Band ﬁlling with free charge carriers in organometal halide perovskites. Nat. Photonics 8, 737–743 (2014). 15. Xing, G. et al. Long-range balanced electron- and hole-transport lengths in organic-inorganic CH3NH3PbI3. Science 342, 344–347 (2013). 41. Wang, Q. et al. Large ﬁll-factor bilayer iodine perovskite solar cells fabricated by a low-temperature solution-process. Energy Environ. Sci. 7, 2359–2365 (2014). 16. Stranks, S. D. et al. Electron-hole diffusion lengths exceeding 1 micrometer in an organometal trihalide perovskite absorber. Science 342, 341–344 (2013). 17. Sheng, R. et al. Methylammonium lead bromide perovskite-based solar cells by vapor-assisted deposition. J. Phys. Chem. C 119, 3545–3549 (2015). 42. Chen, Q. et al. Controllable self-induced passivation of hybrid lead iodide perovskites toward high performance solar cells. Nano Lett. 14, 4158–4163 (2014). 18. Tanaka, K. et al. Comparative study on the excitons in lead-halide-based perovskite-type crystals CH3NH3PbBr3 CH3NH3PbI3. Solid State Commun. 127, 619–623 (2003). Acknowledgements 19. Elliott, R. J. Intensity of optical absorption by excitons. Phys. Rev. 108, 1384–1389 (1957). This work was supported by the Division of Chemical Sciences, Geosciences and Biosciences, Ofﬁce of Basic Energy Sciences of the US Department of Energy through the Solar Photochemistry Program under contract No. DE-AC36-08GO28308 to NREL. We thank Philip A. Parilla for help in X-ray diffraction analysis and thank Wan-jian Yin for useful discussion on the surface defects. X-ray diffraction measurement was supported by the NREL LDRD program. This work was supported by the Division of Chemical Sciences, Geosciences and Biosciences, Ofﬁce of Basic Energy Sciences of the US Department of Energy through the Solar Photochemistry Program under contract No. DE-AC36-08GO28308 to NREL. We thank Philip A. Parilla for help in X-ray diffraction analysis and thank Wan-jian Yin for useful discussion on the surface defects. X-ray diffraction measurement was supported by the NREL LDRD program. 20. Saba, M. et al. Correlated electron-hole plasma in organometal perovskites. Nat Commun 5, 5049 (2014). 21. Sabbah, A. J. & Riffe, D. M. Femtosecond pump-probe reﬂectivity study of silicon carrier dynamics. Phys. Rev. B 66, 165217 (2002). y y 22. Chemla, D. S., Miller, D. A. B., Smith, P. W., Gossard, A. C. & Wiegmann, W. Room temperature excitonic nonlinear absorption and refraction in GaAs/AlGaAs multiple quantum well structures. IEEE J. Quant. Electron 20, 265–275 (1984). Author contributions Y.Y. and M.B. conceived the original experimental ideas. Y.Y. carried out the transient reﬂectance measurements and analysed the data. Y.Yan and M.Y. prepared the sample. S.K. carried out the ellipsometry characterization. J.L. carried out the X-ray measurement. Y.Y. and M.B. prepared the manuscript. K.Z. and S.K. discussed the results and commented on the manuscript. Y.Y. and M.B. conceived the original experimental ideas. Y.Y. carried out the transient reﬂectance measurements and analysed the data. Y.Yan and M.Y. prepared the sample. S.K. carried out the ellipsometry characterization. J.L. carried out the X-ray measurement. Y.Y. and M.B. prepared the manuscript. K.Z. and S.K. discussed the results and commented on the manuscript. 23. Schmitt-Rink, S., Chemla, D. S. & Miller, D. A. B. Theory of transient excitonic optical nonlinearities in semiconductor quantum-well structures. Phys. Rev. B 32, 6601–6609 (1985). 24. Becker, P. C. et al. Femtosecond dynamics of resonantly excited room-temperature excitons in II-VI CdZnTe/ZnTe quantum wells. Phys. Rev. Lett. 68, 1876–1879 (1992). 25. Huang, D., Chyi, J.-I. & Morkoc¸, H. Carrier effects on the excitonic absorption in GaAs quantum-well structures: phase-space ﬁlling. Phys. Rev. B 42, 5147–5153 (1990). References 1. Gray, J. L. in Handbook of Photovoltaic Science and Engineering 61–112 (John Wiley & Sons, Ltd, 2005). 2. Green, M. A. The path to 25% silicon solar cell efﬁciency: history of silicon cell evolution. Prog. Photovolt: Res. Appl 17, 183–189 (2009). 3. Sopori, B. in Handbook of Photovoltaic Science and Engineering 307–357 (John Wiley & Sons, Ltd, 2005). 3. Sopori, B. in Handbook of Photovoltaic Science and Engineering 307–357 (John Wiley & Sons, Ltd, 2005). 4. Nie, W. et al. High-efﬁciency solution-processed perovskite solar cells with millimeter-scale grains. Science 347, 522–525 (2015). 4. Nie, W. et al. High-efﬁciency solution-processed perovskite solar cells with millimeter-scale grains. Science 347, 522–525 (2015). 5 2015 Macmillan Publishers Limited. All rights reserved. & 2015 Macmillan Publishers Limited. All rights reserved. Additional information Supplementary Information accompanies this paper at http://www.nature.com/ naturecommunications 26. Hoffman, C. A., Jarasˇiu¯nas, K., Gerritsen, H. J. & Nurmikko, A. V. Measurement of surface recombination velocity in semiconductors by diffraction from picosecond transient free-carrier gratings. Appl. Phys. Lett. 33, 536–539 (1978). Competing ﬁnancial interests: The authors declare no competing ﬁnancial interests. Competing ﬁnancial interests: The authors declare no competing ﬁnancial interests. Competing ﬁnancial interests: The authors declare no competing ﬁnancial interests. Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ 27. Vaitkus, J. The nonequilibrium hall effect and related transport phenomena in semiconductors under inhomogeneous excitation by a laser pulse. Phys. Status Solidi A 34, 769–775 (1976). How to cite this article: Yang, Y. et al. Low surface recombination velocity in solution-grown CH3NH3PbBr3 perovskite single crystal. Nat. Commun. 6:7961 doi: 10.1038/ncomms8961 (2015). How to cite this article: Yang, Y. et al. Low surface recombination velocity in solution-grown CH3NH3PbBr3 perovskite single crystal. Nat. Commun. 6:7961 doi: 10.1038/ncomms8961 (2015). 28. Tanaka, T., Harata, A. & Sawada, T. Subpicosecond surface-restricted carrier and thermal dynamics by transient reﬂectivity measurements. J. Appl. Phys. 82, 4033–4038 (1997). 29. Beck, S. M. & Wessel, J. E. Picosecond transient reﬂectivity of unpinned gallium arsenide (100) surfaces. Appl. Phys. Lett. 50, 149–151 (1987). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ pp y 30. Beard, M. C., Turner, G. M. & Schmuttenmaer, C. A. Transient photoconductivity in GaAs as measured by time-resolved terahertz spectroscopy. Phys. Rev. B 62, 15764–15777 (2000). 30. Beard, M. C., Turner, G. M. & Schmuttenmaer, C. A. Transient photoconductivity in GaAs as measured by time-resolved terahertz spectroscopy. Phys. Rev. B 62, 15764–15777 (2000). p py y 31. Gershenzon, M. & Mikulyak, R. M. Radiative Pair Recombination and Surface Recombination in GaP Photoluminescence. Appl. Phys. Lett. 8, 245–247 (1966). 31. Gershenzon, M. & Mikulyak, R. M. Radiative Pair Recombination and Surface Recombination in GaP Photoluminescence. Appl. Phys. Lett. 8, 245–247 (1966). 6 NATURE COMMUNICATIONS \| 6:7961 \| DOI: 10.1038/ncomms8961 \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| 6:7961 \| DOI: 10.1038/ncomms8961 \| www.nature.com/naturecommunications & 2015 Macmillan Publishers Limited. All rights reserved.
https://openalex.org/W2969634344	https://www.biorxiv.org/content/biorxiv/early/2020/02/17/741249.full.pdf	English	null	Adapting the Eysenck Personality Questionnaire-Revised Neuroticism scale for use in epidemiologic studies: A psychometric evaluation using item response theory in the UK Biobank	bioRxiv (Cold Spring Harbor Laboratory)	2,019	cc-by	15,157	Bauermeister & Gallacher: Neuroticism . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint Bauermeister & Gallacher: Neuroticism . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint Bauermeister & Gallacher: Neuroticism . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint Bauermeister & Gallacher: Neuroticism Bauermeister & Gallacher: Neuroticism A psychometric evaluation of the 12-item EPQ-R neuroticism scale in 502,591 UK Biobank participants using item response theory (IRT) Sarah Bauermeister1 and John Gallacher1 1Department of Psychiatry and on behalf of Dementias Platform UK, Warneford Hospital, University of Oxford, Oxford, OX3 7JX 1Department of Psychiatry and on behalf of Dementias Platform UK, Warneford Hospital, University of Oxford, Oxford, OX3 7JX Sarah Bauermeister (corresponding author) sarah.bauermeister@psych.ox.ac.uk John Gallacher john.gallacher@psych.ox.ac.uk 1 Bauermeister & Gallacher: Neuroticism . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint Bauermeister & Gallacher: Neuroticism Bauermeister & Gallacher: Neuroticism Background 2 Background 2 Neuroticism has been described as a broad and pervasive personality dimension or 3 ‘heterogeneous’ trait measuring components of mood instability such as worry; anxiety; 4 irritability; moodiness; self-consciousness; sadness and irritabililty. Consistent with 5 depression and anxiety-related disorders, increased neuroticism places an individual 6 vulnerable for other unipolar and bipolar mood disorders. However, the measurement 7 of neuroticism remains a challenge. Our aim was to identify psychometrically efficient 8 items and inform the inclusion of redundant items across the 12-item EPQ-R 9 Neuroticism scale using Item Response Theory (IRT). 10 11 Methods 12 The 12-item binary EPQ-R Neuroticism scale was evaluated by estimating a two- 13 parameter (2-PL) IRT model on data from 502,591 UK Biobank participants aged 37 to 14 73 years (M = 56.53 years; SD = 8.05), 54% female. Models were run listwise (n= 15 401,648) and post-estimation mathematical assumptions were computed. All analyses 16 were conducted in STATA 16 SE on the Dementias Platform UK (DPUK) Data Portal. 17 18 Results 19 A plot of θ values (Item Information functions) showed that most items clustered 20 around the mid-range where discrimination values ranged from 1.34 to 2.28. Difficulty 21 values for individual item θ scores ranged from -0.13 to 1.41. A Mokken analysis 22 Neuroticism has been described as a broad and pervasive personality dimension or 3 ‘heterogeneous’ trait measuring components of mood instability such as worry; anxiety; 4 irritability; moodiness; self-consciousness; sadness and irritabililty. Consistent with 5 depression and anxiety-related disorders, increased neuroticism places an individual 6 vulnerable for other unipolar and bipolar mood disorders. However, the measurement 7 of neuroticism remains a challenge. Our aim was to identify psychometrically efficient 8 items and inform the inclusion of redundant items across the 12-item EPQ-R 9 Neuroticism scale using Item Response Theory (IRT). 10 Neuroticism has been described as a broad and pervasive personality dimension or 3 ‘heterogeneous’ trait measuring components of mood instability such as worry; anxiety; 4 irritability; moodiness; self-consciousness; sadness and irritabililty. Consistent with 5 depression and anxiety-related disorders, increased neuroticism places an individual 6 vulnerable for other unipolar and bipolar mood disorders. However, the measurement 7 of neuroticism remains a challenge. Our aim was to identify psychometrically efficient 8 items and inform the inclusion of redundant items across the 12-item EPQ-R 9 Neuroticism scale using Item Response Theory (IRT). 10 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint Methods 12 The 12-item binary EPQ-R Neuroticism scale was evaluated by estimating a two- 13 parameter (2-PL) IRT model on data from 502,591 UK Biobank participants aged 37 to 14 73 years (M = 56.53 years; SD = 8.05), 54% female. Models were run listwise (n= 15 401,648) and post-estimation mathematical assumptions were computed. All analyses 16 were conducted in STATA 16 SE on the Dementias Platform UK (DPUK) Data Portal. 17 18 Results 19 A plot of θ values (Item Information functions) showed that most items clustered 20 around the mid-range where discrimination values ranged from 1.34 to 2.28. Difficulty 21 values for individual item θ scores ranged from -0.13 to 1.41. A Mokken analysis 22 The 12-item binary EPQ-R Neuroticism scale was evaluated by estimating a two- 13 parameter (2-PL) IRT model on data from 502,591 UK Biobank participants aged 37 to 14 73 years (M = 56.53 years; SD = 8.05), 54% female. Models were run listwise (n= 15 401,648) and post-estimation mathematical assumptions were computed. All analyses 16 were conducted in STATA 16 SE on the Dementias Platform UK (DPUK) Data Portal. 17 2 Bauermeister & Gallacher: Neuroticism . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint Bauermeister & Gallacher: Neuroticism Bauermeister & Gallacher: Neuroticism suggested a weak to medium level of monotonicity between the items, no items reach 23 strong scalability (H=0.35-0.47). Systematic item deletions and rescaling found that an 24 7-item scale is more efficient and with information (discrimination) ranging from 1.56 25 to 2.57 and stronger range of scalability (H=0.47-0.52). A 3-item scale is highly 26 discriminatory but offers a narrow range of person ability (difficulty). A logistic 27 regression differential item function (DIF) analysis exposed significant gender item bias 28 functioning uniformly across all versions of the scale. Methods 12 It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint Bauermeister & Gallacher: Neuroticism Bauermeister & Gallacher: Neuroticism Background 42 Neuroticism has been described as a broad and pervasive personality dimension with 43 influences beyond its own limited definition (1). Operationally, it has been defined as a 44 personality trait assessed by items referencing to instances of worry; anxiety; 45 irritability; moodiness; self-consciousness; sadness and irritabililty (2-4). The NEO-PI 46 (Neuroticism-Extraversion-Openess Personality Inventory) operationalises neuroticism 47 as a combination of individual behavioural traits which may also be measured as 48 isolated components of mood state e.g., anxiety; hostility; depression; self-consiousness; 49 impulsiveness and vulnerability (1). Also defined as a ‘heterogeneous’ trait possessing 50 significant overlap with depression and anxiety, neuroticism places an individual 51 vulnerable for other unipolar and bipolar mood disorders (3). Moreover, increased 52 levels of neuroticism places an individual vulnerable to other neurotic disorders, 53 psychological distress and ‘emotional instability’ (5). There is also consistent research 54 suggesting a positive relationship between neuroticism and negative effect (6) 55 notwithstanding neurotism essentially existing as a dimension of negative affect (7). 56 Eysenck has further argued that neuroticism is a direct reaction to the autonomic 57 nervous system (8, 9), findings supported finding increased neuroticism correlated with 58 tolerance to a highly stressed environment, suggesting a habituation relationship with 59 everyday stressors (10, 11). 60 Neuroticism has been described as a broad and pervasive personality dimension with 43 influences beyond its own limited definition (1). Operationally, it has been defined as a 44 personality trait assessed by items referencing to instances of worry; anxiety; 45 irritability; moodiness; self-consciousness; sadness and irritabililty (2-4). The NEO-PI 46 (Neuroticism-Extraversion-Openess Personality Inventory) operationalises neuroticism 47 as a combination of individual behavioural traits which may also be measured as 48 isolated components of mood state e.g., anxiety; hostility; depression; self-consiousness; 49 impulsiveness and vulnerability (1). Also defined as a ‘heterogeneous’ trait possessing 50 significant overlap with depression and anxiety, neuroticism places an individual 51 vulnerable for other unipolar and bipolar mood disorders (3). Moreover, increased 52 levels of neuroticism places an individual vulnerable to other neurotic disorders, 53 psychological distress and ‘emotional instability’ (5). There is also consistent research 54 suggesting a positive relationship between neuroticism and negative effect (6) 55 notwithstanding neurotism essentially existing as a dimension of negative affect (7). Methods 12 29 30 Conclusions 31 Across 401,648 UK Biobank participants, the 12-item EPQ-R neuroticism scale exhibited 32 psychometric inefficiency with poor discrimination at the extremes of the scale-range. 33 High and low scores are relatively poorly represented and uninformative suggesting 34 that high neuroticism scores derived from the EPQ-R are a function of cumulative mid- 35 range values. The scale also shows evidence of gender item bias and future scale 36 development should consider the former along with item deletions. 37 38 Keywords 39 Item Response Theory; IRT; neuroticism; psychometric; EPQ-R; UK Biobank; 40 epidemiology 41 suggested a weak to medium level of monotonicity between the items, no items reach 23 strong scalability (H=0.35-0.47). Systematic item deletions and rescaling found that an 24 7-item scale is more efficient and with information (discrimination) ranging from 1.56 25 to 2.57 and stronger range of scalability (H=0.47-0.52). A 3-item scale is highly 26 discriminatory but offers a narrow range of person ability (difficulty). A logistic 27 regression differential item function (DIF) analysis exposed significant gender item bias 28 functioning uniformly across all versions of the scale. 29 Across 401,648 UK Biobank participants, the 12-item EPQ-R neuroticism scale exhibited 32 psychometric inefficiency with poor discrimination at the extremes of the scale-range. 33 High and low scores are relatively poorly represented and uninformative suggesting 34 that high neuroticism scores derived from the EPQ-R are a function of cumulative mid- 35 range values. The scale also shows evidence of gender item bias and future scale 36 development should consider the former along with item deletions. 37 Across 401,648 UK Biobank participants, the 12-item EPQ-R neuroticism scale exhibited 32 psychometric inefficiency with poor discrimination at the extremes of the scale-range. 33 High and low scores are relatively poorly represented and uninformative suggesting 34 that high neuroticism scores derived from the EPQ-R are a function of cumulative mid- 35 range values. The scale also shows evidence of gender item bias and future scale 36 development should consider the former along with item deletions. 37 Item Response Theory; IRT; neuroticism; psychometric; EPQ-R; UK Biobank; 40 epidemiology 41 Item Response Theory; IRT; neuroticism; psychometric; EPQ-R; UK Biobank; 40 epidemiology 41 3 3 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Background 42 56 Eysenck has further argued that neuroticism is a direct reaction to the autonomic 57 nervous system (8, 9), findings supported finding increased neuroticism correlated with 58 tolerance to a highly stressed environment, suggesting a habituation relationship with 59 everyday stressors (10, 11). 60 61 Eysenck’s attempts to define neuroticism and evaluate the measurement items thereof 62 resulted in an original version of the Eysenck neuroticism scale existing as a component 63 of the Maudsley Medical Questionnaire (12). Assessment outcomes of this scale were 64 reported in the Manual for the Maudsley Personality Inventory (MPI) where gender 65 4 Bauermeister & Gallacher: Neuroticism . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint Bauermeister & Gallacher: Neuroticism Bauermeister & Gallacher: Neuroticism Bauermeister & Gallacher: Neuroticism differences were found across the psychiatric patients and soldiers, on whom the data 66 were derived (13). Later versions of the MPI were revised to remove gender-specific 67 items although to our knowledge, details of the method of their removal are not 68 available. The revised neuroticism scale became a component of the Eysenck 69 Personality Questionnaire (EPQ-R: 14) and thereby exists as a culmination of attempts 70 to select the relevant items through multiple revisions of the MPI. Although the EPQ-R 71 neuroticism scale is reported to have been developed through clinical judgement and, 72 multiple cluster and factor analyses, reasons for acceptance or rejection of items were 73 complex, unclear and are not ‘objectified’ (13, 15). 74 75 Using factor analysis and correlations for item deletion whilst widely used, have a bias 76 towards identifying closely associated items as being informative and is opaque to the 77 individual item contribution or person ability. The process is commonly known as 78 classical test theory (CTT) whereby a summated score is computed from individual item 79 scoring. We investigated the psychometric efficiency of the 12-item EPQ-R neuroticism scale - 86 hereafter ‘EPQ-R’ (14) as a widely used measurement of neuroticism. We applied item 87 response theory (IRT) to psychometrically evaluate the EPQ-R using data from UK 88 Biobank (19), a large population study which assessed neuroticism at baseline. Our 89 Background 42 The EPQ-R neuroticism scale has been found to lack items to identify 80 respondents who would normally endorse items at the extreme ends of the trait 81 continuum, e.g. high vs. low neuroticism (5). Furthermore, the scale maintains gender- 82 specific items, females consistently scoring higher (14, 16), a difference which has been 83 reported cross-culturally (17) and across the age range (e.g., 18). 84 Using factor analysis and correlations for item deletion whilst widely used, have a bias 76 towards identifying closely associated items as being informative and is opaque to the 77 individual item contribution or person ability. The process is commonly known as 78 classical test theory (CTT) whereby a summated score is computed from individual item 79 scoring. The EPQ-R neuroticism scale has been found to lack items to identify 80 respondents who would normally endorse items at the extreme ends of the trait 81 continuum, e.g. high vs. low neuroticism (5). Furthermore, the scale maintains gender- 82 specific items, females consistently scoring higher (14, 16), a difference which has been 83 reported cross-culturally (17) and across the age range (e.g., 18). 84 We investigated the psychometric efficiency of the 12-item EPQ-R neuroticism scale - 86 hereafter ‘EPQ-R’ (14) as a widely used measurement of neuroticism. We applied item 87 response theory (IRT) to psychometrically evaluate the EPQ-R using data from UK 88 Biobank (19), a large population study which assessed neuroticism at baseline. Our 89 5 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint B i t & G ll h N ti i . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. Background 42 ; https://doi.org/10.1101/741249 doi: bioRxiv preprint Bauermeister & Gallacher: Neuroticism Bauermeister & Gallacher: Neuroticism expectation was that the large sample size and balanced gender ratio (54% female) 90 would provide valuable item-level information for assessing the informativeness of 91 individual items and overall psychometric reliability of the scale. This assessment may 92 have important implications in clinical settings and for epidemiological research where 93 it is widely utilised. 94 95 Methods 96 Participants 97 The UK Biobank is a large population-based prospective cohort study of 502,665 98 participants. Invitations to participate in the UK Biobank study were sent to 9.2 million 99 community-dwelling persons in the UK who were registered with the UK National 100 Health Service (NHS) aged between 37 and 73 years. A response rate of 5.5% was 101 recorded. Ethical approval was granted to Biobank from the Research Ethics Committee 102 - REC reference 11/NW/0382 (19). The analysis here was applied to the whole cohort 103 of 502,591 participants after withdrawals were considered. 104 105 Procedure 106 Assessments took place at 22 centres across the UK where participants completed an 107 informed consent and undertook comprehensive mental health, cognitive, lifestyle, 108 expectation was that the large sample size and balanced gender ratio (54% female) 90 would provide valuable item-level information for assessing the informativeness of 91 individual items and overall psychometric reliability of the scale. This assessment may 92 have important implications in clinical settings and for epidemiological research where 93 it is widely utilised. 94 expectation was that the large sample size and balanced gender ratio (54% female) 90 would provide valuable item-level information for assessing the informativeness of 91 individual items and overall psychometric reliability of the scale. This assessment may 92 have important implications in clinical settings and for epidemiological research where 93 it is widely utilised. 94 p p g p g it is widely utilised. 94 95 Methods 96 Participants 97 The UK Biobank is a large population-based prospective cohort study of 502,665 98 participants. Invitations to participate in the UK Biobank study were sent to 9.2 million 99 community-dwelling persons in the UK who were registered with the UK National 100 Health Service (NHS) aged between 37 and 73 years. A response rate of 5.5% was 101 recorded. Ethical approval was granted to Biobank from the Research Ethics Committee 102 - REC reference 11/NW/0382 (19). Background 42 The analysis here was applied to the whole cohort 103 of 502,591 participants after withdrawals were considered. 104 105 Procedure 106 6 it is widely utilised. 94 95 Methods 96 Participants 97 The UK Biobank is a large population-based prospective cohort study of 502,665 98 participants. Invitations to participate in the UK Biobank study were sent to 9.2 million 99 community-dwelling persons in the UK who were registered with the UK National 100 Health Service (NHS) aged between 37 and 73 years. A response rate of 5.5% was 101 recorded. Ethical approval was granted to Biobank from the Research Ethics Committee 102 - REC reference 11/NW/0382 (19). The analysis here was applied to the whole cohort 103 of 502,591 participants after withdrawals were considered. 104 105 Procedure 106 Assessments took place at 22 centres across the UK where participants completed an 107 informed consent and undertook comprehensive mental health, cognitive, lifestyle, 108 biomedical and physical assessments. The selection of mental health assessments were 109 completed on a touchscreen computer, including the 12-item EPQ-R neuroticism scale 110 (14) where participants were required to answer, ‘yes’, ‘no’, ‘I don’t know’ or ‘I do not 111 wish to answer’ in response to the 12 questions: ‘Does your mood often go up and 112 The UK Biobank is a large population-based prospective cohort study of 502,665 98 participants. Invitations to participate in the UK Biobank study were sent to 9.2 million 99 community-dwelling persons in the UK who were registered with the UK National 100 Health Service (NHS) aged between 37 and 73 years. A response rate of 5.5% was 101 recorded. Ethical approval was granted to Biobank from the Research Ethics Committee 102 - REC reference 11/NW/0382 (19). The analysis here was applied to the whole cohort 103 of 502,591 participants after withdrawals were considered. 104 The UK Biobank is a large population-based prospective cohort study of 502,665 98 participants. Invitations to participate in the UK Biobank study were sent to 9.2 million 99 community-dwelling persons in the UK who were registered with the UK National 100 Health Service (NHS) aged between 37 and 73 years. A response rate of 5.5% was 101 recorded. Ethical approval was granted to Biobank from the Research Ethics Committee 102 - REC reference 11/NW/0382 (19). The analysis here was applied to the whole cohort 103 of 502,591 participants after withdrawals were considered. 104 6 . Background 42 CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint Bauermeister & Gallacher: Neuroticism Bauermeister & Gallacher: Neuroticism For each item, an item response function (IRF) may be calculated which calibrates the 133 responses of an individual against each item. A calibrated standardised score for trait 134 severity θ is returned and may be plotted as an item characteristic curve (ICC) along a 135 standardised scale with a mean of 0 (Figure 1a). From the ICC two parameters may be 136 estimated. The first is the value of θ at which the likelihood of item endorsement is 0.5, 137 interpreted as ‘expressed trait severity’. The second is the slope of the curve from the 138 point at which the likelihood of item endorsement is 0.5, interpreted as ‘expressed item 139 discrimination’ i.e., the ability to discriminate between greater and lesser severity 140 scores. The IRF may also be expressed as an item information curve (IIF) which displays 141 the relationship between severity and discrimination (Figure 1b). The apex of the curve 142 for any IIC indicates the value of θ at which there is maximum discrimination. By 143 convention, scales expressing a range of θ values are more informative than those with 144 items clustering around a single value and items with a discrimination of score of >1.7 145 are considered informative, although lower values are considered contributory within 146 context (20). Statistical assumptions underlying the IRT principles of scalability, 147 unidimensionality and item-independence are examined. UK Biobank data for this 148 analysis (application 15008) were uploaded onto the Dementias Platform UK (DPUK) 149 Data Portal (21) and analysed using STATA SE 16.1 (22) 150 For each item, an item response function (IRF) may be calculated which calibrates the 133 responses of an individual against each item. Background 42 CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint Bauermeister & Gallacher: Neuroticism Bauermeister & Gallacher: Neuroticism down?’; ‘Do you ever feel just miserable for no reason?’; ‘Are you an irritable person?’; 113 ‘Are your feelings easily hurt?’; ‘Do you often feel fed-up?’; ‘Would you call yourself a 114 nervous person?’; ‘Are you a worrier?’; ‘Would you call yourself tense or highly strung?’; 115 ‘Do you worry too long after an embarrassing experience?’; ‘Do you suffer from 116 nerves?’; ‘Do you often feel lonely?’; ‘Are you often troubled by feelings of guilt?’. The 117 aim of the study was to investigate informativeness and reliability of the 12-item scale, 118 and to explore revised versions using psychometric methodologies such as Item 119 Response Theory (IRT). 120 For these binary response data a 2 parameter logistic (2-PL) IRT model is appropriate: 123 For these binary response data a 2 parameter logistic (2-PL) IRT model is appropriate: 123 1, , exp 1 exp The dependent variable is the dichotomous response (yes/no), the independent 124 variables are the person’s trait level, theta (θ) and item difficulty ( . The independent 125 variables combine accumulatively and the item’s difficulty is subtracted from θ. That is, 126 the ratio of the probability of success for a person on an item to the probability of 127 failure, where a logistic function provides the probability that solving any item ( is 128 independent from the outcome of any other item, controlling for person parameters (θ), 129 and item parameters. The 2-PL model includes two parameters to represent the item 130 properties (difficulty and discrimination) in the exponential form of the logistic model. 131 132 132 7 7 Bauermeister & Gallacher: Neuroticism . igure 1. Item Characteristic Curve (ICC) and Item Information Function (IIF) graphs Background 42 A calibrated standardised score for trait 134 severity θ is returned and may be plotted as an item characteristic curve (ICC) along a 135 standardised scale with a mean of 0 (Figure 1a). From the ICC two parameters may be 136 estimated. The first is the value of θ at which the likelihood of item endorsement is 0.5, 137 interpreted as ‘expressed trait severity’. The second is the slope of the curve from the 138 point at which the likelihood of item endorsement is 0.5, interpreted as ‘expressed item 139 discrimination’ i.e., the ability to discriminate between greater and lesser severity 140 scores. The IRF may also be expressed as an item information curve (IIF) which displays 141 the relationship between severity and discrimination (Figure 1b). The apex of the curve 142 for any IIC indicates the value of θ at which there is maximum discrimination. By 143 convention, scales expressing a range of θ values are more informative than those with 144 items clustering around a single value and items with a discrimination of score of >1.7 145 are considered informative, although lower values are considered contributory within 146 context (20). Statistical assumptions underlying the IRT principles of scalability, 147 unidimensionality and item-independence are examined. UK Biobank data for this 148 analysis (application 15008) were uploaded onto the Dementias Platform UK (DPUK) 149 Data Portal (21) and analysed using STATA SE 16.1 (22) 150 Figure 1. Item Characteristic Curve (ICC) and Item Information Function (IIF) graphs 152 153 Results 154 Sample 155 Figure 1. Item Characteristic Curve (ICC) and Item Information Function (IIF) graphs 152 8 8 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint Bauermeister & Gallacher: Neuroticism . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Background 42 It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint Bauermeister & Gallacher: Neuroticism The whole sample available after withdrawals were considered was 502,591 UK 156 Biobank participants aged 37 to 73 years (M = 56.53 years; SD = 8.05), 54% female. 157 Models were run listwise and the number of participants included in analyses were: 158 401,648 (12-item); 434,693 (7-item); 473,940 (3-item). 159 160 IRT analysis 161 A 2-PL IRT model was estimated whereby difficulty and discrimination parameters 162 were computed (Table 1). The discrimination (item-information) parameters across the 163 scale range between 1.34 and 2.28. The item measuring ‘Does your mood often go up 164 and down?’ exhibits the highest level of discrimination at 2.28, suggesting that this 165 ‘mood’ question possesses the highest amount of information synonymous with the 166 neurotic trait. In contrast, the item ‘Are you an irritable person?’, 1.34, is the lowest, and 167 below the suggested recommended level of 1.7 for an ideal discrimination level for 168 items measuring trait values (20). The items, ‘Are you a worrier?’; ‘Do you suffer from 169 nerves’; ‘Do you ever feel just miserable for no reason?’; ‘Do you often feel fed-up?’ and 170 ‘Would you call yourself tense or highly strung’ also have discrimination values of above 171 1.7. 172 173 The whole sample available after withdrawals were considered was 502,591 UK 156 Biobank participants aged 37 to 73 years (M = 56.53 years; SD = 8.05), 54% female. 157 Models were run listwise and the number of participants included in analyses were: 158 401,648 (12-item); 434,693 (7-item); 473,940 (3-item). 159 9 IRT analysis 161 A 2-PL IRT model was estimated whereby difficulty and discrimination parameters 162 were computed (Table 1). The discrimination (item-information) parameters across the 163 scale range between 1.34 and 2.28. The item measuring ‘Does your mood often go up 164 and down?’ exhibits the highest level of discrimination at 2.28, suggesting that this 165 ‘mood’ question possesses the highest amount of information synonymous with the 166 neurotic trait. In contrast, the item ‘Are you an irritable person?’, 1.34, is the lowest, and 167 below the suggested recommended level of 1.7 for an ideal discrimination level for 168 items measuring trait values (20). Background 42 ; https://doi.org/10.1101/741249 doi: bioRxiv preprint Bauermeister & Gallacher: Neuroticism Bauermeister & Gallacher: Neuroticism Bauermeister & Gallacher: Neuroticism the discrimination curve and position of the difficulty value on the M continuum. For 179 example, for the item ‘Does your mood often go up and down?’, there is a 50% 180 probability that someone with a M of 0.21 (someone who does experience neurotic trait 181 characteristics) would endorse this item, therefore it is considered an item 182 characteristic of neuroticism, albeit low. On contrary, for the item ‘’Are you a worrier?”, 183 there is a 50% chance of someone with a M of -0.13 endorsing this item, therefore, 184 someone who does not experience neurotic trait charateristics. 185 Figure 2. ICC graph for the 12-item scale 187 188 Additional item discrimination is available by graphing the IIF curves (see Figure 3). 189 The IIF curves thereby display the relationship between difficulty (trait level) and 190 discrimination (information), and an important feature of this graph is also the position 191 on the continuum from which the point is drawn perpendicular from the apex of each 192 item curve. The items which have their maximum curvature positioned along the M 193 continuum in the positive half provide information about the neurotic trait when there 194 is an endorsement (presence) of the trait characteristic. For example, the item ‘Do you 195 often feel lonely?’ is an endorsement of neuroticism if a respondent endorses it, as its 196 apex is positioned in positive M and is more likely to be endorsed by someone with a 197 higher level of neuroticism (1.41) than a person endorsing the item ‘Does your mood 198 often go up and down?’ which is also positioned in the positive M but has a lower 199 difficulty value (0.21). Therefore, although the ‘mood’ item has the highest 200 discrimination value (see previous), it does not provide sufficient information about 201 respondents who possess a high level (presence) of the trait (+1 to +4) or a low level 202 Additional item discrimination is available by graphing the IIF curves (see Figure 3). 189 The IIF curves thereby display the relationship between difficulty (trait level) and 190 discrimination (information), and an important feature of this graph is also the position 191 on the continuum from which the point is drawn perpendicular from the apex of each 192 item curve. Background 42 The items, ‘Are you a worrier?’; ‘Do you suffer from 169 nerves’; ‘Do you ever feel just miserable for no reason?’; ‘Do you often feel fed-up?’ and 170 ‘Would you call yourself tense or highly strung’ also have discrimination values of above 171 1.7. 172 173 Table 1. 2-PL IRT model item parameters for the 12-item scale 174 175 The difficulty parameter functions as a probability scale with the item position on M 176 indicating the probability value of a respondent endorsing an item. Figure 2 shows the 177 item characteristic curves (ICCs) for each of the items, presenting both the steepness of 178 A 2-PL IRT model was estimated whereby difficulty and discrimination parameters 162 were computed (Table 1). The discrimination (item-information) parameters across the 163 scale range between 1.34 and 2.28. The item measuring ‘Does your mood often go up 164 and down?’ exhibits the highest level of discrimination at 2.28, suggesting that this 165 ‘mood’ question possesses the highest amount of information synonymous with the 166 neurotic trait. In contrast, the item ‘Are you an irritable person?’, 1.34, is the lowest, and 167 below the suggested recommended level of 1.7 for an ideal discrimination level for 168 items measuring trait values (20). The items, ‘Are you a worrier?’; ‘Do you suffer from 169 nerves’; ‘Do you ever feel just miserable for no reason?’; ‘Do you often feel fed-up?’ and 170 ‘Would you call yourself tense or highly strung’ also have discrimination values of above 171 1.7. 172 The difficulty parameter functions as a probability scale with the item position on M 176 indicating the probability value of a respondent endorsing an item. Figure 2 shows the 177 item characteristic curves (ICCs) for each of the items, presenting both the steepness of 178 9 Bauermeister & Gallacher: Neuroticism . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. Background 42 The items which have their maximum curvature positioned along the M 193 continuum in the positive half provide information about the neurotic trait when there 194 is an endorsement (presence) of the trait characteristic. For example, the item ‘Do you 195 often feel lonely?’ is an endorsement of neuroticism if a respondent endorses it, as its 196 apex is positioned in positive M and is more likely to be endorsed by someone with a 197 higher level of neuroticism (1.41) than a person endorsing the item ‘Does your mood 198 often go up and down?’ which is also positioned in the positive M but has a lower 199 difficulty value (0.21). Therefore, although the ‘mood’ item has the highest 200 discrimination value (see previous), it does not provide sufficient information about 201 respondents who possess a high level (presence) of the trait (+1 to +4) or a low level 202 10 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint Bauermeister & Gallacher: Neuroticism . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint Bauermeister & Gallacher: Neuroticism Bauermeister & Gallacher: Neuroticism (absence) of the trait (-1 to -4), instead it provides the most information for 203 respondents who possesses an average (M=0) to a minimal amount of the neuroticism 204 trait (see Table 1). The item which possesses the least trait characteristic 205 discrimination is the item, ‘Are you an irritable person?’, Although the IIF curve apex is 206 positioned over a positive M (0.95), and may be endorsed by a respondent possessing 207 an amount of the trait characteristic, the discrimination value is low (1.34). 208 209 Figure 3. Background 42 IIF graph for the 12-item scale 210 211 In summary, the overall pattern of item distribution across the M continuum suggests 212 that across the 12-item EPQ-R neuroticism scale there are no items which measure an 213 extreme level of neurotic trait characteristics or an extreme level of non-neurotic trait 214 characteristics. It suggests that the questions are mostly measuring the neurotic trait 215 characteristics which have a higher probability of endorsement by individuals who are 216 experiencing a minimal to no level of neuroticism (M = -0.13 to 1.41). 217 218 Reliability 219 In IRT, reliability may be calculated at multiple point values of M along the continuum 220 rather than a single reliability score as in CTT. Reliability is defined at different points of 221 M with the mean of M fixed at 0 and the variance at 1, facilitating identification of the 222 model and reliability for all points along the M continuum, distinguishing respondents 223 according to specific values of M (23). For the 12-item scale there is reliable information 224 (absence) of the trait (-1 to -4), instead it provides the most information for 203 respondents who possesses an average (M=0) to a minimal amount of the neuroticism 204 trait (see Table 1). The item which possesses the least trait characteristic 205 discrimination is the item, ‘Are you an irritable person?’, Although the IIF curve apex is 206 positioned over a positive M (0.95), and may be endorsed by a respondent possessing 207 an amount of the trait characteristic, the discrimination value is low (1.34). 208 In summary, the overall pattern of item distribution across the M continuum suggests 212 that across the 12-item EPQ-R neuroticism scale there are no items which measure an 213 extreme level of neurotic trait characteristics or an extreme level of non-neurotic trait 214 characteristics. It suggests that the questions are mostly measuring the neurotic trait 215 characteristics which have a higher probability of endorsement by individuals who are 216 experiencing a minimal to no level of neuroticism (M = -0.13 to 1.41). 217 11 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint . Background 42 CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint Bauermeister & Gallacher: Neuroticism Bauermeister & Gallacher: Neuroticism Bauermeister & Gallacher: Neuroticism information (M=0; 0.87 and M=1; 0.88), considered very good for reliability However, 226 reliability then decreases (M=2; 0.76 and M=-1; 0.71) suggesting that the highest 227 reliability of measuring the neurotic trait is at normal or a minimal amount of 228 neuroticism, M=0 or 1. Thereafter, reliability reduces so that the extreme end of the 229 continuum, M=3; 4; -2; -3; -4, is no longer reliably measured (See Table 2) . 230 231 Table 2. Reliability for values of ( from a 2-PL IRT model fit for the 12-item scale 232 233 Statistical assumptions 234 1. Item independence 235 A correlation analysis assessed initial item independency and all items were 236 significantly correlated (p <.000) but the majority of values were lower than 0.50, 237 suggesting basic local item independence. A residual coefficient matrix, requested after 238 estimation of a single-factor model showed that no residuals were too highly correlated, 239 R >0.20 (24), suggesting basic item independence. 240 241 2. Monotonicity 242 A Mokken analysis produced a Loevinger H coefficient (25) which measures the 243 scalable quality of items expressed as a probability measure independent of a 244 information (M=0; 0.87 and M=1; 0.88), considered very good for reliability However, 226 reliability then decreases (M=2; 0.76 and M=-1; 0.71) suggesting that the highest 227 reliability of measuring the neurotic trait is at normal or a minimal amount of 228 neuroticism, M=0 or 1. Thereafter, reliability reduces so that the extreme end of the 229 continuum, M=3; 4; -2; -3; -4, is no longer reliably measured (See Table 2) . 230 neuroticism, M=0 or 1. Thereafter, reliability reduces so that the extreme end of the 229 continuum, M=3; 4; -2; -3; -4, is no longer reliably measured (See Table 2) . 230 231 Table 2. Reliability for values of ( from a 2-PL IRT model fit for the 12-item scale 232 233 Statistical assumptions 234 1. Background 42 Item independence 235 A correlation analysis assessed initial item independency and all items were 236 significantly correlated (p <.000) but the majority of values were lower than 0.50, 237 suggesting basic local item independence. A residual coefficient matrix, requested after 238 estimation of a single-factor model showed that no residuals were too highly correlated, 239 R >0.20 (24), suggesting basic item independence. 240 241 2. Monotonicity 242 A Mokken analysis produced a Loevinger H coefficient (25) which measures the 243 scalable quality of items, expressed as a probability measure, independent of a 244 respondent’s M. These coefficients ranged between 0.35 and 0.47 (Table 3), suggesting a 245 weak (H=0.3-0.4) to moderate (H=0.4-0.5) monotonicity, no items reached strong 246 scalability (H≥0.5) (25). 247 A correlation analysis assessed initial item independency and all items were 236 significantly correlated (p <.000) but the majority of values were lower than 0.50, 237 suggesting basic local item independence. A residual coefficient matrix, requested after 238 estimation of a single-factor model showed that no residuals were too highly correlated, 239 R >0.20 (24), suggesting basic item independence. 240 12 12 Neuroticism: An IRT analysis 13 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint Neuroticism: An IRT analysis 13 Neuroticism: An IRT analysis 13 248 249 Table 3. Mokken analysis with Loevinger H coefficients for the 12-item scale 250 251 3. Unidimensionality 252 A principal component analysis (PCA) shows that a single major factor is responsible 253 for 36% of the variance and a second factor responsible for 11% of the variance, the 254 difference of which is above the suggested 20% indicating a single major factor is being 255 measured (26). A post-IRT estimation model measure of unidimensionality was also 256 computed using a semi-partial correlation controlling for M. This analysis provides 257 individual item variance contribution after adjusting for all the other variables including 258 M. It demonstrates the relationship between local independence and unidimensionality, 259 reflecting a conservative assessment whereby the desired R 2 should ideally be zero or 260 as close to zero as possible (27). Items ranged between 0.01 and 0.02, suggesting 261 unidimensionality. . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint Background 42 To our knowledge, there is still no standardised cut-off criterium for 262 assessing this value (i.e., how close to zero all items should be across a scale). 263 264 IRT revised analysis 265 To assess a revised scale, items were systematically removed from the scale according 266 248 249 13 251 3. Unidimensionality 252 A principal component analysis (PCA) shows that a single major factor is responsible 253 for 36% of the variance and a second factor responsible for 11% of the variance, the 254 difference of which is above the suggested 20% indicating a single major factor is being 255 measured (26). A post-IRT estimation model measure of unidimensionality was also 256 computed using a semi-partial correlation controlling for M. This analysis provides 257 individual item variance contribution after adjusting for all the other variables including 258 M. It demonstrates the relationship between local independence and unidimensionality, 259 reflecting a conservative assessment whereby the desired R 2 should ideally be zero or 260 as close to zero as possible (27). Items ranged between 0.01 and 0.02, suggesting 261 unidimensionality. To our knowledge, there is still no standardised cut-off criterium for 262 assessing this value (i.e., how close to zero all items should be across a scale). 263 264 IRT revised analysis 265 To assess a revised scale, items were systematically removed from the scale according 266 to discrimination value with the lowest discrimating item removed first (‘Are you an 267 irritable person?’, 1.34) whereafter a 11-item 2-PL IRT model was esitmated with the 268 remaining items and the process repeated, removing the lowest discrimating item, 269 below 1.7. In order of removal, the items systematically removed thereafter were: ‘Do 270 A principal component analysis (PCA) shows that a single major factor is responsible 253 for 36% of the variance and a second factor responsible for 11% of the variance, the 254 difference of which is above the suggested 20% indicating a single major factor is being 255 measured (26). A post-IRT estimation model measure of unidimensionality was also 256 computed using a semi-partial correlation controlling for M. This analysis provides 257 individual item variance contribution after adjusting for all the other variables including 258 M. It demonstrates the relationship between local independence and unidimensionality, 259 reflecting a conservative assessment whereby the desired R 2 should ideally be zero or 260 as close to zero as possible (27). Background 42 Items ranged between 0.01 and 0.02, suggesting 261 unidimensionality. To our knowledge, there is still no standardised cut-off criterium for 262 assessing this value (i.e., how close to zero all items should be across a scale). 263 13 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint Neuroticism: An IRT analysis 14 Neuroticism: An IRT analysis 14 you often feel lonely?’; ‘Are you often troubled by feelings of guilt?’; ‘Do you worry too 271 long after an embarrassing experience?’ and ‘Are your feelings easily hurt?’ at which 272 stage the 7 remaining items were maintained as most were > 1.70 on 434,693 273 individuals. 274 you often feel lonely?’; ‘Are you often troubled by feelings of guilt?’; ‘Do you worry too 271 long after an embarrassing experience?’ and ‘Are your feelings easily hurt?’ at which 272 stage the 7 remaining items were maintained as most were > 1.70 on 434,693 273 individuals. 274 275 The item parameters for the 7-item scale are presented in Table 4. Statistical 276 assumptions were computed on the revised scale of 7 items (Table 4) and importantly a 277 Mokken analysis suggests improved scalability (monotonicity) compared to the full 12- 278 item scale with two items reaching values >0.50 (Table 5). Reliability across the scale 279 is marginally improved compared to the full scale suggesting redundancy of the 280 removed items (Table 6). Acceptable metrics for unidimensionality and item 281 independence were achieved for this revised scale. The ICC and IIF graphs for the 282 revised 7-item scale are presented in Figures 4 and 5 where improved item information 283 over the 12-item scale is evident. 284 285 Table 4. 2-PL IRT model item parameters for the 7-item scale 286 287 Table 5. Mokken analysis with Loevinger H coefficients for the 7-item scale 288 289 Table 6. Reliability for values of ( from a 2-PL IRT model fit for the 7-item scale 290 291 Figure 4. ICC graph for the 7-item scale 292 14 14 Neuroticism: An IRT analysis 15 Neuroticism: An IRT analysis 15 293 Figure 5. . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint Figure 5. IIF graph for the 7-item scale 294 Background 42 IIF graph for the 7-item scale 294 295 296 Further item reduction was explored to investigate a ‘minimal’ scale. After systematic 297 item-removal, three items remained when the 2-PL was estimated on sample of 473,940 298 individuals. The scale parameters suggest those items which possessed high 299 discrimination and positive difficulty values, ‘Does your mood often go up and down?’ 300 (3.44; 0.14); ‘Do you ever feel just miserable for no reason?’ (2.79; 0.22) and ‘Do you 301 often feel fed-up?’ (2.92; 0.28) (Table 7). A Mokken analysis suggests that scalability is 302 strong (H≥ 0.50) across all items (Table 8), a semi-partial correlation analysis 303 controlling for M showed all values were 0.00. Reliability is only good at M = 0 304 suggesting this scale is only reliable to measure those with an average trait (Table 9). 305 The ICC and IIF graphs suggest the three-item scale may present an efficient, alternative 306 and highly informative scale, however, the scale is narrow in range and does not 307 possess items measuring neurotic traits above or below average M, at the extreme ends 308 of the trait spectrum (Figure 6). 309 310 293 Further item reduction was explored to investigate a ‘minimal’ scale. After systematic 297 item-removal, three items remained when the 2-PL was estimated on sample of 473,940 298 individuals. The scale parameters suggest those items which possessed high 299 discrimination and positive difficulty values, ‘Does your mood often go up and down?’ 300 (3.44; 0.14); ‘Do you ever feel just miserable for no reason?’ (2.79; 0.22) and ‘Do you 301 often feel fed-up?’ (2.92; 0.28) (Table 7). A Mokken analysis suggests that scalability is 302 strong (H≥ 0.50) across all items (Table 8), a semi-partial correlation analysis 303 controlling for M showed all values were 0.00. Reliability is only good at M = 0 304 suggesting this scale is only reliable to measure those with an average trait (Table 9). 305 The ICC and IIF graphs suggest the three-item scale may present an efficient, alternative 306 and highly informative scale, however, the scale is narrow in range and does not 307 possess items measuring neurotic traits above or below average M, at the extreme ends 308 of the trait spectrum (Figure 6). 309 Table 7. 2-PL IRT model item parameters for the 3-item scale 311 312 Table 8. Background 42 Mokken analysis with Loevinger H coefficients for the 3-item scale 313 314 314 15 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint Neuroticism: An IRT analysis 16 Table 9. Reliability for values of ( from a 2-PL IRT model fit for the 3-item scale 315 316 Figure 6. ICC and IIF graphs for the 3-item scale 317 318 Differential-Item Functioning (DIF) Analysis 319 Table 9. Reliability for values of ( from a 2-PL IRT model fit for the 3-item scale 315 316 Figure 6. ICC and IIF graphs for the 3-item scale 317 318 Differential-Item Functioning (DIF) Analysis 319 To investigate gender differences in item functioning, a logistic DIF analysis was 320 conducted across all three versions of the scale with gender as the observed group. A 321 uniform and nonuniform DIF assessed whether specific items favoured one group over 322 the other (male vs. female) for all values of the latent trait (uniform) or just selected 323 values of the latent trait (nonuniform). The output of these analyses are presented in 324 Table 10 where evidence of significant uniform DIF for gender was found across all 325 three versions. 326 327 Table 10. Logistic regression differential item function (DIF) analysis across 12, 7 and 3- 328 item scales 329 330 Discussion 331 Table 9. Reliability for values of ( from a 2-PL IRT model fit for the 3-item scale 315 Table 9. Reliability for values of ( from a 2-PL IRT model fit for the 3-item scale 315 To investigate gender differences in item functioning, a logistic DIF analysis was 320 conducted across all three versions of the scale with gender as the observed group. A 321 uniform and nonuniform DIF assessed whether specific items favoured one group over 322 the other (male vs. female) for all values of the latent trait (uniform) or just selected 323 values of the latent trait (nonuniform). The output of these analyses are presented in 324 Table 10 where evidence of significant uniform DIF for gender was found across all 325 three versions. 326 In a large population cohort of 502,591 adults aged 37-73 years, limitations in the range 332 and reliability of item trait characteristics were found across the 12-item EPQ-R 333 neuroticism scale when a 2PL IRT model was estimated listwise on 401,648 individuals. 334 Our findings suggest that the 12-item scale is inefficient with poor discrimination at the 335 extreme ends of the scale-range, such that high and low scores are relatively poorly 336 16 Bauermeister & Gallacher: Neuroticism . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint Bauermeister & Gallacher: Neuroticism Bauermeister & Gallacher: Neuroticism Bauermeister & Gallacher: Neuroticism represented and uninformative. A reliability function analysis also suggests there is 337 poor reliability at the extremes of the scale score and high neuroticism scores derived 338 from the EPQ-R are a function of accumulative mid-range values. In a revised 7-item 339 version of the scale, greater item-discrimination and reliability was found across the 340 scale suggesting that selective items within the 12-item version are redundant. A 341 further reduced 3-item version was investigated but although this scale possesses items 342 of high discrimination and scalability, range is very narrow and lacks reliability beyond 343 an average trait value. Table 9. Reliability for values of ( from a 2-PL IRT model fit for the 3-item scale 315 A DIF analysis with gender as a group outcome suggests the scale 344 exhibits significant gender differential item functionting across all versions of the scale. 345 346 To our knowledge, this is the first study to conduct a comprehensive psychometric scale 347 assessment applying IRT to the EPQ-R on such a large population. Furthermore, 348 although the assumption values and parameter output of the 12-item IRT calibration 349 were mostly acceptable according to established psychometric standards, an 350 examination of individual items suggests that there were items of low discrimination 351 and the scale could benefit from revisions based on psychometric methodologies such 352 as those presented here, and as evidenced in the scale-revision analysis. 353 354 It is of fundamental importance that health measurement scales are reliable and valid 355 measures of the construct of interest. Utilising psychometric methodologies to analyse 356 psychosocial and health related outcomes has important implications for assessing 357 represented and uninformative. A reliability function analysis also suggests there is 337 poor reliability at the extremes of the scale score and high neuroticism scores derived 338 from the EPQ-R are a function of accumulative mid-range values. In a revised 7-item 339 version of the scale, greater item-discrimination and reliability was found across the 340 scale suggesting that selective items within the 12-item version are redundant. A 341 further reduced 3-item version was investigated but although this scale possesses items 342 of high discrimination and scalability, range is very narrow and lacks reliability beyond 343 an average trait value. A DIF analysis with gender as a group outcome suggests the scale 344 exhibits significant gender differential item functionting across all versions of the scale. 345 represented and uninformative. A reliability function analysis also suggests there is 337 poor reliability at the extremes of the scale score and high neuroticism scores derived 338 from the EPQ-R are a function of accumulative mid-range values. In a revised 7-item 339 version of the scale, greater item-discrimination and reliability was found across the 340 scale suggesting that selective items within the 12-item version are redundant. A 341 further reduced 3-item version was investigated but although this scale possesses items 342 of high discrimination and scalability, range is very narrow and lacks reliability beyond 343 an average trait value. Table 9. Reliability for values of ( from a 2-PL IRT model fit for the 3-item scale 315 Furthermore, it 363 is also suggested that using an IRT derived M in longitudinal studies, over the summated 364 score, may be preferable with reducing overestimation of the repeated measure 365 variance and underestimation of the between-person variance (30). 366 latent metric predictive of individual M scores on the fitted IRT model. This M metric 361 may then be used as a latent construct in assessing longitudinal change (28) which may 362 be a more reliable measure compared to a single summated score (29). Furthermore, it 363 is also suggested that using an IRT derived M in longitudinal studies, over the summated 364 score, may be preferable with reducing overestimation of the repeated measure 365 variance and underestimation of the between-person variance (30). 366 A further advantage of utilising psychometric methodologies in an epidemiological 368 context is that IRT extends the opportunity to utilise, computer adaptive testing (CAT) 369 for both scale development and for efficient test delivery. During CAT administration, M 370 is automatically computed in response to the trait (M) of the respondent and it is 371 therefore not necessary to present the full range of items as the response scale is 372 adaptive to individual performance (trait level), the items underlying the trait and a 373 stopping rule (31). The potential to reduce a scale so that only the most reliable and 374 informative questions are presented to participants is essential in clinical settings and 375 for epidemiological research. This is important to consider when working with 376 individuals who are older or who have co comorbid psychiatric disorders. Moreover, 377 focused, reliable and user-friendly scales in a research setting increases user 378 satisfaction, reduces participant burden and maintains long-term participant retention. 379 380 A further advantage of utilising psychometric methodologies in an epidemiological 368 context is that IRT extends the opportunity to utilise, computer adaptive testing (CAT) 369 for both scale development and for efficient test delivery. During CAT administration, M 370 is automatically computed in response to the trait (M) of the respondent and it is 371 therefore not necessary to present the full range of items as the response scale is 372 adaptive to individual performance (trait level), the items underlying the trait and a 373 stopping rule (31). Table 9. Reliability for values of ( from a 2-PL IRT model fit for the 3-item scale 315 ; https://doi.org/10.1101/741249 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint Bauermeister & Gallacher: Neuroticism Bauermeister & Gallacher: Neuroticism latent metric predictive of individual M scores on the fitted IRT model. This M metric 361 may then be used as a latent construct in assessing longitudinal change (28) which may 362 be a more reliable measure compared to a single summated score (29). Furthermore, it 363 is also suggested that using an IRT derived M in longitudinal studies, over the summated 364 score, may be preferable with reducing overestimation of the repeated measure 365 variance and underestimation of the between-person variance (30). 366 367 A further advantage of utilising psychometric methodologies in an epidemiological 368 context is that IRT extends the opportunity to utilise, computer adaptive testing (CAT) 369 for both scale development and for efficient test delivery. During CAT administration, M 370 is automatically computed in response to the trait (M) of the respondent and it is 371 therefore not necessary to present the full range of items as the response scale is 372 adaptive to individual performance (trait level), the items underlying the trait and a 373 stopping rule (31). The potential to reduce a scale so that only the most reliable and 374 informative questions are presented to participants is essential in clinical settings and 375 for epidemiological research. This is important to consider when working with 376 individuals who are older or who have co comorbid psychiatric disorders. Moreover, 377 focused, reliable and user-friendly scales in a research setting increases user 378 satisfaction, reduces participant burden and maintains long-term participant retention. 379 380 Participants who display or possess the extreme trait characteristics are rare, however, 381 h i l h ld i f hi li b l i l latent metric predictive of individual M scores on the fitted IRT model. This M metric 361 may then be used as a latent construct in assessing longitudinal change (28) which may 362 be a more reliable measure compared to a single summated score (29). Table 9. Reliability for values of ( from a 2-PL IRT model fit for the 3-item scale 315 A DIF analysis with gender as a group outcome suggests the scale 344 exhibits significant gender differential item functionting across all versions of the scale. 345 346 To our knowledge, this is the first study to conduct a comprehensive psychometric scale 347 assessment applying IRT to the EPQ-R on such a large population. Furthermore, 348 although the assumption values and parameter output of the 12-item IRT calibration 349 were mostly acceptable according to established psychometric standards, an 350 examination of individual items suggests that there were items of low discrimination 351 and the scale could benefit from revisions based on psychometric methodologies such 352 as those presented here, and as evidenced in the scale-revision analysis. 353 To our knowledge, this is the first study to conduct a comprehensive psychometric scale 347 assessment applying IRT to the EPQ-R on such a large population. Furthermore, 348 although the assumption values and parameter output of the 12-item IRT calibration 349 were mostly acceptable according to established psychometric standards, an 350 examination of individual items suggests that there were items of low discrimination 351 and the scale could benefit from revisions based on psychometric methodologies such 352 as those presented here, and as evidenced in the scale-revision analysis. 353 It is of fundamental importance that health measurement scales are reliable and valid 355 measures of the construct of interest. Utilising psychometric methodologies to analyse 356 psychosocial and health related outcomes has important implications for assessing 357 longitudinal change both in clinical settings and epidemiological research. An IRT 358 analysis provides item-level information and scale characteristics through the further 359 computation of post-estimation assumptions including the estimation of an individual M 360 It is of fundamental importance that health measurement scales are reliable and valid 355 measures of the construct of interest. Utilising psychometric methodologies to analyse 356 psychosocial and health related outcomes has important implications for assessing 357 longitudinal change both in clinical settings and epidemiological research. An IRT 358 analysis provides item-level information and scale characteristics through the further 359 computation of post-estimation assumptions including the estimation of an individual M 360 17 Bauermeister & Gallacher: Neuroticism . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. Table 9. Reliability for values of ( from a 2-PL IRT model fit for the 3-item scale 315 The potential to reduce a scale so that only the most reliable and 374 informative questions are presented to participants is essential in clinical settings and 375 for epidemiological research. This is important to consider when working with 376 individuals who are older or who have co comorbid psychiatric disorders. Moreover, 377 focused, reliable and user-friendly scales in a research setting increases user 378 satisfaction, reduces participant burden and maintains long-term participant retention. 379 380 Participants who display or possess the extreme trait characteristics are rare, however, 381 the potential should exist for this eventuality, but many scales are simply not 382 adequately designed to do so (28). Moreover, previous research suggests that both the 383 12 and 3-item EPQ-R neuroticism scales may have reduced power to discriminate 384 Participants who display or possess the extreme trait characteristics are rare, however, 381 the potential should exist for this eventuality, but many scales are simply not 382 adequately designed to do so (28). Moreover, previous research suggests that both the 383 12 and 3-item EPQ-R neuroticism scales may have reduced power to discriminate 384 18 Bauermeister & Gallacher: Neuroticism . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint Bauermeister & Gallacher: Neuroticism . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint Bauermeister & Gallacher: Neuroticism between low and high scoring individuals (5); we found evidence of this in the 12-item 385 scale. Table 9. Reliability for values of ( from a 2-PL IRT model fit for the 3-item scale 315 399 400 Declarations 401 Ethics approval and consent to participate: 402 Analysis of secondary data only with ethical approval in place from source cohort, UK 403 Biobank Research Ethics Committee - REC reference 11/NW/0382. 404 405 Consent for publication: 406 Table 9. Reliability for values of ( from a 2-PL IRT model fit for the 3-item scale 315 It is important in both clinical and research settings that scales are designed to 386 measure across the trait spectrum and this is possible if scales are developed using 387 psychometric methodologies such as those described here and elsewhere (e.g., 32, 33). 388 389 Conclusions 390 The 12-item neuroticism EPQ-R scale lacks item reliability and neurotic trait-specific 391 information at the extreme ends of the neurotic continuum when a 2-PL IRT model is 392 estimated. A secondary analysis suggests that systematic item-elimination and re- 393 estimation of the 2-PL model produces a 7-item with higher levels of item information 394 and reliability. This study suggests that the 12-item EPQ-R scale could benefit from item 395 revisions and updating including item deletions and validation of replacement items 396 which consider gender item bias. Strengths of this study were the large population 397 cohort available for a comprehensive IRT analysis and the psychometric methodologies 398 which were applied to the data. 399 400 Declarations 401 between low and high scoring individuals (5); we found evidence of this in the 12-item 385 scale. It is important in both clinical and research settings that scales are designed to 386 measure across the trait spectrum and this is possible if scales are developed using 387 psychometric methodologies such as those described here and elsewhere (e.g., 32, 33). 388 19 measure across the trait spectrum and this is possible if scales are developed using 387 psychometric methodologies such as those described here and elsewhere (e.g., 32, 33). 388 389 Conclusions 390 The 12-item neuroticism EPQ-R scale lacks item reliability and neurotic trait-specific 391 information at the extreme ends of the neurotic continuum when a 2-PL IRT model is 392 estimated. A secondary analysis suggests that systematic item-elimination and re- 393 estimation of the 2-PL model produces a 7-item with higher levels of item information 394 and reliability. This study suggests that the 12-item EPQ-R scale could benefit from item 395 revisions and updating including item deletions and validation of replacement items 396 which consider gender item bias. Strengths of this study were the large population 397 cohort available for a comprehensive IRT analysis and the psychometric methodologies 398 which were applied to the data. Conclusions 390 The 12-item neuroticism EPQ-R scale lacks item reliability and neurotic trait-specific 391 information at the extreme ends of the neurotic continuum when a 2-PL IRT model is 392 estimated. A secondary analysis suggests that systematic item-elimination and re- 393 estimation of the 2-PL model produces a 7-item with higher levels of item information 394 and reliability. This study suggests that the 12-item EPQ-R scale could benefit from item 395 revisions and updating including item deletions and validation of replacement items 396 which consider gender item bias. Strengths of this study were the large population 397 cohort available for a comprehensive IRT analysis and the psychometric methodologies 398 which were applied to the data. 399 Analysis of secondary data only with ethical approval in place from source cohort, UK 403 Biobank Research Ethics Committee - REC reference 11/NW/0382. 404 19 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint Bauermeister & Gallacher: Neuroticism . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint Bauermeister & Gallacher: Neuroticism SB and JG give full consent for publication 407 408 Availability of data and materials: 409 The dataset(s) supporting the conclusions of this article is(are) available in the 410 Dementias Platform UK (DPUK) Data Portal repository, 411 https://portal.dementiasplatform.uk/. 412 413 Competing interests: 414 SB and JG declare no competing interests 415 416 Funding: 417 The Medical Research Council supports DPUK through grant MR/L023784/2 418 419 Authors' contributions: 420 SB and JG conceptualised the idea. SB analysed and interpreted the data, and wrote the 421 manuscript. JG edited and proofread the manuscript. Both authors read and approved 422 the final manuscript. . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint References 438 1. Costa PT, Jr., McCrae RR. Neuroticism, somatic complaints, and disease: is the bark 439 worse than the bite? J Pers. 1987;55(2):299-316. 440 2. Costa PT, Jr., McCrae RR. Influence of extraversion and neuroticism on subjective well- 441 being: happy and unhappy people. J Pers Soc Psychol. 1980;38(4):668-78. 442 3. Lahey BB. Public health significance of neuroticism. Am Psychol. 2009;64(4):241-56. 443 4. Costa PT, Jr., McCrae RR. Four ways five factors are basic. Personality and Individual 444 Differences. 1992;13(6):653-65. 445 5. Birley AJ, Gillespie NA, Heath AC, Sullivan PF, Boomsma DI, Martin NG. Heritability and 446 nineteen-year stability of long and short EPQ-R neuroticism scales. Personality and Individual 447 Differences. 2006;40(4):737-47. 448 6. Rusting CL. Personality, mood, and cognitive processing of emotional information: three 449 conceptual frameworks Psychol Bull 1998;124(2):165-96 450 1. Costa PT, Jr., McCrae RR. Neuroticism, somatic complaints, and disease: is the bark 439 worse than the bite? J Pers. 1987;55(2):299-316. 440 2. Costa PT, Jr., McCrae RR. Influence of extraversion and neuroticism on subjective well- 441 being: happy and unhappy people. J Pers Soc Psychol. 1980;38(4):668-78. 442 3. Lahey BB. Public health significance of neuroticism. Am Psychol. 2009;64(4):241-56. 443 4. Costa PT, Jr., McCrae RR. Four ways five factors are basic. Personality and Individual 444 Differences. 1992;13(6):653-65. 445 5. Birley AJ, Gillespie NA, Heath AC, Sullivan PF, Boomsma DI, Martin NG. Heritability and 446 nineteen-year stability of long and short EPQ-R neuroticism scales. Personality and Individual 447 Differences. 2006;40(4):737-47. 448 6. Rusting CL. Personality, mood, and cognitive processing of emotional information: three 449 conceptual frameworks. Psychol Bull. 1998;124(2):165-96. 450 7. Watson D, Clark LA. Negative affectivity: the disposition to experience aversive 451 emotional states. Psychol Bull. 1984;96(3):465-90. 452 8. Eysenck HJ. The biological basis of personality. London: Springfield, III: Charles C. 453 Thomas; 1967. 454 9. Eysenck HJ. Personality; biological foundation. the neurophysiology of indivdual 455 difference. New York: Academic Press; 1994. 456 10. Farrington D, Jolliffe D. Personality and Crime. In: N. J. Smelser PBB, editor. International 457 Encyclopedia of the Social & Behavioral Sciences. 1st ed. USA: Elsevier; 2001. 458 10. Farrington D, Jolliffe D. Personality and Crime. In: N. J. Smelser PBB, editor. International 457 Encyclopedia of the Social & Behavioral Sciences. 1st ed. USA: Elsevier; 2001. 458 Encyclopedia of the Social & Behavioral Sciences. 1st ed. USA: Elsevier; 2001. 458 11. LeBlanc J, Ducharme MB, Thompson M. Conclusions 390 423 424 Acknowledgements: 425 This is a Dementias Platform UK (DPUK) supported project with all analyses conducted 426 on the DPUK Data Portal constituting part 1 of DPUK Application 0169 427 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made av The copyright holder for this preprint (wh this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint This is a Dementias Platform UK (DPUK) supported project with all analyses conducted 426 on the DPUK Data Portal, constituting part 1 of DPUK Application 0169. 427 20 Bauermeister & Gallacher: Neuroticism . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint Bauermeister & Gallacher: Neuroticism 428 Abbreviations 429 DPUK Dementias Platform UK 430 EPQ-R Eysenck Personality Questionnaire-Revisied 431 ICC Item Characteristic Curve 432 IIF Item Information Function 433 IRF Item Response Function 434 IRT Item Response Theory 435 PCA Principal Component Analysis 436 437 428 21 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint Bauermeister & Gallacher: Neuroticism References 438 Study on the correlation of the autonomic 459 nervous system responses to a stressor of high discomfort with personality traits. Physiol 460 Behav. 2004;82(4):647-52. 461 12. Faulwasser H, Kittlaus H. [Economy of the Maudsley Medical Questionnaire (MMQ)]. 462 Psychiatr Neurol Med Psychol (Leipz). 1973;25(5):276-81. 463 22 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint Bauermeister & Gallacher: Neuroticism Bauermeister & Gallacher: Neuroticism 13. Francis LJ. The Dual Nature of the Eysenckian Neuroticism Scales - a Question of Sex- 464 Differences. Personality and Individual Differences. 1993;15(1):43-59. 465 14. Eysenck SB, Eysenck HJ, Barrett P. A revised version of the psychoticism scale. 466 Personality and Individual Differences. 1985;6:21-9. 467 15. Eysenck HJ, Eysenck SBG. Psychoticism as a dimension of personality. London: Hodder & 468 Stoughton; 1976. 469 16. Allsop J, Eysenck HJ, Eysenck SBG. Machiavellianism as a component in psychoticism and 470 extraversion. Personality and Individual Differences. 1991;12:29-41. 471 17. Eysenck HJ, Eysenck SBG. Recent advances in the cross-cultural study of personality. In: 472 Spielberger CD, Butcher JN, editors. Advances in personality development. Hillsdale, USA.: 473 17. Eysenck HJ, Eysenck SBG. Recent advances in the cross-cultural study of personality. In: 472 17. Eysenck HJ, Eysenck SBG. Recent advances in the cross-cultural study of personality. In: 472 Spielberger CD, Butcher JN, editors. Advances in personality development. Hillsdale, USA.: 473 Erlbaum; 1982. p. 41-69. 474 18. Eysenck SB, Abdel-Khalek AM. A cross-cultural study of personality: egyptian and 475 english children. Int J Psychol. 1989;24(1-5):1-11. 476 19. Sudlow C, Gallacher J, Allen N, Beral V, Burton P, Danesh J, et al. UK Biobank: An Open 477 Access Resource for Indentifying the Causes of a Wide Range of Complex Diseases of Middle and 478 Old Age. 2015;12(3). 479 20. Baker FB. The basics of item response theory. References 438 Orignal work published in 1985 480 http://echo.edres.org:8080/irt/baker/final.pdf: College Park, DM: ERIC Clearinghouse on 481 Assessment and Evaluation; 2001. 482 21. Bauermeister S, Orton C, Thompson S, Barker RA, Bauermeister JR, Ben-Shlomo Y, et al. 483 21. Bauermeister S, Orton C, Thompson S, Barker RA, Bauermeister JR, Ben-Shlomo Y, et al. 483 Data Resource Profile: The Dementias Platform UK (DPUK) Data Portal. BioRxiv. Preprint. 484 21. Bauermeister S, Orton C, Thompson S, Barker RA, Bauermeister JR, Ben Shlomo Y, et al. 483 Data Resource Profile: The Dementias Platform UK (DPUK) Data Portal. BioRxiv. Preprint. 484 2. StataCorp L. Stata SE 16.1: StataCorp LLC; 2019 [Available from: stata@stata.com. 23. Thissen. Reliability and measurement precision. In: Wainer H, editor. Computerized 486 Adaptive Testing: A primer. London: Lawrence Erlbaum: Lawrence Erlbaum; 2000. p. 159-84. 487 24. Yen WM. Scaling performance assessments: Strategies for managing local item 488 dependence. Journal of Educational Measurement. 1993;30:187-213. 489 23 . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint . CC-BY 4.0 International license under a not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available The copyright holder for this preprint (which was this version posted February 17, 2020. ; https://doi.org/10.1101/741249 doi: bioRxiv preprint Bauermeister & Gallacher: Neuroticism Bauermeister & Gallacher: Neuroticism 25. Sijtsma K, Molenaar IW. Introduction to nonparametric item response theory. London: 490 Sage Publications; 2002. 491 Sage Publications; 2002. 491 26. Reeve BB, Hays RD, Bjorner JB, Cook KF, Crane PK, Teresi JA, et al. Psychometric 492 evaluation and calibration of health-related quality of life item banks: plans for the Patient- 493 Reported Outcomes Measurement Information System (PROMIS). Med Care. 2007;45(5 Suppl 494 1):S22-31. 495 27. De Mars C. Item Response Theory. New York, USA: Oxford University Press; 2010. 496 28. Acock AC. A Gentle Introduction to Stata. 5th ed. Texax, USA: A Stata Press Publication; 497 2016. 498 29. Lu IRR. Embedding IRT in structural equation models: A comparison with regression 499 based on IRT scores. Struct Equ Modeling. 2005;12(2):263-77. 500 30. Gorter R, Fox JP, Twisk JW. References 438 Why item response theory should be used for longitudinal 501 questionnaire data analysis in medical research. BMC Med Res Methodol. 2015;15:55. 502 31. Wainer H, Dorans NJ, Flaugher R, Green BF, Mislevy RJ, Steinberg L, et al. Computerized 503 Adaptive Testing: A Primer. 2nd ed. New York, USA: Routledge; 2014. 335 p. 504 32. de Ayala RJ. The Theory and Practice of Item Response Theory. USA: The Guildford 505 Press; 2009. 506 33. Streiner DL, Norman GR, Cairney J. Health Measurement Scales. 5th ed. Great Britain: 507 26. Reeve BB, Hays RD, Bjorner JB, Cook KF, Crane PK, Teresi JA, et al. Psychometric 492 evaluation and calibration of health-related quality of life item banks: plans for the Patient- 493 Reported Outcomes Measurement Information System (PROMIS). Med Care. 2007;45(5 Suppl 494 1):S22-31. 495 27. De Mars C. Item Response Theory. New York, USA: Oxford University Press; 2010. 496 28. Acock AC. A Gentle Introduction to Stata. 5th ed. Texax, USA: A Stata Press Publication; 497 2016. 498 29. Lu IRR. Embedding IRT in structural equation models: A comparison with regression 499 based on IRT scores. Struct Equ Modeling. 2005;12(2):263-77. 500 30. Gorter R, Fox JP, Twisk JW. Why item response theory should be used for longitudinal 501 questionnaire data analysis in medical research. BMC Med Res Methodol. 2015;15:55. 502 31. Wainer H, Dorans NJ, Flaugher R, Green BF, Mislevy RJ, Steinberg L, et al. Computerized 503 Adaptive Testing: A Primer. 2nd ed. New York, USA: Routledge; 2014. 335 p. 504 Adaptive Testing: A Primer. 2nd ed. New York, USA: Routledge; 2014. 335 p. 504 Adaptive Testing: A Primer. 2nd ed. New York, USA: Routledge; 2014. 335 p. 504 32. de Ayala RJ. The Theory and Practice of Item Response Theory. USA: The Guildford 505 Press; 2009. 506 33. Streiner DL, Norman GR, Cairney J. Health Measurement Scales. 5th ed. Great Britain: 507 33. Streiner DL, Norman GR, Cairney J. Health Measurement Scales. 5th ed. Great Britain: 507 O f d U i it P 2015 508 Oxford University Press; 2015. 508 509 24 evity, p see t p < .0 sm 25 Table 1. 2-PL IRT model item parameters for the 12-item scale Parameter B SE Z 95% CI Discrimination 2.28 0.01 222.03 (2.26 2.30 Difficulty 0.21 0.00 84.64 (0.21 0.2 Discrimination 1.60 0.01 230.62 (1.59 1.62 Difficulty -0.13 0.00 -44.94 (-0.14 -0.1 Discrimination 1.85 0.01 229.06 (1.83 1.87 Difficulty -0.13 0.00 -49.27 (-0.14 -0.1 Discrimination 1.85 0.01 210.41 (1.83 1.87 Difficulty 1.03 0.00 268.82 (1.03 1.05 Discrimination 1.97 0.01 228.19 (1.95 1.99 Difficulty 0.29 0.00 106.76 (0.28 0.29 Discrimination 2.09 0.01 244.94 (2.07 2.11 Difficulty 0.36 0.00 135.93 (0.36 0.37 Discrimination 2.05 0.01 203.01 (2.03 2.07 Difficulty 1.23 0.00 299.42 (1.22 1.24 Discrimination 1.47 0.01 198.86 (1.46 1.49 Difficulty 1.41 0.01 256.50 (1.40 1.42 Discrimination 1.34 0.01 211,83 (1.33 1.35 Difficulty 0.95 0.00 217.40 (0.95 0.96 25 Discrimination 1.85 0.01 229.06 (1.83 1.87 Difficulty -0.13 0.00 -49.27 (-0.14 -0.1 Discrimination 1.85 0.01 210.41 (1.83 1.87 Difficulty 1.03 0.00 268.82 (1.03 1.05 Discrimination 1.97 0.01 228.19 (1.95 1.99 Difficulty 0.29 0.00 106.76 (0.28 0.29 Discrimination 2.09 0.01 244.94 (2.07 2.11 Difficulty 0.36 0.00 135.93 (0.36 0.37 Discrimination 2.05 0.01 203.01 (2.03 2.07 Difficulty 1.23 0.00 299.42 (1.22 1.24 Discrimination 1.47 0.01 198.86 (1.46 1.49 Difficulty 1.41 0.01 256.50 (1.40 1.42 Discrimination 1.34 0.01 211,83 (1.33 1.35 Difficulty 0.95 0.00 217.40 (0.95 0.96 4 -0 3 1.8 4 -0 3 1.8 3 1.0 5 1.9 8 0.2 7 2.1 6 0.3 3 2.0 2 1.2 6 1.4 0 1.4 3 1.3 5 0.9 1. 2-PL IRT model item parameters for the 12-item scale Parameter B SE Z Discrimination 2.28 0.01 222.03 Difficulty 0.21 0.00 84.64 208.65 (1.65 1.69) 266.14 (1.16 1.18) 226.58 (1.44 1.46) 48.19 (0.14 0.15) 220.37 (1.53 1.56) 224.84 (0.85 0.86) nts; SE=standard error 208 266 226 48. 220 224 SE=sta 27 of from a 2-PL IRT model fit for the 12-item scale IF TIF SE Reliability 02 0.99 0.02 10 0.95 0.09 52 0.81 0.34 43 0.54 0.71 96 0.35 0.87 04 0.35 0.88 11 0.49 0.76 77 0.75 0.44 16 0.93 0.14 formation Function; SE=standard error 28 0.44 0.47 0.39 0.35 0.41 ience? 0.39 0.39 s truncated for brevity, see text 28 inger H coefficients for the 12-item scale H 0.46 0.44 0.47 0.43 0.43 0.44 0.47 0.39 0.35 0.41 ience? 0.39 0.39 s truncated for brevity, see text forma 0 0 0 0 0 0 beta 0.02 0.00 coef 29 item parameters for the 7-item scale B SE Z 95% CI 2.57 0.01 195.10 (2.54 2.59) 0.19 0.00 82.07 (0.18 0.19) 1.56 0.01 215.41 (1.54 1.57) -0.16 0.00 -57.07 (-0.17 -0.16) 1.79 0.01 196.09 (1.77 1.81) 1.04 0.00 265.19 (1.03 1.05) 2.15 0.01 205.06 (2.00 2.04) 0.29 0.00 105.39 (0.28 0.29) 2.17 0.01 209.88 (2.17 2.21) 0.34 0.00 136.47 (0.34 0.35) 1.98 0.01 194.87 (1.96 2.00) 1.25 0.00 297.66 (1.24 1.26) 1.70 0.01 199.42 (1.69 1.72) 1.15 0.00 269.35 (1.15 1.16) see text; B=standardised beta coefficients; SE=standard error p < .000 for all B values. m nam 30 0.50 0.49 0.47 0.47 0.52 0.47 ncated for brevity, see text. 31 of from a 2-PL IRT model fit for the 7-item scale TIF SE Reliability 1.00 1.01 0.98 0.04 0.90 0.20 0.65 0.58 0.40 0.84 0.42 0.83 0.60 0.65 0.85 0.27 0.97 0.06 nformation Function; SE=standard error forma r the 3-item scale SE Z 95% CI 0.03 128.71 (3.39 3.49) 0.00 74.48 (0.14 0.15) 0.02 182.49 (2.76 2.82) 0.00 103.80 (0.21 0.22) 0.02 173.92 (2.89 2.96) 0.00 133.95 (0.28 0.28) ed beta coefficients; SE=standard error 96) 28) -item SE .03 .00 .02 .00 .02 .00 coeff r the 3-item scale text. 34 ility for values of from a 2-PL IRT model fit for the 3-item scale TIF TIF SE Reliability 1.00 1.00 0.00 1.00 1.00 0.00 1.03 0.98 0.03 1.66 0.76 0.40 7.39 0.37 0.86 3.09 0.57 0.68 1.13 0.94 0.11 1.01 1.00 0.01 1.00 1.00 0.00 Note: TIF=Test Information Function; SE=standard error 34 y 1.00 1.00 0.00 1.00 1.00 0.00 1.03 0.98 0.03 1.66 0.76 0.40 7.39 0.37 0.86 3.09 0.57 0.68 1.13 0.94 0.11 1.01 1.00 0.01 1.00 1.00 0.00 Note: TIF=Test Information Function; SE=standard error Note: sion -ite m 35 ion differential item function (DIF) analysis across 12, 7 and 3-item scales item scale 8-item scale 3-item scale m Uniform Nonuniform Uniform Nonuniform Uniform P Chi2 P Chi2 P 2268.50* 76.58* 1518.61* 5.10* 1623.9 5355.91* 2502.24* 66.89* 4628.85* 74.00* 11.22 31.54* * 1152.08* 26.20* 2426.29* 16.19* 5131.04 1267.51* 119.35* 659.99* 53.50* 931.90* 152.88* 70.14* 22.37* 149.19* 8256.58* 3286.63* 792.74* 3174.96* 1385.12* 1991.85* 623.9 131.0 31.90 623.9 131.0 31.90 131.0 31.90 604 d Item Information Function (IIF) graphs 37 Figure 2. ICC graph for the 12-item scale m scale 38 38 39 Figure 4. ICC graph for the 7-item scale 39 39 9 m 40 Figure 5. IIF graph for the 7-item scale 40 40 40 and IIF graphs for the 3-item scale 41 CC and IIF graphs for the 3-item scale 41
https://openalex.org/W3126680696	https://www.frontiersin.org/articles/10.3389/fbioe.2021.624503/pdf	English	null	Advances in the Microbial Synthesis of 5-Hydroxytryptophan	Frontiers in bioengineering and biotechnology	2,021	cc-by	5,044	Advances in the Microbial Synthesis of 5-Hydroxytryptophan Xin-Xin Liu 1, Bin Zhang 2 and Lian-Zhong Ai 1* 1 Shanghai Engineering Research Center of Food Microbiology, School of Medical Instrument and Food Engineering, University of Shanghai for Science and Technology, Shanghai, China, 2 College of Bioscience and Bioengineering, Jiangxi Agricultural University, Nanchang, China 5-Hydroxytryptophan (5-HTP) plays an important role in the regulation of emotion, behavior, sleep, pain, body temperature, and other physiological functions. It is used in the treatment of depression, insomnia, migraine, and other diseases. Due to a lack of effective biosynthesis methods, 5-HTP is mainly obtained by natural extraction, which has been unable to meet the needs of the market. Through the directed evolution of enzymes and the introduction of substrate supply pathways, 5-HTP biosynthesis and yield increase have been realized. This review provides examples that illustrate the production mode of 5-HTP and the latest progress in microbial synthesis. Keywords: 5-hydroxytryptophan, tryptophan hydroxylase, L-trp, biosynthesis, tetrahydrobiopterin Reviewed by: Reviewed by: Zhongyang Qiu, Huaiyin Normal University, China K. Madhavan Nampoothiri, National Institute for Interdisciplinary Science and Technology (CSIR), India Correspondence: Lian-Zhong Ai ailianzhong1@126.com Correspondence: Lian-Zhong Ai ailianzhong1@126.com Specialty section: This article was submitted to Industrial Biotechnology, a section of the journal Frontiers in Bioengineering and Biotechnology Specialty section: This article was submitted to Industrial Biotechnology, a section of the journal Frontiers in Bioengineering and Biotechnology At present, 5-HTP is mainly extracted from natural products, speciﬁcally from the seeds of the African plant Griﬀonia Simplicifolia, but this method of extraction is unable to meet the market demand due to high cost and a lack of raw materials. Chemical synthesis does not depend on natural products. However, it is not currently possible to synthesize 5-HTP economically and eﬀectively due to the tedious steps involved and harsh conditions required. With the development of biotechnology in bioinformatics, genetics, metabolic engineering, biochemistry, protein engineering, and so on, new strategies are available for the use of microorganisms to synthesize 5-HTP. The production of 5-HTP by the microbial method has the advantages of short production cycle, continuous production and mild reaction conditions. Among the organisms used for this method, Escherichia coli is a model strain of prokaryotes with clear genetic information and well-established fermentation conditions, making it particularly well-suited for use as a host cell for the study of 5-HTP biosynthesis. Received: 31 October 2020 Accepted: 04 January 2021 Published: 03 February 2021 Edited by: Yi-Rui Wu, Shantou University, China Edited by: Yi-Rui Wu, Shantou University, China 5-Hydroxytryptophan (5-HTP) is a natural amino acid (AA) that does not participate in protein synthesis. It is derived from tryptophan (trp), and the hydrogen atoms at the 5′-position on the benzene ring of trp are replaced by hydroxyl groups. 5-HTP appears as a ﬁne white powder that is insoluble in water, but is soluble in alcohol. In mammals, 5-HTP is the precursor of the neurotransmitter serotonin and the amine hormone melatonin. It has been successfully used in the treatment of depression, insomnia, migraines, and other diseases due to its regulatory eﬀects on sleep, pain, appetite, and other physiological functions (Figure 1) (Birdsall, 1998). 5-HTP is widely used for psychotropic drugs and weight loss in developed countries. Health-care drugs with 5-HTP as the main ingredient are used in 20 countries. To date, 44 preparation types have been developed worldwide mainly in the form of capsules with 100–150 mg content, as well as in the form of tablets, powders, and sustained-release agents (JunDe et al., 2014). According to the 2014 Thomson Reuters market research report, the annual global sales of 5-HTP were $7.5 million, an increase of 50% over the same period the previous year, with an annual consumption of 1.6 tons. REVIEW published: 03 February 2021 doi: 10.3389/fbioe.2021.624503 REVIEW published: 03 February 2021 doi: 10.3389/fbioe.2021.624503 Citation: Liu X-X, Zhang B and Ai L-Z (2021) Advances in the Microbial Synthesis of 5-Hydroxytryptophan. Front. Bioeng. Biotechnol. 9:624503. doi: 10.3389/fbioe.2021.624503 Liu X-X, Zhang B and Ai L-Z (2021) Advances in the Microbial Synthesis of 5-Hydroxytryptophan. Front. Bioeng. Biotechnol. 9:624503. doi: 10.3389/fbioe.2021.624503 February 2021 \| Volume 9 \| Article 624503 Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org Liu et al. Liu et al. Biosynthesis of 5-Hydroxytryptophan FIGURE 1 \| The 5-HTP metabolic pathway and its function. rapid eye movement sleep period to improve sleep quality for the treatment of insomnia. Natural Extraction of 5-HTP 5-HTP is widely present in the seeds of legumes, with the seeds of African plant Griﬀonia Simplicifolia haing the highest content. The leaves and seeds of the Ghanaian tree have been used as medicine in Africa since ancient times to treat wounds, kidney disease, and for enemas. The paste made from its bark is also used to treat skin diseases, in which the main active ingredient is 5-HTP. Lemaire and Adosraku extracted 5-HTP using the alcohol method, and found that it constituted 20.83% of the fresh weight of the seeds (Lemaire and Adosraku, 2010). After optimization of the extraction temperature, the content of 5-HTP in the extract increased from 6.37 to 8.98% and the purity reached 92% (Addotey, 2009). Using ultraﬁltration membrane separation technology, the 5-HTP transfer rate was found to be 83.5%, and purity reached 90.5% (Qin et al., 2014). In addition, high-purity 5-HTP was also obtained from ﬂower beans by ultrasonic method (Duan et al., 2017). FIGURE 1 \| The 5-HTP metabolic pathway and its function. PRODUCTION OF 5-HTP The anabolic-metabolic engineering design for 5-HTP has gained considerable attention and has been widely studied because of its important physiological functions and huge market demand. At present, the main methods for producing 5-HTP are natural product extraction, chemical synthesis, and microbial fermentation. Among these methods, natural product extraction remains the primary method for the commercial production of 5-HTP. While microbial synthesis and catalysis provide a fast and environmentally friendly alternative to produce natural compounds of medical value. PHYSIOLOGICAL FUNCTION OF 5-HTP As shown in Figure 1, 5-HTP has been widely studied for its important role in the treatment of depression and for weight loss. Depression is a common mental disorder with high levels of disability and stress. More than 350 million people worldwide suﬀer from depression, and ∼1 million people commit suicide due to depression every year (World Health Organization, 2017). It has become the second most important disease worldwide, posing a serious burden on human beings. Dysfunction of serotonin in the brain is thought to be a major cause of depression. 5-HTP is a natural and safe antidepressant because it can increase serotonin levels in the brain. In a clinical trial of 107 patients with depression, 69% of symptoms improved through the daily intake of 50–300 mg of 5-HTP. The response rate to this drug was signiﬁcantly faster than that of ordinary drugs (Sano, 1972). In addition, the content of 5-hydroxyindoleacetic acid (a serotonin-decomposition product) in the cerebrospinal ﬂuid of the patients signiﬁcantly increased after 5-HTP intake, indicating that 5-HTP was successfully converted to serotonin after entering the central nervous system (Takahashi et al., 1975). However, the natural source of 5-HTP is relatively singular. There is a shortage of raw materials and costs are continually increasing with the increase of exploitation. The increasing market demand is impossible to meet through natural extraction alone. Therefore, there has recently been an increasing number of studies regarding the production of 5-HTP via chemical synthesis and biological fermentation. Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org Chemical Synthesis of 5-HTP TPH2 is mainly responsible for the synthesis of serotonin in the central nervous system, including the frontal area, thalamus, hippocampus, amygdala, and hypothalamus, whereas TPH1 is mainly expressed in the pineal gland and the gut and is responsible for the synthesis of serotonin in other parts of the body, such as the heart, lungs, and kidneys (Walther and Bader, 2003; Walther et al., 2003; Patel et al., 2004; Zhang et al., 2004; Sakowski et al., 2006). FIGURE 2 \| The biosynthesis pathway of 5-HTP catalyzed by TPH using BH4 as a cofactor. The blue line represents the BH4 synthesis pathway. The black line represents BH4 regeneration pathway. FIGURE 2 \| The biosynthesis pathway of 5-HTP catalyzed by TPH using BH4 as a cofactor. The blue line represents the BH4 synthesis pathway. The black line represents BH4 regeneration pathway. First, TPH was heterologous expressed in E. coli, and its structure, enzymatic properties, and catalytic mechanism were studied. Windahl et al. (2008) studied the active center structure of chicken TPH1. The Fe2+ coordination structure was found to belong to the heme-independent general Fe2+ coordination structure. The Fe2+ coordination is a distorted trigonal bipyramidal coordination with His273, His278, Glu318, and an imidazole ligand. The substrate trp binds to the hydrophobic pocket of the active center. This hydrophobic pocket is composed of Tyr236, Thr266, Pro267, Glu268, Pro269, His273, Phe314, Phe319, and Lie367 (Windahl et al., 2008). McKinney et al. (2004) expressed human TPH in E. coli and yeast expression system. They found that the soluble expression of TPH could be enhanced by fusion expression with maltose- binding protein. The fusion expressed TPH’s enzyme activity and aﬃnity with L-trp was signiﬁcantly improved. Moran et al. (1998) expressed rabbit-derived TPH in E. coli. It was found that the soluble expression was signiﬁcantly increased after removing 101 AAs at the N-terminal and 28 AAs at the C-terminal. Interestingly, this mutated TPH exists in the form of a monomer rather than a tetramer. Kino et al. (2009) found that Leu101 and Trp180 from the active center of Pseudomonas aeruginosa PAH exerted eﬀects on substrate speciﬁcity and hydroxylase activity. The enzyme catalytic rate constant, Kcat, increased 5.2 times after mutation at these two sites (Kino et al., 2009). (ethyl) ester hydrochloride. 5-HTP was obtained after desalting, acetylation, redox, deacetylation, and other reactions. After cooling and crystallization, 5-HTP crystals were obtained. Chemical Synthesis of 5-HTP y 5-HTP is synthesized by Michael addition reaction using 5-bromoindole 3-bromo-2-hydroxyimino-propionate as the substrate. The addition reaction connects the side chain of 2-hydroxyimino-propionate to the 3-position of the indole. Subsequently, the reduction reaction, hydrolysis reaction, and resolution steps are introduced to obtain 5-HTP (Fuchun et al., 2013). In an invention patent, Raney-Ni and ZnO were added to an autoclave as catalysts. Trp, glycolic acid, and hydrochloric acid were reacted in the autoclave for 0.25–6 h, then ﬁltered and dried to obtain poly-hydroxy-trp drying substance. The dried substance was then reacted with trp, sodium hydroxide, and deionized water. The 5-HTP crystals were ﬁnally obtained after ﬁltration, chromatography and cooling crystallization (Rihe, 2010). Wenhui and colleagues ﬁrst demonstrated the methyl (ethyl) esteriﬁcation of L-trp to form tryptophan methyl y Dieting leads to a sharp decrease in serotonin levels in the serum and brain, and a decrease in serotonin leads to gluttonous gluttony. 5-HTP can prevent dieting induced decrease in serotonin in patients with obesity, thus reducing appetite and assisting with weight loss (Ceci et al., 1989; Cangiano et al., 1991, 1992). In addition, 5-HTP can also improve the symptoms of ﬁbromyalgia, including pain, morning stiﬀness, anxiety, and fatigue (Caruso et al., 1990; Sarzi Puttini and Caruso, 1992; Nicolodi and Sicuteri, 1996). Chronic headaches are caused by reduced serotonin levels in the body. 5-HTP has successfully been used to prevent various types of chronic headaches, including migraines, tension headaches, and adolescent headaches (Bono et al., 1982; Longo et al., 1984; Benedittis and Massei, 1985; Titus et al., 1986; De Giorgis et al., 1987; Maissen and Ludin, 1991; Nicolodi and Sicuteri, 1996). In addition, 5-HTP can increase the February 2021 \| Volume 9 \| Article 624503 Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org 2 Biosynthesis of 5-Hydroxytryptophan Liu et al. in mammals: TPHI and TPH2 (Walther et al., 2003). TPH1 was discovered ﬁrst and studied in depth. In adults, TPH1 is mainly expressed in non-nerve cells (Murphy et al., 2008). Walther et al. found that the brains of mice could still produce serotonin normally after knocking out the TPH1 gene. Further studies found another TPH, TPH2, in the brains of mice (Walther et al., 2003). TPH1 and TPH2 have 71% AA sequence homology. Biosynthesis of 5-HTP 5-HTP production by biological methods is favored due to its advantages, such as a short production cycle, continuous production, and mild reaction conditions. In vivo, 5-HTP is obtained by L-trp hydroxylation catalyzed by trp hydroxylase (TPH) using L-trp as a substrate (Figure 2). TPH is a monooxygenase that uses trp and oxygen as substrates, and tetrahydrobiopterin (BH4) and Fe2+ are required as cofactors in its catalytic process (Kappock and Caradonna, 1996; Fitzpatrick, 1999; Olsson et al., 2010; Roberts and Fitzpatrick, 2013). Chemical Synthesis of 5-HTP The purity of the product was 99.2% and the overall yield was 45% (Wenhui et al., 2013). The chemical method for the synthesis of 5-HTP is tedious, harsh, and costly. It uses a variety of organic reagents, resulting in serious environmental pollution. Therefore, this method is not suitable for the large-scale production of 5-HTP. Synthesis and Regeneration of BH4 y g 4 One diﬃculty in using E. coli to produce 5-HTP is that it does not synthesize the coenzyme BH4, which is essential for TPH. E. coli can synthesize analogs of BH4, tetrahydromonapterin (MH4) (Ikemoto et al., 2002). PAH of Pseudomonas aeruginosa can use MH4 as a coenzyme to hydroxylate L-phenylalanine to L-tyrosine. Zhang et al. (2016) expressed mutant PAH or TPH in Saccharomyces cerevisiae to catalyze the production of 5-HTP from L-trp. The activities of PAH and TPH in the hydroxylation of trp with MH4 and BH4 as cofactors were compared. The results showed that the hydroxylation activity of TPH using BH4 as a cofactor was 17 times higher than that of PAH using MH4 (Zhang et al., 2016). In vivo, 5-HTP is produced from L-trp and the reaction is catalyzed by TPH, which uses L-trp and O2 as substrates and requires BH4 and Fe2+ as cofactors. The activity of TPH, the supply of L-trp, and the synthesis and regeneration of BH4 are three key factors that restrict restricting 5-HTP synthesis. L-Tryptophan Hydroxylase (TPH) coli. Additionally, 5 mM trp could be transformed into 2.5 mM of 5-HTP by shaking ﬂask culture for 24 h. After converting this pathway into L-trp producing bacteria, the synthesis of 5-HTP from glucose was realized. The ﬂask yield was 100 mg/L in 60 h, and the batch fermentation yield was 962 mg/L (Mora-Villalobos and Zeng, 2018). BH4 is shown in Figure 2. The production capacity of GTP (the precursor of BH4) was increased by mutation breeding (Perkins et al., 1999). By optimizing the activity of GCHI (the enzyme that catalyzes the ﬁrst step of BH4 biosynthesis) from diﬀerent sources, 4 g/L BH4 was obtained under fed-batch fermentation (Yamamoto et al., 2003). Kino et al. (2009) ﬁrst expressed the mutagenic TPH in E. coli and synthesized 0.8 mM of 5-HTP by adding BH4 as a substrate. Subsequently, the BH4 regeneration pathway and glucose dehydrogenase from Bacillus subtilis were introduced to increase the utilization rate of BH4. The yield of 5-HTP increased to 2.5 mM under these conditions (Table 1) (Kino et al., 2009; Hara and Kino, 2013). Knight et al. (2013) introduced the mammalian BH4 synthesis pathway and regeneration pathway into E. coli and co-expressed it with rabbit-derived TPH1. When L-trp was used as the substrate, the yield of 5-HTP reached 198 mg/L (Knight et al., 2013). Although 5-HTP synthesis was achieved by introducing the synthesis and regeneration pathway of BH4, the yield of 5-HTP was low and additional L- trp was needed, indicating that it is not a suitable method for mass production. ) Wang et al. (2018) introduced human TPH into E. coli BL21, and co-expressed it with the synthetic and regenerative pathways of human BH4. The engineered bacteria hydroxylated 2 g/L trp to produce 1.24 g/L 5-HTP. The authors further introduced the trp synthesis pathway into the recombinant E. coli to realize the biosynthesis of 5-HTP. After the optimization of culture conditions, the yield of 5-HTP was further increased by 13 times to 314.8 mg/L. After module optimization, which included: (a)enzymatic modiﬁcation to improve the hydroxylation activity of TPH, (b)reduction of the copy number of the trp synthesis gene, and (c) regulation of the promoter strength of genes involved in BH4 synthesis and regeneration, the yield of 5-HTP in the modiﬁed recombinant strain HTPL01-LMT was 1.29 g/L, 3.1-fold increase (Wang et al., 2018). L-Tryptophan Hydroxylase (TPH) L-Tryptophan Hydroxylase (TPH) TPH, phenylalanine hydroxylase (PAH), and tyrosine Hydroxylase (TH) are pterin-dependent aromatic AA hydroxylases (AAAHs) (Windahl et al., 2008; Olsson et al., 2010). Moreover, these three hydroxylases have substrate interconnectedness, and each hydroxylase can catalyze the hydroxylation of these three aromatic AAs (Olsson et al., 2010; Roberts and Fitzpatrick, 2013). There are two subtypes of TPH With an in-depth understanding of the biosynthetic pathway of BH4, Yamamoto et al. (2003) synthesized BH4 through the heterologous expression in E. coli. The synthetic pathway of February 2021 \| Volume 9 \| Article 624503 Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org 3 Biosynthesis of 5-Hydroxytryptophan Liu et al. TABLE 1 \| Overview of 5-HTP production by microorganism. Strains Modulations Titer (g/L) Cultivation References E. coli Overexpression of mutant PAH, 0.1762 Shake ﬂask; Supplementation of BH4 and 5 mM L-trp Kino et al., 2009; Hara and Kino, 2013 E. coli Overexpression of mutant PAH; Insertion of BH4 regeneration pathway; Insertion of glucose dehydrogenase from Bacillus subtilis 0.55 Shake ﬂask; Supplementation of 5 mM L-Trp Hara and Kino, 2013 E. coli Mutation of PAH from Xanthomonas campestris; Co-expression of MH4 regeneration pathway and L-trp synthesis pathway 0.1529 Shake ﬂask; Supplementation of glucose Lin et al., 2014 E. coli Mutation of the PAH from Xanthomonas campestris; Co-expression of MH4 regeneration and the L-trp synthesis pathway; Transformation into an L-trp high-yield strain 0.962 Fed-batch Mora-Villalobos and Zeng, 2018 E. coli Mutation of phenylalanine-4-hydroxylases (P4Hs); Deletion of the pheA, tyrA, and tnaA genes 1.1–1.2 Shake ﬂask; Supplementation of 2 g/L L-Trp Lin et al., 2014 E. coli Mutation of AAAH; Insertion of the human BH4 regeneration pathway; Disruption of tryptophanase 0.55 Supplementation of 1 g/L L-Trp Mora-Villalobos and Zeng, 2017 E. coli Expression of a truncated human TPH2; Reconstitution of the BH4 synthesis and regeneration pathway; Modulation of the plasmid copy number and promoter strength; Modulation of different modules’ expression levels 1.3 Shake ﬂask; Glycerol as carbon source Wang et al., 2018 E. coli Same as the previous line 5.1 Fed-batch; Glycerol as carbon source Wang et al., 2018 E. coli Expression of a truncated human TPH2; Reconstitution of the BH4 synthesis and regeneration pathway; Modulation of the plasmid copy number and promoter strength; Modulation of the relative expression levels among different modules; Designing promoter strength to increase tryptophan production 1.61 Shake ﬂask; Glycerol as carbon source Xu et al., 2020 BH4 regeneration pathway in E. L-Tryptophan Hydroxylase (TPH) To improve the stability of this system, the authors further integrated the L-trp biosynthesis pathway into E. coli genome and designed the promoter strength of the enzyme-coding gene, which catalyzes the ﬁrst step of L- trp biosynthesis. To regulate the production of 5-HTP, they also regulated the copy number of the L-TPH coding gene plasmid. After these optimization steps, the amount of 5-HTP of shake ﬂask fermentation increased to 1.61 g/ml, which was 24% higher than that of the original strain (Table 1) (Xu et al., 2020). REFERENCES Fuchun, G., Can, L., and Yan, X. (2013). A New Simple Method for Synthesis of L-5-hydroxytryptophan, CN. Patent CN103554005A. Addotey, J. (2009). Local production of 5-HTP from the seeds of Griﬀonia simplicifolia. World J. Pharm. Pharm. 5. Hara, R., and Kino, K. (2013). Enhanced synthesis of 5-hydroxy-l- tryptophan through tetrahydropterin regeneration. AMB Express 3:70. doi: 10.1186/2191-0855-3-70 Benedittis, G. D., and Massei, R. (1985). Serotonin precursors in chronic primary headache. A double-blind cross-over study with L-5-hydroxytryptophan vs. placebo. J. Neurosurg. Sci. 29, 239–248. Ikemoto, K., Sugimoto, T., Murata, S., Tazawa, M., Nomura, T., Ichinose, H., et al. (2002). (6R)-5,6,7,8-tetrahydro-L-monapterin from Escherichia coli, a novel natural unconjugated tetrahydropterin. Biol. Chem. 383, 325–330. doi: 10.1515/BC.2002.035 Birdsall, T. C. (1998). 5-Hydroxytryptophan: a clinically-eﬀective serotonin precursor. Altern. Med. Rev. 3, 271–280. Bono, G., Criscuoli, M., Martignoni, E., Salmon, S., and Nappi, G. (1982). Serotonin precursors in migraine prophylaxis. Adv. Neurol. 33:357. JunDe, L., Shaohua, L., and Mi, T. (2014). Research progress of 5-hydroxytryptophan. Fine Specialized Chem. 22, 36–39. Kappock, T. J., and Caradonna, J. P. (1996). Pterin-dependent amino acid hydroxylases. Chem. Rev. 96, 2659–2756. doi: 10.1021/cr9402034 Cangiano, C., Ceci, F., Cairella, M., Cascino, A., Ben, M. D., Laviano, A., et al. (1991). Eﬀects of 5-Hydroxytryptophan on Eating Behavior and Adherence to Dietary Prescriptions in Obese Adult Subjects. New York, NY: Springer. doi: 10.1007/978-1-4684-5952-4_73 Kino, K., Hara, R., and Nozawa, A. (2009). Enhancement of L-tryptophan 5- hydroxylation activity by structure-based modiﬁcation of L-phenylalanine 4- hydroxylase from Chromobacterium violaceum. J. Biosci. Bioeng. 108, 184–189. doi: 10.1016/j.jbiosc.2009.04.002 Cangiano, C., Ceci, F., Cascino, A., Del Ben, M., Laviano, A., Muscaritoli, M., et al. (1992). Eating behavior and adherence to dietary prescriptions in obese adult subjects treated with 5-hydroxytryptophan. Am. J. Clin. Nutr. 56, 863–867. doi: 10.1093/ajcn/56.5.863 Knight, E. M., Zhu, J., Förster, J., and Hao, L. (2013). Microorganisms for the production of 5-hydroxytryptophan. Nippon Hinyokika Gakkai Zasshi the Japanese J. Urol. 56, 317–330. doi: 10.1093/mnras/stt2484 Caruso, I., Sarzi Puttini, P., Cazzola, M., and Azzolini, V. (1990). Double- blind study of 5-hydroxytryptophan versus placebo in the treatment of primary ﬁbromyalgia syndrome. J. Int. Med. Res. 18, 201–209. doi: 10.1177/030006059001800304 Lemaire, P. A., and Adosraku, R. K. (2010). An HPLC method for the direct assay of the serotonin precursor, 5-hydroxytrophan, in seeds of Griﬀonia simplicifolia. Phytochem. Anal. 13, 333–337. doi: 10.1002/pca.659 Lin, Y., Sun, X., Yuan, Q., and Yan, Y. (2014). ACKNOWLEDGMENTS We would like to thank Editage (www.editage.cn) for English language editing. Optimization of trp Supply 5-HTP yield can be increased by increasing TPH activity and introducing BH4 synthesis and regeneration pathways. Mora-Villalobos and Zeng (2017) mutated the aromatic amino acid hydroxylase of Cupriavidus taiwanensis and obtained the trp preference mutant enzyme CtAAAH- W192F. The mutated enzyme was co-expressed with the February 2021 \| Volume 9 \| Article 624503 Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org 4 Biosynthesis of 5-Hydroxytryptophan Liu et al. FUNDING This work was sponsored by the Shanghai Sailing Program (20YF1433500); the National Key R&D Program of China [Grant No. 2018YFC1604305]; the Shanghai Agriculture Applied Technology Development Program, China [Grant No. 2019-02-08-00-07-F01152]; the Natural Science Foundation of China [Grant No. 31871757]. Shanghai Technical Standard Program, China [18DZ2200200]; Shanghai Engineering Research Center of food microbiology program [19DZ22 81100]. Although 5-HTP production has increased 10-fold, remains insuﬃcient for large-scale commercial production. The metabolic network is a complex system, and the eﬃciency of the target metabolic pathway is often aﬀected by other metabolic pathways. Therefore, strain evolution and breeding for global metabolism may eﬀectively improve the yield of the target product. Besides, high-density cell culture is another strategy for increasing the yield. Combined with the optimization of fermentation conditions and the improvement of cell culture density, the yield of 5-HTP could be further improved. PROSPECTS X-XL wrote the manuscript. BZ and L-ZA revised the Metabolic engineering strategy for 5-HTP synthesis section. All the authors contributed to the literature collection and data analysis, and approved it for publication. This review assesses the synthesis and research progress of 5- HTP. Compared with natural product extraction and chemical synthesis, biosynthesis has the advantages of a short cycle, continuous production and mild reaction conditions; thus, it has garnered considerable research attention. In the biosynthesis process, three aspects of optimization have been implemented to increase the output of 5-HTP; (a) improving the hydroxylation activity of the TPH enzyme by directed evolution, (b) introducing BH4 synthesis and the regeneration pathway, (c) introducing the trp synthesis pathway. To date, the yield of shake ﬂask culture has reached 1.61 g /L. REFERENCES Engineering bacterial phenylalanine 4-hydroxylase for microbial synthesis of human neurotransmitter precursor 5-hydroxytryptophan. ACS Synth. Biol. 3, 497–505. doi: 10.1021/sb5002505 Ceci, F., Cangiano, C., Cairella, M., Cascino, A., Del Ben, M., Muscaritoli, M., et al. (1989). The eﬀects of oral 5-hydroxytryptophan administration on feeding behavior in obese adult female subjects. J. Neural Transm. 76, 109–117. doi: 10.1007/BF01578751 Longo, G., Rudoi, I., Iannuccelli, M., Strinati, R., and Panizon, F. (1984). Treatment of essential headache in developmental age with L-5-HTP (cross over double- blind study versus placebo). Pediatr. Med. Chir. 6, 241–245. De Giorgis, G., Miletto, R., Iannuccelli, M., Camuﬀo, M., and Scerni, S. (1987). Headache in association with sleep disorders in children: a psychodiagnostic evaluation and controlled clinical study–L-5-HTP versus placebo. Drugs Exp. Clin. Res 13:425. Maissen, C. P., and Ludin, H. P. (1991). Comparison of the eﬀect of 5- hydroxytryptophan and propranolol in the interval treatment of migraine. Schwzerische Medizinische Wochenschrift 121, 1585–1590. Duan, G., Mingbo, G., Qiao, C., Xiaochen, W., and Yining, L. (2017). Ultrasonic extraction of 5-hydroxytryptophan from adzuki bean. Guangzhou Chem. Indus. 45, 52–54. McKinney, J., Knappskog, P. M., Pereira, J., Ekern, T., Toska, K., Kuitert, B. B., et al. (2004). Expression and puriﬁcation of human tryptophan hydroxylase from Escherichia coli and Pichia pastoris. Protein Expr. Purif. 33, 185–194. doi: 10.1016/j.pep.2003.09.014 Fitzpatrick, P. F. (1999). Tetrahydropterin-dependent amino acid hydroxylases. Annu. Rev. Biochem. 68, 355–381. doi: 10.1146/annurev.biochem.68.1.355 February 2021 \| Volume 9 \| Article 624503 Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org 5 Liu et al. Biosynthesis of 5-Hydroxytryptophan Moran, G. R., Daubner, S. C., and Fitzpatrick, P. F. (1998). Expression and characterization of the catalytic core of tryptophan hydroxylase. J. Biol. Chem. 273, 12259–12266. doi: 10.1074/jbc.273.20.12259 Titus, F., Dávalos, A., Alom, J., and Codina, A. (1986). 5-Hydroxytryptophan versus methysergide in the prophylaxis of migraine. Randomized clinical trial. Eur. Neurol. 25, 327–329. doi: 10.1159/000116030 Walther, D. J., and Bader, M. (2003). A unique central tryptophan hydroxylase isoform. Biochem. Pharmacol. 66, 1673–1680. doi: 10.1016/S0006-2952(03)00556-2 Mora-Villalobos, J.-A., and Zeng, A. P. (2017). Protein and pathway engineering for the biosynthesis of 5-hydroxytryptophan in Escherichia coli. Eng. Life Sci. 17, 892–899. doi: 10.1002/elsc.201700064 Mora-Villalobos, J. A., and Zeng, A. P. (2018). Synthetic pathways and processes for eﬀective production of 5-hydroxytryptophan and serotonin from glucose in Escherichia coli. J. Biol. Eng. 12:3. doi: 10.1186/s13036-018-0094-7 Walther, D. J., Peter, J. U., Bashammakh, S., Hörtnagl, H., Voits, M., Fink, H., et al. (2003). REFERENCES Synthesis of serotonin by a second tryptophan hydroxylase isoform. Science 299:76. doi: 10.1126/science.1078197 Wang, H., Liu, W., Shi, F., Huang, L., Lian, J., Qu, L., et al. (2018). Metabolic pathway engineering for high-level production of 5-hydroxytryptophan in Escherichia coli. Metab. Eng. 48, 279–287. doi: 10.1016/j.ymben.2018.06.007 Murphy, K. L., Zhang, X., Gainetdinov, R. R., Beaulieu, J. M., and Caron, M. G. (2008). A regulatory domain in the n terminus of tryptophan hydroxylase 2 controls enzyme expression. J. Biol. Chem. 283, 13216–13224. doi: 10.1074/jbc.M706749200 Wenhui, H., Xing, T., Xiaobing, L., and Jiajin, Y. (2013). A Method for Preparing L- 5-Hydroxytryptophan. Patent CN201110272240.3. Nicolodi, M., and Sicuteri, F. (1996). Fibromyalgia and migraine, two faces of the same mechanism. Serotonin as the common clue for pathogenesis and therapy. Adv. Exp. Med. Biol. 398, 373–379. doi: 10.1007/978-1-4613-0381-7_58 Windahl, M. S., Petersen, C. R., Christensen, H. E., and Harris, P. (2008). Crystal structure of tryptophan hydroxylase with bound amino acid substrate. Biochemistry 47, 12087–12094. doi: 10.1021/bi8015263 Olsson, E., Teigen, K., Martinez, A., and Jensen, V. R. (2010). The aromatic amino acid hydroxylase mechanism: a perspective from computational chemistry. Adv. Inorg. Chem. 62, 437–500. doi: 10.1016/S0898-8838(10)62011-9 World Health Organization (2017). Depression and Other Common Mental Disorders: Global Health Estimates. Geneva: World Health Organization. Patel, P. D., Pontrello, C., and Burke, S. (2004). Robust and tissue-speciﬁc expression of TPH2 versus TPH1 in rat raphe and pineal gland. Biol. Psychiatry 55, 428–433. doi: 10.1016/j.biopsych.2003.09.002 Xu, D., Fang, M., Wang, H., Huang, L., Xu, Q., and Xu, Z. (2020). Enhanced production of 5-hydroxytryptophan through the regulation of L- tryptophan biosynthetic pathway. Appl. Microbiol. Biotechnol. 104, 2481–2488. doi: 10.1007/s00253-020-10371-y Perkins, J. B., Sloma, A., Hermann, T., Theriault, K., Zachgo, E., Erdenberger, T., et al. (1999). Genetic engineering of Bacillus subtilis for the commercial production of riboﬂavin. J. Indus. Microbiol. Biotechnol. 22, 8–18. doi: 10.1038/sj.jim.2900587 Yamamoto, K., Kataoka, E., Miyamoto, N., Furukawa, K., Ohsuye, K., and Yabuta, M. (2003). Genetic engineering of Escherichia coli for production of tetrahydrobiopterin. Metab. Eng. 5, 246–254. doi: 10.1016/S1096-7176(03)00046-6 Qin, Z., Xuesong, H., Ling, Q., and Yanjie, K. (2014). Study on separation and puriﬁcation of 5-hydroxytryptophan from Ghanaian seeds by ultraﬁltration membrane. Res. Dev. Nat. Products 26, 2033–2036. Zhang, J., Wu, C., Sheng, J., and Feng, X. (2016). Molecular basis of 5- hydroxytryptophan synthesis in Saccharomyces cerevisiae. Mol. Biosyst. 12, 1432–1435. doi: 10.1039/C5MB00888C Rihe, S. (2010). Synthesis of L-5-Hydroxytryptophan. Patent CN101323586B. Zhang, X., Beaulieu, J. Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org REFERENCES M., Sotnikova, T. D., Gainetdinov, R. R., and Caron, M. G. (2004). Tryptophan hydroxylase-2 controls brain serotonin synthesis. Science 305:217. doi: 10.1126/science.1097540 Roberts, K. M., and Fitzpatrick, P. F. (2013). Mechanisms of tryptophan and tyrosine hydroxylase. IUBMB Life 65, 350–357. doi: 10.1002/iub.1144 Sakowski, S. A., Geddes, T. J., Thomas, D. M., Levi, E., Hatﬁeld, J. S., and Kuhn, D. M. (2006). Diﬀerential tissue distribution of tryptophan hydroxylase isoforms 1 and 2 as revealed with monospeciﬁc antibodies. Brain Res. 1085, 11–18. doi: 10.1016/j.brainres.2006.02.047 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. 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. Sano, I. (1972). L-5-hydroxytryptophan(L-5-HTP) therapy in endogenous depression. Munch. Med. Wochenschr. 114, 1713–1716. doi: 10.1111/j.1440-1819.1972.tb01107.x Copyright © 2021 Liu, Zhang and Ai. 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. Sarzi Puttini, P., and Caruso, I. (1992). Primary ﬁbromyalgia syndrome and 5- hydroxy-L-tryptophan: a 90-day open study. J. Int. Med. Res. 20, 182–189. doi: 10.1177/030006059202000210 Takahashi, S., Kondo, H., and Kato, N. (1975). Eﬀect of l-5-hydroxytryptophan on brain monoamine metabolism and evaluation of its clinical eﬀect in depressed patients. J. Psychiatr. Res. 12, 177–187. doi: 10.1016/0022-3956(75)90025-4 February 2021 \| Volume 9 \| Article 624503 Frontiers in Bioengineering and Biotechnology \| www.frontiersin.org 6
https://openalex.org/W3000426034	https://www.periodicos.udesc.br/index.php/palindromo/article/download/15166/10914	Portuguese	null	A imagem do ânus e os provérbios neerlandeses	Palíndromo	2,020	cc-by	6,265	Palíndromo, v. 12, n. 26, p. 94-109, jan - abr 2020 Kethlen Kohl 1 Kethlen Kohl 1 1 Doutoranda em Teoria e História da Arte, na Universidade do Estado de Santa Catarina (CEART-UDESC). Mestre em Teoria e História da Arte pela Universidade do Estado de Santa Catarina (CEART-UDESC). Pós-Graduação Especialização em História da Arte pela Universidade da Região de Joinville - UNIVILLE. Graduada em História pela Universidade da Região de Joinville – UNIVILLE. kethlenkohl@gmail.com http://lattes.cnpq.br/9425972290083804 https://orcid.org/0000-0003-0781-1028 A imagem do ânus e os provérbios neerlandeses A imagem do ânus e os provérbios neerlandeses Resumo O presente artigo procura fazer uma reflexão sobre um conjunto de imagens que tem como ponto comum o corpo grotesco. Em seus detalhes são exploradas narrativas sobre o ânus e as narrativas escatológicas A partir das imagens analisadas, problematizo questões sobre a relação entre imagem e escrita em dois aspectos: a primeira compreende o jogo entre escrita e imagem presente em um díptico anôni- mo do século XVI, onde a escrita procura alertar o expectador a não abrir o díptico; a segunda questão analisada volta-se à presença dos provérbios que se traduzem em imagens nas obras de Pieter Bruegel e Hendrick Avercamp. alavras-chave: Imagem do ânus; provérbios neerlandeses; escrita e imagem. Keywords: Image of the anus; Dutch proverbs; writing and image Keywords: Image of the anus; Dutch proverbs; writing and image. A imagem do ânus e os provérbios neerlandeses Palíndromo, v. 12, n. 26, p. 94-109, jan - abr 2020 94 Resumen Este artículo busca reflexionar sobre un conjunto de imágenes que tienen en común el cuerpo grotesco. Se exploran las narrativas sobre el ano y las narrativas escatológicas. A partir de las imágenes analizadas, cuestiono preguntas sobre la relación entre imagen y escritura en dos aspectos: el prime- ro comprende el juego entre escritura e imagen presente en un díptico anónimo del siglo XVI, donde la escritura busca alertar al espectador para que no abra el díptico; La segunda pregunta analizada se refiere a la presencia de proverbios que se traducen en imágenes en las obras de Pieter Bruegel y Hendrick Avercamp. Palabras claves: Imagen del ano; Proverbios holandeses; escritura e imagen. alabras claves: Imagen del ano; Proverbios holandeses; escritura e imagen. Abstract This article seeks to reflect on a set of images that have as common the grotesque body. Narratives about the anus and eschatological narratives are explored. From the images analyzed, I discuss questions about the relationship between image and writing in two aspects: the first comprises the game between writing and image present in an anonymous diptych of the 16th century, where writing seeks to alert the viewer not to open the diptych; The second question analyzed concerns the presence of proverbs that translate into images in the works of Pieter Bruegel and Hendrick Avercamp. 1. A escrita e a imagem Durante o século XX, muitos artistas trouxeram à tona obras com provocações que dizem respeito tanto à escrita quanto à imagem. Obras como de Marcel Duchamp feita com um cartão postal de Mona Lisa e o jogo de siglas L. H. O. Q que, lidas em voz alta em francês, soam como “elle a chaud au cul (Ela tem fogo no cú)” (TOMKINS, 2013 p. 245). Ou o famoso cachimbo de Rene Magritte e seu enigmático enunciado “Ceci n’est pas une pipe”, obra que rendeu um belíssimo ensaio de Michel Foucault sobre as possíveis grandes “armadilhas” com relação à intepretação no diálogo entre imagem e escrita. Não podemos esquecer de Joseph Kosuth, que incorpora a escrita junto ao objeto da arte transformando-a em nuances tautológicas complexas. Es- paços expositivos foram inteiramente preenchidos com palavras sobre imagens por Barbara Kruger, para pensar a questão de como somos constituídos por determina- dos discursos. A relação entre imagem e escrita na arte podem ir das provocações mais racionais para as mais emocionais como são os bordados de Leonilson, onde linhas e figuras se embaraçam criando uma espécie de caligrama em tecido. Sabemos que a relação entre imagem e escrita não é um advento do século XX. Papiros egípcios de 1285 a.C. possuem uma narrativa escrita pelos hieróglifos e figuram imagens ao modo canônico de sua civilização. As iluminuras da idade média ilustram os manuscritos e as letras capitulares são constituídas de pequenas figuras. Se pensarmos nas obras do oriente, encontraremos diversos pergaminhos do perío- do Edo com imagens de samurais, todos acompanhados de textos. Na América, os Maias criaram o Códice de Dresden, livros com imagens e textos ritualísticos e astrológicos. Podemos citar diversos exemplos da relação entre imagem e escrita, juntas fun- cionam tão bem quanto separadas. Em algumas dessas obras, descritas acima, existe uma equidade entre escrita e imagem, não há uma operação hierárquica entre elas. Nessas obras atuam uma simultaneidade entre o dizer para ver; a escrita incentiva o olhar e a imagem quer fazer ler a escrita ou vice-versa. Dessa maneira, a proposta desse texto é apresentar uma breve análise sobre uma tradição muito peculiar entre artistas flamengos que zamba da relação entre imagem e palavra. Essa tradição en- volve os provérbios Neerlandeses e as imagens feitas a partir dessa herança cultural dos ditos populares. 1 Nesse ponto o texto se caracteriza como um desdobramento de um capítulo de minha dissertação de mestrado. KOHL; Kethlen. Consentimento e Interdição sobre o corpo nas Artes Visuais: Implicações e Desdobramentos dos (Contra) Dispositivos. 2017. Dissertação, (Mestrado em Artes Visuais), programa de pós-gra- duação em Artes Visuais, Universidade do Estado de Santa Catarina, Florianópolis. A imagem do ânus e os provérbios neerlandeses A imagem do ânus e os provérbios neerlandeses A imagem do ânus e os provérbios neerlandeses ISSN: 2175-2346 95 Palíndromo, v. 12, n. 26, p. 95-109, jan - abr 2020 Kethlen Kohl A imagem do ânus e os provérbios neerlandeses nos provérbios, torna-se imagens e, na maioria das vezes, essas imagens são pontu- adas por formas humoradas e cômicas. nos provérbios, torna-se imagens e, na maioria das vezes, essas imagens são pontu- adas por formas humoradas e cômicas. 2. Um peculiar díptico flamengo Figura 1 - Artista flamengo anônimo, parte 1, díptico satírico. Século XVI. Óleo sobre madeira. 58,5 x 44 cm (cada painel) University of Liége coleção artistas (galeria Wittert). Bélgica. (fonte: http://www.wittert.ulg.ac.be/fr/flori/opera/anonyme1/anonyme1_notice.html, com acesso em 12.09.2019) 1. A escrita e a imagem O recorte, em específico, procura salientar as imagens e provér- bios que destacam o escatológico e o ânus1. Primeiramente será apresentado um díptico do século XVI de um pintor neer- landês anônimo que procura transformar um provérbio em um jogo entre a escrita, o ver e o não ver. A questão dos provérbios neerlandeses vai muito além de uma he- rança escrita, ela procura estar relacionada a uma tradição da imagem. Os provérbios se desdobram nas obras de Pieter Bruegel e Hendrick Avercamp, sempre mantendo uma relação com a figura do camponês, a escatologia e o ânus. O grotesco, presente 96 96 Palíndromo, v. 12, n. 26, p. 96-109, jan - abr 2020 Kethlen Kohl Kethlen Kohl 2 Tradução de: “ Laisse ce panneau fermé, sinon tu seras fâché contre moi.” (MARTIN, 2016, p. 13, tradução informal da autora) 3 Traduzido de: “Ce ne sera pas de ma faute car je t’avais prévenu” (MARTIN, 2016, p. 13, tradução informal da autora) 4 Traduzido de: ‘Et plus nous voudrons te mettre en garde, plus tu auras envie de sauter par la fenêtre” (MARTIN, 2016, p. 13, tradução informal da autora) 2. Um peculiar díptico flamengo 2. Um peculiar díptico flamengo Figura 1 - Artista flamengo anônimo, parte 1, díptico satírico. Século XVI. Óleo sobre madeira. 58,5 x 44 cm (cada painel) University of Liége coleção artistas (galeria Wittert). Bélgica. (fonte: http://www.wittert.ulg.ac.be/fr/flori/opera/anonyme1/anonyme1_notice.html, com acesso em 12.09.2019) Este díptico de madeira apresenta-se fechado, na imagem. A obra retrata a pin- tura de um homem que veste roupa vermelha e um chapéu na cabeça, típica roupa dos camponeses da região de flandres no século XVI, também presentes nas obras de Bruegel. As bordas arredondadas em dourado criam aspecto de estarmos observan- do o buraco de uma fechadura ou uma janela muito pequena. Uma das mãos do ho- mem encontra-se atrás de suas costas, parece esconder algum segredo. A outra mão aponta para um recado escrito em um pequeno pergaminho que se estende na parte inferior da moldura, nele segue a descrição: Deixe este painel fechado, para que não tenhas raiva de mim2. O camponês alerta o espectador para que não seja enxerido, ele nos incentiva a não ver o que está escondido, obviamente isso acaba despertan- do-lhe curiosidade e questionamentos. O que possui no interior deste díptico? Por que o personagem está nos alertando para não abrir? O que pode ter de perigoso? 2 Tradução de: “ Laisse ce panneau fermé, sinon tu seras fâché contre moi.” (MARTIN, 2016, p. 13, tradução informal da autora) 97 Palíndromo, v. 12, n. 26, p. 97-109, jan - abr 2020 Kethlen Kohl A imagem do ânus e os provérbios neerlandeses 3 Traduzido de: “Ce ne sera pas de ma faute car je t’avais prévenu” (MARTIN, 2016, p. 13, tradução informal da autora) 4 Traduzido de: ‘Et plus nous voudrons te mettre en garde, plus tu auras envie de sauter par la fenêtre” (MARTIN, 2016, p. 13, tradução informa 5 Tradução informal da autora: Leck mire den A ... recht schon, fein sauber Lecke ihn, fein sauber Lecke, leck mire den A ... Das ist ein fettigs Begehren, nur gut mit Butter geschmiert, den das lecken der Braten mein tagliches Thun. Drei lecken mehr als Zweie, nur ela, machet die Prob ‘ und leckt, leckt, leckt. Jeder leckt sein A ... fur sich. Disponível em: < http://www.gutenberg.us/articles/leck_mir_den_arsch_fein_recht_sch%C3%B6n_sauber > Data de acesso em: 12, mai, 2019. A imagem do ânus e os provérbios neerlandeses O díptico ilustra, através da pintura, um provérbio neerlandês que trabalha a questão do ver de forma cômica e obscena. O ânus representa a parte grotesca do corpo, segundo Mikhail Bakhtin: Em oposição aos cânones modernos, o corpo grotesco não está separado do resto do mundo, não está isolado, acabado nem perfeito, mas ultrapassa-se a si mesmo, franqueia seus próprios limites. Coloca-se ênfase nas partes do corpo em que ele se abre ao mundo exterior, isto é, onde o mundo penetra nele ou dele sai ou ele mesmo sai para o mundo, através de orifícios, protu- berâncias, ramificações e excrescências, tais como boca aberta, os órgãos genitais, seios, falo, barriga e nariz. É em atos tais como o coito, a gravidez, o parto, a agonia, o comer, o beber, e a satisfação de necessidades naturais, que o corpo revela sua essência como princípio em crescimento que ultra- passa seus limites. (BAKHTIN, 1999, p.23) Ao mostrar o orifício, o artista anônimo procura punir o espectador, ele figura aquilo que fere os olhos, tange a moral e a índole daquele que o vê. Por isso, tam- bém pode ser encarado como uma brincadeira: uma cena cômica ou uma peça a ser pregada a um curioso. Os provérbios aparecem nessa obra em forma de imagem e escrita, no entanto, essas mesmas sátiras do ânus estão presentes em um imaginário social e são exibidas em diversas obras seja como escrita ou como imagem. Figura 2 - Artista flamengo anônimo, parte 1, díptico satírico. Século XVI. Óleo sobre madeira. 58,5 x 44 cm (cada painel) University of Liége coleção artistas (galeria Wittert). Bélgica. (fonte: http://www.wittert.ulg.ac.be/fr/flori/opera/anonyme1/anonyme1_notice.html, com acesso em 12.09.2019) Figura 2 - Artista flamengo anônimo, parte 1, díptico satírico. Século XVI. Óleo sobre madeira. 58,5 x 44 cm (cada painel) University of Liége coleção artistas (galeria Wittert). Bélgica. (fonte: http://www.wittert.ulg.ac.be/fr/flori/opera/anonyme1/anonyme1_notice.html, com acesso em 12.09.2019) Ao abrir o díptico, o espectador se depara com duas nádegas brancas com algu- mas feridas. A sua mão, que estava escondida, agora puxa uma das nádegas para que o ânus cabeludo fique exageradamente à mostra. As vestes íntimas estão na altura de suas coxas e próximo à região anal se avistam duas belas flores. Abaixo uma nova legenda diz: Eu não tenho culpa, eu avisei para não abrir3. Em seguida, na terceira parte, outro personagem aparece fazendo uma careta e mostrando a língua. A des- crição conclui a charada Quanto mais nós queremos avisá-lo para não ver, mais você vai querer olhar4. Figura 3 - Artista flamengo anônimo, parte 1, díptico satírico. Século XVI. Óleo sobre madeira. 58,5 x 44 cm (cada painel) University of Liége coleção artistas (galeria Wittert). Bélgica. (fonte: http://www.wittert.ulg.ac.be/fr/flori/opera/anonyme1/anonyme1_notice.html, com acesso em 12.09.2019) Figura 3 - Artista flamengo anônimo, parte 1, díptico satírico. Século XVI. Óleo sobre madeira. 58,5 x 44 cm (cada painel University of Liége coleção artistas (galeria Wittert). Bélgica. (fonte: http://www.wittert.ulg.ac.be/fr/flori/opera/anonyme1/anonyme1_notice.html, com acesso em 12.09.2019) Figura 3 - Artista flamengo anônimo, parte 1, díptico satírico. Século XVI. Óleo sobre madeira. 58,5 x 44 cm (cada painel) University of Liége coleção artistas (galeria Wittert). Bélgica. (fonte: http://www.wittert.ulg.ac.be/fr/flori/opera/anonyme1/anonyme1_notice.html, com acesso em 12.09.2019) 98 98 Palíndromo, v. 12, n. 26, p. 98-109, jan - abr 2020 Kethlen Kohl (...) em Rebelais, as imagens grotescas conservam uma natureza original, di- ferenciam-se claramente das imagens da vida cotidiana, preestabelecidas e perfeitas. São imagens ambivalentes e contraditórias que parecem disfor- mes, monstruosas e horrendas, se consideradas do ponto de vista da estética “clássica”, isto é, da vida cotidiana preestabelecida e completa. A nova percep- ção histórica que as trespassa, confere-lhes um sentido diferente, embora conservando seu conteúdo e matéria tradicional: o coito, a gravidez, o parto, o crescimento corporal, a sua materialidade imediata, continuam sendo os elementos fundamentais do sistema de imagens grotescas. (BAKHTIN, 1999, p. 22) 3. Os provérbios neerlandeses: a presença da escrita através da imagem Os ditos populares sobre excremento e ânus não eram temas apenas da pintu- ra, elas fizeram parte do repertório de alguns músicos da idade moderna. Wolfgang Amadeus Mozart (1756 -1791) produziu algumas músicas e textos falando sobre ânus e escatologias, entre outras ações obscenas. A música mais conhecida delas é Leck mich im Arsch (lamba-me na bunda) que reproduzia os seguintes versos: Lamba minha bunda bem, lambe ela agradável e limpo, agradável e limpo, deve lamber minha bunda. Isso é um desejo gorduroso, bem amanteiga- do, como a lambida de carne assada, minha atividade diária. Três vai lamber mais de dois, vamos lá, basta experimentá-lo, e lamber, lamber, lamber. Todo mundo lambe seu traseiro para si5. Essas músicas de Mozart foram encontradas na Universidade de Harvard nos anos 90 e, desde então, estão sendo estudadas a fim de descobrirem sua veracidade. Michael Ochs, o estudioso das obras de Mozart, diz ser difícil ter certeza se os textos desses dois volumes são de Mozart pois, quando o músico morreu, elas foram publi- cadas por Breitkopf & Hartel. Os dois alegaram que tinham recebido os manuscritos da viúva de Mozart, Constanze (KOZINN, 1991). No entanto, tudo indica que tenham sido feitas por ele pois, além dessas músicas, foram encontradas diferentes cartas de amigos e da família de Mozart contendo frases obscenas e escatologias. Parece que 99 Palíndromo, v. 12, n. 26, p. 99-109, jan - abr 2020 Kethlen Kohl A imagem do ânus e os provérbios neerlandeses era um costume entre amigos e família, o ato de mandar cartas com ofensas e brin- cadeiras um para o outro. era um costume entre amigos e família, o ato de mandar cartas com ofensas e brin- cadeiras um para o outro. O corpo grotesco e escatológico também fez parte da literatura e ilustrações das obras de François Rebelais (1494 – 1553). Na obra Gargântua, enreda-se uma his- tória das aventuras de um gigante alegre e glutão que vivia a chafurdar nos prazeres mundanos da vida. As narrativas chegam ao limite do absurdo explorando o grotesco do corpo: (...) em Rebelais, as imagens grotescas conservam uma natureza original, di- ferenciam-se claramente das imagens da vida cotidiana, preestabelecidas e perfeitas. São imagens ambivalentes e contraditórias que parecem disfor- mes, monstruosas e horrendas, se consideradas do ponto de vista da estética “clássica”, isto é, da vida cotidiana preestabelecida e completa. A nova percep- ção histórica que as trespassa, confere-lhes um sentido diferente, embora conservando seu conteúdo e matéria tradicional: o coito, a gravidez, o parto, o crescimento corporal, a sua materialidade imediata, continuam sendo os elementos fundamentais do sistema de imagens grotescas. (BAKHTIN, 1999, p. 22) Em uma das passagens do livro Gargantua, perdura uma longa conversa entre Grandgousier e seu filho Gargantua sobre qual é a melhor maneira de limpar o ânus. Os dois trocam experiências sobre todas as coisas que usaram para limpá-lo, além de tecerem conversas sobre algumas experiências escatológicas. O costume de mostrar o ânus é um aspecto presente também nas esculturas de algumas culturas germânicas do fim da Idade Média. Observa-se a escultura abaixo de uma antiga construção da cidade de Colônia, na Alemanha. Figura 4- Escultura da “Cologne City Hall” em Colônia Alemanha, século XI. (fonte:https://www.reddit.com/r/cologne/comments/38z870/hello_good_people_of_rcologne_i_came_here_seeking/ com acesso em 12.09.2019) Figura 4- Escultura da “Cologne City Hall” em Colônia Alemanha, século XI. (fonte:https://www.reddit.com/r/cologne/comments/38z870/hello_good_people_of_rcologne_i_came_here_seeking/ com acesso em 12.09.2019) Estas esculturas estão expostas no exterior da “Cologne City Hall”. Cada uma dessas esculturas representa uma pessoa célebre da história de Colônia, abaixo delas 100 Palíndromo, v. 12, n. 26, p. 100-109, jan - abr 2020 Kethlen Kohl A imagem do ânus e os provérbios neerlandeses fundo na direita, dando dimensões diferentes aos personagens. Todos juntos pare- cem uma emaranhada multiplicidade de corpos que se comportam em desordem. fundo na direita, dando dimensões diferentes aos personagens. Todos juntos pare- cem uma emaranhada multiplicidade de corpos que se comportam em desordem. Os provérbios que estão representados na obra eram de conhecimento popular no período. “As recolhas de provérbios fazem parte dos numerosos inventários do século XV. A iniciativa foi tomada pelo grande humanista Erasmo de Roterdão que, em 1500, publicou os provérbios ou fórmulas célebres de autores latinos. Relaciona- das a esta obra surgiram antologias flamengas e alemãs” (HAGEN, 1995, p.34). Brue- gel trabalhou nos mínimos detalhes desses provérbios, transformando-os em uma obra sofisticada e recheada de pequenos espetáculos. “São identificados mais de cem provérbios e ditos, dos quais, muitos já não são usados no holandês moderno. A maior parte deles descreve atitudes, imorais ou insensatas” (HAGEN, 1995, p.34). É É possível que muitas destas cenas dos provérbios também tenham sido inspira- das nas festas de carnaval que eram muito frequentes nos países baixos8. A obra “Pro- vérbios Neerlandeses” foi minuciosamente estudada pelos historiadores Rose-Marie e Rainer Hagen que identificaram e catalogaram 118 provérbios (HAGEN, 1995, p.37). Dentre essas proeminências, destacam-se, nesta análise, aqueles provérbios sobre o ânus e o ato de defecar pensados em relação ao primeiro díptico. Figura 6 - Pieter Brueghel .Detalhe 1, Provérbios Neerlandeses.1559. (fonte: http://cargocollective.com/spreekwoorden/obras-e-artistas com acesso em 12.09.2019) Destaca-se, primeiramente, Detalhe 1 (Figura 6) um homem pendurado em uma janela com as calças arriadas. Ele está a defecar, sobre o mundo, de cabeça para bai- xo. Este detalhe indica dois provérbios: primeiro o mundo está de cabeça para baixo e no segundo ele defeca para o mundo. O mundo de ponta cabeça tinha o sentido da desordem, da inversão dos preceitos morais e também da subversão de todas as Figura 6 - Pieter Brueghel .Detalhe 1, Provérbios Neerlandeses.1559. (fonte: http://cargocollective.com/spreekwoorden/obras-e-artistas com acesso em 12.09.2019) Destaca se primeiramente Detalhe 1 (Figura 6) um homem pendura Figura 6 - Pieter Brueghel .Detalhe 1, Provérbios Neerlandeses.1559. (fonte: http://cargocollective.com/spreekwoorden/obras-e-artistas com acesso em 12.09.2019) Destaca-se, primeiramente, Detalhe 1 (Figura 6) um homem pendurado em uma janela com as calças arriadas. Ele está a defecar, sobre o mundo, de cabeça para bai- xo. A imagem do ânus e os provérbios neerlandeses estão cravados seus nomes. Além disso, as esculturas apresentam outro detalhe em sua parte inferior. Um dos detalhes que chama mais a atenção é aquele que está na escultura de Konrad Von Hochstaden, onde se avista um homenzinho de cócoras mostrando seus ânus e tentando abocanhar seu pênis. Este detalhe fica escondido, é impossível ver do solo. É perceptível através da aproximação com o zoom de câmeras fotográficas ou outra forma de visualização próxima. O grande prédio histórico onde estão as esculturas foi construído no sécu- lo XI, ele é um dos marcos da Idade Média, pois representa um momento em que as cidades começaram a criar autonomia. Mas, diferente do que era possível entender, esse detalhe não foi feito no mesmo período da construção do prédio. No período da Segunda Guerra Mundial esse prédio foi bombardeado e sua es- trutura foi danificada6. Após o final da Segunda Guerra ele foi reconstruído e, nesse período, o restaurador das esculturas resolveu colocar esse detalhe obsceno. É pos- sível deduzir que a escultura teria sido uma homenagem a uma tradição medieval das arquiteturas de Colônia. A tradição se refere a um costume de colocar esculturas de pessoas defecando no exterior da casa, isso era chamado de Kallendresser7. Outra relação que percebemos entre a escrita e imagem, é sobre a obra de Pie- ter Brueghel. O artista elencou diversos Provérbios Neerlandeses através de uma de- talhada pintura. Figura 5 - Pieter Brueghel. Provérbios Neerlandeses. 1559. Óleo sobre madeira. 117 x 163. Gemalde galerie. Berlin. (fonte: http://cargocollective.com/spreekwoorden/obras-e-artistas com acesso em 12.09.2019) Figura 5 - Pieter Brueghel. Provérbios Neerlandeses. 1559. Óleo sobre madeira. 117 x 163. Gemalde galerie. Berlin. (fonte: http://cargocollective.com/spreekwoorden/obras-e-artistas com acesso em 12.09.2019) Figura 5 - Pieter Brueghel. Provérbios Neerlandeses. 1559. Óleo sobre madeira. 117 x 163. Gemalde galerie. Be (fonte: http://cargocollective.com/spreekwoorden/obras-e-artistas com acesso em 12.09.2019) Em meio ao caos humano, estão postas diversas representações de provérbios neerlandeses, que nesta obra se misturam a uma personificação da vida dos aldeões do século XVI. A cena é construída em diagonal, da ponta da esquerda, indo para o Palíndromo, v. 12, n. 26, p. 101-109, jan - abr 2020 101 Kethlen Kohl Este detalhe indica dois provérbios: primeiro o mundo está de cabeça para baixo e no segundo ele defeca para o mundo. O mundo de ponta cabeça tinha o sentido da desordem, da inversão dos preceitos morais e também da subversão de todas as 102 Palíndromo, v. 12, n. 26, p. 102-109, jan - abr 2020 Kethlen Kohl Kethlen Kohl coisas. Segundo Peter Burke: coisas. Segundo Peter Burke: coisas. Segundo Peter Burke: O Carnaval era uma representação do ‘mundo virado de cabeça para baixo’, tema favorito na cultura popular dos inícios da Europa moderna; le monde renversé, il mondo alla rovescia, die verkehrte Welt. O ‘mundo de ponta-cabeça’ prestava-se a ilustrações, dos meados do século XVI em diante foi um tema predileto em estampas populares (BURKE, 2010, p.324). Defecar sobre o mundo define-se como o ato de dar a mínima importância para ele. Já que está de cabeça para baixo, defeca-se sobre ele para transformá-lo em uma verdadeira imundície. Este detalhe apresenta apenas um pedaço da nádega do personagem; não existe um ânus explícito, no entanto, percebe-se um dejeto líquido que cai em direção ao globo. O ato de defecar apresenta-se como um ato pejorativo, pois ‘cagar para o mundo’ é um nível alto de desprezo. Isso pode ser uma forma do artista explicitar o seu descontentamento com o que acontecia ao seu redor. Peter Bruegel morava em Bruxelas, em 1567, com a sua esposa e filhos, era um momento penoso para se viver nas regiões dos países baixos. Bruegel residia em Bruxelas quando, em agosto de 1567, o duque de Alba chegou à frente das suas tropas. Era enviado pelo rei de Espanha, Felipe II, cujo império compreendia também as províncias dos Países Baixos. O co- mandante, encarregado de converter os protestantes pela força, condenou à morte várias milhares de pessoas durante os anos que se seguiram. Esta excepcional dureza levou à revolta e depois à guerra que viria a durar oitenta anos e terminar com a divisão das províncias em dois blocos: a (futura) Bél- gica católica ao Sul e os Países Baixos protestantes ao Norte (HAGEN, 1995, p. 7). Em meio aos conflitos, Bruegel nunca deixou totalmente explícito em suas obras se ele era protestante ou católico. A sua crítica em relação à reforma religiosa tam- bém se torna evidente na tela A luta entre o Carnaval e a Quaresma (1559.118 × 164 cm). É possível que por trás desses provérbios hajam diversas críticas pontuais, que estão relacionadas a estes embates políticos e religiosos emergentes em seu período. No Detalhe 2 (Figura 7) encontra-se um puxadinho de madeira em uma torre, nela existe um buraco, uma latrina onde, entre a fenda, revelam-se dois sujeitos que defecam. O detalhe refere-se ao provérbio ‘eles defecam porcaria pelo mesmo bura- co’. A imagem do ânus e os provérbios neerlandeses coisas. Segundo Peter Burke: Os dois são inseparáveis, fazem as coisas mundanas juntos, e, outra vez, defecar é mostrado como algo vilipendioso. Em meio aos conflitos, Bruegel nunca deixou totalmente explícito em suas obras se ele era protestante ou católico. A sua crítica em relação à reforma religiosa tam- bém se torna evidente na tela A luta entre o Carnaval e a Quaresma (1559.118 × 164 cm). É possível que por trás desses provérbios hajam diversas críticas pontuais, que estão relacionadas a estes embates políticos e religiosos emergentes em seu período. No Detalhe 2 (Figura 7) encontra-se um puxadinho de madeira em uma torre, nela existe um buraco, uma latrina onde, entre a fenda, revelam-se dois sujeitos que defecam. O detalhe refere-se ao provérbio ‘eles defecam porcaria pelo mesmo bura- co’. Os dois são inseparáveis, fazem as coisas mundanas juntos, e, outra vez, defecar é mostrado como algo vilipendioso. Palíndromo, v. 12, n. 26, p. 103-109, jan - abr 2020 103 Kethlen Kohl A imagem do ânus e os provérbios neerlandeses A imagem do ânus e os provérbios neerlandeses Figura 7 - Pieter Brueghel .Detalhe 2, Provérbios Neerlandeses.1559. (fonte: http://cargocollective.com/spreekwoorden/obras-e-artistas com acesso em 12.09.2019) Figura 7 - Pieter Brueghel .Detalhe 2, Provérbios Neerlandeses.1559. (fonte: http://cargocollective.com/spreekwoorden/obras-e-artistas com acesso em 12.09.2019) Figura 7 - Pieter Brueghel .Detalhe 2, Provérbios Neerlandeses.1559. (fonte: http://cargocollective.com/spreekwoorden/obras-e-artistas com acesso em 12.09.2019) Figura 8 - Pieter Brueghel .Detalhe 3, Provérbios Neerlandeses.1559. (fonte: http://cargocollective.com/spreekwoorden/obras-e-artistas com acesso em 12.09.2019) Figura 8 - Pieter Brueghel .Detalhe 3, Provérbios Neerlandeses.1559. g g (fonte: http://cargocollective.com/spreekwoorden/obras-e-artistas com acesso em 12.09.2019) 104 Palíndromo, v. 12, n. 26, p. 104-109, jan - abr 2020 Kethlen Kohl Kethlen Kohl Kethlen Kohl A imagem do ânus e os provérbios neerlandeses de modo a indicar alguma coisa ao seu colega de branco. No canto inferior esquerdo, um homem está de cócoras defecando próximo à forca. O homem é quase invisível na pintura, defeca em um canto escondido fazendo parte do primeiro plano da pintu- ra. O personagem parece fazer parte do adornado de árvores que percorre as bordas da pintura. de modo a indicar alguma coisa ao seu colega de branco. No canto inferior esquerdo, um homem está de cócoras defecando próximo à forca. O homem é quase invisível na pintura, defeca em um canto escondido fazendo parte do primeiro plano da pintu- ra. O personagem parece fazer parte do adornado de árvores que percorre as bordas da pintura. Segundo Rose Marie e Rainer Hagen, “A pega representava as más línguas, boas para a forca... Foi, de facto, sobre a delação que o duque de Alba construiu seu regi- me de terror” (HAGEN, 1995, p.12). Dito isso, as representações da forca e do pássaro estão relacionadas com o genocídio dos protestantes. Esse é mais um dos indícios de que Bruegel trazia, à tona, questões políticas em suas obras. Os provérbios são cenas humorísticas; o ânus e fezes são postos em cena como elementos de grandes trocadilhos. Seus trabalhos se concentram entre a alusão po- lítica e a ironia moral. Aspectos comuns ao de Bruegel estão presentes também nas obras de Hendrick Avercamp (1585- 1639). Figura 10 - Hendrick Avercamp. Winter Landscape with Ice Skaters .1608. Òleo sobre madeira. 77 x 131 cm. (fonte: https://commons.wikimedia.org/wiki/File:Hendrick_Avercamp_-_Winterlandschap_met_ijsvermaak.jpg com acesso em 12.09.2019) Figura 10 - Hendrick Avercamp. Winter Landscape with Ice Skaters .1608. Òleo sobre madeira. 77 x 131 cm. /commons.wikimedia.org/wiki/File:Hendrick_Avercamp_-_Winterlandschap_met_ijsvermaak.jpg com acesso em 12.09.2019) Avercamp representou a vida cotidiana dos holandeses no período de inverno. Nesta obra, o pintor não teve a intenção de representar os provérbios neerlandeses. O sentido aqui é demonstrar a paisagem de inverno e a vida após a República das províncias unidas da Holanda. A visão panorâmica da obra permite que o cotidiano seja contado em seus detalhes, até mesmo os mais sórdidos e mais virtuosos dessa população. Apesar de Avercamp ter como inspiração principal de sua obra os traba- lhos de Bruegel, esta pintura não possui o mesmo intuito. Avercamp procura mostrar uma comunidade mais organizada praticando atividades sadias no inverno holandês. A imagem do ânus e os provérbios neerlandeses O último Detalhe 3 (Figura 8) encontra-se, ao fundo da pintura, no canto supe- rior esquerdo: uma pessoa defeca na forca. Sobre ela estão os pássaros pretos e ou- tros que sobrevoam o território. Os pássaros significam que ‘Onde tem carniça, voam os corvos’, ou seja, onde as pessoas podem lucrar, lá estarão a rondar. Obstante a forca e os corvos remetem também à morte e às punições aos que em um mundo caótico só satisfazem aos corvos. O homem a purgar na forca poderia ser interpretado de duas formas: se ele de- feca na forca é porque é inexorável a qualquer penalidade e não teria medo nem da morte, um típico libertino que não se importa com as leis. Em outro sentido, defecar na forca pode ser interpretado como uma pessoa que tem medo de ser penalizado, medo do seu destino, então, quando chega próximo à forca, borra-se. A cena se re- pete em outra obra de Bruegel O pêga na Forca. Figura 9 - Pieter Bruegel. O pega na forca. 1568. Óleo sobre Madeira. 45,9 x 50,8 cm. Museu Land de Hesse, Amsterdã. fonte: http://theredlist.com/wiki-2-351-861-414-398-1233-view-northern-europe-profile-bruegel-the-elder-pieter.htm com acesso em 12.09.2019) Figura 9 - Pieter Bruegel. O pega na forca. 1568. Óleo sobre Madeira. 45,9 x 50,8 cm. Museu Land de Hesse, Amsterdã. (fonte: http://theredlist.com/wiki-2-351-861-414-398-1233-view-northern-europe-profile-bruegel-the-elder-pieter.htm com acesso em 12.09.2019) A cena é construída na vertical, partindo do primeiro plano, no alto de uma montanha, para o fundo, onde se encontram casas, castelos, árvores e um horizonte que segue o rio. No primeiro plano e ao centro da tela, destacam-se uma forca torta e um Pêga (pássaro da família dos corvídeos). O pássaro pousa sobre ela, enquanto os aldeões dançam em sua proximidade. Ao meio, um homem aponta para o pássaro 105 Palíndromo, v. 12, n. 26, p. 105-109, jan - abr 2020 Kethlen Kohl In- clusive, o ato de defecar pode apresentar sentidos distintos daqueles da obra de Bruegel. 106 Palíndromo, v. 12, n. 26, p. 106-109, jan - abr 2020 Kethlen Kohl Kethlen Kohl A imagem do ânus e os provérbios neerlandeses A imagem do ânus e os provérbios neerlandeses A imagem do ânus e os provérbios neerlandeses A imagem do ânus e os provérbios neerlandeses A imagem do ânus e os provérbios neerlandeses 4. Conclusão O presente artigo propôs duas problemáticas entre a escrita e a imagem que ca- racterizam uma fusão indissociável: a primeira sobre o jogo entre a imagem e escrita pintadas e a segunda sobre a presença do proverbio que incide sobre a imagem. O vínculo entre escrita e imagem no díptico flamengo é muito peculiar, ele apresenta um jogo entre escrita e imagem que é próprio do costume neerlandês. A inscrição da imagem é um aviso para que o espectador não veja porque, caso assim decida, será punido: o provérbio sugere “não ver”. A potência dos imperativos “não veja”, “não abra” faz com que a imagem se torne vigorosa, energética, assim almejamos ver aqui- lo que não podemos. A interdição contida nesse discurso é transgredida pelo desejo de olhar, pois, segundo Bataille “o interdito existe para ser violado” (1987, p. 60). Ao abrirmos o díptico algo nos atinge, um grande ânus, ao estilo daqueles presentes no imaginário da cultura popular, um elemento para fazer rir. A palavra é introduzida novamente, e a piada se conclui, pois, “Quanto mais nós queremos avisá-lo para não ver, mais você vai querer olhar” Os jogos entre imagem e escrita estão presentes em toda a relação entre os pro- vérbios. Um provérbio não existe sem um texto, ele é um texto que se constitui entre as comunidades através da oralidade, mas o discurso também circula como escrita. Mesmo que as imagens de Brueguel e Avercamp não possuam o provérbio escrito na imagem, a imagem aprisiona a tautologia do texto. Não conseguimos ver um ho- mem que dê rosa aos porcos sem associarmos diretamente ao ditado popular. Isso porque esse provérbio é comum à língua portuguesa, essas imagens estão presentes na construção a várias expressões idiomáticas, mas a imagem não possui uma língua específica, ela é traduzível a todas as línguas. Obras como estas eternizaram uma tradição da escrita, passaram os provérbios para um patamar da memória de diversos povos através da imagem. Elas também fazem viver uma forma de lidar com os desejos humanos (como querer ver) de uma maneira bem-humorada. Esse humor se torna um refúgio para lidar com embates políticos do período, bem como questões éticas e morais. A imagem do ânus e os provérbios neerlandeses mente fazem rir, basta pensar na satisfação com que as crianças apreciam dizer ou ouvir piadas sobre excrementos” (ECO, 2015, p.131). As sátiras com ânus e excremen- tos são muito exploradas pelos artistas europeus desse período e não procuram estar presentes apenas nas tradições escritas, mas também através da imagem. mente fazem rir, basta pensar na satisfação com que as crianças apreciam dizer ou ouvir piadas sobre excrementos” (ECO, 2015, p.131). As sátiras com ânus e excremen- tos são muito exploradas pelos artistas europeus desse período e não procuram estar presentes apenas nas tradições escritas, mas também através da imagem. . Figura 11 - Hendrick Avercamp. Detalhe 1, Winter Landscape with Ice Skaters.1608 (fonte: https://commons.wikimedia.org/wiki/File:Hendrick_Avercamp_-_Winterlandschap_met_ijsvermaak.jpg com acesso em 12.09.2019) Figura 11 - Hendrick Avercamp. Detalhe 1, Winter Landscape with Ice Skaters.1608 s.wikimedia.org/wiki/File:Hendrick_Avercamp_-_Winterlandschap_met_ijsvermaak.jpg com acesso em 12.09.2019) Figura 11 - Hendrick Avercamp. Detalhe 1, Winter Landscape with Ice Skaters.1608 iki di / iki/Fil H d i k A Wi t l d h t ij k j Figura 11 - Hendrick Avercamp. Detalhe 1, Winter Landscape with Ice Skaters.1608 : https://commons wikimedia org/wiki/File:Hendrick Avercamp - Winterlandschap met ijsvermaak jpg com Figura 11 - Hendrick Avercamp. Detalhe 1, Winter Landscape with Ice Skaters.1608 (fonte: https://commons.wikimedia.org/wiki/File:Hendrick_Avercamp_-_Winterlandschap_met_ijsvermaak.jpg com acesso em 12.09.2019) Figura 11 Hendrick Avercamp. Detalhe 1, Winter Landscape with Ice Skaters.1608 (fonte: https://commons.wikimedia.org/wiki/File:Hendrick_Avercamp_-_Winterlandschap_met_ijsvermaak.jpg com acesso em 12.09.2019) g p , p (fonte: https://commons.wikimedia.org/wiki/File:Hendrick_Avercamp_-_Winterlandschap_met_ijsvermaak.jpg com Chamo atenção para esse detalhe que está ao canto esquerdo, escondido entre as construções. Ali, dois camponeses defecam: um pela latrina e, abaixo dela, avista- -se um morro de fezes e o outro evacua próximo à árvore. Os dois homens defecam simultaneamente, o que ressalta a ideia de que evacuar é humano, todos defecam inclusive ao mesmo tempo em lugares diferentes. A cena também pode ser compa- rada ao provérbio presente na obra de Bruegel onde os dois defecam juntos, o que significa que eles “cometem os mesmos erros”. Avercamp mostra pessoas defecando de uma forma menos explícita que Brue- gel, dispondo os personagens escondidos no canto esquerdo. Esses dois persona- gens também não foram colocados na cena de forma estratégica a fim de criticar uma questão política, eles estão defecando em lugares próprios para isso. Claro que isso não impede que a cena tenha um sentido cômico do cotidiano. Todos esses detalhes possuem um sentido cômico, tanto nas obras de Bruegel como de Avercamp. Estas obras também tinham o intuito de provocar o riso no es- pectador, pois: “Pode-se exibir comportamentos obscenos por raiva ou por provoca- ção, mas, com muita frequência, a linguagem ou comportamento obscenos simples- 107 Palíndromo, v. 12, n. 26, p. 107-109, jan - abr 2020 Kethlen Kohl 9 DIDI-HUBERMAN, Georges. A imagem sobrevivente: História da arte e tempo dos fantasmas segundo Aby Warburg. Rio de Janeiro: Contraponto, 2013. BATAILLE, Georges. O erotismo. Porto Alegre: L&PM, 1987. BATAILLE, Georges. O erotismo. Porto Alegre: L&PM, 1987. BAKHTIN, Mikhail. A cultura popular na Idade Média e no Renascimento: o contexto de François Rebelais. São Paulo: Hucitec, 1999. 4. Conclusão Essas obras demonstram que a escrita e a imagem não são opostas, nem possuem hierarquias, pois surgem de um mesmo princípio, a necessidade de marcar, expelir o pensamento, o que faz com que ele sobreviva (Nacheleben)9 ao tempo. ERMAN, Georges. A imagem sobrevivente: História da arte e tempo dos fantasmas segundo Aby Warburg. Rio de Janeiro: Contraponto, 2013. 108 Kethlen Kohl Palíndromo, v. 12, n. 26, p. 108-109, jan - abr 2020 Kethlen Kohl TOMKINS, Calvin. Duchamp: uma biografia. São Paulo: Cosac Naify, 2013. BURKE, Peter. Cultura Popular na idade moderna (1500-1800). São Paulo: Compa- nhia das letras, 2010. BURKE, Peter. Cultura Popular na idade moderna (1500-1800). São Paulo: Compa- nhia das letras, 2010. DIDI-HUBERMAN, Georges. A imagem sobrevivente: História da arte e tempo dos fantasmas segundo Aby Warburg. Rio de Janeiro: Contraponto, 2013. ECO, Umberto. História da feiura. Rio de Janeiro: Record, 2015. MARTIN, Jean-Humbert. Dossier Pédagogique: Carambolages. Paris: RmnGP, 2016 KOHL; Kethlen. Consentimento e Interdição sobre o corpo nas Artes Visuais: Impli- cações e Desdobramentos dos (Contra) Dispositivos.2017. Dissertação, (Mestrado em Artes Visuais), programa de pós-graduação em Artes Visuais, Universidade do Estado de Santa Catarina, Florianópolis. KOZINN, Allan. Three Naughty Mozart Texts Are Found. The New York times. 2 de mar, 1991. Disponível em:< http://www.nytimes.com/1991/03/02/arts/three-naugh- ty-mozart-texts-are-found.html.> Data de acesso: 10 abril, 2019. HAGEN, Rainer; HAGEN, Rose Marie. Pieter Bruegel o velho: camponeses, loucos e demónios. Taschen, 1995. A imagem do ânus e os provérbios neerlandeses Submetido em: 20/04/2019 Aceito em: 15/08/2019 Submetido em: 20/04/2019 Aceito em: 15/08/2019 109 Palíndromo, v. 12, n. 26, p. 109-109, jan - abr 2020 Palíndromo, v. 12, n. 26, p. 109-109, jan - abr 2020 Kethlen Kohl
https://openalex.org/W2793535520	https://europepmc.org/articles/pmc5828443?pdf=render	English	null	Precision (repeatability and reproducibility) of ocular parameters obtained by the Tomey OA-2000 biometer compared to the IOLMaster in healthy eyes	PloS one	2,018	cc-by	6,669	RESEARCH ARTICLE Precision (repeatability and reproducibility) of ocular parameters obtained by the Tomey OA-2000 biometer compared to the IOLMaster in healthy eyes Yanjun Hua, Wei Qiu, Qiuyi Xiao, Qiang Wu Department of Ophthalmology, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Xuhui District, Shanghai, China Yanjun Hua, Wei Qiu, Qiuyi Xiao, Qiang Wu Department of Ophthalmology, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Xuhui District, Shanghai, China Yanjun Hua, Wei Qiu, Qiuyi Xiao, Qiang Wu Department of Ophthalmology, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Xuhui District, Shanghai, China * hyj1860@hotmail.com (YH); qiang.Wu@shsmu.edu.cn (QW) a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Hua Y, Qiu W, Xiao Q, Wu Q (2018) Precision (repeatability and reproducibility) of ocular parameters obtained by the Tomey OA-2000 biometer compared to the IOLMaster in healthy eyes. PLoS ONE 13(2): e0193023. https://doi.org/ 10.1371/journal.pone.0193023 Purpose To assess the precision (repeatability and reproducibility) of ocular parameters measured by the Tomey OA-2000 biometer, and to compare them with those measured by the IOLMaster. Methods In this prospective study, the right eyes of 108 healthy subjects were included. Three conse- cutive scans were obtained by 2 observers using the Tomey OA-2000, and in the same ses- sion one observer used the IOLMaster (version 5.4.4.0006) for the measurements. About 1 week later, 3 scans were obtained by one observer using the Tomey OA-2000. The axial length (AL), central corneal thickness (CCT), anterior chamber depth (ACD), lens thickness (LT), keratometer readings, pupil diameter (PD) and corneal diameter (CD) values mea- sured by the Tomey OA-2000 and IOLMaster were analyzed. The coefficient of variation (CoV), intraclass correlation coefficient (ICC), within subject standard deviation (Sw) and 2.77Sw were calculated to assess the repeatability and reproducibility. The paired t test and Bland-Altman plots were used to analyze the differences and agreements of parameters measured by the two devices, respectively. Editor: Manuel Garza Leo´n, Universidad de Monterrey Division de Ciencias de la Salud, MEXICO Copyright: © 2018 Hua 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. Introduction Accurate ocular biometry is essential for predicting the intraocular lens (IOL) power before refractive and cataract surgery[1]. Generally for cataract surgery, precise ocular biometry can decrease refractive error and achieve the predicted postoperative outcome. Ultrasound-based axial length (AL) and anterior chamber depth (ACD, from corneal epithelium to anterior sur- face of crystalline lens) measurements have been the benchmark for a long period of time. However, the contact measurement is accompanied by the risk of infection and indentation compelling technicians and clinicians to seek safer and more comfortable techniques for ocu- lar biometry. In 1999, the introduction of the partial coherence interferometry (PCI) based IOLMaster (Carl Zeiss Meditec AG) marked a great improvement due to its non-contact nature and higher resolution of AL measurement (about ±0.02 mm compared to ±0.15 mm by ultrasound)[2–4]. Furthermore, the inclusion of the corneal curvature, ACD and horizontal corneal diameter (CD) measurements in the same device, minimized the time for all measure- ments and improved the calculation of IOL power. In the last decade, the precision of the IOL- Master in both cataract biometry[5–7] and the study of refractive error evaluation[8, 9] set a new standard for ocular biometry. However, only AL measurement is based on PCI principle for the IOLMaster. The corneal curvature, ACD and CD are obtained from image analysis. Nevertheless, in IOLMaster 500 and older models, such as the model(software version: 5.4.4.0006) used in the present study, the corneal, crystalline lens or retinal thickness are not evaluated[10]. Additionally, realignment is required before each parameter is measured[11]. Several new devices based on optical low coherence reflectometry (OLCR)[12], such as Len- star LS900 (Haag-Streit Ko¨niz, Switzerland), Aladdin (Topcon, Japan), AL-Scan (Nidek, Japan) and OA-2000 (Tomey, Japan) are now commercially available for ocular biometry. In these devices, the AL, central corneal thickness (CCT), ACD (from corneal epithelium to ante- rior surface of crystalline lens), pupil diameter (PD) and crystalline lens thickness (LT) were measured automatically without the need of realignment. The Tomey OA-2000 biometer, which was an upgraded device of its predecessor OA-1000[13, 14], was developed to perform keratometry measurement based on Placido disc-based topography techniques with 9 rings each 256 points in a 5.5-mm zone projected on the anterior corneal surface[15]. Several stud- ies[11, 16–24] have reported that the ocular parameters measured by Lenstar LS 900, Aladdin and AL-Scan biometers have excellent precision, and agreed well with those measured by the IOLMaster. Conclusion Except for the PD and CD, the ocular parameters measured by the Tomey OA-2000 were highly repeatable and reproducible. Except for the CD value, there was good agreement of ocular parameters measured by the Tomey OA-2000 and the IOLMaster in healthy eyes. Results Intraobserver repeatability, and interobserver and intersession reproducibility of the AL, CCT, ACD, LT, Kf, Ks, Km, PD and CD values measured by the Tomey OA-2000 biometer showed a CoV of less than 1% except that for PD, and an ICC of more than 0.97 except that for PD and CD. The AL, Kf, Ks, Km and CD values measured by the Tomey OA-2000 were 0.058 ± 0.094 mm, 0.088± 0.150 diopters (D), 0.163 ± 0.170 D, 0.127 ± 0.117 D and 0.171 ± 0.217 mm lower than those measured by the IOLMaster, respectively (all Ps < 0.05). How- ever, the ACD values from the two devices were comparable (P = 0.169). The 95% linite of Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: The authors received no specific funding for this work. Competing interests: The authors have declared that no competing interests exist. PLOS ONE \| https://doi.org/10.1371/journal.pone.0193023 February 27, 2018 1 / 11 Precision of the Tomey OA-2000 biometer and agreement with the IOLMaster agreement (LoA) of the AL, ACD, CD and all keratometer readings were no more than 0.24 mm, 0.14 mm 0.60 mm and 0.5 D, respectively. PLOS ONE \| https://doi.org/10.1371/journal.pone.0193023 February 27, 2018 Subjects and methods The study was conducted between July and September 2015 at the Department of Ophthalmol- ogy, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital. The study protocol fol- lowed the principles of the Declaration of Helsinki and was approved by the Office of Research Ethics Committee, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital. All sub- jects provided written informed consent after the purpose of the study had been explained to them in detail. One-hundred and eight subjects (64 women), with a mean age of 27.43±7.37 years (range, 18–48 years) participated in this study. The mean spherical equivalent refraction was -3.24 ± 2.30 diopters (D; range, -8.88 to +2.50 D). All subjects could communicate well and cooperate with good fixation ability. The inclusion criteria were healthy subjects with a best corrected distance visual acuity equal to or better than 20/20 and an intraocular pressure of 10 to 21 mmHg. The exclusion criteria included 1) history of ocular pathology, or corneal or intraocular trauma; 2) previous ocular surgery; 3) wearing soft contact lenses within 2 weeks or rigid contact lenses within 4 weeks; 4) dry eye syndrome (with subjective dry eye symptoms, tear film break-up time shorter than 5 seconds). Only the right eye for each subject was selected for all measurements. Repeatability, reproducibility and agreement were assessed based on standards adopted by the British Standards Institute and the International Organization for Standardization[25]. In the first session, 2 observers obtained 3 consecutive scans using the Tomey OA-2000 biometer, respectively, to assess intraobserver repeatability and interobserver reproducibility; ocular parameters were also measured by the IOLMaster (version 5.4.4.0006) in the same session. The average of AL, Keratometer readings, PD and CD obtained by the Tomey OA-2000 biometer by the first observer were used to compared with those obtained by IOLMaster. In the second session, about 1 week later, 1 observer obtained another 3 consecutive scans using the Tomey OA-2000 biometer to assess intersession reproducibility. All measurements were performed at least 3 hours after waking between 10 AM and 5 PM to minimize variations in the results. During the measurements, all the subjects sat in front of the devices, put their chin on the chinrest, focused on the target accordingly, opened their eyes wide after blinking before each scan. The whole procedure was completed within 15 minutes and only qualified measure- ments were adopted. Subjects and methods All the subjects were confirmed to have avoided substantial reading before the measurements were performed. Introduction To the best of our knowledge, few reports have been published to assess the preci- sion (repeatability and reproducibility) of ocular parameters measured by Tomey OA-2000 biometer. The purpose of this study was to evaluate the precision of the Tomey OA-2000 biometer for assessing ocular biometry, and to compare the parameters measured by the new device with those by IOLMaster. PLOS ONE \| https://doi.org/10.1371/journal.pone.0193023 February 27, 2018 2 / 11 Precision of the Tomey OA-2000 biometer and agreement with the IOLMaster PLOS ONE \| https://doi.org/10.1371/journal.pone.0193023 February 27, 2018 Intraobserver repeatability of ocular parameters measured by the Tomey OA-2000 biometer Table 1 shows the repeatability of the AL, CCT, ACD, LT, Kf, Ks, Km, PD and CD values mea- sured by the Tomey OA-2000 by the 2 observers. All CoV values were lower than 1.0%, except that of PD (5.298% and 4.889%). All ICC values were no less than 0.987, except that of PD (0.943 and 0.954) and CD (0.916 and 0.936). For both observers, the 2.77Sw for PD were 1.001 mm and 0.893 mm, while the 2.77Sw were 0.547 mm and 0.473 mm. Instruments and measurements protocol The Tomey OA-2000 biometer measures distances in the eye based on the OLCR principle using a superluminescent diode at a wavelength of 820 μm[15]. The AL, CCT, ACD, PD and LT can be measured using OLCR[22]. To measure the corneal curvature, Placido-disc based topography techniques with 9 rings each 256 points in a 5.5-mm zone are projected onto the cornea, and corneal curvature of 2-mm, 2.5-mm and 3-mm central corneal zone can be obtained[15]. The standard keratometric index of 1.3375 is used for calculating the kerat- ometer readings[26, 27]. In the present study, the AL, CCT, ACD, PD, LT, CD and kerat- ometer readings data of the 2.5 mm zone for each scan were collected. The IOLMaster measures AL based on the PCI principle, which generates infrared light with a wavelength of 780 μm[10]. The AL is measured from the anterior corneal surface to reti- nal pigment epithelium. To measure the corneal curvature, the IOLMaster reflects 6 points of light from the tear film surface at a hexagonal pattern of approximately 2.3 mm diameter. The separation of opposite pairs of lights is measured by the internal software and the surface cur- vatures are calculated from 3 fixed meridians. The standard keratometric index of 1.3375 is PLOS ONE \| https://doi.org/10.1371/journal.pone.0193023 February 27, 2018 3 / 11 Precision of the Tomey OA-2000 biometer and agreement with the IOLMaster used for calculating the Keratometer readings. To measure the ACD, the device directs a 0.7-mm wide slit of light beam through the anterior segment of the eye at an angle of 38 degrees to visual axis, and the distance between the anterior corneal pole and the anterior crys- talline lens surface is calculated as the ACD[10]. In the present study, the AL, ACD, Kerat- ometer readings and CD data were collected. Statistical analysis Both SPSS software for Windows version 17 (SPSS Inc., Chicago, IL, U.S.) and MedCalc Statis- tical Software version 11.0 (MedCalc Software, Inc., Mariakerke, Belgium) were applied for statistical analyses. A P value of less than 0.05 was considered to have statistical significance. The distribution of all the datasets were analyzed for normality using Kolmogorov-Smirnov tests. No sample size calculation was made because statistical methods for agreement and pre- cision studies recommend that the sample size should be at least 100 subjects[28]. To deter- mine the intraobserver repeatability, interobserver and intersession reproducibility, within- subject standard deviation (Sw), test-retest repeatability (TRT), within-subject coefficient of variation (CoV), and intraclass correlation coefficient (ICC)[28] were calculated for the 3 con- secutive measurements obtained during the first session. The test-retest repeatability was defined as 2.77Sw, which indicated the interval within which 95% of the differences between measurements are expected to lie. The CoV was calculated as the ratio of the Sw to the overall mean. A smaller CoV means that the repeatability was higher. The ICC (ranging from 0 to 1) assess the consistency for data sets of repeated measurements. The closer the ICC is to 1, the better the measurement consistency, and a value more than 0.9 indicates acceptable clinical reliability[29]. To compare ocular parameters obtained by the 2 devices, a paired t test was applied to identify pairs that had significant differences. Bland-Altman plots[25] were con- structed to assess the agreement of measurements between the devices. The 95% limits of agreement (LoA) were defined as ±1.96 standard deviation. A narrower 95% LoA indicated better agreement between measurements. Precision of the Tomey OA-2000 biometer and agreement with the IOLMaster Table 1. Intraobserver repeatability of ocular parameters measured by Tomey OA-2000 biometer (n = 108). Parameter Observer Mean ± SD CoV (%) Sw 2.77Sw ICC AL (mm) 1st 24.56 ± 1.30 0.034 0.013 0.037 1.000 2nd 24.56 ± 1.30 0.035 0.011 0.030 1.000 CCT (μm) 1st 520.48 ± 31.63 0.668 5.08 14.06 0.991 (0.988–0.994) 2nd 521.05 ± 31.69 0.591 4.75 13.17 0.993 (0.990–0.995) ACD (mm) 1st 3.59 ± 0.32 0.426 0.020 0.057 0.999 (0.998–0.999) 2nd 3.58 ± 0.32 0.385 0.016 0.045 0.999 (0.998–0.999) LT (mm) 1st 3.72 ± 0.29 0.651 0.055 0.152 0.990 (0.987–0.993) 2nd 3.73 ± 0.30 0.686 0.063 0.175 0.987 (0.982–0.991) Kf (D) 1st 42.81 ± 1.44 0.141 0.070 0.200 0.999 (0.999–0.999) 2nd 42.81 ± 1.41 0.140 0.077 0.214 0.999 (0.999–0.999) Ks (D) 1st 43.84 ± 1.61 0.220 0.134 0.372 0.998 (0.997–0.998) 2nd 43.84 ± 1.63 0.183 0.134 0.372 0.998 (0.997–0.998) Km (D) 1st 43.32 ± 1.48 0.174 0.055 0.152 1.000 (0.999–1.000) 2nd 43.32 ± 1.49 0.113 0.070 0.196 0.999 (0.999–1.000) PD (mm) 1st 5.53 ± 0.88 5.298 0.363 1.001 0.943 (0.922–0.960) 2nd 5.49 ± 0.87 4.885 0.322 0.893 0.954 (0.936–0.967) CD (mm) 1st 11.88 ± 0.39 0.829 0.197 0.547 0.916 (0.885–0.940) 2nd 11.87 ± 0.39 0.575 0.170 0.472 0.936 (0.912–0.954) AL: axial length, CCT: central corneal thickness, ACD: anterior chamber depth, LT: lens thickness, Kf: flat keratometry, Ks: steep keratometry, Km: mean keratometry, PD: pupil diameter, CD: white to white, D: diopter, SD: standard deviation, CoV: coefficient of variance, Sw: within-subject standard deviation, ICC: intraclass correlation coefficient. Table 1. Intraobserver repeatability of ocular parameters measured by Tomey OA-2000 biometer (n = 108). AL: axial length, CCT: central corneal thickness, ACD: anterior chamber depth, LT: lens thickness, Kf: flat keratometry, Ks: steep keratometry, Km: mean keratometry, PD: pupil diameter, CD: white to white, D: diopter, SD: standard deviation, CoV: coefficient of variance, Sw: within-subject standard deviation, ICC: intraclass correlation coefficient. https://doi.org/10.1371/journal.pone.0193023.t001 Interobserver reproducibility of ocular parameters measured by the Tomey OA-2000 biometer Table 2 shows the high interobserver reproducibility of the AL, CCT, ACD, LT, Kf, Ks, Km and CD values measured by the Tomey OA-2000, excluding PD. All CoV values were lower than 1% and all the ICC value were higher than 0.972, except that of PD (CoV = 3.855%, ICC = 0.944). The 2.77Sw values were 0.023 mm for AL, 8.355μm for CCT, 0.160 mm for ACD, 0.248 mm for CD and less than 0.210 D for all keratometer readings, while the 2.77Sw value for PD was 1.020 mm. PLOS ONE \| https://doi.org/10.1371/journal.pone.0193023 February 27, 2018 4 / 11 https://doi.org/10.1371/journal.pone.0193023.t002 keratometry, Ks: steep keratometry, Km: mean keratometry, PD: pupil diameter, CD: white to white, D: diopter, SD: standard deviation, CoV: coefficient of variance, Sw: within-subject standard deviation, ICC: intraclass correlation coefficient. PLOS ONE \| https://doi.org/10.1371/journal.pone.0193023 February 27, 2018 AL: axial length, CCT: central corneal thickness, ACD: anterior chamber depth, LT: lens thickness, Kf: flat Intrasession reproducibility of ocular parameters measured by Tomey OA- 2000 biometer Intrasession reproducibility of ocular parameters measured by Tomey OA-2000 biometer (n = 108). AL: axial length, CCT: central corneal thickness, ACD: anterior chamber depth, LT: lens thickness, Kf: flat keratometry, Ks: steep keratometry, Km: mean keratometry, PD: pupil diameter, CD: white to white, D: diopter, SD: standard deviation, CoV: coefficient of variance, Sw: within-subject standard deviation, ICC: intraclass correlation coefficient. https://doi.org/10.1371/journal.pone.0193023.t003 Comparison of ocular parameters measured by the Tomey OA-2000 biometer and IOLMaster The average of the 3 measurements of AL, Keratometer readings, PD and CD by the Tomey OA-2000 were used to compared with those by IOLMaster. All ocular parameters measured by the Tomey OA-2000 were statistically smaller than those measured by the IOLMaster, exclud- ing the ACD (Table 4). The AL and CD values measured by the Tomey OA-2000 were 0.058 ± 0.094 mm and 0.171 ± 0.217 mm smaller, respectively, than those measured by the IOL- Master, and the Kf, Ks and Km values measured by the Tomey OA-2000 were 0.088± 0.150 D, 0.163 ± 0.170 D and 0.127 ± 0.117 D lower, respectively, than those measured by the IOLMaster (all Ps < 0.05). However, the ACD values from the 2 devices were comparable (P = 0.169). The 95% LoA of the AL, ACD, CD and Keratometer readings were 0.24 mm, 0.14 mm, 0.60 mm and no more than 0.5 D, respectively (Fig 1). This indicates that agreements of the AL, ACD and all the keratometer readings measured by the two devices were relatively good. https://doi.org/10.1371/journal.pone.0193023.t004 Intrasession reproducibility of ocular parameters measured by Tomey OA- 2000 biometer Table 3 shows the high intrasession reproducibility of the AL, CCT, ACD, LT, Kf, Ks, Km and CD values measured by the Tomey OA-2000, excluding PD. All CoV values were lower than 1% and the ICC values were higher than 0.97, except that of PD (CoV = 4.787%, ICC = 0.903). Table 2. Interobserver reproducibility of ocular parameters measured by Tomey OA-2000 biometer (n = 108). Table 2. Interobserver reproducibility of ocular parameters measured by Tomey OA-2000 biometer (n = 108). Parameter CoV (%) Sw 2.77Sw ICC AL (mm) 0.022 0.008 0.023 1.000 (1.000–1.000) CCT (μm) 0.423 3.016 8.355 0.995 (0.993–0.997) ACD (mm) 0.332 0.022 0.061 0.998 (0.997–0.998) LT (mm) 0.550 0.058 0.160 0.981 (0.972–0.987) Kf (D) 0.082 0.045 0.124 0.999 (0.999–0.999) Ks (D) 0.110 0.073 0.202 0.999 (0.999–0.999) Km (D) 0.068 0.045 0.124 0.999 (0.999–0.999) PD (mm) 3.855 0.370 1.020 0.944 (0.918–0.962) CD (mm) 0.378 0.090 0.248 0.973 (0.961–0.982) AL: axial length, CCT: central corneal thickness, ACD: anterior chamber depth, LT: lens thickness, Kf: flat keratometry, Ks: steep keratometry, Km: mean keratometry, PD: pupil diameter, CD: white to white, D: diopter, SD: standard deviation, CoV: coefficient of variance, Sw: within-subject standard deviation, ICC: intraclass correlation coefficient. AL: axial length, CCT: central corneal thickness, ACD: anterior chamber depth, LT: lens thickness, Kf: flat keratometry, Ks: steep keratometry, Km: mean keratometry, PD: pupil diameter, CD: white to white, D: diopter, SD: standard deviation, CoV: coefficient of variance, Sw: within-subject standard deviation, ICC: intraclass correlation coefficient. PLOS ONE \| https://doi.org/10.1371/journal.pone.0193023 February 27, 2018 5 / 11 Precision of the Tomey OA-2000 biometer and agreement with the IOLMaster Table 3. Intrasession reproducibility of ocular parameters measured by Tomey OA-2000 biometer (n = 108). Parameter CoV (%) Sw 2.77Sw ICC AL (mm) 0.061 0.072 0.199 0.999 (0.998–0.999) CCT (μm) 0.466 3.213 8.900 0.995 (0.992–0.996) ACD (mm) 0.374 0.023 0.065 0.997 (0.996–0.998) LT (mm) 0.725 0.066 0.184 0.974 (0.962–0.982) Kf (D) 0.082 0.050 0.139 0.999 (0.999–0.999) Ks (D) 0.119 0.073 0.201 0.999 (0.999–0.999) Km (D) 0.103 0.042 0.116 0.999 (0.999–0.999) PD (mm) 4.787 0.365 1.010 0.903 (0.858–0.934) CD (mm) 0.388 0.082 0.228 0.976 (0.964–0.983) AL: axial length, CCT: central corneal thickness, ACD: anterior chamber depth, LT: lens thickness, Kf: flat keratometry, Ks: steep keratometry, Km: mean keratometry, PD: pupil diameter, CD: white to white, D: diopter, SD: standard deviation, CoV: coefficient of variance, Sw: within-subject standard deviation, ICC: intraclass correlation coefficient. Table 3. Discussion With the development of phacoemulsification and IOL implantation in cataract and refractive lens surgery, accurate ocular biometry is essential for achieving the predicted postopervative Table 4. Comparison of ocular parameters measured by Tomey OA-2000 biometer and IOLMaster (n = 108). Pairings Mean Difference ± SD 95% CI t Value p Value AL -0.058 ± 0.094 -0.076 to -0.040 -6.405 < 0.05 Kf -0.088 ± 0.150 -0.117 to -0.060 -6.107 < 0.05 Ks -0.163 ± 0.170 -0.195 to -0.130 -9.978 < 0.05 Km -0.127 ± 0.117 -0.150 to -0.105 -11.281 < 0.05 ACD 0.010 ± 0.075 -0.004 to 0.024 1.382 0.169 CD -0.171 ± 0.217 -0.213 to -0.130 -8.188 < 0.05 AL: axial length, CCT: central corneal thickness, ACD: anterior chamber depth, LT: lens thickness, Kf: flat keratometry, Ks: steep keratometry, Km: mean keratometry, PD: pupil diameter, CD: white to white, D: diopter, SD: standard deviation, CI: confidence interval, : two tailed. Table 4. Comparison of ocular parameters measured by Tomey OA-2000 biometer and IOLMaster (n = 108). AL: axial length, CCT: central corneal thickness, ACD: anterior chamber depth, LT: lens thickness, Kf: flat keratometry, Ks: steep keratometry, Km: mean keratometry, PD: pupil diameter, CD: white to white, D: diopter, SD: standard deviation, CI: confidence interval, : two tailed. PLOS ONE \| https://doi.org/10.1371/journal.pone.0193023 February 27, 2018 6 / 11 Precision of the Tomey OA-2000 biometer and agreement with the IOLMaster Fig 1. Bland-Altman plots present the mean plotted against the differences in values of AL (A), Kf (B), Ks (C), Km (D), ACD (E) and CD (F) for a comparison between the Toomey OA-2000 biometer and IOLMaster. The solid line indicates the mean difference. The interval between upper and lower lines represent the 95% LoA. Fig 1. Bland-Altman plots present the mean plotted against the differences in values of AL (A), Kf (B), Ks (C), Km (D), ACD (E) and CD (F) for a comparison between the Toomey OA-2000 biometer and IOLMaster. The solid line indicates the mean difference. The interval between upper and lower lines represent the 95% LoA. https://doi.org/10.1371/journal.pone.0193023.g001 refractive outcomes. In the present study, we evaluated the precision of ocular parameters measured by the Tomey OA-2000 biometer and found excellent intraobserver repeatability and interobserver and intersession reproducibility for all parameters except for PD and CD. PLOS ONE \| https://doi.org/10.1371/journal.pone.0193023 February 27, 2018 Discussion In Goebels S et al.’s study, the PLOS ONE \| https://doi.org/10.1371/journal.pone.0193023 February 27, 2018 7 / 11 Precision of the Tomey OA-2000 biometer and agreement with the IOLMaster corneal radii measured from the 3 devices were compared, and we could calculated kerat- ometer readings accordingly. Following the formula: K = (1.3375–1.000)/corneal radii[30], The flat K measured by Tomey OA-2000, Lenstar and IOLMaster were 43.66 D, 43.27 D and 43.38 D; and the steep K measured by Tomey OA-2000, Lenstar and IOLMaster were 44.18 D, 44.41 D and 44.41 D. It indicated that Tomey OA-2000 showed the highest values for the flat K and lowest values for the steep K. This was different from the results from the present study in which for all the keratometer readings including Kf, Ks and Km Tomey OA-2000 showed lower values compared with IOLMaster. Besides, for ACD measurements Tomey OA-2000 was comparable with IOLMaster in the present study, while it showed higher values than IOL- Master in Goebels S et al.’s study. They might be attributed to the different subjects included in the studies. In the present study, we included normal subject with healthy eyes and the mean age of 27.43±7.37 years, while in Goebels S et al.’s study the patients, who were scheduled for cataract surgery, with the mean age of 71±12 years were enrolled. However, in the present study, for AL measurements the difference between Tomey OA- 2000 and IOLMaster was rather small (on average -0.058 ± 0.094 mm, 95%LoA -0.24 to 0.13 mm) but statistically significant. Several studies referred to the comparison of OLCR and PCI biometers have had similar results. In Holzer et al.’s study[18], AL measurements by Lenstar were highly correlated to those by IOLMaster. In Liampa et al.’s study[31], similar AL values were measured by Lenstar and IOLMaster. Hoffer et al.[32] found that for AL measurements IOLMaster showed small but significantly difference compared with Lenstar. For corneal curvature measurement, Tomey OA-2000 biometer possesses Placido-disc based topography with 9 rings in a 5.5 mm zone, and the corneal curvature of 2 mm, 2.5 mm and 3 mm central corneal zone can be measured. In the present study, we collected the data from 2.5 mm zone, and we found that all the keratometer readings (including Kf, Ks and Km) measured by Tomey OA-2000 were significantly lower than those measured by IOLMaster. Discussion Moreover, we compared the AL, keratometer readings, ACD and CD measured by the new device with those measured by the IOLMaster and found that the differences were very small but statistically significant. To the best of our knowledge, there is few study to investigate the precision (repeatability and reproducibility) of ocular parameters measured by Tomey OA-2000 biometer in healthy eyes, comprehensively. However, several studies have assessed the precision of ocular biometers based on the OLCR principle. Goebels SC et al.[14] evaluated the reproducibility of the Tomey OA-1000 (the predecessor of the Tomey OA-2000) for measuring the AL, ACD and CCT, and the Cronbach’s α was 1.000 for AL, 0.979 for ACD and 0.999 for CCT. Shammas HJ et al.[16] evaluated the repeatability and reproducibility of the Lenstar LS 900, and reported that all ICCs were more than 0.9, except that for CD (0.849). In Huang J et al.’s study[23], for evaluating the repeatability and reproducibility of AL-Scan biometer, all the ICCs were more than 0.96 except for CD (0.834 and 0.843 for both observers), and all the CoV values were less than 0.5% except that for PD (5.46% and 4.79%, respectively) and CD (1.70% and 1.69%, respectively). Similar results were observed in Kola M et al.’s study[24] with all the ICCs of more than 0.93 except for PD (0.872 and 0.905) for the repeatability assessment. In Huang J et al.’s another study[21], the repeatability and reproducibility of parameters measured by Aladdin ocular biometer in healthy and cataract eyes were evaluated, all the ICCs were more than 0.94 except that for CD in cataract eyes (0.814 and 0.795, respectively). In the present study, we compared the parameters measured by the new device with those measured by the IOLMaster. It was observed that AL, keratometer readings and CD measured by Tomey OA-2000 biometer were significantly lower than those measured by IOLMaster (all Ps < 0.05), except for ACD (P = 0.169). Goebels S et al.[15] measured 138 cataract eyes of 74 patients using Tomey OA-2000, Lenstar and IOLMaster. They found that AL with Tomey OA- 2000 showed smaller mean values compared with IOLMaster and higher values compared with Lenstar (P < 0.001), and for ACD measurements Tomey OA-2000 showed higher mean values compared with Lenstar and IOLMaster (P < 0.001). Discussion The differences were 0.09 D for Kf, 0.16 D for Ks and 0.12 D for Km, respectively. As we know that a difference of 1.0 D in keratometer readings leads to a difference of approximately 1.4 D in IOL power prediction. So, the difference of about 0.1 D in K value would result in a differ- ence of 0.14 D in IOL power prediction, which is clinically negligible. In the present study, the ACD measured by Tomey OA-2000 and IOLMaster were compa- rable with the 95% LoA of -0.14 mm to 0.16 mm. This was different from previous studies. In Goebels S et al.’s studies[13, 15], the differences of ACD measurements between Tomey OA- 1000 and IOLMaster, Tomey OA-2000 and IOLMaster were 0.4 mm and 0.2 mm, respectively. Liampa et al.[31] and Holzer et al.[18] reported 0.2 mm and 0.16 mm differences of ACD mea- surements between Lenstar LS 900 and IOLMaster. In Hoffer KJ et al’s study[33], the Aladdin biometer obtained 0.16 mm (U.S) and 0.05 mm (China) deeper ACD than IOLMaster 500, In the present study, the ACD measured by Tomey OA-2000 and IOLMaster were compa- rable with the 95% LoA of -0.14 mm to 0.16 mm. This was different from previous studies. In Goebels S et al.’s studies[13, 15], the differences of ACD measurements between Tomey OA- 1000 and IOLMaster, Tomey OA-2000 and IOLMaster were 0.4 mm and 0.2 mm, respectively. Liampa et al.[31] and Holzer et al.[18] reported 0.2 mm and 0.16 mm differences of ACD mea- surements between Lenstar LS 900 and IOLMaster. In Hoffer KJ et al’s study[33], the Aladdin biometer obtained 0.16 mm (U.S) and 0.05 mm (China) deeper ACD than IOLMaster 500, and these differences were statistically significant. In Hoffer KJ et al’s another study[34], IOL- Master 700 obtained 0.03 mm deeper ACD than Lenstar LS 900. In Huang J et al’s latest study [35], Tomey OA-2000 was used to measure 65 right eyes of 65 healthy people, and the results showed that the repeatability and reproducibility of Tomey OA-2000 were excellent for AL, keratometer readings, ACD, LT and CD values and good agreement with IOLMaster for most parameters. And these findings were similar to the present study. and these differences were statistically significant. In Hoffer KJ et al’s another study[34], IOL- Master 700 obtained 0.03 mm deeper ACD than Lenstar LS 900. PLOS ONE \| https://doi.org/10.1371/journal.pone.0193023 February 27, 2018 References References 1. Norrby S. Sources of error in intraocular lens power calculation. J Cataract Refract Surg. 2008; 34 (3):368–76. https://doi.org/10.1016/j.jcrs.2007.10.031 PMID: 18299059. 2. Hill W, Angeles R, Otani T. Evaluation of a new IOLMaster algorithm to measure axial length. J Catarac Refract Surg. 2008; 34(6):920–4. https://doi.org/10.1016/j.jcrs.2008.02.021 PMID: 18498996. 3. Hitzenberger CK, Drexler W, Dolezal C, Skorpik F, Juchem M, Fercher AF, et al. Measurement of the axial length of cataract eyes by laser Doppler interferometry. Invest Ophthalmol Vis Sci. 1993; 34 (6):1886–93. PMID: 8491541. 4. Haigis W, Lege B, Miller N, Schneider B. Comparison of immersion ultrasound biometry and partial coherence interferometry for intraocular lens calculation according to Haigis. Graefes Arch Clin Exp Ophthalmol. 2000; 238(9):765–73. PMID: 11045345. 5. Dulku S, Smith HB, Antcliff RJ. Keratometry obtained by corneal mapping versus the IOLMaster in the prediction of postoperative refraction in routine cataract surgery. Clin Experiment Ophthalmol. 2013; 41(1):12–8. https://doi.org/10.1111/j.1442-9071.2012.02816.x PMID: 22594695. 6. Knox Cartwright NE, Johnston RL, Jaycock PD, Tole DM, Sparrow JM. The Cataract National Dataset electronic multicentre audit of 55,567 operations: when should IOLMaster biometric measurements be rechecked? Eye (Lond). 2010; 24(5):894–900. https://doi.org/10.1038/eye.2009.196 PMID: 19680278. 7. Freeman G, Pesudovs K. The impact of cataract severity on measurement acquisition with the IOLMas ter. Acta Ophthalmol Scand. 2005; 83(4):439–42. https://doi.org/10.1111/j.1600-0420.2005.00473.x PMID: 16029267. 8. Bueno-Gimeno I, Espana-Gregori E, Gene-Sampedro A, Lanzagorta-Aresti A, Pinero-Llorens DP. Relationship among corneal biomechanics, refractive error, and axial length. Optom Vis Sci. 2014; 91 (5):507–13. https://doi.org/10.1097/OPX.0000000000000231 PMID: 24705484. 1. Norrby S. Sources of error in intraocular lens power calculation. J Cataract Refract Surg. 2008; 34 (3):368–76. https://doi.org/10.1016/j.jcrs.2007.10.031 PMID: 18299059. 2. Hill W, Angeles R, Otani T. Evaluation of a new IOLMaster algorithm to measure axial length. J Cataract Refract Surg. 2008; 34(6):920–4. https://doi.org/10.1016/j.jcrs.2008.02.021 PMID: 18498996. 3. Hitzenberger CK, Drexler W, Dolezal C, Skorpik F, Juchem M, Fercher AF, et al. Measurement of the axial length of cataract eyes by laser Doppler interferometry. Invest Ophthalmol Vis Sci. 1993; 34 (6):1886–93. PMID: 8491541. 4. Haigis W, Lege B, Miller N, Schneider B. Comparison of immersion ultrasound biometry and partial coherence interferometry for intraocular lens calculation according to Haigis. Graefes Arch Clin Exp Ophthalmol. 2000; 238(9):765–73. PMID: 11045345. 5. Dulku S, Smith HB, Antcliff RJ. Keratometry obtained by corneal mapping versus the IOLMaster in the prediction of postoperative refraction in routine cataract surgery. Clin Experiment Ophthalmol. 2013; 41(1):12–8. https://doi.org/10.1111/j.1442-9071.2012.02816.x PMID: 22594695. 6. Conceptualization: Yanjun Hua. Conceptualization: Yanjun Hua. Data curation: Wei Qiu, Qiuyi Xiao. Formal analysis: Yanjun Hua, Wei Qiu. Methodology: Yanjun Hua. Project administration: Yanjun Hua. Supervision: Qiang Wu. Supervision: Qiang Wu. Writing – original draft: Yanjun Hua. Writing – original draft: Yanjun Hua. Writing – review & editing: Yanjun Hua. Writing – review & editing: Yanjun Hua. Discussion In Huang J et al’s latest study [35], Tomey OA-2000 was used to measure 65 right eyes of 65 healthy people, and the results showed that the repeatability and reproducibility of Tomey OA-2000 were excellent for AL, keratometer readings, ACD, LT and CD values and good agreement with IOLMaster for most parameters. And these findings were similar to the present study. There are some limitations for this study. First, we only included normal subjects with healthy eyes for the precision and agreement assessment, and the results might be different in eyes with cataract. Second, we only compared the new device with IOLMaster for AL, kerat- ometer readings, ACD and CD measurements, but the comparisons of parameters measured by the new device and the ultrasonic equipment were not mentioned. Third, we did not com- pare the predicted outcomes of IOL powers by the new device with those by IOLMaster, and this would be our further work in the future. Forth, the model of the IOLMaster used in the PLOS ONE \| https://doi.org/10.1371/journal.pone.0193023 February 27, 2018 8 / 11 Precision of the Tomey OA-2000 biometer and agreement with the IOLMaster present study was relatively old, and different results maybe generated for the new models of IOLMaster (500 and 700). In conclusion, the ocular parameters measured by Tomey OA-2000 biometer were highly repeatable and reproducible, except for PD and CD. Good agreements of ocular parameters (except for CD values) measured by Tomey OA-2000 biometer and IOLMaster were found in healthy eyes. S1 Data. Precision of the Tomey OA-2000 biometer. (XLSX) S2 Data. Comparison between the Tomey OA-2000 and the IOLMaster. (XLSX) PLOS ONE \| https://doi.org/10.1371/journal.pone.0193023 February 27, 2018 Author Contributions Author Contributions Conceptualization: Yanjun Hua. Data curation: Wei Qiu, Qiuyi Xiao. Formal analysis: Yanjun Hua, Wei Qiu. Methodology: Yanjun Hua. Project administration: Yanjun Hua. Supervision: Qiang Wu. Writing – original draft: Yanjun Hua. Writing – review & editing: Yanjun Hua. Author Contributions Conceptualization: Yanjun Hua. Data curation: Wei Qiu, Qiuyi Xiao. Formal analysis: Yanjun Hua, Wei Qiu. Methodology: Yanjun Hua. Project administration: Yanjun Hua. Supervision: Qiang Wu. Writing – original draft: Yanjun Hua. Writing – review & editing: Yanjun Hua References Knox Cartwright NE, Johnston RL, Jaycock PD, Tole DM, Sparrow JM. The Cataract National Dataset electronic multicentre audit of 55,567 operations: when should IOLMaster biometric measurements be rechecked? Eye (Lond). 2010; 24(5):894–900. https://doi.org/10.1038/eye.2009.196 PMID: 19680278. 7. Freeman G, Pesudovs K. The impact of cataract severity on measurement acquisition with the IOLMas- ter. Acta Ophthalmol Scand. 2005; 83(4):439–42. https://doi.org/10.1111/j.1600-0420.2005.00473.x PMID: 16029267. 8. Bueno-Gimeno I, Espana-Gregori E, Gene-Sampedro A, Lanzagorta-Aresti A, Pinero-Llorens DP. Relationship among corneal biomechanics, refractive error, and axial length. Optom Vis Sci. 2014; 91 (5):507–13. https://doi.org/10.1097/OPX.0000000000000231 PMID: 24705484. PLOS ONE \| https://doi.org/10.1371/journal.pone.0193023 February 27, 2018 9 / 11 Precision of the Tomey OA-2000 biometer and agreement with the IOLMaster 9. Tay E, Li X, Gimbel HV, Kaye G. Assessment of axial length before and after myopic LASIK with the IOLMaster. J Refract Surg. 2013; 29(12):838–41. https://doi.org/10.3928/1081597X-20130924-01 PMID: 24088060. 10. Santodomingo-Rubido J, Mallen EA, Gilmartin B, Wolffsohn JS. A new non-contact optical device for ocular biometry. Br J Ophthalmol. 2002; 86(4):458–62. PMID: 11914218. 11. Buckhurst PJ, Wolffsohn JS, Shah S, Naroo SA, Davies LN, Berrow EJ. A new optical low coherence reflectometry device for ocular biometry in cataract patients. Br J Ophthalmol. 2009; 93(7):949–53. https://doi.org/10.1136/bjo.2008.156554 PMID: 19380310. 12. Rohrer K, Frueh BE, Walti R, Clemetson IA, Tappeiner C, Goldblum D. Comparison and evaluation of ocular biometry using a new noncontact optical low-coherence reflectometer. Ophthalmology. 2009; 116(11):2087–92. https://doi.org/10.1016/j.ophtha.2009.04.019 PMID: 19744720. 13. Goebels SC, Seitz B, Langenbucher A. Comparison of the new biometer OA-1000 with IOLMaster and Tomey AL-3000. Curr Eye Res. 2013; 38(9):910–6. https://doi.org/10.3109/02713683.2013.788722 PMID: 23841799. 14. Goebels SC, Seitz B, Langenbucher A. Reproducibility of the optical Biometer OA-1000 (Tomey). Biomed Res Int. 2014; 2014:814761. https://doi.org/10.1155/2014/814761 PMID: 24818155. 15. Goebels S, Pattmoller M, Eppig T, Cayless A, Seitz B, Langenbucher A. Comparison of 3 biometry devices in cataract patients. J Cataract Refract Surg. 2015; 41(11):2387–93. https://doi.org/10.1016/j. jcrs.2015.05.028 PMID: 26703487. 16. Shammas HJ, Hoffer KJ. Repeatability and reproducibility of biometry and keratometry measurements using a noncontact optical low-coherence reflectometer and keratometer. Am J Ophthalmol. 2012; 153 (1):55–61 e2. https://doi.org/10.1016/j.ajo.2011.06.012 PMID: 21907967. 17. Bjelos Roncevic M, Busic M, Cima I, Kuzmanovic Elabjer B, Bosnar D, Miletic D. Intraobserver and interobserver repeatability of ocular components measurement in cataract eyes using a new optical low coherence reflectometer. Graefes Arch Clin Exp Ophthalmol. 2011; 249(1):83–7. https://doi.org/10. 1007/s00417-010-1546-z PMID: 20981435. 18. Holzer MP, Mamusa M, Auffarth GU. References Accuracy of a new partial coherence interferometry analyser for biometric measurements. Br J Ophthalmol. 2009; 93(6):807–10. https://doi.org/10.1136/bjo.2008. 152736 PMID: 19289385. 19. Jasvinder S, Khang TF, Sarinder KK, Loo VP, Subrayan V. Agreement analysis of LENSTAR with other techniques of biometry. Eye (Lond). 2011; 25(6):717–24. https://doi.org/10.1038/eye.2011.28 PMID: 21394115. 20. Mandal P, Berrow EJ, Naroo SA, Wolffsohn JS, Uthoff D, Holland D, et al. Validity and repeatability of the Aladdin ocular biometer. Br J Ophthalmol. 2014; 98(2):256–8. https://doi.org/10.1136/bjophthalmol- 2013-304002 PMID: 24227803. 21. Huang J, Savini G, Wu F, Yu X, Yang J, Yu A, et al. Repeatability and reproducibility of ocular biometry using a new noncontact optical low-coherence interferometer. J Cataract Refract Surg. 2015; 41 (10):2233–41. https://doi.org/10.1016/j.jcrs.2015.10.062 PMID: 26703300. 22. Cruysberg LP, Doors M, Verbakel F, Berendschot TT, De Brabander J, Nuijts RM. Evaluation of the Lenstar LS 900 non-contact biometer. Br J Ophthalmol. 2010; 94(1):106–10. https://doi.org/10.1136/ bjo.2009.161729 PMID: 19692383. 23. Huang J, Savini G, Li J, Lu W, Wu F, Wang J, et al. Evaluation of a new optical biometry device for mea- surements of ocular components and its comparison with IOLMaster. Br J Ophthalmol. 2014; 98 (9):1277–81. https://doi.org/10.1136/bjophthalmol-2014-305150 PMID: 24795336. 24. Kola M, Duran H, Turk A, Mollamehmetoglu S, Kalkisim A, Erdol H. Evaluation of the Repeatability and the Reproducibility of AL-Scan Measurements Obtained by Residents. J Ophthalmol. 2014; 2014:739652. https://doi.org/10.1155/2014/739652 PMID: 25136453. 25. Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet. 1986; 1(8476):307–10. PMID: 2868172. 26. Hua Y, Stojanovic A, Utheim TP, Chen X, Raeder S, Huang J, et al. Keratometric index obtained by Fourier-domain optical coherence tomography. PLoS One. 2015; 10(4):e0122441. https://doi.org/10. 1371/journal.pone.0122441 PMID: 25886489. 27. Ho JD, Tsai CY, Tsai RJ, Kuo LL, Tsai IL, Liou SW. Validity of the keratometric index: evaluation by the Pentacam rotating Scheimpflug camera. J Cataract Refract Surg. 2008; 34(1):137–45. https://doi.org/ 10.1016/j.jcrs.2007.09.033 PMID: 18165094. 28. McAlinden C, Khadka J, Pesudovs K. Statistical methods for conducting agreement (comparison of clin- ical tests) and precision (repeatability or reproducibility) studies in optometry and ophthalmology. Oph- thalmic Physiol Opt. 2011; 31(4):330–8. https://doi.org/10.1111/j.1475-1313.2011.00851.x PMID: 21615445. 10 / 11 PLOS ONE \| https://doi.org/10.1371/journal.pone.0193023 February 27, 2018 Precision of the Tomey OA-2000 biometer and agreement with the IOLMaster 29. Bland JM, Altman DG. Measurement error. BMJ. 1996; 313(7059):744. PMID: 8819450. 30. Hua Y, Xu Z, Qiu W, Wu Q. PLOS ONE \| https://doi.org/10.1371/journal.pone.0193023 February 27, 2018 References Precision (Repeatability and Reproducibility) and Agreement of Corneal Power Measurements Obtained by Topcon KR-1W and iTrace. PLoS One. 2016; 11(1):e0147086. https://doi.org/10.1371/journal.pone.0147086 PMID: 26752059. 31. Liampa Z, Kynigopoulos M, Pallas G, Gerding H. Comparison of two partial coherence interferometry devices for ocular biometry. Klin Monbl Augenheilkd. 2010; 227(4):285–8. https://doi.org/10.1055/s- 0029-1245182 PMID: 20408075. 32. Hoffer KJ, Shammas HJ, Savini G. Comparison of 2 laser instruments for measuring axial length. J Cat- aract Refract Surg. 2010; 36(4):644–8. Epub 2010/04/07. https://doi.org/10.1016/j.jcrs.2009.11.007 PMID: 20362858. 33. Hoffer KJ, Shammas HJ, Savini G, Huang J. Multicenter study of optical low-coherence interferometry and partial-coherence interferometry optical biometers with patients from the United States and China. J Cataract Refract Surg. 2016; 42(1):62–7. https://doi.org/10.1016/j.jcrs.2015.07.041 PMID: 26948779. 34. Hoffer KJ, Hoffmann PC, Savini G. Comparison of a new optical biometer using swept-source optical coherence tomography and a biometer using optical low-coherence reflectometry. J Cataract Refract Surg. 2016; 42(8):1165–72. https://doi.org/10.1016/j.jcrs.2016.07.013 PMID: 27531293. 35. Huang J, Savini G, Hoffer KJ, Chen H, Lu W, Hu Q, et al. Repeatability and interobserver reproducibility of a new optical biometer based on swept-source optical coherence tomography and comparison with IOLMaster. Br J Ophthalmol. 2017; 101(4):493–8. https://doi.org/10.1136/bjophthalmol-2016-308352 PMID: 27503393. 11 / 11
https://openalex.org/W4386659375	https://zenodo.org/record/8340184/files/PANDEM-Source%2C%20a%20tool%20to%20collect%20or%20generate%20surveillance%20indicatorsfor%20pandemic%20management:%20A%20use%20case%20with%20COVID-19%20data.pdf	English	null	PANDEM-Source, a tool to collect or generate surveillance indicators for pandemic management: A use case with COVID-19 data	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	7,088	Methods A requirement gathering process with EU pandemic managers in the consortium was performed with the objective of identifying and prioritising a list of variables and indicators useful for surveillance and pandemic management. Using COVID-19 pandemic as a use case, we developed PS, a tool to map, collect, and integrate data used for surveillance from different data sources with the purpose of feeding all necessary data to be displayed in the PANDEM-2 dashboard. Introduction PANDEM-Source (PS) is a tool to collect and integrate openly available public health related data from heterogeneous data sources to support infectious diseases surveillance for pandemic management. The tool may be used also for pandemic preparedness by collecting or generating surveillance data for training purposes. It was developed as part of the EU funded Horizon 2020 PANDEM-2 project during the COVID-19 pandemic as result of a close collaboration in a consortium of nineteen partners including six European public health agencies, one hospital, and three first responders organisations. PANDEM-Source, a tool to collect or generate surveillance indicators for pandemic management: A use case with COVID-19 data Francisco Orchard1, Charline Clain1, William Madie1, Jessica S. Hayes2, Máire A. Connolly2, Etienne Sevin1, Alexis Sentís1 1 Epiconcept, Paris, France 2University of Galway, Ireland 1 Epiconcept, Paris, France 2University of Galway, Ireland 1 Epiconcept, Paris, France 2University of Galway, Ireland Correspondence: Francisco Orchard f.orchard@epiconcept.fr Conclusion PS design provides flexibility to collect heterogeneous data from open data sources or to upload end users' own data and customise surveillance indicators. These features are supplemented by the ability to generate realistic epidemiological synthetic datasets that can be used for training purposes. All these features make PS a unique and valuable tool for pandemic management. With the lessons learnt during the COVID-19 pandemic, it is important to keep building capacity to monitor potential threats and develop tools that can facilitate training all the necessary aspects to manage future pandemics. The open source availability of PS1 and the supporting documentation to facilitate easy adaptation to future threats or different training scenarios may represent a cost efficient impact tool for pandemic management. Results PS routinely monitors, collects, and standardises data from from open or restricted heterogeneous data sources (users can upload their own data). It supports traditional surveillance indicators and health resources reported by national and international agencies, and non-traditional surveillance indicators such as those captured in social and mass media, participatory surveillance and sero-prevalence studies. The tool can also calculate indicators and be used to produce data for training purposes by generating synthetic data from a minimal set of indicators to simulate pandemic 1 scenarios. PS is currently set up for COVID-19 surveillance at European level but can be adapted to other diseases or threats and regions. scenarios. PS is currently set up for COVID-19 surveillance at European level but can be adapted to other diseases or threats and regions. 1 https://github.com/pandem2/pandem-source 1 Introduction PANDEM-2 is an Horizon 2020 EU-funded project aiming to develop and demonstrate innovative solutions to strengthen pandemic preparedness and response at EU Level for public health emergencies at subnational, national, EU and global level. The IT tools are accessible through an interactive decision support dashboard that encompasses data from a variety of domains including traditional epidemiological surveillance, non-pharmaceutical interventions, contact tracing, and hospital resources, but also non-traditional surveillance data such as social or mass media analysis and participatory surveillance data and flights. An integrated epidemiological and hospital resource capacity modelling is also available to support planning and what-if scenarios. The PANDEM-2 consortium includes partners that cover key aspects of pandemic preparedness and response including six National Public Health Agencies in the EU (RKI in Germany, FOHM in Sweden, THL in Finland, INSA in Portugal, NIPH in Romania and RIVM in the Netherlands), first responders organisations (Austrian Red Cross, Italian Red Cross, INEM Portugal and the Radboud Medical Centre, the Netherlands). All the tools developed were designed and validated in close collaboration with the consortium partners and are distributed using open source licences. PANDEM-Source (PS) is an IT surveillance tool to collect and integrate openly available public health related data from heterogeneous data sources to better support communicable disease surveillance for pandemic preparedness and response. PS main objective is to identify, map and integrate pandemic-related data from multiple sources into a coherent pandemic-management database so it can provide all the necessary data to feed the PANDEM-2 dashboard with, when available, near real time data. Its data model was developed in close coordination with the consortium partners aiming to address the challenge of monitoring the COVID-19 pandemic response, but it is flexible and can be adapted to other diseases or new threats, variables and indicators by changing source description files without changes in code. Effective training of public health professionals is an essential element of pandemic management [1] which is targeted by the PANDEM-2 project by developing training scenarios and simulation 2 exercises [2]. During the simulation exercises the PANDEM-2 dashboard displays realistic information matching a specific designed scenario for training. exercises [2]. During the simulation exercises the PANDEM-2 dashboard displays realistic information matching a specific designed scenario for training. Collecting and producing the required data can be a challenge due to the broad scope of information available on the dashboard for pandemic management. 1 Introduction To ensure quality and facilitate processes PS includes monitoring systems/visualisation of data collection by source and features to detect missing data on an initial set of indicators to create a realistic full synthetic dataset covering all necessary variables to perform simulation exercises. These features were used during the PANDEM-2 simulation exercise which focused on assessing PANDEM-2 tools on Public Health Emergency Operation Centres in Germany and the Netherlands during an influenza pandemic scenario. The initial set of indicators were produced using the PANDEM-2 modelling tools and included confirmed cases, deaths, and vaccination status by age group for Germany and the Netherlands. Subsequently, PS generated data for the remaining variables in the dashboard for all EU/EEA countries, including subnational level and distribution by age group, gender, and comorbidities. These variables included population level data on contact tracing, hospital resources and social media analysis trends (sentiment, emotion and suggestion). After data generation, PS performed the calculations for those indicators that need to be computed such as hospital occupancy, incidence rates, mortality rates, vaccination coverage, etc. In this paper we will describe PS features and design with the aim of disseminating its characteristics and capabilities to strengthen pandemic preparedness and response. 2.2 Design goals PS was designed to achieve the following design goals. Adding new sources and variables should be possible without changes in code. ● Adding new sources and variables should be possible without changes in c ● Keep a track of the reporting institution and methodology applied. ● Capability of integrating data from a wide variety of sources and formats. ● Automatic data standardisation based the source description having capacity from transcoding from multiple coding schemas e.g. the region name in local languages to the region code. ● Automatic data standardisation based the source description having capacity from transcoding from multiple coding schemas e.g. the region name in local languages to the region code. I t ti t t d di ti i f d t th d t ● Automatic data standardisation based the source description having capacity from multiple coding schemas e.g. the region name in local languages to the r ● Automatic data standardisation based the source description having capacity from transcoding from multiple coding schemas e.g. the region name in local languages to the region code. ● Integration, type or standardisation errors are informed to the data manager. from multiple coding schemas e.g. the region name in local languages to the region code. ● Integration, type or standardisation errors are informed to the data manager. p g g g g g ● Integration, type or standardisation errors are informed to the data manager. ● The data integration process should be fault tolerant. Errors after data submissions should not lead to data loss. ● The data integration process should be fault tolerant. Errors after data submissions should not lead to data loss. 2.1 Identification of relevant variables and indicators for pandemic management The PANDEM-2 project commenced twelve months after the onset of the COVID-19 pandemic in the EU. The initial work involved a requirement gathering process with the European public health agencies and first responders on the PANDEM-2 consortium. We conducted a structured process to identify the most important variables and indicators to be included into a dashboard that aimed to be useful to cover current and future needs in pandemic management. The process started with a web and literature search and meetings with experts which allowed us to identify relevant data sources for the project as well as which ones were open access. . In parallel, all consortium participants were invited to provide a list of data requirements according to their ideal dashboard to be used for pandemic management. The list of requirements and meeting outputs were analysed to produce an initial list of categorised variables [2]. A data survey was distributed to end users to score the importance and to provide details on data priorities and availability for all the identified variables focusing on COVID-19 use case. These outputs were used to accomplish a final refinement list of variables taking in consideration PANDEM-2 partners assessment on importance, priorities and data availability. We defined an automatic data collection process for variables available in open data sources and data generation for non available variables. We generated synthetic data for missing relevant variables to showcase the full potential of the PANDEM-2 dashboard and to introduce it as a training resource for pandemic management. The resulting dataset is described in [3]. The figure 1 schematises this approach by stating tasks, outputs and dependencies. 3 Figure 1: Process followed for defining pandemic management variables and indicators to be collect or generate Figure 1: Process followed for defining pandemic management variables and indicators to be collect or generate 2.3 Assumptions In order to generate a generic approach for data integration we defined the following assumptions: In order to generate a generic approach for data integration we defined the following assumptions: ● A variable is a label indicating general concept and some variables are computed indicators that are expected to be collected directly from different data sources e.g. number of cases, incidence rate, symptom name, etc. Names are previously defined and are associated with a particular definition, the type (numeric, text), coding schema and/or calculation method. y g ● Limited variables types: integer, numeric, date, datetime, string. ● Variables can be grouped in observations and attributes. Observations contain mainly epidemiological information like “number of cases” or “incidence rate”. Attributes provide extra information or characteristic details associated with a particular observation e.g., the age group of the observed cases. ● Variables can be tied together in as tuples containing an unique measure, a date, a source, and several attributes: an be tied together in as tuples containing an unique measure, a date, a source, and ibutes: s can be tied together in as tuples containing an unique measure, a date, a source, a attributes: ○ Confirmed cases:13 ○ Pathogen: dengue ○ Source: COVID-19 ecdc-dataset. 4 ● Time series are built using observation values in time for tuples with the same attributes e.g. the evolution of confirmed cases for a given age group and city . Indicators can be calculated using functions at the time series level. e.g. the time series for rt_number[] is obtained based on the time series of the number of confirmed cases over time for a disease and the given population on that geographical location. p g g p r a given set of attributes and an observation there can be only a single unique valu ● Data sources provide stable identifiers that can be used for unequivocally retrieving the associated data. ● Data sources provide stable identifiers that can be used for unequivocally retrieving the associated data. ● Data source resources can be read in a tabular format. ● Data source resources can be read in a tabular format. 2.4 Data pipeline design To tackle the issue of data collection and generation we developed the data labelling schema (DLS), a declarative methodology based on text files, for documenting, standardising and integrating surveillance data sources and producing homogeneous and comparable time series. This methodology provides a common approach to address the heterogeneity of formats, sources and types of data found during the COVID-19 pandemic. The DLS integration pipeline uses a set of source description files providing information including ownership, how to detect changes to trigger an import, the files format, how to read the files in a tabular format, how to map columns to PS variables and the applicability of data generation. The list of PS variables contains functions for calculated indicators such as incidence or mortality rate so they can be automatically calculated when necessary parameters are present. Such functions are defined in R language, a widely used language in the domain of epidemiology. Advanced calculations can be performed by integrating external algorithms. For instance, social media analysis data is obtained thanks to natural language processing algorithms developed by the University of Galway. A final step is performed of aggregation up to country level if not already provided by the source. Synthetic data generation was addressed by defining data generation formulas on the list of variables. PS will automatically detect which variables are missing when integrating data and will evaluate the synthetic formulas to generate them. When partitioned data was missing e.g. deaths by comorbidity, a weighted distribution function was applied. When base data was missing e.g. number of people traced for contact tracing, we created hypothetical estimations based on the number of confirmed cases. Data generated in such a way is tagged in the resulting dataset as ‘synthetic’. The resulting data is stored as json files and available using a REST API [4] allowing to query the integration process and obtain the results. The figure 2 shows the entire PS integration pipeline. 5 5 Figure 2: PANDEM-Source (PS) integration pipeline Figure 2: PANDEM-Source (PS) integration pipeline Figure 2: PANDEM-Source (PS) integration pipeline 3.1 The list of variables The process of defining pandemic management variables produced a list of 126 observations and indicators which we present here grouped into 29 main variables. Variables are also grouped into families associated with different aspects of pandemic management. Main variable variation are indicators calculated as rates of another variable or stratification by attributes. This structure is presented in table 1. Data family Main variable Variations cases confirmed cases by variant, by gender, by comorbidity, rate in population (incidence) number of notifications rate in population active cases rate in population recovered cases rate in population rt number 6 deaths deaths infected by bed type (ICU, ward), rate in population, rate in hospitalised, alert excess mortality hospital capacity hospitalised infected by bed type, by comorbidity, by comorbidity and bed type, rate in capacity, rate in population, rate in comorbidities, alert average length of stay by bed type admissions by bed type, by comorbidity, rate in capacity, rate in population available staff by staff type, rate in population beds capacity by bed type, rate in population syndromic surveillance primary care cases by ILI vs SARI primary care positivity rate by ILI vs SARI testing and lab performed tests by variant, by test type, rate in cases (positivity), rate in population sequenced samples by mutation, by variant, rate in population vaccination doses injected by dose number, rate in population (vaccination coverage) people fully vaccinated rate in population (vaccination coverage) contact tracing contact tracing cases by being already a contact, by reached status, by reach in a day contact tracing contacts by being already a contact, by reached status, by reach in a day participatory surveillance participants declaring symptoms alert number of participants estimated incidence with confidence interval 7 number of visits by visit type non pharmaceutical interventions implemented_measure by measure type (e.g. mask wearing) and measure group (e.g. contact tracing, testing, etc) population studies seroprevalence by study name, by study type studied population by study name, by study type transport Number of incoming flights by country of origin social and mass media article count by topic, sentiment, emotion and suggestion referential population by population type Table 1 list of PANDEM-Source (PS) variables 3.2 Integrated sources PS uses a set of source description JSON files containing necessary information for integrating data and calculating time series. During the PANDEM-2 project we developed source description files for European Union countries using data from the COVID-19 pandemic. It is important to highlight that PS follows an all-hazard approach [5] and COVID-19 is only a use case that can be easily extended to support other diseases or threats, with their variables and indicators, as well as other geographical scopes. Sources used to build the PANDEM-2 COVID-19 dataset [3] can be grouped on those used for indicators and those for standardisation. The list of sources implemented are the following: ● Sources used for Indicators ● Sources used for Indicators ○ ECDC COVID-19 datasets \|6]: Selection of datasets describing the EU members surveillance and response to the pandemic published by the European Center for Disease Prevention and Control (ECDC). ○ COVID-19-Datahub [7]: A unified dataset collecting global fine-grained case data on the pandemic surveillance and response. This dataset is used for indicators that are not available at subnational level on ECDC datasets. ○ Our World in Data [8]: Our world in data is a scientific online publication that focuses on global problems. It provides several COVID-19 related datasets. This source was used to obtain extracted excess mortality data since this indicator was missing from previous sources... ○ Influenzanet [9]: a Europe-wide network to monitor the activity of influenza-like-illness (ILI) with the aid of volunteers via the Internet. It is operational in twelve countries. Influenza.net publishes datasets on participants declaring ○ Influenzanet [9]: a Europe-wide network to monitor the activity of influenza-like-illness (ILI) with the aid of volunteers via the Internet. It is operational in twelve countries. Influenza.net publishes datasets on participants declaring 8 COVID-19 symptoms and healthcare seeking behaviour as well as provide estimated incidence for the participating countries. COVID-19 symptoms and healthcare seeking behaviour as well as provide estimated incidence for the participating countries. ○ Twitter, via the Panacea Lab COVID-19 tweet collection [10]: A large-scale COVID-19 Twitter dataset for open science starting in march 2020. This dataset contains COVID-19 related tweet IDs. We obtained the list of IDS from the data sharing platform Zenodo.org[] and downloaded the tweet texts using the Twitter API. The tweets were annotated using Natural Language Processing models to produce dedicated time series by country. ○ Twitter, via the Panacea Lab COVID-19 tweet collection [10]: A large-scale COVID-19 Twitter dataset for open science starting in march 2020. This dataset contains COVID-19 related tweet IDs. We obtained the list of IDS from the data sharing platform Zenodo.org[] and downloaded the tweet texts using the Twitter API. The tweets were annotated using Natural Language Processing models to produce dedicated time series by country. ○ MediSys [11]: The Medical Information System MedISys is an internet monitoring and analysis system developed at the JRC in collaboration with EC Directorate General SANCO to rapidly identify potential threats to public health using information from the internet. MedISys continuously monitors about 900 specialist medical sites plus all the generic EMM news, i.e. ● Sources used for Indicators over 20000 RSS feeds and HTML pages sites from 7000 generic news portals and 20 commercial news wires in altogether 70 languages. We generated a connector for Medisys capable of extracting the last 30 days of articles for predefined topics but due to the lack of historic data collection this source could not be included on the COVID-19 dataset. ○ OpenSky Network [12]: OpenSky Network is a non-profit association that provides open access of flight tracking control data. It was set up as a research project by several universities and government entities with the goal to improve the security, reliability and efficiency of the airspace. The used dataset is a derived version from the full dataset published on Zenodo covering the period from 2019 to 2022. ○ Eurostat [13]: Eurostats is the statistical office of the European Union and publishes a wide range of datasets & statistics concerning European countries. We obtained information about available beds and hospital staff. ○ Eurostat [13]: Eurostats is the statistical office of the European Union and publishes a wide range of datasets & statistics concerning European countries. We obtained information about available beds and hospital staff. p ○ OECD [14]: The OECD (Organisation for Economic Co-operation and Development). The OECD regularly publishes comparable statistics on numerous subjects. In the case of COVID-19 it was the selected source for getting an estimation on the evolution of the number of Intensive Care Unit (ICU) operational beds by country. ○ SeroTracker [15]: SeroTracker is a dashboard and data platform for SARS-CoV-2 serosurveys. They conduct an ongoing systematic review to track serosurveys (antibody testing-based surveillance efforts) around the world. Seroprevalence studies results were integrated. g urces used for standardisation ● Sources used for standardisation ● Sources used for standardisation ostat: [16] The NUTS classification (Nomenclature of territorial units for statistics) hierarchical system for dividing up the economic territory of the EU and the UK. ○ Eurostat: [16] The NUTS classification (Nomenclature of territorial units for statistics) is a hierarchical system for dividing up the economic territory of the EU and the UK. Standard region codes and names were obtained. ○ Geonames.org [17]: GeoNames is a geographical database distributed under the creative commons attribution licence. It contains over 27 million geographical names. We used this source to extract ISO2 and ISO3 code equivalences and to obtain multilingual aliases for countries in the world and and regions in Europe. g g p ○ ICD-10-CM [18]: The ICD-10 Clinical Modification (ICD-10-CM) is a modification of the ICD-10 (International classification of diseases) used as a source for diagnosis codes in the United States of America.This source is used to get the list of pathogens. g g ○ ISCO-08 [19]: ISCO-08 ISCO-08 is a four-level hierarchically structured classification that allows all jobs in the world to be classified into 436 unit groups. We used this codification to store information about resource types in hospitals such as doctors or nurses. 9 ○ Our airports [20]: OurAirports is a free site where visitors can explore the world's airports, read other people's comments, and leave their own. This site provides freely available files with the list of world airports. ○ Our airports [20]: OurAirports is a free site where visitors can explore the world's airports, read other people's comments, and leave their own. This site provides freely available files with the list of world airports. 3.3.2 Data Acquisition The source descriptor file of each source must also define all necessary information to monitor each source, trigger automatic updates when changes are performed and how to interpret file format to acquire data. Multiple acquisition channels were implemented including git repositories, URL and predefined Application Programming Interfaces (APIs). If a new channel is required, the user can provide custom R or python scripts to perform the data acquisition. PS checks for updates on a regular predefined basis using when available versioning methods to avoid downloading data to detect changes. The source descriptor files also define the format of target files, including Excel, CSV, JSON and XML. Each format has its own formatting properties to interpret the provided data. If the source is too complicated or needs to be cleaned before integration, PS supports the usage of dedicated Python scripts to pre-process datasets. 3.3.1 Source description The Data Labelling Schema (DLS) is a declarative approach that requires a detailed description of the sources using JSON files in complement with a list of variables, mappings and indicator formulas. Based on the source description, PS takes care of all transformations to perform the data acquisition, validation, standardisation, and, if necessary, the calculation indicators or data generation. Each data unit goes through a standardised integration pipeline (Figure 2) which reduces risk of errors and ensures updated metadata is kept of the whole database. Each variable is linked to a unit, a source, a date of integration and and mapped to a target “pre-defined” PS variable providing a description, a referential (if transcoded was necessary) and associated formulas. If new variables need to be added, the list of variables can be directly modified to include new concepts without changes in code. A CSV file can be easily modified by a data manager allowing complete autonomy on variable definitions which is a key feature to allow adaptation to unknown or novel threats. The file is publicly available on https://github.com/pandem2/pandem-source/blob/main/pandemsource/data/list-of-variables.csv 3.3.3 Standardisation A number of well-defined standards [21, 22] are included in PS and the tool automatically monitors and updates these references from public data sources to compute specific indicators, from NUTS, ISO country codes, ICD-10 diseases, or geonames. When input data does not match the expected format or referential data, PS provides a list of integration issues allowing the data manager to visualise and fix them. For instance, a source provides the number of confirmed cases for an unknown country, the data is ignored and details of integration issues are reported. The user can 10 define mappings using json files to support transcoding between different codification systems such as ISO3 or region names to NUTS. 3.3.5 Data generation In the list of variable definitions, PS also includes formulas for data generation allowing creating the entire PS datasets with a minimal set of input variables. This data generation feature was designed to generate training datasets that can be used during simulation exercises. This feature was used during the PANDEM-2 simulation exercise, where an Influenza pandemic scenario initiated in two European countries using the PANDEM-2 dashboard was simulated. The PANDEM-2 modelling tools [23] were used to produce the time series for the number of cases, deaths hospitalisations and vaccinations and PS used its data generation functions for generating plausible data about social media posts, participatory surveillance, contact tracing, public health staff variations, syndromic surveillance and stratification by comorbidities age groups. This feature reduced the amount of effort needed for preparing the data for the simulation exercise. 3.3.4 Calculated metrics and indicators Calculated indicators such as incidence rates, effective reproduction number (Rt) are produced by R scripts included in PS that can be also modified by any user with basic knowledge of R language. PS proceeds automatically to indicator calculation whenever all available parameters are provided by a source or already provided by a related source. Computing indicators instead of directly collecting it from different data sources ensures that the same methodology is used, thus supporting comparability. Aggregation from subnational to national level is also performed automatically. 3 https://pykka.org/en/latest/ 2 https://ec.europa.eu/info/european-union-public-licence_en 2 https://ec.europa.eu/info/european-union-public-licence_en 3 https://pykka.org/en/latest/ p p p p 3 https://pykka.org/en/latest/ p p Figure 3: PANDEM-Source (PS) actor dependencies Figure 3: PANDEM-Source (PS) actor dependencies 3.4 PANDEM-Source (PS) architecture PS is a python package that implements the DLS with out of the box definitions for integrating a wide range of indicators from heterogeneous surveillance data sources. It has been published on Pypi https://pypi.org/project/pandem-source/ and its code is open under the EUPL2 licence and it is available on GitHub https://github.com/pandem2/pandem-source. PS has been implemented following a micro services architecture using the Actor Model and using the package with pykka3. Each actor receives messages, processes them one by one and can send messages to other actors. Messages can be any python object. This programming pattern allows to achieve a good level of parallelization of tasks while keeping a simple programing model and file access. For external algorithm integration we have used docker containers and REST APIs. The following classes of actors have been defined: ● Orchestrator: Launch actors, manage docker encapsulation, close actors ● Storage: Keep persistent information of the integration process and process all data storage operations. ● Acquisition: Triggers data integration of known data source files 11 ● Data pipeline: Ensures that integration is performed and ensures that the process will run until an end (error, warning, success) ● Data pipeline: Ensures that integration is performed and ensures that the process will run until an end (error, warning, success) ● Algorithms: Execute a particular algorithm during the pipeline ● Format readers: Transform input files into dataframes ● Dataframe reader: Transform data frames into a list of non-standard tuples ● Standardisation: Transform a list of non-standard tuples into a list of standard tuples. ● Variables: Reads and writes standardised variable values. Provides a necessary mapping information to standardisation actor in order to standardise variable values e.g., Country Name => Code ISO 3.5 Integrating social media analysis components The integration of social media components necessary to classify tweets required the execution of Social Media Analysis (SMA) components developed as part of PANDEM-2. These components were packaged and exposed as an API and utilised a TensorFlow Serving Docker to facilitate its integration. PS includes all necessary parameters to automatically launch the right docker tensor flow 12 server locally if not already running on the configured URL, the only information that is needed is the folder where the models are saved. The models are launched using configuration files, so adding new models does not require changes on its code. Once the models are running, PS will evaluate any source including the variable article text and add the resulting model outputs as new attributes of the related tuple. After annotation the article text is removed, and PS will calculate the aggregations for each used algorithm and produce the related time series. 3.7 PANDEM-Source (PS) python package PS includes a Shiny4 app for validating the integration and to visualise the integrated time series. The integration dashboard is structured as follows: 3.6 Integrating the Next Generation Sequencing (NGS) Simulator Another external algorithm utilised is the Multiparametric Next Generation Sequencing (NGS) simulator [24]. This tool used to generate realistic time series by variant and mutation not being publicly available. The simulator can combine data from real data sources such as cases by variants, and number of hospitalisations by age group and vaccination status. The resulting datasets are built using machine learning approaches to find a realistic combination of these variables. This simulator is written in R. PS uses the simulator which needs to be installed as an R package. It uses git to locally check if there have been changes on the input files before launching the simulator. 4 https://shiny.rstudio.com/ 3.7.1 Data integration page List of integrated sources showing current integration status, next expected check for changes, the history of data sets collection executed, and issues found see Figure 5. This page refreshes automatically. 13 Figure 4 The PANDEM-Source (PS) data integration page Figure 4 The PANDEM-Source (PS) data integration page 14 3.7.2 Available sources page List of defined data sources including information about the source descriptor files such as acquisition channel and variable mappings see Figure 5. Figure 5 The PANDEM-Source (PS) data source page Figure 5 The PANDEM-Source (PS) data source page Figure 5 The PANDEM-Source (PS) data source page 15 The entire list of variables defined on PS including all metadata and formula definitions, see Figure 6. The entire list of variables defined on PS including all metadata and formula definitions, see Figure 6. The entire list of variables defined on PS including all metadata and formula definitions, see Figure 6. 6. Figure 6 The PANDEM-Source (PS) variable list Figure 6 The PANDEM-Source (PS) variable list 3.7.4 Time series page Displays all the integrated time series. A dynamic filter system and the count of matching time series helps the user to explore and understand the underlying data see Figure 7. Time series from different sources can be easily compared. 16 Figure 7 The PANDEM-Source (PS) time series exploration report Figure 7 The PANDEM-Source (PS) time series exploration report 5 https://shiny.rstudio.com/ Figure 8 The PANDEM-Source (PS) time series exploration report Figure 8 The PANDEM-Source (PS) time series exploration report 3.7.5 Exporting data Data processed by PS is available via a REST API. Which can be regularly called to get the most current data. The REST API is also the way of acquiring data for the Shiny5 Integration Dashboard, so any functionality on the integration dashboard can be replicated on the PANDEM-2 database. Figure 8 displays the implemented endpoints of the REST API. 17 Figure 8 The PANDEM-Source (PS) time series exploration report 4 Conclusions In the context of the COVID-19 pandemic and within the umbrella of PANDEM-2 project, PS has been developed with the advice of a large variety of health and public health experts to include relevant indicators for infectious disease surveillance and to monitor the health response that feeds the PANDEM-2 dashboard. During the PANDEM-2 simulation exercises, PS and PANDEM-2 dashboard have proven useful to facilitate training for pandemic management. The design provides flexibility to collect data from open data sources or to upload end users own data and customise indicators. PS can support public health agencies and first responders in developing their own data collection tools according to their specific training and response needs and reduce the development effort of building them from scratch. This flexibility and easy to customise feature is supplemented by the capacity to generate realistic epidemiological synthetic datasets that can be used for training purposes. All these features make PS a unique and valuable tool for pandemic management. To our knowledge, although some data collection tools have been developed during the COVID-19 18 pandemic [7, 25, 26], PS is unique in terms of flexibility and customisation to be adapted to different data sources or diseases or to develop new training. It also differs from by its broader approach, the heterogeneity of the data sources and collected data and its flexibility allowing it to quickly adapt to emergent threats imposing new data needs. We believe that these characteristics make the tool useful not only for training purposes but also, with further development and adaptation according to the context, to be deployed to support monitoring and managing an emergent epidemic or pandemic. At the end of integration, PS also allows users to visualise the uploaded data as time series by time and geographical level or other shared attributes such as variant, or age group which may facilitate monitoring and validate the surveillance results before being captured in the dashboard (or just to visualise some results from some variables not incorporated in the dashboard). This approach also leverages visualisation and comparison of any kind of indicator independent of their nature. For instance, it allows visualisation of social media emotion trends together with the number of cases of a particular mutation or the evolution of the seroprevalence in a specific population or the evolution of people’s opinions to specific public health related topics based on social media analysis. 4 Conclusions PS is a ready to use open-source tool allowing pandemic managers to collect and harmonise multiple surveillance data on specific pathogens from traditional and non-traditional publicly available or restricted data sources. It currently includes a wide list of COVID-19 sources to collect data from different domains (cases, deaths, ICU occupancy, social media analysis, Lab and NGS data, non-pharmaceutical interventions) from different data sources such as ECDC or other public health agencies websites - via the COVID-19 Datahub scripts, Influenzanet, Twitter, MediSYS, etc. In summary, PS contribute to the pandemic management community through: - Out of the box data collection for pandemic preparedness and response. - Flexibility and customisation on data collection - Data can be visualised in the tool - Exploited directly through the PS API - Visualised through the PANDEM-2 dashboard - Simplifies cross domain data collection for epidemiological surveillance - Foundations for a multi-source multi threat early warning system - Proposes a standard methodology for collecting surveillance data and computing indicators that could support standardisation and data sharing among countries. - Data generation for training purposes. - Data generation for training purposes. It is relevant to highlight some potential limitations when using the tool: First, the data availability is a limitation factor. It is possible, as may happen at the beginning of an epidemic or pandemic, that data is still scarce. Second, we can find that, although some data may be potentially available from the health provision services, there are data sharing limitations in different scenarios; to share data with the general public or with other countries but also it may happen that there are restrictions to 19 share personal data for public health surveillance purposes without patient consent. Related with first and second, it is worth mentioning that often the most useful data to respond to at local level is not available or if available is sensitive due to data protection issues (possibility to identify individuals). Third, the list of data sources required (open or restricted) may substantially change over time but also for different threats or pathogens so the current data sources included in PS may need to substantially change according to the epidemiological need. Four, some users may need some training to install and use the tool (not always possible to have assistance). No conflicts of interests No conflicts of interests 6 Author Contributions Francisco Orchard: Manuscript writing, software design and implementation. Co-coordination of PANDEM-2 surveillance workpackage. Alexis Sentís: Epidemiological support, co-coordination of PANDEM-2 surveillance workpackage, and manuscript review Alexis Sentís: Epidemiological support, co-coordination of PANDEM-2 surveillance workpackage, and manuscript review 8 Acknowledgments We would like to acknowledge the PANDEM-2 consortium for their invaluable input and support, especially the tech team devoted to development of the tool. 9 Supplementary Material No supplementary material provided 7 Funding The project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 883285. The material presented and views expressed here are the responsibility of the author(s) only. The EU Commission takes no responsibility for any use made of the information set out. 10 Data Availability Statement The data generated with the tools is subject to a different publication. The data generated with the tools is subject to a different publication 4 Conclusions Despite these limitations, we believe that the fact of PS is a flexible and easy to customise tool that will facilitate its use and will represent a useful tool for preparedness and response related activities. IT solutions to facilitate and strengthen disease prevention and control are needed and will be developed in the coming years and PS , as well as other tools developed under the PANDEM-2 project, can be useful prototypes to be further developed according to future needs and threats. 20 11 References 21 1 Frieden TR, Buissonnière M, McClelland A. The world must prepare now for the next pandemic. BMJ Global Health. 2021 Mar;6(3):e005184. 2 Tighe C, Ngongalah L, Sentís A, Orchard F, Pacurar GA, Hayes C, Hayes JS, Adrian Toader A, Connolly MA. Development of the PANDEM-2 Dashboard: A Novel Approach to Strengthen Pandemic Preparedness and Response. JMIR Preprints. 25/08/2023:52119 3 Chen H, Gala JL, Bonjean M, Ambroise J, Zayed O, Buitelaar P, Connoly MA, Hayes JS, Sevin E, Sentis A, Orchard F. A COVID-19 European data set to support training in pandemic management. Zenodo [prepriny]; 2023 10.5281/zenodo.8339303. 4 RedHat. What is a REST API?. https://www.redhat.com/en/topics/api/what-is-a-rest-api (accessed on August 20, 2023) 5 Adini B, Goldberg A, Cohen R, Laor D, Bar-Dayan Y. Evidence-based support for the all-hazards approach to emergency preparedness. Israel Journal of Health Policy Research. 2012 Oct 25;1(1). 6 European Centre for Disease Prevention and Control. COVID-19 Datasets https://www.ecdc.europa.eu/en/covid-19/data (accessed on August 20, 2023) 7 Guidotti E. A worldwide epidemiological database for COVID-19 at fine-grained spatial resolution. Scientific Data. 2022 Mar 29;9(1). 8 Mathieu, E. et al. Coronavirus pandemic (covid-19). Our World Data. https://ourworldindata.org/coronavirus (2020) (accessed on August 20, 2023) 9 Paolotti D, Carnahan A, Colizza V, Eames K, Edmunds J, Gomes G, et al. Web-based participatory surveillance of infectious diseases: the Influenzanet participatory surveillance experience. Clinical Microbiology and Infection. 2014 Jan;20(1):17–21. 10 Banda JM, Tekumalla R, Wang G, Yu J, Liu T, Ding Y, et al. A Large-Scale COVID-19 Twitter Chatter Dataset for Open Scientific Research—An International Collaboration. Epidemiologia. 2021 Aug 5;2(3):315–24. 11 Linge JP, Steinberger R, Flavio Fuart, Bucci S, Jenya Belyaeva, Monica G, et al. MedISys. IGI Global eBooks. 2011 May 24;131–42. 12 Strohmeier M, Olive X, Lübbe J, Schäfer M, Lenders V. Crowdsourced air traffic data from the OpenSky Network 2019–2020. Earth System Science Data. 2021 Feb 11;13(2):357–66. 13 Eurostats. Eurostat Health care resources https://ec.europa.eu/eurostat/cache/metadata/en/hlth_res_esms_fr.htm (accessed on August 20, 2023) 14 Organisation for Economic Co-operation and Development. Beyond Containment: Health Systems Responses to COVID-19 in the OECD. OECD Publishing. 2020. 22 15 Arora RK, Joseph A, Wyk JV, Rocco S, Atmaja A, May E, et al. SeroTracker: a global SARS-CoV-2 seroprevalence dashboard. The Lancet Infectious Diseases. 2020 Aug 4; 21 ‌ 16 European Commission. Eurostat, Statistical regions in the European Union and partner countries : NUTS and statistical regions 2021 : 2022 edition. Publications Office of the European Union. 2022 17 Geonames. 11 References Geonames.org (accessed on August 20, 2023) 7 Geonames. Geonames.org (accessed on August 20, 2023) 18 National Center for Health Statistics (NCHS). International Classification of Diseases, Tenth Revision, Clinical Modification (ICD-10-CM) https://ftp.cdc.gov/pub/Health_Statistics/NCHS/Publications/ICD10CM (accessed on August 20, 2023) 19 International Labour Office. International standard classification of occupations ISCO-08 https://www.ilo.org/public/english/bureau/stat/isco/isco08/ (accessed on August 20, 2023) 20 David Megginson. Ourairports https://ourairports.com/data (accessed on August 20, 2023) 21 Cai W, Tolksdorf K, Hirve S, Schuler E, Zhang W, Haas W, et al. Evaluation of using ICD‐10 code data for respiratory syncytial virus surveillance. Influenza and Other Respiratory Viruses. 2019 Jun 17; 22 Costa C, Freitas Â, Stefanik I, Krafft T, Pilot E, Morrison J, et al. Evaluation of data availability on population health indicators at the regional level across the European Union. Population Health Metrics. 2019 Aug 7;17(1).‌ 23 Jair A, Green C, Carlos T, & Duggan J. (2023). PANDEM-2 Simulator (v1.0). Zenodo. https://doi.org/10.5281/zenodo.8263512‌ 24 Bonjean, M., Ambroise, J., Connolly, M. A., Hayes, J. S., Hurel, J., Sentis, A., Orchard, F., Gala, J.L. Design and use of a multiparametric simulator for integrating patient metadata with genetic data on pathogens with pandemic potential: A step forward in pandemic preparedness and response. Preprint at bioRxiv, 2023 25 Reinhart A, Brooks L, Jahja M, Rumack A, Tang J, Agrawal S, et al. An open repository of real-time COVID-19 indicators. Proceedings of the National Academy of Sciences. 2021 Dec 13;118(51). 26 Dong E, Du H, Gardner L. An interactive web-based dashboard to track COVID-19 in real time. The Lancet Infectious Diseases [Internet]. 2020 Feb;20(5). Available from: https://www.thelancet.com/journals/laninf/article/PIIS1473-3099(20)30120-1/fulltext 23
https://openalex.org/W4313904647	https://ejurnal.seminar-id.com/index.php/bits/article/download/1981/1350	Indonesian	null	Penerapan Metode Hybrid Case Base Pada Sistem Pakar Diagnosa Penyakit Jantung	Building of Informatics, Technology and Science	2,022	cc-by	5,245	Penerapan Metode Hybrid Case Base Pada Sistem Pakar Diagnosa Penyakit Jantung Ayu Handayani, Jeperson Hutahaean, Akmal Nasution Prodi Sistem Informasi, STMIK ROYAL, Kisaran, Indonesia Email: 1ayuhandayani438@gmail.com, 2,jepersonhutahean@gmail.com,3nst.akmal@gmail.com Email Penulis Korespondensi: jepersonhutahean@gmail.com Submitted:26/07/2022; Accepted:15/08/2022; Published: 30/09/2022 Ayu Handayani, Jeperson Hutahaean, Akmal Nasution Prodi Sistem Informasi, STMIK ROYAL, Kisaran, Indonesia Email: 1ayuhandayani438@gmail.com, 2,jepersonhutahean@gmail.com,3nst.akmal@gmail.com Email Penulis Korespondensi: jepersonhutahean@gmail.com Submitted:26/07/2022; Accepted:15/08/2022; Published: 30/09/2022 Abstrak-Banyak orang yang meninggal karena serangan jantung mendadak. Peran dokter spesialis sangat diperlukan untuk mengatasi hal tersebut, tetapi peran dokter juga terbentur karena keterbatasan dalam segi jumlah dan jam kerja. Selain itu, pada umumnya penderita penyakit jantung sering mengabaikan serta kurang memahami penyebab dan gejala terjadinya penyakit jantung. Kebanyakan pasien enggan memeriksakan kesehatan jantungnya karena terkendala biaya pengobatan yang mahal, serta kurangnya pelayanan terhadap pasien. Dalam proses konsultasi penyakit jantung, pasien harus datang ke rumah sakit dan melakukan pemeriksaan secara langsung yang tentu saja harus melalui serangkaian prosedur mulai dari pendaftaran dan seterusnya untuk mendapatkan hasil. Untuk mengatasi permasalahan tersebut maka dibutuhkan sebuah aplikasi sistem pakar untuk diagnosa awal penyakit jantung. Tujuan dari penelitian ini adalah untuk membangun aplikasi sistem pakar diagnosa penyakit jantung berbasis web. Aplikasi sistem pakar ini menggunakan metode Hybrid Case Base yang merupakan kombinasi dari sistem Rule Based Reasoning (RBR) dan Case Based Reasoning (CBR). Sistem RBR maupun sistem CBR memiliki kelebihan dan kekurangan masing-masing. Analisa dan perancangan sistem yang digunakan adalah UML dan Entity Relationship Diagram. Pada aplikasi sistem pakar ini, pasien akan berkonsultasi dengan cara menjawab beberapa pertanyaan mengenai gejala penyakit jantung yang ditampilkan oleh sistem dengan cara mencentang gejala-gejala yang dialaminya. Kemudian sistem akan memberikan hasil diagnosa berdasarkan gejala-gejala yang dialami pasien. Hasil dari penelitian ini mampu melakukan diagnosa jenis penyakit jantung yang dialami pasien dan solusi pengobatannya serta berapa persen kemungkinan pasien menderita penyakit tersebut dengan cepat. Kata Kunci: Sistem Pakar; Diagnosa; Penyakit; Jantung; Hybrid Case Base Abstract-Many people have died of sudden heart attacks. The role of a specialist doctor is very necessary to overcome this, but the role of the doctor is also bumped due to limitations in terms of numbers and hours worked. In addition, in general, people with heart disease often ignore and do not understand the causes and symptoms of heart disease. Most patients are reluctant to have their heart health checked because they are constrained by expensive medical expenses, as well as a lack of service to patients. Penerapan Metode Hybrid Case Base Pada Sistem Pakar Diagnosa Penyakit Jantung In the process of consulting for heart disease, the patient must come to the hospital and do an examination in person which of course must go through a series of procedures starting from registration onwards to get results. To overcome this problem, an expert system application is needed for the early diagnosis of heart disease. The purpose of this study was to build a web-based system of heart disease diagnosis expert applications. This expert system application uses the Hybrid Case Base method which is a combination of the Rule-Based Reasoning (RBR) and Case-Based Reasoning (CBR) systems. The RBR system and the CBR system have their advantages and disadvantages. The analysis and design of the systems used are UML and Entity Relationship Diagrams. In this expert system application, patients will consult by answering several questions about the symptoms of heart disease displayed by the system by checking the symptoms they experience. Then the system will provide diagnostic results based on the symptoms experienced by the patient. The results of this study can diagnose the type of heart disease experienced by patients and their treatment solutions and what percentage of the patient is likely to suffer from the disease quickly. Keywords: Expert System; Diagnosis; Disease; Heart; Hybrid Case Base Building of Informatics, Technology and Science (BITS) Volume 4, No 2, September 2022 Page: 537−544 ISSN 2684-8910 (media cetak) ISSN 2685-3310 (media online) DOI 10.47065/bits.v4i2.1981 Copyright © 2022 Ayu Handayani, Page 537 BITS is licensed under a Creative Commons Attribution 4.0 International License 1. PENDAHULUAN Banyak orang yang meninggal karena serangan jantung mendadak. Peran dokter spesialis sangat diperlukan untuk mengatasi hal tersebut, tetapi peran dokter juga terbentur karena keterbatasan dalam segi jumlah dan jam kerja dimana dalam melakukan konsultasi penyakit jumlah pasien yang datang begitu banyak sedangkan dokter spesialis yang menangani hanya satu atau dua orang saja. Selain itu, pada umumnya penderita penyakit jantung sering mengabaikan serta kurang memahami penyebab dan gejala terjadinya penyakit jantung. Kebanyakan pasien enggan memeriksakan kesehatan jantungnya karena terkendala biaya pengobatan yang mahal, serta kurangnya pelayanan terhadap pasien. Dalam proses konsultasi penyakit jantung, pasien harus datang ke rumah sakit dan melakukan pemeriksaan secara langsung yang tentu saja harus melalui serangkaian prosedur mulai dari pendaftaran dan seterusnya untuk mendapatkan hasil. Hambatan-hambatan yang menyebabkan sulitnya melakukan konsultasi penyakit jantung dapat diatasi dengan adanya kemajuan teknologi, yaitu melalui pembuatan atau pengembangan aplikasi sistem pakar untuk diagnosa awal penyakit jantung pada manusia dimana pengetahuan para pakar dalam hal ini dokter spesialis penyakit jantung dan di adopsi ke teknologi computer. Sistem pakar adalah sistem yang memanfaatkan pengetahuan manusia. Pengetahuan ini dimasukkan ke komputer dan biasanya digunakan untuk memecahkan masalah yang membutuhkan keahlian manusia atau kepakaran [1]. Terdapat beberapa metode yang digunakan dalam penyelesayan sistem kepakaran yatu Backward Chainning, Forward Chainning, Certainly Factor, Depth First Search, Dempster Shafer dan Hybrid Case Base [2]. Dalam penelitian ini metode yang digunakan dalam penggunaan sistem pakar yaitu metode Hybrid Case Base. Metode Hybrid Case Base digunakan dalam pengambilan keputusan terbaik dari sejumlah gejala atau alternatif dari penyakit yang Copyright © 2022 Ayu Handayani, Page 537 BITS is licensed under a Creative Commons Attribution 4.0 International License Building of Informatics, Technology and Science (BITS) Volume 4, No 2, September 2022 Page: 537−544 ISSN 2684-8910 (media cetak) ISSN 2685-3310 (media online) DOI 10.47065/bits.v4i2.1981 didiagnosa. Adapun cara melakukan diagnosa yaitu menggabungkan (hybrid) dua teknik yaitu case based reasoning dan rule based reasoning[3]. didiagnosa. Adapun cara melakukan diagnosa yaitu menggabungkan (hybrid) dua teknik yaitu case based reasoning dan rule based reasoning[3]. Untuk mendukung penelitian ini, maka penulis mengambil acuan dari beberapa penelitian terdahulu yang mencakup penyakit jatung dan siatem pakar menerapkan metode Hybrid Case Base. Penelitian yang dilakauna oleh Fajar Agung Nugroho pada tahun 2018 tentang diagnosa penyakit jantung dapat melakukan diagnose gejala awal penyakit dengan menerapkan metode Forward Chainning [4]. 2.1 Tahapan Penelitian Adapun tahapan dalam penelitian diagnosa penyakit jantung dengan menggunakan metode Hybrid Case Base dapat dilihat pada gambar 1 dibawah ini: Gambar 1. Tahapan Penelitian Identifikasi Masalah Wawancara Studi Literatur Penerapan Metode Identifikasi Masalah Wawancara Studi Literatur Penerapan Metode Gambar 1. Tahapan Penelitian Pada gambar 1 tahapan penelitian dimulai dari : Pada gambar 1 tahapan penelitian dimulai dari : Pada gambar 1 tahapan penelitian dimulai dari : a. Identifikasi masalah, tahap awal penelitian yang merupakan upaya untuk menemukan masalah terkait dengan proses diagnosa penyakit jantung dan mendefinisikan masalah penelitian Pada gambar 1 tahapan penelitian dimulai dari : a Identifikasi masalah tahap awal penelitian yang merupakan upaya untuk menemukan masalah terkait dengan a. Identifikasi masalah, tahap awal penelitian yang merupakan upaya untuk menemukan masalah te proses diagnosa penyakit jantung dan mendefinisikan masalah penelitian. b. Wawancara (interview), untuk memperoleh informasi yang dibutuhkan dalam rangka mencapai tujuan penelitian maka seorang peneliti membutuhkan pengumpulan data untuk diteliti secara lebih mendalam melalui proses wawancara terkait jenis penyakit jantung, gejala dan nilai bobot kepastian secara langsung kepada pakar dokter spesialis penyakit jantung. b. Wawancara (interview), untuk memperoleh informasi yang dibutuhkan dalam rangka mencapai tujuan penelitian maka seorang peneliti membutuhkan pengumpulan data untuk diteliti secara lebih mendalam melalui proses wawancara terkait jenis penyakit jantung, gejala dan nilai bobot kepastian secara langsung kepada pakar dokter spesialis penyakit jantung. c. Studi literatur, proses selanjutnya ialah membaca literatur yang relevan dari penelitian-penelitian sebelumnya, diantaranya review jurnal dan buku tentang sistem pakar untuk mendiagnosa penyakit jantung menggunakan metode Hybrid Case Base. c. Studi literatur, proses selanjutnya ialah membaca literatur yang relevan dari penelitian-penelitian sebelumnya, diantaranya review jurnal dan buku tentang sistem pakar untuk mendiagnosa penyakit jantung menggunakan metode Hybrid Case Base. d. Penerapan metode, sistem pakar untuk mendiagnosa penyakit jantung menggunakan metode Hybrid Case Base yang dapat mendefinisikan ukuran kepastian terhadap fakta atau aturan untuk menggambarkan keyakinan seorang pakar terhadap masalah yang sedang dihadapi. pakar terhadap masalah yang sedang dihadapi. 1. PENDAHULUAN Penelitian yang dilakuan oleh Dona, Hendri Maradona dan Masdewi pada tahun 2018 tentang diagnosa penyakit jantung mendapatka tingkat akurasi dognosa kemeripan kegajala penyakit dengan penyakit yang di derita berdasarkan metode CBR [5]. Penelitian yang dilakaukan oleh Cindy Pamela Cornelia Munaiseche dkk pada tahun 2018 tentang sistem pakar diagnosa penyakit jantung dapat mengetahua jenis penyakit jantuang berdasrkan gejala yang di derita menerapkaan forward chaining [6]. Penelitian yang dilakukan pada tahun 2020 oleh Muhammad Fauzan menyimpulkan hasil diagnosa dengan pesentasi sebesar 72,83% berdasrkan sampel gejala user padan penyakit kandung kemih menerapkan Hybrid Case Base[7]. Penelitian yang dilakukan pada tahun 2020 oleh Agung Laksamana SP meyimpulkan hasil diagnose penyakit kolera sebesar 93.02 % berdasakan uji sampel user dengan menerpkam metode Hybrid Case Base[8]. Serta penelitian yang dilakukan pada tahun 2019 oleh Rizky Delilah Rambe menyimpulkan hasil diagnosa penyakit usus besar dengan presentasi 44% berdasarkan sampel user yang digunkan dengan menerpkan metode Hybrid Case Base [9]. Tujuan dari penelitian ini adalah untuk membangun aplikasi sistem pakar diagnosa penyakit jantung menggunakan metode Hybrid Case Base berbasis web. Aplikasi sistem pakar ini diharapkan dapat membantu pasien dalam melakukan diagnosa penyakit jantung, mengetahui jenis penyakit jantung dialaminya dan solusi pengobatannya, mengetahui informasi terkait penyakit jantung dan merupakan alat bantu bagi dokter spesialis untuk melakukan diagnosa awal pasien secara cepat. 2.4 Metode Hybrid Case Base Metode Hybrid Case Base digunakan dalam pengambilan keputusan terbaik dari sejumlah gejala atau alternatif dari penyakit yang didiagnosa. Adapun cara melakukan diagnosa yaitu menggabungkan (hybrid) dua teknik yaitu case based reasoning dan rule based reasoning[3]. Metode Hybrid Case Based ini perlu diterapkan selain mendapatkan akurasi lebih dibandingkan metode yang berdiri sendiri. Metode Rule Based Reasoning dan Case Based Reasoning memiliki kelebihan dan kekurangan masing-masing. Namun kedua sistem tersebut sangat mungkin digabungkan (hybrid) untuk mendapatkan sebuah sistem yang baik dengan gabungan kelebihan keduanya, serta untuk menutupi kekurangan masing-masing[16]. Adapun tahapan dalam penerapan metode Hybrid Case Base dalam sistempakar yaitu[17]: tukan gejala-gejala pada penyakit y a. Menetukan gejala-gejala pada penyakit y a. Menetukan gejala-gejala pada penyakit a. Menetukan gejala-gejala pada penyakit a. Menetukan gejala-gejala pada penyakit at pertanyaan kepada user terhadap gejala yang dialami b. Mebuat pertanyaan kepada user terhadap gejala yang dialami b. Mebuat pertanyaan kepada user terhadap gejala yang dialami c. Mencari nilai tertinggi dari nilai kepercayaan terhadap gejala penyakit yang dialami dengan pengukuran Similarity Adapun rumus pengukuran Similarity yaitu [18]: Similarit y(𝐴𝑖.𝐵𝑖) = 𝐴𝑖.𝐵𝑖 \|𝐴𝑖\|.\|𝐵𝑖\| = ∑ (𝐴𝑖∗𝐵𝑖) 𝑛 𝑖=1 √∑ 𝐴𝑖 𝑛.∑ =𝐵𝑖 𝑛 𝑛 𝑖=1 𝒏 𝒊=𝟏 Similarit y(𝐴𝑖.𝐵𝑖) = 𝐴𝑖.𝐵𝑖 \|𝐴𝑖\|.\|𝐵𝑖\| = ∑ (𝐴𝑖∗𝐵𝑖) 𝑛 𝑖=1 √∑ 𝐴𝑖 𝑛.∑ =𝐵𝑖 𝑛 𝑛 𝑖=1 𝒏 𝒊=𝟏 (1) Dimana: Dimana: Ai = Bobot i pada A Bi = Bobot i pada B N = Jumlah vektor A = Vektor (Kasus Baru) B = Vektor (kasus lama) Dimana: Ai = Bobot i pada A Bi = Bobot i pada B N = Jumlah vektor A = Vektor (Kasus Baru) B = Vektor (kasus lama) 2.2 Sistem Pakar Sistem pakar adalah sistem yang memanfaatkan pengetahuan manusia. Pengetahuan ini dimasukkan ke komputer dan biasanya digunakan untuk memecahkan masalah yang membutuhkan keahlian manusia atau kepakaran [10][11]. Dengan menggunakan sistem pakar ini, bahkan rata-rata orang bisa memecahkan masalah-masalah yang sangat kompleks yang tidak dapat diselesaikan tanpa bantuan seorang pakar. Tujuan pengembangan sistem pakar sistem y g y g p Dengan menggunakan sistem pakar ini, bahkan rata-rata orang bisa memecahkan masalah-masalah yang sangat kompleks yang tidak dapat diselesaikan tanpa bantuan seorang pakar. Tujuan pengembangan sistem pakar sistem Copyright © 2022 Ayu Handayani, Page 538 BITS is licensed under a Creative Commons Attribution 4.0 International License Copyright © 2022 Ayu Handayani, Page 538 BITS is licensed under a Creative Commons Attribution 4.0 International License Building of Informatics, Technology and Science (BITS) Volume 4, No 2, September 2022 Page: 537−544 ISSN 2684-8910 (media cetak) ISSN 2685-3310 (media online) DOI 10.47065/bits.v4i2.1981 pakar betujuan untuk mempermudah seseorang dalam menggunkan suatu perangkat lunak yang di adopsi dari seorang pakar tanpa menggantikan peran seorang pakar dengan baiya relative kecil[12]. Building of Informatics, Technology and Science (BITS) Volume 4, No 2, September 2022 Page: 537−544 ISSN 2684-8910 (media cetak) ISSN 2685-3310 (media online) DOI 10.47065/bits.v4i2.1981 pakar betujuan untuk mempermudah seseorang dalam menggunkan suatu perangkat lunak yang di adopsi dari seorang pakar tanpa menggantikan peran seorang pakar dengan baiya relative kecil[12]. 2.3 Penyakit Jantung Penyakit jantung adalah suatu kondisi ketika jantung terganggu. Bentuk kelainan itu sendiri bermacam-macam, bisa berupa kelainan pada pembuluh darah jantung, katup jantung atau miokardium. Penyakit jantung juga bisa disebabkan oleh infeksi atau cacat lahir [13] [14]. Penyakit jantung merupakan salah satu penyaki yang sangat tinggi reseko kematinya. Dimana angka kematia yang dialami para penderita penyakit jantung sangat meningkat hal ini dikarenakan sangat minimnya pengetahuan tentang penanganan awala seperti gejala yang diderita serta kurangnya fasiltas kesehatan untuk penanganan penyakit jantung di Indonesia yang masih sangat terbatas[15]. 3.1 Analisa Masalah Analisis masalah dalam studi sistem pakar untuk diagnosis penyakit jantung dilakukan dengan mempelajari dan mengumpulkan data dan pengetahuan yang diperoleh dari seorang spesialis atau pakar. Dimana hasil dari sistem pakar ini diharapkan terdapat hasil dan analisis yang jelas dan terstruktur. Sistem ini dirancang untuk menentukan sifat penyakit melalui konsultasi antara ahli dan peneliti. Dengan berkonsultasi dengan dokter spesialis, kita akan memahami gejala penyakit jantung dan membuat diagnosa dari gejala yang didapat. Untuk mendiagnosis penyakit jantung, seseorang harus mengetahui terlebih dahulu gejala-gejala yang telah terjadi, kemudian para ahli dapat menarik kesimpulan tentang penyakit yang dideritanya. Tabel 1. Penyakit Pada Jantung No Kode Penyakit Nama Penyakit 1 KPT01 Jantung Koroner 2 KPT02 Gagal Jantung 3 KPT04 Perikarditis 4 KPT05 Aritmia 5 KPT06 Heart Valve Disease Tabel 1. Penyakit Pada Jantung Tabel 1 merupakan data jenis penyakit jantung yang dijadikan sampel penelitian, meliputi 5 jenis penyakit jantung. Data tersebut akan dijadikan sebagai input bagi sistem pakar yang selanjutnya akan disimpan didalam database. Copyright © 2022 Ayu Handayani, Page 539 BITS is licensed under a Creative Commons Attribution 4.0 International License Tabel 2. Daftar Gejala Penyakit Pada Jantung Kode Gejala Nama Gejala Bobot Copyright © 2022 Ayu Handayani, Page 539 BITS is licensed under a Creative Commons Attribution 4.0 International License Tabel 2. Daftar Gejala Penyakit Pada Jantung Kode Gejala Nama Gejala Bobot Tabel 2. Daftar Gejala Penyakit Pada Jantung Bobot Copyright © 2022 Ayu Handayani, Page 539 Building of Informatics, Technology and Science (BITS) Volume 4, No 2, September 2022 Page: 537−544 ISSN 2684-8910 (media cetak) ISSN 2685-3310 (media online) DOI 10.47065/bits.v4i2.1981 KG01 Dada terasa penuh 5 KG02 Detak jantung cepat (tachycardia) 5 KG03 Detak jantung lambat (bradycardia) 3 KG04 Denyut nadi yang lemah dan cepat 5 KG05 Nyeri pada dada sebelah kiri 5 KG06 Sesak napas 5 KG07 Demam tinggi dan menggigil 5 KG08 Katub jantung tidak bekerja dengan baik 5 KG09 Bunyi jantung abnormal 3 KG10 Penyempitan pada dada 5 KG11 Pusing 1 KG12 Mual dan muntah 3 KG13 Pingsan 3 KG14 Berat badan menurun 3 KG15 Sulit tidur 5 KG16 Denyut nadi yang lemah dan cepat 5 KG17 Mudah lelah 5 Tabel 2 merupakan data gejala penyakit jantung, dari 5 jenis penyakit jantung ada 17 gejala yang disertai dengan nilai bobot kepercayaan yang diberikan oleh pakar. Data gejala penyakit jantung tersebut akan dijadikan sebagai input bagi sistem pakar yang selanjutnya akan disimpan didalam database Tabel 3. Terminologi Bobot Kepercayaan Kepercayaan Bobot Sangat Yakin 5 Yakin 3 Sedikit yakin 1 Tabel 3 merupakan terminologi bobot kepercayaan dalam melakukan konsultasi system dimana setiap pengguna diberikan jawaban dengan bobot tersebut. Tabel 4. Nilai Presentasi Kepastian Tingkat Presentasi Nilai Kemungkinan 100% Positip Terkena Penyakit 80% - 99% Kemungkinan besar tejadi 51% - 79% Kemungkinan 0% - 50% Sedikit kemungkinan / kemungkinan kecil Tabel 4 merupakan nilai presentase kepastian, dimana proses untuk melakukan perhitungan suatu interval presentasi kepercayaan dimulai dengan pemecahan dari sebuah rule. 3.2 Penerapan Metode Hybrid Case Base 3.2 Penerapan Metode Hybrid Case Base Sebelum menerapkan metode Hybrid Case Base, terlebih dahulu melakukan pencocokan gejala pasien yang terindikasi penyakit jantung. Adapun bentuk rule sistem pakar untuk mendiagnosa penyakit pada jantung dapat dilihat pada tabel dibawah ini: Tabel 5. Data Gejala Penyakit Pada Jantung Berdasarkan Rule Tabel 5. Data Gejala Penyakit Pada Jantung Berdasarkan Rule RULE IF THEN Rule 1 KG01; KG05;KG06; KG08;KG14 KPT01 Rule 2 KG01; KG04;KG05;KG07;KG08;KG09;KG11;KG12;KG15;KG17 KPT02 Rule 3 KG01;KG05; KG06; KG07; KG09; KG10;KG11;KG12;KG15; KG17 KPT03 Rule 4 KG01;KG04;KG06;KG11 KPT04 Rule 5 KG02; KG03;KG04; KG06; KG08; KG09; KG11;KG13;KG14 KPT05 Tabel 5 merupakan rule yang mencocokan antara data gejala dengan penyakit. Adapun bentuk rule diagnosa penyakit penderita jantung berupa pertanyaan berdasarkan gejala-gejala yang diderita. Selanjutnya berdasarkan rule yang sudah tersimpan di dalam knowledge database, maka selanjutnya melakukan pengukuran similarity pada tiap jenis penyakit jantung dengan rumus persamaan 1. a. Perhitungan untuk penyakit aritmia sesuai rule adalah sebagai berikut: Perhitungan untuk penyakit aritmia sesuai rule adalah sebagai berikut: a. Perhitungan untuk penyakit aritmia sesuai rule adalah sebagai berikut: 1. Dada terasa penuh : 5 2. Denyut nadi yang lemah dan cepat : 5 3 S k 5 1. Dada terasa penuh : 5 2. Denyut nadi yang lemah dan cepat : 5 3. Sesak napas : 5 Building of Informatics, Technology and Science (BITS) Volume 4, No 2, September 2022 Page: 537−544 ISSN 2684-8910 (media cetak) ISSN 2685-3310 (media online) DOI 10.47065/bits.v4i2.1981 4. Pusing : 1 Similarity= (15)+(15)+(15)+(11) 5+5+5+1 Similarity= 5+5+5+1 5+5+5+1 Similarity= 16 16 Similarity=1 Building of Informatics, Technology and Science (BITS) Volume 4, No 2, September 2022 Page: 537−544 ISSN 2684-8910 (media cetak) ISSN 2685-3310 (media online) DOI 10.47065/bits.v4i2.1981 Building of Informatics, Technology and Science (BITS) Volume 4, No 2, September 2022 Page: 537−544 ISSN 2684-8910 (media cetak) ISSN 2685-3310 (media online) DOI 10.47065/bits.v4i2.1981 4. Pusing : 1 Similarity= (15)+(15)+(15)+(11) 5+5+5+1 Similarity= 5+5+5+1 5+5+5+1 Similarity= 16 16 Similarity=1 4. Pusing : 1 Similarity= (15)+(15)+(15)+(11) 5+5+5+1 Similarity= 5+5+5+1 5+5+5+1 Similarity= 16 16 Similarity=1 Similarity=1 Begitu seterusnya untuk pengukuran similarity pada tiap jenis penyakit jantung. Adapun hasil dari pengukuran Similarity pada tiap jenis penyakit terhadap pengujian pada data sampel dapat dilihat pada tabel dibawah ini: Begitu seterusnya untuk pengukuran similarity pada tiap jenis penyakit jantung. Adapun hasil dari pengukuran Similarity pada tiap jenis penyakit terhadap pengujian pada data sampel dapat dilihat pada tabel dibawah ini: Tabel 6. Hasil Perhitungan Sampel Data Gejala Sesuai Rule Tabel 6. Hasil Perhitungan Sampel Data Gejala Sesuai Rule Tabel 6. 3.2 Penerapan Metode Hybrid Case Base Nyeri pada dada sebelah kiri : 5 Copyright © 2022 Ayu Handayani, Page 541 BITS is licensed under a Creative Commons Attribution 4.0 International License BITS is licensed under a Creative Commons Attribution 4.0 International License Building of Informatics, Technology and Science (BITS) Volume 4, No 2, September 2022 Page: 537−544 ISSN 2684-8910 (media cetak) ISSN 2685-3310 (media online) DOI 10.47065/bits.v4i2.1981 Building of Informatics, Technology and Science (BITS) Volume 4, No 2, September 2022 Page: 537−544 ISSN 2684-8910 (media cetak) ISSN 2685-3310 (media online) DOI 10.47065/bits.v4i2.1981 Building of Informatics, Technology and Science (BIT 4. Demam tinggi dan menggigil : 5 gg gg g 5. Katup jantung tidak bekerja dengan baik : 5 6. Bunyi jantung abnormal : 3 7. Pusing : 1 7. Pusing : 1 8. Mual dan muntah : 3 Similarity= (15)+(05)+(05)+(05)+(05)+(03)+(01)+(03)+(05)+(05) 5+5+5+5+5+3+1+3+3+5 Similarity= 5+0+0+0+0+0+0+0+0+0 5+5+5+5+5+3+1+3+5+5 e. Perhitungan untuk penyakit perikarditis adalah sebagai berikut: e. Perhitungan untuk penyakit perikarditis adalah sebagai berikut: . Perhitungan untuk penyakit perikarditis adalah sebagai berikut: 1. Dada terasa penuh : 5 2. Nyeri pada dada sebelah kiri : 5 3. Sesak napas : 5 4. Demam tinggi dan menggigil : 5 5. Katup jantung tidak bekerja dengan baik : 5 6. Bunyi jantung abnormal : 3 7. Penyempitan pada dada : 5 8. Pusing : 1 9. Mual dan muntah : 3 10. Sulit tidur : 5 11. Mudah lelah : 5 Similarity= (15)+(05)+(15)+(05)+(03)+(05)+(01)+(03)+(05)+(05) 5+5+5+5+3+5+1+3+5+5 Similarity= 5+0+5+0+0+0+0+0+0+0 5+5+5+5+3+5+1+3+5+5 Similarity= 10 42 Similarity=0,2381 1. Dada terasa penuh : 5 1. Dada terasa penuh : 5 2. Nyeri pada dada sebelah kiri : 5 3. Sesak napas : 5 4. Demam tinggi dan menggigil : 5 5. Katup jantung tidak bekerja dengan baik : 5 6. Bunyi jantung abnormal : 3 11. Mudah lelah : 5 Similarity= (15)+(05)+(15)+(05)+(03)+(05)+(01)+(03)+(05)+(05) 5+5+5+5+3+5+1+3+5+5 Similarity= 5+0+5+0+0+0+0+0+0+0 5+5+5+5+3+5+1+3+5+5 f. Perhitungan untuk penyakit aritmia adalah sebagai berikut: f. Perhitungan untuk penyakit aritmia adalah sebagai berikut: . Perhitungan untuk penyakit aritmia adalah sebagai berikut: 1. Dada terasa penuh : 5 2. Denyut nadi yang lemah dan cepat : 5 3. Sesak napas : 5 4. Pusing : 1 Similarity= (15)+(05)+(15)+(01) 5+5+5+1 Similarity= 5+0+5+0 5+5+5+1 Similarity= 10 16 Similarity=0,625 . Perhitungan untuk penyakit aritmia adalah sebagai berik 1. Dada terasa penuh : 5 2. Denyut nadi yang lemah dan cepat : 5 3. Sesak napas : 5 4. 3.2 Penerapan Metode Hybrid Case Base Hasil Perhitungan Sampel Data Gejala Sesuai Rule No Kasus Jml Gejala Sama Jml Gejala Kasus Jml Gejala Dipilih Bobot Gejala Sama Bobot Gejala Kasus Hasil Hasil (Dalam Persen) 1 Aritmia 4 4 4 16 16 1 100 % 2 Jantung Koroner 2 5 4 10 23 0,43478 43,4783 % 3 Heart Valve Disease 3 9 4 11 33 0,33333 33,3333 % 4 Gagal jantung 3 10 4 11 42 0,26190 26,1905 % 5 Perikarditis 3 10 4 11 42 0,26190 26,1905 % Hasil pengujian berdasarkan data gejala yang dialami oleh pasien pada sampel data didapatkan hasil persentasi dalam pengukuran similarity dapat dilihat pada tabel 6 dengan hasil diagnosa penyakit paling besar presentasi nya adalah penyakit aritmia dengan presentasi sebesar 100% maka kemungkinan pasien mengalami penyakit tersebut. b. Perhitungan dari pengukuran Similarity pada data sampel gejala secara acak adalah sebagai berikut: g p g y p p g j g Dari gejala-gejala penyakit pada penderita jantung yang dijawab pasien misalnya dipilih 4 gejala secara acak sebagai berikut : Tabel 7. Daftar Gejala Yang Dipilih Oleh Pasien Kode Nama Gejala 1 Dada terasa penuh 2 Detak jantung cepat (tachycardia) 3 Detak jantung lambat (bradycardia) 4 Sesak napas Tabel 7. Daftar Gejala Yang Dipilih Oleh Pasien Kemudian proses selanjutnya melakukan pengukuran similarity pada tiap jenis penyakit jantung. c. Perhitungan untuk penyakit jantung koroner adalah sebagai berikut: 1. Dada terasa penuh : 5 2. Nyeri pada dada sebelah kiri : 5 3. Sesak napas : 5 4. Katup jantung tidak bekerja dengan baik : 5 5. Berat badan menurun : 3 5. Berat badan menurun : 3 Kemudian kasus lama akan dicocokkan dengan kasus yang baru. Jika gejala tersebut ada maka akan di berikan nilai 1, jika tidak ada akan di berikan nilai 0. Selanjutnya akan dikalikan dengan bobot yang diberikan oleh pakar. Similarity= (15)+(05)+(15)+(05)+(03) 5+ 5+5+ 5+3 Similarity= 5+0+5+0+0 5+ 5+5+ 5+3 Similarity= 10 23 Similarity=0,43478 Similarity= (15)+(05)+(15)+(05)+(03) 5+ 5+5+ 5+3 Similarity= 5+0+5+0+0 5+ 5+5+ 5+3 Similarity= 10 23 Similarity=0,43478 Similarity=0,43478 Similarity=0,43478 d. Perhitungan untuk penyakit gagal jantung adalah sebagai berikut: d. Perhitungan untuk penyakit gagal jantung adalah sebagai berikut: 1. Dada terasa penuh : 5 2. Denyut nadi yang lemah dan cepat : 5 3. Similarity= (15)+(13)+(05)+(15)+(05)+(03)+(01)+(03)+(03) 5+3+5+5+5+3+1+3+3 Similarity= (15)+(13)+(05)+(15)+(05)+(03)+(01)+(03)+(03) 5+3+5+5+5+3+1+3+3 Similarity= 5+3+0+5+0+0+0+0+0 5+3+5+5+5+3+1+3+3 Similarity= 13 33 Similarity=0,3939 un hasil dari pengukuran Similarity pada tiap jenis penyakit terhadap pengujian pada data sampel dapat tabel dibawah ini: Adapun hasil dari pengukuran Similarity pada tiap jenis penyakit terhadap pengujian pada data sampel dapat dilihat pada tabel dibawah ini: Adapun hasil dari pengukuran Similarity pada tiap jenis penyakit terhadap pengujian pada data sampel dapat dilihat pada tabel dibawah ini: Tabel 8. Hasil Perhitungan Sampel Data Gejala Secara Acak Tabel 8. Hasil Perhitungan Sampel Data Gejala Secara Acak Tabel 8. Hasil Perhitungan Sampel Data Gejala Secara Acak No Kasus Jml Gejala Sama Jml Gejala Kasus Jml Gejala Dipilih Bobot Gejala Sama Bobot Gejala Kasus Hasil Hasil (Dalam Persen) 1 Aritmia 2 4 4 10 16 0,625 62,5 % 2 Jantung Koroner 2 5 4 10 23 0,43478 43,4783% 3 Heart Valve Disease 3 9 4 13 33 0,39394 39,3939% 4 Perikarditis 2 10 4 10 42 0,238095 23,8095% 5 Gagal jantung 1 10 4 5 42 0,119048 11,9048% Hasil pengujian berdasarkan data gejala yang dialami oleh pasien pada sampel data didapatkan hasil persentasi dalam pengukuran similarity dapat dilihat pada tabel 8 dengan hasil diagnosa penyakit paling besar presentasi nya adalah penyakit aritmia dengan presentasi sebesar 62,5% maka kemungkinan pasien mengalami penyakit tersebut 4. KESIMPULAN Berdasarkan hasil dan pembahasan maka dapat diambil kesimpulan yaitu penerapan metode Hybrid Case Base pada sistem pakar dapat digunakan dalam menentukan tingkat presentasi kemiripan gejala penyakit yang di alami oleh pasien. Hasil dari penelitian ini mampu melakukan diagnosa jenis penyakit jantung yang dialami pasien dan solusi pengobatannya serta berapa persen kemungkinan pasien menderita penyakit tersebut dengan cepat. REFERENCES [1] D. I. W. Yuanita and D. O. Marleen, “PEMBUATAN SITUS SISTEM PAKAR UNTUK MENDIAGNOSA GANGGUAN SISTEM PENCERNAAN PADA MANUSIA,” 2014. [1] D. I. W. Yuanita and D. O. Marleen, “PEMBUATAN SITUS SISTEM PAKAR UNTUK MENDIAGNOSA GANGGUAN SISTEM PENCERNAAN PADA MANUSIA,” 2014. [2] A. P. Purnama and R. Kurniawan, “Kajian Literatur Metode Sistem Pakar pada Penanganan Kesehatan Gigi dan Mulut,” 2019. [1] D. I. W. Yuanita and D. O. Marleen, “PEMBUATAN SITUS SISTEM PAKAR UNTUK MENDIAGNOSA GANGGUAN SISTEM PENCERNAAN PADA MANUSIA,” 2014. [2] A. P. Purnama and R. Kurniawan, “Kajian Literatur Metode Sistem Pakar pada Penanganan Kesehatan Gigi dan Mulut,” 2019. SISTEM PENCERNAAN PADA MANUSIA,” 2014. [2] A. P. Purnama and R. Kurniawan, “Kajian Literatur Metode Sistem Pakar pada Penanganan Kesehatan Gigi dan Mulut,” 2019. , [2] A. P. Purnama and R. Kurniawan, “Kajian Literatur Metode Sistem Pakar pada Penanganan Kesehatan Gigi dan Mulut,” 2019 [3] A. Imran, “IMPLEMENTASI SISTEM PAKAR DIAGNOSA PENYAKIT EPISTAKSIS PADA MANUSIA MENGGUNAKAN METODE HYBRID CASE BASED DAN RULE BASED REASONING,” J. Maj. Ilm. Inf. dan Teknol. Ilm., vol. 7, no. 1, pp. 85–92, 2019. [3] A. Imran, “IMPLEMENTASI SISTEM PAKAR DIAGNOSA PENYAKIT EPISTAKSIS PADA MANUSIA MENGGUNAKAN METODE HYBRID CASE BASED DAN RULE BASED REASONING,” J. Maj. Ilm. Inf. dan Teknol. Ilm., vol. 7, no. 1, pp. 85–92, 2019. pp [4] N. FAJAR, AGUNG, “PERANCANGAN SISTEM PAKAR DIAGNOSA PENYAKIT JANTUNG DENGAN METODE FORWARD CHAINING,” J. Inform. Univ. PAMULANG, vol. 3, no. 2, pp. 75–79, 2018. pp [4] N. FAJAR, AGUNG, “PERANCANGAN SISTEM PAKAR DIAGNOSA PENYAKIT JANTUNG DENGAN METODE FORWARD CHAINING,” J. Inform. Univ. PAMULANG, vol. 3, no. 2, pp. 75–79, 2018. [5] Dona, H. Maradona, and Masdewi, “SISTEM PAKAR DIAGNOSA PENYAKIT JANTUNG DENGAN METODE CASE BASED REASONING (CBR),” Zo. Junarl Sist. Inf., vol. 3, no. 1, pp. 1–12, 2021. Cindy, Pamela Cornelia, R. Vivi, Peggie, M. Hiskia, Kamang, and B. Nancy, Silvia, “Implementasi Sistem Pak gnosa Penyakit Jantung Pada Manusia,” J. Sains dan Teknol., vol. 1, no. 2, pp. 201–206, 2018. [7] M. Fauzan, “Sistem Pakar Mendiagnosa Penyakit Kandungan Kemih Dengan Menerapkan Metode Hybrid Case Base,” J. Inf. DAN Teknol. Ilm., vol. 7, no. 3, pp. 264–268, 2020. pp [8] A. L. SP, “Sistem Pakar Mendiagnosa Penyakit Kolera Menerapkan Metode Hybrid Case Based,” Heal. Contemp. Technol. J., vol. 1, no. 1, pp. 13–19, 2020. pp elilah Rambe, “Sistem Pakar Mendiagnosa Penyakit Kanker Usus Besar Pada Manusia Dengan Menerapkan Metode Case Based,” J. Ris. Komput., vol. 3.2 Penerapan Metode Hybrid Case Base Pusing : 1 Similarity= (15)+(05)+(15)+(01) 5+5+5+1 Similarity= 5+0+5+0 5+5+5+1 Similarity= 10 16 Similarity=0,625 g p y g 1. Dada terasa penuh : 5 2. Denyut nadi yang lemah dan cepat : 5 3. Sesak napas : 5 3. Sesak napas : 5 4. Pusing : 1 Similarity= (15)+(05)+(15)+(01) 5+5+5+1 Similarity= 5+0+5+0 5+5+5+1 Similarity= 10 16 Similarity=0,625 g. Perhitungan untuk penyakit heart valve disease adalah sebagai berikut: 1. Detak jantung cepat (tachycardia) g. Perhitungan untuk penyakit heart valve disease adalah sebagai berikut: 1 Detak jantung cepat (tachycardia) 1. Detak jantung cepat (tachycardia) 2. Detak jantung lambat (bradycardia) : 3 3. Denyut nadi yang lemah dan cepat : 5 4. Sesak napas : 5 4. Sesak napas : 5 5. Katup jantung tidak bekerja dengan baik : 5 6. Bunyi jantung abnormal : 3 7. Pusing : 1 7. Pusing : 1 8. Pingsan (syncope) : 3 9. Berat badan menurun : 3 Copyright © 2022 Ayu Handayani, Page 542 BITS is licensed under a Creative Commons Attribution 4.0 International License Building of Informatics, Technology and Science (BITS) Volume 4, No 2, September 2022 Page: 537−544 ISSN 2684-8910 (media cetak) ISSN 2685-3310 (media online) DOI 10.47065/bits.v4i2.1981 Copyright © 2022 Ayu Handayani, Page 544 BITS is licensed under a Creative Commons Attribution 4.0 International License [17] E. D. Simanjuntak, H. Sunandar, and R. K. Hondro, “Implementasi Metode Hybrid Case-Based Reasoning Untuk Mendiagnosa Pengidap Penyakit Post-Traumatic Stress Disorder(PTSD),” JURIKOM (Jurnal Ris. Komputer), vol. 7, no. 2, pp. 256–263, 2020. [18] P. Tarigan, “Sistem Pakar Untuk Mendiagnosa Penyakit Disentri Dengan Menggunakan Metode Hybrid Case Based,” J. Tek. Inform. Kaputama, vol. 2, no. 1, 2018. py g y y , g BITS is licensed under a Creative Commons Attribution 4.0 International License REFERENCES 6, no. 6, pp. 606–611, 2019. pp [9] Rizky Delilah Rambe, “Sistem Pakar Mendiagnosa Penyakit Kanker Usus Besar Pada Hybrid Case Based,” J. Ris. Komput., vol. 6, no. 6, pp. 606–611, 2019. [9] Rizky Delilah Rambe, “Sistem Pakar Mendiagnosa Penyakit Kanker Usus Besar Pada Manusia Dengan Menerapkan Metode Hybrid Case Based,” J. Ris. Komput., vol. 6, no. 6, pp. 606–611, 2019. [10] R Wahyuni “Jurnal APLIKASI SISTEM PAKAR UNTUK DIAGNOSA PENYAKIT CAMPAK DAN PENCEGAHAN yuni, “Jurnal APLIKASI SISTEM PAKAR UNTUK DIAGNOSA PENYAKIT CAMPAK DAN PENCEGAHAN AN METODE FORWARD CHAINING BERBASIS WEB,” J. Ilmu Komput., vol. 8, no. 2, 2019. [11] D. Aldo, “Sistem Pakar Diagnosis Hama Dan Penyakit Bawang Merah Menggunakan Metode Dempster Shafer,” KOMPUTIKA J. Sist. Komput., vol. 9, no. 2, 2020. [12] S. Nelly Astuti Hasibuan, Hery Sunandar, Senanti Alas, “SISTEM PAKAR MENDIAGNOSA PENYAKIT KAKI GAJAH MENGGUNAKAN METODE CERTAINTY FACTOR,” JURASIK (Jurnal Ris. Sist. Inf. dan Tek. Inform., vol. 2, no. 1, 2017. [13] N. Lannywati, Ghani; Made, Dewi, Susilawati; Harli, “Faktor Risiko Dominan Penyakit Jantung Koroner di Indonesia,” Bul. Penelit. Kesehat., vol. 44, no. 3, pp. 153–164, 2016. pp [14] A. Vicky, Agnes, F. Iskandar, and M. Eri, “Implementasi Metode Penalaran CBR dalam Mengidentifikasi 14] A. Vicky, Agnes, F. Iskandar, and M. Eri, “Implementasi Metode Penalaran CBR dalam Mengidentifikasi Gejala Awal Copyright © 2022 Ayu Handayani, Page 543 BITS is licensed under a Creative Commons Attribution 4.0 International License BITS is licensed under a Creative Commons Attribution 4.0 International License Building of Informatics, Technology and Science (BITS) Volume 4, No 2, September 2022 Page: 537−544 ISSN 2684-8910 (media cetak) ISSN 2685-3310 (media online) DOI 10.47065/bits.v4i2.1981 [15] M. Sita and A. Sigit, “Sistem Diagnosa Penyakit Jantung Berbasis Case Based Reasoning (CBR),” in Seminar Nasional Hasil Penelitian dan Pengabdian Masyarakat 2021, 2021, pp. 1–11. DOI 10.47065/bits.v4i2.1981 Penyakit Jantung menggunakan Algoritma Sorensen Coeffient,” J. JTIK (Jurnal Teknol. Inf. dan Komunikasi), vol. 5, no. 306–313, 2021. Penyakit Jantung menggunakan Algoritma Sorensen Coeffient,” J. JTIK (Jurnal Teknol. Inf. dan Komunikasi), vol. 5, no. 306–313, 2021. [15] M. Sita and A. Sigit, “Sistem Diagnosa Penyakit Jantung Berbasis Case Based Reasoning (CBR),” in Seminar Nasional Hasil Penelitian dan Pengabdian Masyarakat 2021, 2021, pp. 1–11. [16] A R dh S S dh h d M A I f di “I l i Si P k Di P ki Gi i D M l [16] A. Romadhony, S. Saadhah, and M. A. Irfandi, “Implementasi Sistem Pakar Diagnosa Penyakit Gigi Dan Mulut Menggunakan Metode Hybrid Case Based dan Rule Based Reasoning,” in Indonesia Symposium On Computing, 2015, pp. 2460–3295. [17] E. D. Simanjuntak, H. Sunandar, and R. K. Hondro, “Implementasi Metode Hybrid Case-Based Reasoning Untuk Mendiagnosa Pengidap Penyakit Post-Traumatic Stress Disorder(PTSD),” JURIKOM (Jurnal Ris. Komputer), vol. 7, no. 2, pp. 256–263, 2020. [18] P. Tarigan, “Sistem Pakar Untuk Mendiagnosa Penyakit Disentri Dengan Menggunakan Metode Hybrid Case Based,” J. Tek. Inform. Kaputama, vol. 2, no. 1, 2018.
https://openalex.org/W2046065118	https://zenodo.org/records/2221894/files/article.pdf	English	null	Discussion on Is the Intercepting Trap a Failure ?	Journal - Royal Sanitary Institute	1,906	public-domain	12,811	185 185 DISCUSSION ON IS THE INTERCEPTING TRAP A FAILURE P Opened by WILLIAM BUTLER, M.B., D.P.H., Medical Officer of Health, Willesden. (FELLOW.) And R. READ, A.M.I.C.E., City Surreyor, Gloueester. (MEMBER.) Opened by WILLIAM BUTLER, M.B., D.P.H., Medical Officer of Health, Willesden. (FELLOW.) And R. READ, A.M.I.C.E., City Surreyor, Gloueester. (MEMBER.) At Sessional Meeting, London, February 14th, 1906. W. BUTLER, M.B., D.P.H. EF BE i ) , EFORE answering such L as this, it will be necessary to BEFORE answering such of the iiitereeptoi- it will be SN-st(,I&dquo;l of i ) consider the purpose of the interceptor in a modern system of drainage. And it may be desirable to go to the root of the matter and consider first the primary hygienic and physical principles which underlie modern methods of sanitation us applied to the removal of human excreta and slop waters by a water-borne system. It may be said that they simply ailll at the removal, as rapidly and emciently as possible, of all foul or waste decomposable organic matter from the neighbourhood of human dwellings; and that equally important with the rapid removal of sewage matter is the preB’ention of any accumulation and escape uf the gases of decomposition or of polluted waters near tu the house. Whatever view be taken as to tlle nzodu.s ol~emzzzdi hy which these agencies pt-o(luce disease, it will be conceded that the admission to the house of such gases, or the pollution of the site by liquid filtll, is wont to be followed by outbreaks of disease often explosi,-e in character, and is in any case calculated to Impair health. The aim of modern drainage is simply to secure the dwelling from these untoward results, and upon the degree of success which iu practice attends this aim must the means currently adopted be judged. adopted j g I shall endeavour to show that a drainage scheme in which the drain is disconnected from tlle sewer is as mischievous in practice as it is faulty 186 in theory. It is based on the assumption that it is safe to have a coii- ditions of tllings in the public sewers that it is unsafe to have in the house drains which commullicate with the sewers, and that yon may recognise the existence of a danger zune on one side of a trap against which the other is assumed I to be safely secured by a water seal. W. BUTLER, M.B., D.P.H. safely by It must of course be conceded that the inside of a sewer can never be regarded as a sanitary situatiun, but from this to the assumption that its gaseous contents may be contemplated as so potently charged with danger that they may not even be admitted to a gas-tight system of tubes having no unsealed opening savc to tlle outer air above the housetops, is a most serious confession of sanitary failure. The truth is that. accunlulations of considerable volumes of sewer gas are dangerous and insanitary, whether they be permitted to stagnate in the sewers or in the llousc drains. If the sewers are emciently ventilated, as they should be, there is no ground to suppose that tlle atmosphere of the sewer is more harmful than that of the house drain, and the disconnection of one from the other is not only harmful, but irratiunal also. For it presupposes what sanitarily cannot be presupposed, n:mulv, a dangerous atmosphere in the sewers; .lrld it is an attempted defence, by means of a trap, against this danger, which in its very adoption casts doubt upon the efficiency of the means, since the same are used ia the defence of the house against the still dangerously regarded atmosphere of tlle house drain. But. it may well be urged that academic objections to the Intercepting siphon are little likely to disturb so trusted and respectable a contrivance of the practical sanitarian. I will only answer such an objection by saying that the siphon had its origin less in a practical need than in a faulty theory, and that academic criticism alone should have prevented the extensive adop- tion of what many regard as a mischievous appliance. many regard appliance. Perhaps the most universally objectionable feature of the siphon is that it prevents the cfficiellt ventilation of tlle sewers. That the public sewers are inefficiently ventilated ill those cases where their communication with the soil-pipc ventilators of tlle house drains is intercepted, is shown in many ways. W. BUTLER, M.B., D.P.H. It is a common experience with municipal oflicers that daily complaints are received, during the summer months, of the offensive smells proceeding fr<>in the openings to the sewers in the crown of the roadway, placed there originally with the intention of acting as fresh-air inlets to the sewers, and now iu many districts being sealed off because they are found to act as vcnts for the foul gases of an insufplrielltly ventilated sewer. Visual demonstration of this fact may be had during 187 frosty weather, when a stream of condensed vapour may be observed proceeding from these openings into the sewers. It might also be shown with regard to most servers that the number of upcast shafts which it has been possible to erect are insuflicieiit to cause such a negative pressure in the sewers as shall result in anything like continuous aspiration of air i nto the sewers through the roacl openings,. through ope gs,. But, short uf effecting tllis, the road grids become a nuisance owing to escape from them of the gaseous contents of the sewer at the street level, and under conditions, uwiy to inadeynate sewer ventilation, in which these gaseous contents urc apt to contain a large admixture of sewer gas, I think it will not be clislaltcd that what. is most urgently needed in respect of must public sewers, is au Increased number of exit ventilators Intentionally su acting. And could these be provided in sufficient numbers-as practically at present they cannot bu-the problem of sewer ventilation would be solved, and the atmosphere of the sewers would then be rendered as innocuous as rapid change in the aerial contents of tlie sewers can render it. Such sufficient numbers of llpcast ventilating shafts would be provided &dquo;’ere each house drain aud soil ventilating pipe in direct aerial communi- cation with the sewers. The aspirating effect of so many outlet ventilators to the sewer, up which the lighter air of thu sewer would tend constantl3- to ascend would be a ral)iclly Innowing current of fresh air to the sewers at all lower openings; and thus at one anll the same time both the sewers and the house drains would be effectively ventilated and any accumulation of gases of decomposition, either iu the sewei-s or tlie drains or their escape in improper situations, rendered impossible. improper , impossible. W. BUTLER, M.B., D.P.H. The interception of the drains from the sewers, however, lias deprived the sewers of what should be their natural outlet and on occasion inlet ventilators. It has necessitated tllC provision on eal,ch house drain of an untrapped opening, iii a large proportion of cases in immediate proximity to the doors or windows of the house, and acting, at least with a frequency uneontemplated in the theory of its advocates, as an outlet ventilator to tllc drain. Further, to provide access to the segment of the drain between the interceptor and the sewer, another complication in the shape of a raking arm with a readily removable stopper his to be added. An inspel’- tion chamber is a proper equipment of every drain, but the interceptor, necessarily placed as near the sewer as practicable, llas necessitated the placing of the manhole where the drain is deepest, and where, apart from the consequently Increased cost, it necessarily forms a capacious reservoir 188 for the storage of gases at the very site where they are most likely to accumulate owing to the couple of gallons or so of stagnating sewage contained in the trap at the bottom. trap Every une uf these complications introduced into drainage systems to meet the exigencies of the interceptor is extremely apt to go wrong and produce nuisance. The interceptor itself is essentially insanitary. It 1’eti1111S within the precincts of the house premises between twu and three gallons of sewage which is of that aggravated character resulting from the undue retention of solids, and thus breaks with the primal principles of domestic sewage disposal. It occasionally fails of its object of excluding from the house drains the gaseous contents of the sewers ; for it was urged in defence of the system by the late Dr. G. Buchanan, in his Report to the Privy Council and Local Government Board, that .should &dquo; the trap be ever forced by pressure of air in the public sewer, an immediate exit of the sewer air away from the house is afforded &dquo;- by the so-called inlet ventilator to the drains. The plating down of the street gratings to prevent the offensive smells from the sewers, which the introduction of the interceptors has occasioned, will, especially where shafts have not been erected in their place, make this forcing of the trap a more frequent occurrence than was contemplated. In 118 of these cases the manholes were filled with foetid sewage, emitting its foul vapours through the drain inlet ventilator close to the doors and windows of the houses, into which they were duly aspirated. houses, they duly aspirated. In the 170 remaining cases where the drain was Mocked, the manhole remained free of sewage, because of anotlier accident of the system, tlie unstopping of the raking arm, which permitted the escape of tlie drain contents to the sewer, and Incidentally of the sewer gas through the 1I1:l1lhole and drain inlet ventilator,. Iii 654 cases this accident was observed to have occurred. That is to say in nearly 10 per cent. of the drains examined the intereeptor, apart from its incidental drawbacks, failed absolutely of its object, the drains in these cases being in direct communication with the sewers, the sewers being thus provided with exit ventilators in the forecourts of the houses. But for the drains to be accidentally in direct communication with the sewers in only 10 per cent. of the houses is much more serious than for 100 per cent. of the drains to be intentionally directly connected with the sewers. In the one case the sewer is adequately ventilated, in the other the sewer air is presumably 10 times more concentrated than where all the house drains ventilate the sewer. In the one case, moreuver, the sewer air, diluted and comparatively innocuous, finds vent above the roofs, and away from all openings to the houses; In the other, a concentrated sewer air is laid on at the ground level of the houses. The untrapped opening to tlle drain, intended as an inlet ventilator, becomes in these cases a serious danger to health. But to such an extent is the inlet ventilator recognised as a common cause of nuisance, owing to its waywardness of acting as a vent for foul gases, that it lias become common practice to fit it with a mica flap to prevent reflux currents from tlle drain. In ii,1910 cases of those I investigated, however, the fresh air inlet was unprotected by any properly acting appliance. This may appear a trivial matter, since at its inception no valve was considered necessary. But it must be remembered that admission to the house of air from the interior of the drain is still, and I think property, regarded as dangerous. W. BUTLER, M.B., D.P.H. &dquo;’here no facilities even for inadeduate ventilation of the sewers are provided, the daily recurring increases of pressure of the imprisoned gases must necessarily force them through the yielding traps into the house drains. ecessa y through yielding p And this contingency, to which every house drain disconnected from the sewer by a trap is subject, is a greater danger than is incurred when sewers and house drains are the channels for the constant flow of con- tinuously renewecl currents of air. Tllis is a breakdown of the inter- ceptor, the frequency of which it is impossible to estimate. But it fails in other directions. A straight pipe with a proper fall shows little tendency to choke, but if in tlle course of such a pipe al acute kink or bend be introduced, especially if it be of such a character as. to remain filled with floating solids, the tendency to become choked at this point will have increased enormously. During last year, I had a systematic inspection made of all the readily accessible manholes in my district. These amounted to some li,i4~, and l’omprised the inspection chambers of about a third of the houses in Willesden. In no single instance was a drain found to be choked but at tlle interceptor, but no fewer tllan 288 or 4t per cent. of all the drains insloected were dis- covered to be stopped at this point. 189 Most people who would remain unmoved. at the forcing of the intercepting siphon would feel alarm at the unsealing of the yard gully, and a crack in a drain pipe covered with two or three feet of clay is regarded as a most serious insanitary condition by people who are undisturbed at an unprotected gaping opening to their drains a couple of feet below their open window. couple p It might be thought that the result of the inspection of nearly 7,000 190 manholes yielded exceptional results due to accumulation of stoppages long unrecognised. It is true that in many of the stoppages there was evidence of antiquity in the insanitary condition discovered, but in many parts of the district there had been previous systematic inspections yielding results quite as bad, though In less numbers, owing to the more limited area of investigation. Nuisances of the character discovered are, more- over, constantly being abated, being the natural occasion of prompt com- plaillt. I have, however, had a re-inspection made of about :~I11) manholes with a view to seeing whether the results yielded by the first Inspection were confirlnecl. The re-iiispectioii was made within eight months of the first survey, and Included 216 manholes where originally no defects were discovered, and 2(’~S where defects originally found had been made good. Out of the total of ,-,U4 re-inspections, 4H drains were found to be choked at the interceptor, and -11 to have the cap of the raking arm missing. Of those choked, in 11() instances the manholes were more or less filled with sewage, while in 1:~, ill addition to the choking of the drain, the man- hole was in direct aerial communication with tlle sewer, owing to the absence of tlle cap of the raking arm. Altogether in 71 instances the drain was choked, tlie cap of the raking arm missing, or both these con- ditions co-existed. Thus In nearly 1.’) pei’ cent. of the manholes re- examined, the most serious defects were discovered. Both in these and in tllose primarily discovered the defects are traceable to the interceptor, and the modifications which it entails. In an examination of uver 7,000 manholes it is found that In ti-5 per cent. tlle interceptor fails of its object of disconnecting tllu drain from aerial continuity with the sewer. It fails not onl3- of this, but is directly responsible for a high percentage uf blocked drains, and manholes converted into leaking cesspools. , leaking cesspoo s. The untrapped drain-opening in tlle forecourt which it necessitates, normally serves as an outlet for the emanations of the drain, and sets at ridicule all insistence on the need for effectual trapping of yard gullies and tlie elaborate precautions taken to secure a ga,-tight drain. p g , g In tlie very frequent abnormal conditions where the drain is choked, and tlle raking arm upen to the sewer, it is an unmitigated nuisance. It is for these reasons that I am bound to answer the question we are discussing in the affirniatii-e. It is perhaps beyond the range uf this discussion to say what is the counter proposal; but, in coonclusion, I may give what I consider the essential principles of sanitary drainage. A good drain should be so laid as to be self-cleansing; so as to be relied upon to remain gas- tight and water-tight throughout its course. It should have no untrapped 191 opening save at the top of the soil pipe above the roof. Both the drain and the sewer should be effectnally ventilated. Access shuuld bc provided, but the drain should be hermetically senled from the access chamber except when occasion arises for inspection p inspection. These conditions will probably best 1K’ complied with by tlle provision of iron pipes with socketed joints, caulked with blue lead. The inspection chamber can be placed in the shallower part of the drain, in the absence of an interceptor determining its position in the forecotirt, and tlie saying in cost thus effected will generally be more than sufficient to cover the additional expense of llsing iron pipes throughout tllc whole of the drain, Drainage would thus be simplified and cheapened; tlleuretically it would be consistent in detail, and practicallv it would be efficient, little likely to get out of order or to require amendment. The provincial view that you could have a satisfactory drainage system for your house, while the public sewers at your doors must be regarded with suspicion, would give place to the more concrete view which alone is defensible, namely, that the drainage problem is one hygienically, whatever be the need for legal or administrative distinctions between public sewers and private drains. R. READ, A.M.I.C.E. IN the autlmr’s opinion the answer to the above question is yes. Tlie I intercepting trap is intimately connected with the larger question of the ventilation of sewers and drains, which has been the snbject of controversy, more or less for the last :~11 years, commencing after the illness of tlie King when Prince of Wales in 1872. In this controversy the author has taken a somewhat active part at various times. IN I p Tlie intercepting trap was patented by air. 117. P. Buchan, of Glasgow, about 1~7 5, and withuut any special Investigation was adopted by the Local Government Board, and introduced intu their model by-laws in 1a i 7. Ever since then this of~cial recognition has caused it to be taken for granted by large numbers of people, and has deterred many from investigating the question for themselves. est gat g q The controversy above referred to resulted in a general concensus of opinion that &dquo; sewer gas must be cut off from the house,&dquo; and the intercepting trap was adopted by the Lucal Government Board with this 192 object, but this was trying to cure one evil by introducing anoth object, but this was trying to cure one evil by introducing another. In its original form a I}-inch Intercepting trap contained over three gallons of stagnant sewage ; it took the place of and was the same length as an ordinary drain pipe, with from to .’-~ inches difference of level between the inlet and outlet ; its form llas since been modificd by various makers, in the vain attempt to perfect it, chiefly by contracting the body to increase the scour, and the contents are now about 21 gallons or 25 lbs. weight of decomposing sewage, or about an ordinary bucket full. The trap uses up tlle fall required by four or six ordinary pipes, it is inserted at the lower end of the underground portion of the house drain, forming an obstruction therein nearly 8 inches deep to the now of the sewage, it causes the sewage to deposit from 25 per cent. to 35 per cent.. of its solid matter in the trap after each discharge in dry weather, and paralyses the flow of the sewage through the whole length of the underground portion of the house drain. R. READ, A.M.I.C.E. open The trap is introduced into the lower end of the house drain, on the erroneous assumption that it is a safeguard to the house, but it is no protection at all : on the contrary, it is a useless and dangerous ob- struction, which provides at every man’s front door the very conditions and dangers which it is the object of modern sanitation to prevent. dangers object prevent. These traps now form part of many thousands of existing drains, and while they absolutely prevent the adoption of a proper system of ventila- tion, they also provide a reservoir of putrid sewage on every house drain to contaminate everything passing through it, and thus cause noxious gases and smells to be generated in the drains and sewers, which cannot he got rid of by any amount of flushing of the sewers alone, and the more numerous the traps the greater the nuisance. The trap necessitates tlie use of at least :i0 per cent. more water in the flushing cisterns to the w.c.’s, and even a three-gallon flush is not sufficient to prevent stoppages. A series of experiments were carried out by a committee of this Institute in 189i’o, which proved that even with a three-gallon flush, through a properly constructed straight drain 50 ft. long, (i ins. diameter, laid with a fall of 1 in 40, on trestles above ground, as the result of <)00 discharges, 27 per cent. of the solids were left in the intercepting trap after each discharge, and with a two-gallon flush 3.’) per cent., and the committee recommended three gallons as a minimum. More recently the experiments of Dr. Porter on an actual n-illch drain in connection with a factory, proved that tlle Intercepting trap could only be entirely cleared out by a six-gallon flush. by six gallon The great object uf a system of drains and sewers is to discharge the sewage at the outfall in the shortest possible time. ~Vhy, therefore, should we put an obstruction into every drain to defeat this object 0 p y j The intercepting trap is wrong in principle, and is no remedy for either a badly constructed drain, or sewer; it cannot protect the house from the action of a defective drain, and it is liable to be forced at any moment: the true safeguard to the house and its inhabitants being a. R. READ, A.M.I.C.E. During dry weather the contents of the trap are always more or less putrid, and contaminate both the drain and the sewer to which it is connected. , The Intercepting trap necessitates in the front of the house, near the front door or windows, the so-called fresh air inlet, winch acts alternately as an outlet and an inlet with every discharge from the drain. A mica or aluminium flap valve is generally fixcd to these inlets in order to prevent them acting as outlets, but the constant flapping action accom- panying every discharge from the drain, very soon damages the flap to such an extent, that it becomes useless, and the result is that in most cases the householders promptly stop up tlle opening, to get rid of the nuisance. When the houses are built close to the public footpath, the trap has to be fixed below the ground floor or basement of the house, unless the Sanitary Authority allow it to be fixed outside below the public footway. footway. The trap is liable to frequent stoppage, and is provided with all inspection chamber, or man-hole, to facilitate its clearance ; but tilese inspection chambers are, as a rule, only opened when a serious stopl>age occurs, wllich causes the sewage to show above ground. g g . The great majority of stoppages in modern drains occur at the inter- cepting trap, and many are unknown to the householder, because they clear themselves by the accumulation of sewage in the inspection chamber and drain, until a sufficient head of sewage is produced to force the obstruction through the trap; the result of these temporary stoppages being that the brickwork of tlle inspection chamber, of about four to six square yards area, and a considerable length of tlle drain, become 193 plastered with a slimy deposit of decomposing sewage. When the pressure of the head of sewage is not sufficient to force the obstruction, of course the sewage shows at the yard galleys, and men have then to be sent for to open and clear the drain. R. READ, A.M.I.C.E. sound drain laid with a good fall, gas-tight joints, and properly ventilated by a full size pipe to above the roof of the house. On such a drain the intercepting trap is a useless and dangerous obstruction, but its absence allows the drain to be laid with a better fall, to remove the sewage quickly while in a fresh state, and to keep itself clean ; tllis in turn acts 194 upon the sewers in a similar way, enabling the sewage to be discharged at the outfall before decomposition can take place. No sewer gas would then be generated, but only the watery vapour which rises from every wet surface, especially in dry weather, and this could then be easily dealt with Ov the ventilation of the sewers through the ventilated house drains. Neither the drains nor the sewers would be under any pressure, the traps at tlle waterclosets and gullies would be amply sumcient to protect the houses, and a general imIH’üB-el11ent in the health of the community would follow, because the frcsh vapour of the sewage would be promptly oxidised, in its initial stages, before it had time to become putrid or dangerous; the only ohject of sewer and drain ventilation, beyond relieving pressure, should be to deal with this fresh vapour, and it should not be asked to do so after tllis llas been allowed tu develop into sewer ~as. DIr,. H. A]’}~-l&OElig;I> R( 1 E(, II LEt, ( BBu’:1 mina~’r), as one w lm Ililt! had a great deal of experience ill the sewerage of towns, the ventitatiun of sewers, :)nd the drainage of houses, observed that in considering this question two main prill- ciples must 1Iot be lost sight of : that prevention was better than cure, and that of the two evils-l’~(’:111(’ of sewer gas 01’ sewer air in our houses, ’l.G., in confined positions, and escape of sewer air in the streets, ~i.G., in not conuned pusitions-ilm latter was the less. Everybody was agreed on this. In the following remarks he would always assume, both in the case of public sewers and private drains, 1 hat they were properly designed, properly constructed, and properly maintained, as it was absolutely useless to argue the points on any other premises. Those who wished to conn-iuce them that the discon- necting trap was obsolete and had far better he abandoned, must. R. READ, A.M.I.C.E. in order to carry their case prove either the one or tlle other ot’ 1 he following conten- tions. They must either show that sewer gas was beneficial to health, or if that were incapable of pruof, they must clearly demonstrate that the air in the public sewers must always be at least is good as the air in house drains, and further, that the disconnecting trap must in every case prove an unmitigated nuisance. -If it could be shown that sewer gas was beneficial to hoalth-and here the negative proof could not he taken to establish positive evidence-the necessity for the disconnecting trap, and as a matter of fact of all other traps, dis- appeared. The diiference between the disconnecting trap and the traps in the house was one of degree only, the difference consisting in the depth of 195 the seal and the quantity of waste water retained. As the authors (at any rate one of them) llad frankly admitted that sewer gas was dangerous to health, it seemed perfectly logical that tlle3- should do all iu their power to prevent its being laid un systematically to our houses. In the absence of proof of the beneficial character of sewer gas, the opponents of the disconnecting trap had, therefore. to demonstrate that the air in public sewers could never be worse than the air in the house drain., and that further, every disconnecting trap must prove a very serious nuisance. To establish the first contention it would not do tu say tllat the air in the public ~mwl:s >niy7r~ be -is. good as in the house drain,,, as in that case it might also be considerably worse, and as it was generally the unexpected that 11appended, it was plainly our duty to guard against the pollution of the air in the house drains from t]1B’ public sewers. IIe then went. on to discuss the conditions in a public sewer that had been carefully constructed on the separate .system, and was carefully maintained, and showed that owing to the intermittent flow. especially in the more level parts of a. town. one could never make sure of the air in it being perfectly sweet and free from smell. R. READ, A.M.I.C.E. If sewer air carried pathogenic germs such as those of typhoid fever and of other preventable diseases, and if it carried volatile ptomaines, these would be more or less present in the air of public sewers, especially in large towns. With a house drain, especially where baths were in use, the case was altogether different, and llre it was not overstating the case to say that in a carefully constructed and rationally main1:l ined drain the air would be perfectly sweet. This was known to all those who bad frequently examined good house drainage systems. The contention that the air in public sewers must always be at least as good as the air in house drains could, therefore, not be established, and hence it seemed perfectly reasonable to establish n barrier between them. But if it could be shown that the particular form of barrier now universally adopted-the discon- necting tl·Vl)--V’i1S wrong in principle and must always be an unmitigated nuisance, it was high time to abandon it in favour of another form. The dis- connecting trap had now been in existence for about twenty-live years, and there must be at a moderate estimate millions of it in use. Was it imaginable iliat it’ it llad proved a failure in every caw they would still go on employing it’.’ ?~ On the contrary it wuuld have; and that deservedly, disappeared from the scene long ago. This contention was strongly supported by the Willesden experience, mentioned in the first paper. For if lle read the paper aright it was sllowu there that out of every 100 case· the trap had proved perfectly emcient in 9U, so that the chances were as 9 to 1 ia favour of the trap. This was, however, w-e11 on the road to dead certainty that it would prove perfectly satisfactory, and if for this reason they were bound to condemn it they would have to condemn every apparatus now manufactured. For there was nu such thing as an a.ppara- tus, that however improper its use would not get out of order. It was not Ins province to explain the failure in Willesden of every 10 traps out of 100, but he might say from his experience tllat where a carefully constructed disconnecting the seal and the quantity of waste water retained. R. READ, A.M.I.C.E. As the authors (at any rate one of them) llad frankly admitted that sewer gas was dangerous to health, it seemed perfectly logical that tlle3- should do all iu their power to prevent its being laid un systematically to our houses. In the absence of proof of the beneficial character of sewer gas, the opponents of the disconnecting trap had, therefore. to demonstrate that the air in public sewers could never be worse than the air in the house drain., and that further, every disconnecting trap must prove a very serious nuisance. To establish the first contention it would not do tu say tllat the air in the public ~mwl:s >niy7r~ be -is. good as in the house drain,,, as in that case it might also be considerably worse, and as it was generally the unexpected that 11appended, it was plainly our duty to guard against the pollution of the air in the house drains from t]1B’ public sewers. IIe then went. on to discuss the conditions in a public sewer that had been carefully constructed on the separate .system, and was carefully maintained, and showed that owing to the intermittent flow. especially in the more level parts of a. town. one could never make sure of the air in it being perfectly sweet and free from smell. If sewer air carried pathogenic germs such as those of typhoid fever and of other preventable diseases, and if it carried volatile ptomaines, these would be more or less present in the air of public sewers, especially in large towns. With a house drain, especially where baths were in use, the case was altogether different, and llre it was not overstating the case to say that in a carefully constructed and rationally main1:l ined drain the air would be perfectly sweet. This was known to all those who bad frequently examined good house drainage systems. The contention that the air in public sewers must always be at least as good as the air in house drains could, therefore, not be established, and hence it seemed perfectly reasonable to establish n barrier between them. But if it could be shown that the particular form of barrier now universally adopted-the discon- necting tl·Vl)--V’i1S wrong in principle and must always be an unmitigated nuisance, it was high time to abandon it in favour of another form. R. READ, A.M.I.C.E. The dis- connecting trap had now been in existence for about twenty-live years, and there must be at a moderate estimate millions of it in use. Was it imaginable iliat it’ it llad proved a failure in every caw they would still go on employing it’.’ ?~ On the contrary it wuuld have; and that deservedly, disappeared from the scene long ago. This contention was strongly supported by the Willesden experience, mentioned in the first paper. For if lle read the paper aright it was sllowu there that out of every 100 case· the trap had proved perfectly emcient in 9U, so that the chances were as 9 to 1 ia favour of the trap. This was, however, w-e11 on the road to dead certainty that it would prove perfectly satisfactory, and if for this reason they were bound to condemn it they would have to condemn every apparatus now manufactured. For there was nu such thing as an a.ppara- tus, that however improper its use would not get out of order. It was not Ins province to explain the failure in Willesden of every 10 traps out of 100, but he might say from his experience tllat where a carefully constructed disconnecting 196 trap had not fnlfilled its expectii ion tllis was in nl’al’ly ¡’B’ery case du<, t~o gross sanitary neglect Oil tlie part of someone. Granting, however, solely for the sake of argument, tliat a disconnecting trap was not a necessity, his experience told him that the arrangements of a cast iron drainage as suggested in the first paper, and especially as shon-n in the diagrams on the wall, would not give them reasonable security against the entrance of sewer air into their houses. R. READ, A.M.I.C.E. Even if all the house drainage systems in this country could he so well superintended that all the joints both helow and above ground were absolutely gastight and remained so for say 50 years, that all the ventilated ends of soil pipes were carried above the roof without bends so that an accumulation of rust and dirt in them did not block the passage of air through them after a few years, it was well known that the water seals of the traps in their liouses were subject to being broken either by pressure or siphon age, and that sewer air would escape at the point of least resistance wherever that happened to be, eitller inside or outside the house. If, for instance, the air in v long length of soil pipe was too heavy for tlle sewer gas to force, the latter would break through the water seals of the traps inside the house. Or, if the case was taken of a house let out on the Hat system and they assumed that one of them stood empty for some time. which was by no means an unusual thing, would not in such a ease, after tlie water in the traps had evaporated, sewer gas enter and unknown tu the other inhabitants roam through the whole building without check or lcindrance ? ?? But apart from tlii;, what would be the responsibility of the authorities if it could be shown that sewer gas from the public sewers had entered a particular house and there caused serious or fatal illness? A case of tliis kind had been tried in August, 1896, at the Bil’l11ingham Assizes, in which judgment for ~2,875 had been given against an Urban District Council. This was not a discussion on the ventilation of sewers, hence he could not discuss tlie subject, but he might perhaps be permitted to say that it was well known now that there was no such tliing as constancy in the direction of the air currents in sewers. The movement of air i’ would at one time be up-hill and then change to a dOBn1-hill one, and the only thing t hat they could make sure of was continual change. R. READ, A.M.I.C.E. This held good not only for public sewers but also for private house drains, and in districts witllout disconnecting traps it had been amply shown that the air current was not per- manently in tlle direction from tlle public sewer to the llouse, but frequently changed into the opposite direction. In this connection he might mention the Bristol sewers, which were not ventilated at all, and wliere as had been observed there wras a strong down-hill current. It could, tlierefore, not be correct.ly maintained that in the absence of disconnecting traps the public sewers would always be ventilated by the private liouse drains. Was it not further also correct to say that should, in cases, the disconnecting trap prevent the yentila- tion of tlie public sewer through the house drain, its use was more than amply justified by considerations for tlle llealth of the inhabitants. From the fore- going remarks it would be clear, he hoped, that the contentions necessary fur trap had not fnlfilled its expectii ion tllis was in nl’al’ly ¡’B’ery case du<, t~o gross sanitary neglect Oil tlie part of someone. Granting, however, solely for the sake of argument, tliat a disconnecting trap was not a necessity, his experience told him that the arrangements of a cast iron drainage as suggested in the first paper, and especially as shon-n in the diagrams on the wall, would not give them reasonable security against the entrance of sewer air into their houses. Even if all the house drainage systems in this country could he so well superintended that all the joints both helow and above ground were absolutely gastight and remained so for say 50 years, that all the ventilated ends of soil pipes were carried above the roof without bends so that an accumulation of rust and dirt in them did not block the passage of air through them after a few years, it was well known that the water seals of the traps in their liouses were subject to being broken either by pressure or siphon age, and that sewer air would escape at the point of least resistance wherever that happened to be, eitller inside or outside the house. If, for instance, the air in v long length of soil pipe was too heavy for tlle sewer gas to force, the latter would break through the water seals of the traps inside the house. R. READ, A.M.I.C.E. From the fore- going remarks it would be clear, he hoped, that the contentions necessary fur 197 the abandonment of the disconnecting trap could nut be su(-cesSfLI111- maintained, and the position so far taken up 1>3- owners and occupiers of houses, that they with the use of the disconnecting trap should see to the ventilation of the house drainage system, and tliat the authorities must provide for ille ventilation of the street sewers without recourse to the house drains would, therefore, seem per- fuctly logical, hence reasonable and justifiable. Engineers were accustomed to provide in all their constructions a factor of safety, that is, they designed their structures so that they could withstand a strain of from four to six times greater than the calculated one, and this was for the express purpose of meeting unfore- seen accidental conditions over which they had no control. ,Vb.&dquo; should they abandon a factor of safety in sanitary works, where it was doubly necessary ? ’1’be disconnecting trap properly constructed and rationally maintained provided such a factor of safety, which was convenient and cheap, and he hoped it would not be abandoned except under very special circumstances. 1V as not an English- wan’s home liis castle, and why should they allow its invasion by sewer gas any more than by anything else ? Du. CH,BRI,ES SANDERS (-~1’est Ham) said lie came with the object of lending liia support, from the experience of West I-Ial, to the views enunciated by Dr. Butler and Mr. Read, but after the remarks of the last speaker lie felt lie should apologize for representing the views of a large town with apparently very inferior drainage. For if it were true, as stated, that a drain properly laid with an interceptor, inlet and outlet ventilators only contained pure air and did not stink, then lie found it impossible to explain the reason wliy the drains laid by his colleagues the Inspectors of _B ui.ances, whose training the Royal Institute vouched for and whose ability and care he himself could vouch fur, did in numerous cases result in most obnoxious odours at the so-called fresh air inlet- a condition generally remedied IIY the occupier who blocked it up. lie wuuld ask someone to describe the difference between drain air and sewer air. R. READ, A.M.I.C.E. Or, if the case was taken of a house let out on the Hat system and they assumed that one of them stood empty for some time. which was by no means an unusual thing, would not in such a ease, after tlie water in the traps had evaporated, sewer gas enter and unknown tu the other inhabitants roam through the whole building without check or lcindrance ? ?? But apart from tlii;, what would be the responsibility of the authorities if it could be shown that sewer gas from the public sewers had entered a particular house and there caused serious or fatal illness? A case of tliis kind had been tried in August, 1896, at the Bil’l11ingham Assizes, in which judgment for ~2,875 had been given against an Urban District Council. This was not a discussion on the ventilation of sewers, hence he could not discuss tlie subject, but he might perhaps be permitted to say that it was well known now that there was no such tliing as constancy in the direction of the air currents in sewers. The movement of air i’ would at one time be up-hill and then change to a dOBn1-hill one, and the only thing t hat they could make sure of was continual change. This held good not only for public sewers but also for private house drains, and in districts witllout disconnecting traps it had been amply shown that the air current was not per- manently in tlle direction from tlle public sewer to the llouse, but frequently changed into the opposite direction. In this connection he might mention the Bristol sewers, which were not ventilated at all, and wliere as had been observed there wras a strong down-hill current. It could, tlierefore, not be correct.ly maintained that in the absence of disconnecting traps the public sewers would always be ventilated by the private liouse drains. Was it not further also correct to say that should, in cases, the disconnecting trap prevent the yentila- tion of tlie public sewer through the house drain, its use was more than amply justified by considerations for tlle llealth of the inhabitants. 311t. C. CHAMBERS SMITH (Engineer and Surveyor to the Sutton LTrban District Council) said he could not agree with the first two speaker, and after Mr. Roechling’s remarks lie thought it would be agreed that the bottom liad so far been knocked out of their argument. The main objection to the intercepting trap was that it got blocked up. Well, there was no apparatus t hat did not. An intercepting trap was just as necessary as a gully trap, and which of them would say that because that got Mocked up it should bevaholisliecl’? ’? As to the fresh-air inlet venting on to the windows of houses, that objection was met by carrying the pipe higher up the side of the building. Another point was that they had not yet proved the necessity of ventilating sewers. The danger of abolishing intercepting traps was one that they should not cncounter. The risk attendant upon having sewer gas laid (.0iistantly on to houses, which would be the case if intercepting traps were abolished, was too serious to be lightly undertaken, and, as everyone knew, it was impossible to guarantee that a drain passed as sound would remain so for two years; therefore wliere, as fur instance, drains ran under liouses, the escape of sewer air could and would go on until it produced disease and possibly death. It was suggested that with free ventilation of sewers, the offensiveness and danger of sewer air would be practically abolished, hut this could not be supported, and was not in accord- ance B%,itli facts. In the upper reaches of sewers, where tlie sewage was fresh, the odour was not so intolerable ; but when sewage had travelled some dii-5tance in the sev-er it became putrescent. No amount of ventilation could destroy its off(,iisiveii(~.,4s, and one had only to remember as a caw in point the con- dition of an absolutely open sewer, such as the River Aire at Leeds, to rccognise that neither dilution nor oxidation could destroy such conditions. It therefore meant that houses adjacent to such sewers would be-as regards danger and offensiveness from sewer air being laid on to tbem-in an intolerable position. R. READ, A.M.I.C.E. Persun- ally he did not recognise an3- difference, for under the Public IIealth Act, 1875, y soon as two houses were drained by a single pipe, tlm pipe became a sewer ; yet the advocates of the interceptor did not feel it incumbent upon them to place one at the top end of such a sewer, but allowed several houses to drain into one pipe sewer, and placed the interceptor between the sewer in the road and the sewer on private property, thercbv alluwing the sewer air above the interceptor r to escape freely into the house drain at the top end. Dr. Butler liad sliown what actually did happen when intercepting traps were used; the only argument adduced in opposition to the abolition of interceptors consisted of opinions as to what might or would happen if they were abolished ; but no instances of injury to health or nuisance were found in connection with tlie thousands of drains which had no interceptor. He trusted the result of the discussion would be to frighten away the bogey of the assumed danger arising from the ventilation of sewers by means of the house drains. 198 trap was with dimculty cleared by means of the quantity discharged from a t wo-gallon cistern, it must be impossible for this action to take place in regard to tlle largpr intercepting trap placed at so great a distance as :31) feet away, with an approach of 1 in :in instead of a vertical one. The water passed through :t trap by displacement ; if the velocity was small, one could easily imagine the water passing through, and the tloating or solid matter being intercepted or held back by the obstructing trap. Any noxious gas generated by the matter ia the trap would eitlier remain in the house drain or escape by the so-called air inlets or air outlets. He could confirm the writers off the articles In their condemnation of the air inlets, which were placed immediately under windows and in positions where children might use them and tamper witli the mica nap as if it were something placed there for their amusement. The trap inter- cepted the gases from tlle sewer; he believed it did this effectively. If this was the object, then it was not a failure. But if the trap was not in existence, the gas would still be intercepted from the house by tlte trap at the w.c. and gully. Then plice an interceptor in the sewer? The answer to tllis had been that there were people who did not use the gullies, w.e.’s, etc., and the water evapo- rated and tlie trap became an open ventilator, whereas the interceptor would always be full of water, and ofte»t1ul<,s clean water, from the rain-water that entered the drain, so that if the v-.c,’~ were not used, this trap was placed there in a position where it would always have water, and where there could be little or no evaporation. Many said that it was impossible to become infected with disease from sewer gas when it passed through the air, while others had agreed that there v-as danger, so the ordinary man in the street knew not what to do, and he took double precautions-a; in this case he thought two llnterceptors were better than one. ’ 3In. OTILLI_1BI GnLw (hinsbur3yj said Ue advocated the abolitiou of the t rap. Any noxious gas generated by the matter ia the trap would eitlier remain in the house drain or escape by the so-called air inlets or air outlets. He could confirm the writers off the articles In their condemnation of the air inlets, which were placed immediately under windows and in positions where children might use them and tamper witli the mica nap as if it were something placed there for their amusement. The trap inter- cepted the gases from tlle sewer; he believed it did this effectively. If this was the object, then it was not a failure. But if the trap was not in existence, the gas would still be intercepted from the house by tlte trap at the w.c. and gully. Then plice an interceptor in the sewer? The answer to tllis had been that there were people who did not use the gullies, w.e.’s, etc., and the water evapo- rated and tlie trap became an open ventilator, whereas the interceptor would always be full of water, and ofte»t1ul<,s clean water, from the rain-water that entered the drain, so that if the v-.c,’~ were not used, this trap was placed there in a position where it would always have water, and where there could be little or no evaporation. Many said that it was impossible to become infected with disease from sewer gas when it passed through the air, while others had agreed that there v-as danger, so the ordinary man in the street knew not what to do, and he took double precautions-a; in this case he thought two llnterceptors were better than one. ’ soil pipe was desirable and necessary -especially was this the case if there were series of w.c.’s one above the other; if one should be untrapped by the working of the v-.c. on a higher tloor, this trap would prevent the gases entering tlie house. The interceptor at the w.c. was properly worked and was sitisfactoi-3, every time it was used it was emptied of the previous contents. The only methods of clearing an intercepting trap was to send down a la,rge quantity of water in a short time-more than ever would go down from a single w.c. tf the w.c. The objection to intercepting traps had been raised, lie admitted, by municipal engineers in consequence of their inability to ventilate the sewers by them- selves, and medical officers of health had hitherto, and riglitly so, in liis opinion, been adverse to their abolition. It was not, however, the duty uf occupiers and owners to ventilate public sewers, and local authorities should find a proper remedy and not risk the menace to health which would be intru- duc·ed if Intercepting traps on house drains were abolished. ltn. F. Woon ( I&dquo;Lliliam) said that, viewing the matter broadly, he thought t he majority of those present would agree with what the authors of these articles had said. They bad, however, to view this question from the&dquo; exception&dquo; point of view. Intercepting traps, if used properly and judiciously, were not a failure. Whem the trap was a failure, it was for the reason that its application was at fault. The w.c. was a trap, but no one would suggest that this should be dis- pensed with, and the pan left freely open to the main sewer. If the soil pipe from the w.c. had nu nnti-sipl~on pipe, then the interceptor at the foot of the 199 soil pipe was desirable and necessary -especially was this the case if there were series of w.c.’s one above the other; if one should be untrapped by the working of the v-.c. on a higher tloor, this trap would prevent the gases entering tlie house. The interceptor at the w.c. was properly worked and was sitisfactoi-3, every time it was used it was emptied of the previous contents. The only methods of clearing an intercepting trap was to send down a la,rge quantity of water in a short time-more than ever would go down from a single w.c. tf the w.c. trap was with dimculty cleared by means of the quantity discharged from a t wo-gallon cistern, it must be impossible for this action to take place in regard to tlle largpr intercepting trap placed at so great a distance as :31) feet away, with an approach of 1 in :in instead of a vertical one. The water passed through :t trap by displacement ; if the velocity was small, one could easily imagine the water passing through, and the tloating or solid matter being intercepted or held back by the obstructing trap. If the intercepting trap were abolished ventilation could be improved by means of ventilating pipes or soil pipes being anixed to the drain of each house, whereby the gases generated in the sewers by decomposition would not only be carried away more quickly but would be distributed over a greater area. If sewer air was so bad they should protect themselves against it, and this could well be done by having a good drain, tested with water when con- structed. lie did not consider an intercepting trap was a necessary appliance, and if tlie drains were properly ventilated they would be much better without interceptors. 200 (1) 1B1 H. GEO, XI. PHT’riT (Kensington) said he should like to ask a few questions. Would Dr. Butler tell them :--If, in that part of the district where diphtheria was most prevalent, the houses were flooded by storm mater from the sewers during heavy rains. g y Dr. Bl-TLlm replied that that portion of the district referred to was one that was least Hooded by storm water during heavy rains. y (2) In how many cases were the traps that were found to be choked, level traps, or ti-al), that had a cascade i~ lie asked this question because he believed that traps with a cascade impede the velocity and are the cause of many stoppages in the trap, especially in small tenement lcouses where hard paper was used. The paper opened widu in the water in the trap, and the next flush of water instead of sending the paper through the trap, fell on the outer edge of the paper and turned it over and over piece after piece until a ball was formed, with the result that the drain was choked. Many old drain. had been opened up and an old fashioned siphon found placed therein, no one knew it was there until the drain had been exposed for the purpose of fixing a trap in it. This bore out his statement that traps with a cascade were at fault. DR. Bl&dquo;’uF:R replied he was sorry he was unable to supply that information a note not having been made at the time. Dit. SYDNEY DA YIES (Woolwich) who was unable to attend, wrote saying he was decidedly opposed to the general abolition of intercepting traps. Baldwin Latham as follows : If they allowed large volumes of pure air to pass through a sewer in contact with sewage, they had large volumes of foul air escaping at some point, and that the great secret in sewer ventilation was not to encourage these currents of air through sewers, but to give such an amount of vent as should prevent any pressure being exercised upon the traps of their houses, so as not to allow any escape into the hoiise it5elf.&dquo; He did then, and still endorsed this view, which he thought had of late years gained very general acceptance. Dr. Butler assumed just the opposite, that sewer gas could be made innocuous simply by unlimited ventilation. Let him examine any slowly flow-ing dirty ditch which was altogether exposed to the open air, and note whether this complete ventilation abolished smells. No doubt it would be better if intercepting traps could be dispensed with, but as long as the sewers were worse laid and worse t1usl1l’d than the house drains they were a necessity, and to asl; for their removal in order to improve the .sewers was like a man asking lris adversary to lay down his armour in order that the t0er11i111’s wrath may be appeased. 3IR. P. SAL’xuERS C’roydon) said he should like to say a few words on a point not mentioned by the previous speakers. Dr. Butler did not give them. as a result of his very exhaustive inquiries, the number of ventilation pipes that were found stopped ; Dr. Butler was, he understood, an advocate for the abolition of the disconnecting trap, and Dr. Saiiders was of the same opinion, ¡I because they stink.&dquo; Now in his experience, he had invariabl3- found when an oflensive smell was emitted from the fresh air inlet, it was because the ventilation pipe at tlte end of the drain was obstructed, or was too small. Years ago many 2 in. and 2~ in. zinc vent pipes were fixed, these were now frequently found bent over at the top, effectually sealing them ; also the more modern 4 in. iron pipes are found completely stopped with rust, etc , in the bend at tlie foot. ln these cases would it be advisable to do away witli the disconnecting trap ’’ He thought not: u. any defect in the drain under the floor. If all sewers were perfect in construction, perfect in form, and perfectly fiuslled there would be no need for the intercepting trap, which would in that case be nothing but an obstruction to tlie removal of house drainage, but he thought that until that ideal condition of the sewers arrived, of the two evils, escape of sewer gas in the neighbourhood of the houses, and the slight impediment to the getting away of house drainage offered by the trap, the first must be considered decidedly the gr<.ater. Occasionally no doubt some nuisance arose from an intercepting trap, but lie found, on careful euquiry, that in his borough any such nuisance was wcelltional. The Borough engineers who advocated the abolition of tlle trap did so, he understood, not so much because they considered it objectionable in itself, as because they wish to ventilate the sewers through the house drains. Their very reason then for asking for its abolition was an argument against the proposal. If the sewers needed so much ventilation, the sewer gas must be decidedly objectionable, and should not escape in the neigh- bourhood of houses. The object of the Borough Engineers should be not to obtain general ventilation of sewers, but to so improve the sewers, and so flush them, that no ventilation was needed except for those sewers in which men must work These of course must bu separately considered, and special means taken for their ventilation, but not by tapping them at every house. He was surprised to find tlrat Dr. Butler was a convert to the view generally held by the Borough 201 Engineers as to the desirability of making every house assist in ventilating the sewers. About ten years ago he (Dr. Davies) read a paper before the British Medical Association on Ventilation of Sewers, in which it was maintained that more ventilation did not mean less foul gas in the sewers, but rather the contrary. He quoted lB1r. or soil pipes iu t lie house, would certainly be more dangerous. MR. R. RE,B.V (Glouce~ter) remarked that the effect ot the interceptor was to cause an obstruction something like 8 ins. in depth in the flattest length of the drain, the result being that the sewage merely dribbled into one end uf the interceptor and out at the other. There was nu rush because the interceptor was at the end of a comparatively flat gradient, and as long as they had tliat almost stagnant sewage in the trap they would have sewer gas in the drain. The sewers were what the drains made them, and the unfortunate borough engineer lrad to waste water in flushing otherwise clean sewers, to try and 202 get rid of smells created in the drains by the interceptor: i it was like hunting a ghost, or will o’ the wisp, for an hour titter the smell would be as bad as before. If the valve oi’ the so-called air inlet held tight, the drain air was forced through the water seal of the trap by ever3‘ discharge from the Cll’1111, hut if it did not hold tight, a portion of the drain air must be forced through it in front of the door and windows, and the remainder through the trap into the sewer. On the sewer side, if’ the sewage in the sewer rose over the mouth of the drain junction, the sewer air was forced through the water seal of the trap, or blew out tlie stopper of the raking arm, to escape either at the fresh air inlet, at the soil pipe ventilator, or both. But omit 1he trap, and the drain could 11P given a better fall, the sewage would have ;1 clean run through both drain and sewer, with a good velocity, and the ventila- tion could be properly applied and adjusted. The trap in its present position was like a single policeman trying to stop an unruly crowd, he got knocked over, but its prototype the trap of the pan was in its proper position, and like a policeman directing the traflic quietly into its proper channel, that is, it prevented the drain air from passing through the w,c. pan, hy directing it up the soil pipe ventilator. Read had pointed out in his paper) was that upon the trap becoming choked, the head of sewage in the drain and manhole was sufficient after a time automatically to force the stoppage, and thus relieve the condition without anyone being the w iser. But the pollution of the site by the perco- lation of liquid sewage was an unsuspected danger that remained. He was aware of the fallacies that lurked in crude statistics, and when he came to the relative incidence of diphtheria upon the respective areas in his district in which the drains were and were not equipped with interceptors, he wished to give the facts only for what they were worth. But the facts were that in the older part of the district, built for the most part prior to 1875, where the housing con- ditions generally were much lllore insanitary than in anytlling to be found in the never part, and where the general death-rate was considerably higher, the incidence of diphtheria had been materially less. If there was one disease more than another supposed to be associated with emanations from drains and sewers, that disease was diphtheria. And the fact was tllat in Willesden diphtheria bad by far its greatest incidence upon the population living in houses provided with interceptors, notwithsianding that generally their sanitary conditions were otherwise greatly superior to those so unprovided. In the one class, that pro- vided with interceptors, there was a particular provision in the form of an untrapped opening to the drain, situated near the ground level, in the forecourt, by which drain air and, very frequently, sewer air were laid on at the thresholds of the houses. In the other, no such ingenious device for polluting the atmo- sphere of tlle house existed. The different rates of incidence of diphtheria might not be significant of this difference, but their causal correlatiun would only be in accord with prevailing notions as to the relationships of diphtheria and exposure to the emanations of drains and sewrs. What lie wished particularly to insist upon, however, was tluit tlle interceptor and its appurtenances, when critically examined, were found tu be devices for doing what they aimed at avuiding, namely, providing an escape near tlie openings to a house of tlle emanations from drains alld sewers. DIx. BV, D. With the interceptor in its present position they could never secure proper or efficient ventilation, for in order to thoroughly control the ventilation of a sewer, the number of outlets must be largely in excess of that of the inlets, and so distributed over the whole system, that the manhole gratings on the surface of i he road should always be marle to act as inlets, by the combined action of the soil-pipe ventilating outlets. DR. BUTLER wished to join witll )11’, Read in expressing his appreciation of the manner in which their papers had been received. They could not culu- plain of lack of criticism, but that criticism had left him more convinced than ever of the truth of the position he had taken up. It was true that if lB1r. Roechling’s aaudard of what would suflice to condemn the interceptor were adopted (namely, that in cucr~ case it must be proved an unmitigated nuisance) their task was a difficult one. But he submitted that the failure of the dis- connecting trap to achieve its object in so large a proportion of cases as he had been able to establish, and its direct responsibility for the gross insanitary conditions he had observed, constituted a serious impeachment of the system. Mr. Roechling bad urged that in the absence of the trap, sewer gas was system- atically laid on to the houses. His (Dr. Butler’s) position was a denial of this proposition, while he afflrmed that the trap and the complications it necessitated mere a contrii-ance by which sewer ga. actually was laid on to the houses in an ascertained 10 per cent. of the cases. It was a pure assumption that drain air and sewer air were essentially different, and that the one was necessarily less dangerous than the other. As Dr. Sanders in his remarks had pointed out, they were discernibly the same, even while subject to distinction merely by a legal quibble. To admit the aerial contents of the sewer to gas-tight house 203 drains with no untrapped openings was not, Ile maintained, to lay on sewer gas to the hoiis(-.,;, as had been contended. This assumption was at the root of tll objections to doing away with this insanitary contrivance. So far from the intercepting trap being a tell-tale, as had been suggested, what so frequently happened (as Mr. Butler for the investigation at Willesden, which involved no less than 6,745 inspections, it is obvious that even if the number had been doubled it would not have done any- thing 1 o prove tlie emeacy of their alternative proposal. The defective character of one system does not by itself prove the efficacy of another. My own ex- perience now goes back as far as an inspection of the original arrangement by the late Dr. Fergus at his own house in Glasgow, from which -:B11’. Buchan designed Ins wetl-known trap. This established the principle of house discunnec- tion which 1. had already advocated. and which has since been almost universally accepted. Therr is not a shred of evidence in either or the papers i u show that in certain states of the atmosphere there would be any movement whatever through the house drains and ventilating pipe, uf the emanations frum the sewers, or that the street gratings would, under certain conditions prevailing during calm weather, be inlets for fresh air any more than they are at present. Under such conditions the emanations, however objectionable they may be, are at any rate better removed by the width of the pavement and half the width of the street from the houses, than hanging about the roof of the house itself and contaminating the atmosphere in the neighbol1rhood of bedroom windows. It is a matter for regret that Dr. Butler did not take the opportunity presented by the investigation at Willesden to provide information as to the causes of the large percentage of choking which occurred in ilie disconnecting traps. Pre- sumably the houses are occupied by people who are not very particular about their drains, and certainly six-inch pipes are too large for that class of property, The general experience with regard to properly constructed disconnecting traps is that they contain clear water after they have been adequately Hushed, and, in public sewers that is unsafe to haBe in the house drains. Now this is simply nut the case. The unventilated condition of town sewers is universally recog- nised as being fur the most part unsatisfactory, and the trouble is how this defect may be remedied, The problem has proved difficult to solve, and until 1 he remedy has been applied, the practice has been quite a sound one, of making sure that the conditions prevalent in the sewers should not be allowed to invade the interior of the dwelling. This position in the judgment. of tlie great majority of experts is the best available, and it has been developed at an enormous cost to the community. The remedy advocated by the two writers of the papers is that of doing away with disconnecting traps, as an end of the trouble. The question is, have they proved their case ? Proving the existence of defects in the system now in vogue by no means establishes their proposal for the abolition of disconnecting traps as a certain remedy. The problem of sewer ventilation is ~o complicated and su difficult of solution t’hat it will be necessary for the writers to be able to point to a case carried out on a largo .scale not even upon average, but under exceptionally llnfavourable condi- tions. in which the success of their proposals has been proved to the hilt ; so far as the papers are cOl1cl’rned, they have not referred to a single case in which they have even been tried. While giving every credit to Dr. Butler for the investigation at Willesden, which involved no less than 6,745 inspections, it is obvious that even if the number had been doubled it would not have done any- thing 1 o prove tlie emeacy of their alternative proposal. The defective character of one system does not by itself prove the efficacy of another. My own ex- perience now goes back as far as an inspection of the original arrangement by the late Dr. Fergus at his own house in Glasgow, from which -:B11’. Buchan designed Ins wetl-known trap. This established the principle of house discunnec- tion which 1. had already advocated. and which has since been almost universally accepted. 50UTT-OIOB’CltIE1’1’ (London) writes to express his regret tllat lie was unable to be present at the discussion upon the two papers by Dr. Butler and Mr. Read. which he considers at any rate courageous if they are not condu- sive. He thinks that in the first paper there are several statements that may be fairly met by a direct negative and a good many conclusions wbich are il no way justified by tlle facts. Dr. Butler says that the conception of a drainage system in which the drain is disconnected from the sewer is faulty in theory, because it is based on the assumption that it is safe to have a condition of things in the 204 public sewers that is unsafe to haBe in the house drains. Now this is simply nut the case. The unventilated condition of town sewers is universally recog- nised as being fur the most part unsatisfactory, and the trouble is how this defect may be remedied, The problem has proved difficult to solve, and until 1 he remedy has been applied, the practice has been quite a sound one, of making sure that the conditions prevalent in the sewers should not be allowed to invade the interior of the dwelling. This position in the judgment. of tlie great majority of experts is the best available, and it has been developed at an enormous cost to the community. The remedy advocated by the two writers of the papers is that of doing away with disconnecting traps, as an end of the trouble. The question is, have they proved their case ? Proving the existence of defects in the system now in vogue by no means establishes their proposal for the abolition of disconnecting traps as a certain remedy. The problem of sewer ventilation is ~o complicated and su difficult of solution t’hat it will be necessary for the writers to be able to point to a case carried out on a largo .scale not even upon average, but under exceptionally llnfavourable condi- tions. in which the success of their proposals has been proved to the hilt ; so far as the papers are cOl1cl’rned, they have not referred to a single case in which they have even been tried. While giving every credit to Dr. Therr is not a shred of evidence in either or the papers i u show that in certain states of the atmosphere there would be any movement whatever through the house drains and ventilating pipe, uf the emanations frum the sewers, or that the street gratings would, under certain conditions prevailing during calm weather, be inlets for fresh air any more than they are at present. Under such conditions the emanations, however objectionable they may be, are at any rate better removed by the width of the pavement and half the width of the street from the houses, than hanging about the roof of the house itself and contaminating the atmosphere in the neighbol1rhood of bedroom windows. It is a matter for regret that Dr. Butler did not take the opportunity presented by the investigation at Willesden to provide information as to the causes of the large percentage of choking which occurred in ilie disconnecting traps. Pre- sumably the houses are occupied by people who are not very particular about their drains, and certainly six-inch pipes are too large for that class of property, The general experience with regard to properly constructed disconnecting traps is that they contain clear water after they have been adequately Hushed, and, in 205 any case. th<> objectionable contents IIlt’l1ti()nt’d by the writers do not disappear by discharging into tlie sewers, where they will still engender smells and gases, which the writers propose to pass through the soil-pipes and ventilate at points much nearer to windows tlan the street gratings. The disappearance ot’ the plugs from the cleansing eyes of the disconnecting traps to which reference is made is a simple matter of carelessness. The objections raised to large man- holes has long been recognised..md are met by cast-iron manholes with covers at the bottom of the manhole. A much more serious question than the presence of offensive gases in the sewers is the system now in vogue of spraying enormous volumes of sewage into tlie air in districts that are densely populatecl.
https://openalex.org/W2732646669	https://europepmc.org/articles/pmc5499053?pdf=render	English	null	LncRNA-ANCR regulates the cell growth of osteosarcoma by interacting with EZH2 and affecting the expression of p21 and p27	Journal of orthopaedic surgery and research	2,017	cc-by	4,959	© 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. Zhang and Peng Journal of Orthopaedic Surgery and Research (2017) 12:103 DOI 10.1186/s13018-017-0599-7 Zhang and Peng Journal of Orthopaedic Surgery and Research (2017) 12:103 DOI 10.1186/s13018-017-0599-7 Open Access Keywords: LncRNA-ANCR, Osteosarcoma, EZH2, p21, p27 Keywords: LncRNA-ANCR, Osteosarcoma, EZH2, p21, p27 Keywords: LncRNA-ANCR, Osteosarcoma, EZH2, p21, p27 Long non-coding RNAs (lncRNA) are a group of non- protein coding RNAs with a length longer than 200 nucleotides [7], which is different from that of short interfering RNAs (siRNAs), microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs), small nucleolar RNAs (snoRNAs), and other short RNAs. Previous study has shown that the expression levels of many lncRNAs were altered in OS tissue [8]. A recent study reported that downregulation of lncRNA TUG1 reduced the prolifera- tion rate and increased apoptosis rate in OS cell [9], which indicated that lncRNAs may be considered as bio- markers for the diagnosis of OS and potential molecular targets for the treatment. * Correspondence: owujnl@sina.com Department of Orthopaedics Surgery, Ren Min Hospital of Wuhan University, Wuhan 430060, Hubei, China Abstract Background: Osteosarcoma (OS) is one of the most common malignant tumors developed in the bone. EZH2 has been found to play pivotal roles in the development of various cancers. LncRNA-ANCR (anti-differentiation ncRNA) has been reported to interact with EZH2 and regulated osteoblast differentiation. Our study aimed to investigate the effect of lncRNA-ANCR on the tumorigenesis of osteosarcoma and explore the underlying molecular mechanism. Methods: RT-PCR was performed to detect the messenger RNA (mRNA) levels of lncRNA-ANCR, EZH2, p21, and p27 in OS tissues and cell lines. The cell proliferation, transwell invasion, and migration assays were conducted to evaluate the influence of lncRNA-ANCR depletion on the growth of OS cells. RNA pull-down assay was carried out to detect the interaction between lncRNA-ANCR and EZH2. Correlation between the expression of lncRNA-ANCR and the expression of EZH2 were analyzed by cross-tabulation. Results: LncRNA-ANCR is highly expressed in both OS tissues and cell lines. Reduced expression of lncRNA-ANCR inhibited the cell proliferation, invasion, and migration of OS cells. The cell apoptosis rate was also increased with the overexpression of lncRNA-ANCR. Mechanistically, downregulation of lncRNA-ANCR reduced the mRNA level of EZH2 and increased the expression of p21 and p27 at both mRNA and protein levels. LncRNA-ANCR interacted with EZH2 and their expression abundance was positively correlated in OS patients. Conclusion: LncRNA-ANCR inhibited the cell proliferation, migration, and invasion of OS cells possibly through interacting with EZH2 and regulating the expression of p21 and p27. Keywords: LncRNA-ANCR, Osteosarcoma, EZH2, p21, p27 Background Osteosarcoma, which is also called osteogenic sarcoma (OGS), is one of the most common malignant tumors developed in bone. OS accounts for about 60% cases of all the bone cancerous tumors in both adolescence and childhood [1–3]. The patients with OS usually have a high incidence of lung metastasis, and the 5-year sur- vival rate is less than 30% for patients suffering OS com- bined with lung metastasis [4–6]. Therefore, identifying the molecular targets involved in the development of OS and developing treatment strategies is quite necessary. Enhancer of Zeste Homolog 2 (EZH2), a histone meth- yltransferase, has been found to play pivotal roles in the development of various cancers [10, 11]. EZH2 is highly * Correspondence: owujnl@sina.com Department of Orthopaedics Surgery, Ren Min Hospital of Wuhan University, Wuhan 430060, Hubei, China Zhang and Peng Journal of Orthopaedic Surgery and Research (2017) 12:103 Zhang and Peng Journal of Orthopaedic Surgery and Research (2017) 12:103 Page 2 of 7 Mix (Life Technologies, 4486295, USA). GAPDH was used as an endogenous control. The following primers were used: lncRNA-ANCR-forward: 5′-GACATTTCCT GAGTCGTCTTCGAACGGAC and reverse: 5′- TAGT GCGATTTAGAGCTGTACAAGTTTC; p21-forward: 5′- TCTGGGGTVTVACTTCTTGG and reverse: 5′-AT GTGAGGAAGGCTCAGTGG; p27-forward: 5′-GATG GGGTTCACCGTGTTAG and reverse: 5′-CCCTTTCC AAACATCCATTG; EZH2-forward: 5′-TTGTTGGCGG AAGCGTGTAAAATC and reverse: 5′-TCCCTAGTCC CGCGCAATGAGC; GAPDH-forward: 5′-CGAGCCAC ATCGCTCAGACA and reverse: 5′-GTGGTGAAGACG CCAGTGGA. Relative RNA level was calculated using 2ΔΔCt method. expressed in solid tumors to initiate cell proliferation [12], and the high expression of EZH2 is negatively cor- related with the patients’ outcome [13, 14]. It has been reported that overexpression of EZH2 increased the tri- methylation of H3K27 on the promoters of p21, which facilitated the p53 binding on the promoter and acti- vated the expression of p21 [15]. Additionally, p27 was also negatively regulated by EZH2 [16, 17]. These find- ings provided important cues to understand the molecu- lar mechanism of EZH2 in cancer development. expressed in solid tumors to initiate cell proliferation [12], and the high expression of EZH2 is negatively cor- related with the patients’ outcome [13, 14]. It has been reported that overexpression of EZH2 increased the tri- methylation of H3K27 on the promoters of p21, which facilitated the p53 binding on the promoter and acti- vated the expression of p21 [15]. Additionally, p27 was also negatively regulated by EZH2 [16, 17]. These find- ings provided important cues to understand the molecu- lar mechanism of EZH2 in cancer development. Cell culture Human OS cell lines and osteoblast cell line hFOB1.19 were purchased from American Type Culture Collection (Rockville, MD, USA) were cultured in DMEM medium containing 10% FBS at 37 °C in a humidified atmosphere with 5% CO2. In this study, to explore the function of lncRNA- ANCR in OS, the expression level of lncRNA-ANCR in both OS tissues and cell lines were determined. High ex- pression abundance of lncRNA-ANCR in OS was ob- served. Downregulation of lncRNA-ANCR inhibited the cell proliferation of OS cells. Further investigation found that depletion of lncRNA-ANCR suppressed the expres- sion of EZH2 and activated p21 and p27. Positive correl- ation between the expression of lncRNA-ANCR and EZH2 was also observed in OS patients. Transfection MG-63 and UMR-106 cells were seeded into the 6-well plates with 1 × 105 cells per well. Cells were cultured in DMEM medium containing 10% FBS until 70% con- fluence was observed. Transfection was performed with Lipofectamine 3000 (Invitrogen, L3000015, USA) ac- cording to the manufacturer’s instruction. Cells were collected after transfection for 72 h and were subjected to the following experiments. Lnc-ANCR siRNA design and preparation was performed by GenePharma Co. Ltd (Shanghai, China). Cell proliferation assay Cells were seeded in 96-well plates with 3 × 103 cells per well. Cell Counting Kit-8 (CCK-8) was used to detect the viability of cells according to the manufacturer’s in- structions. The abundance at 450 nm was measured. The experiment was performed with three replicates. Clinical patients and OS tissues The tumor specimens were obtained from 20 patients with OS who underwent resection between July 2015 and March 2016 during their hospitalization in Wu Han University affiliated hospital. The tissue samples of OS patients were graded and staged by experienced patholo- gists after tissues were collected. According to the med- ical records, 14 patients have been diagnosed with cancer metastasis among the 20 OS patients. At the same time, 20 patients with lumbar discectomy under- went resection were also selected as the control group. Ethical approval was obtained by Wu Han University affiliated hospital. We had all the necessary consent from any patients involved in the study, including con- sent to participate in the study where appropriate. Western blot Cells were lysed with the RIPA buffer and then centri- fuged at 15,000 g for 15 min at 4 °C. The protein con- centration was measured using BCA protein assay kit (Sigma, pierce23225KIT, USA). Antibodies for EZH2 (#4902), p27 (#2552), p21 (#2947), and GAPDH (#2118) were purchased from Cell Signaling Technology. Background LncRNA-ANCR (anti-differentiation ncRNA) has been demonstrated to be involved in regulating osteoblast dif- ferentiation [18], however, the function of lncRNA- ANCR in osteosarcoma remains largely unknown. Add- itionally, it has been documented that lncRNA-ANCR interacted with EZH2 to regulate the differentiation of osteoblast. In view of the function correlation of EZH2 and lncRNA-ANCR, we hypothesized that lncRNA- ANCR may play critical roles in OS. Cell migration and invasion assay OS cells transfected with the indicated plasmids were collected and suspended in serum-free medium. After that, cells were added to the upper chamber covered with matrix, the lower chamber was filled with medium containing 10% FBS. After incubation at 37 °C for 24 h, cells below the membrane were fixed and stained. The numbers of migrated and invaded cells were counted under a microscope. The procedure of cell migration assay is the same to that of the invasion assay. The only difference is that common transwell chambers were used instead of matrix-coated ones. RT-PCR Total RNA extraction was performed with RMeasy MiNi Kit (Qiagen, 74104, Germany). TransScript-Uni One- Step gDNA Removal and cDNA Synthesis SuperMix (ABSCI, AB452, USA) were used for reverse transcrip- tion. The RT-PCR was carried out with ABI 7500 Real- time PCR instrument and TaqMan Multiplex Master Fluorescence-activated cell sorting (FACS) for apoptosis 3 × 105 of OS cells with the indicated treatment were re- suspended to make the single cell suspension. Cells were stained with Fluorescein isothiocyanate-conjugated Annexin V and 7-AAD (4ABio, FXP027-100, China). Zhang and Peng Journal of Orthopaedic Surgery and Research (2017) 12:103 Page 3 of 7 Page 3 of 7 between lncRNA-ANCR and EZH2. The level of lncRNA-ANCR was examined by PCR analysis. Single staining of FITC and 7-AAD was used to set the parameters and the gate. The cell apoptosis rate was de- tected by the flow cytometer CytoFLEX (Beckman Coulter, Inc., Brea, CA, USA). Statistical analyses Data was analyzed with SPSS 19.0 software. Enumer- ation data were expressed as rate or percentage. Chi- square test was used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. Correlations between the ex- pression of lncRNA-ANCR and the expression of EZH2 were analyzed by cross-tabulation. P < 0.05 was consid- ered to be statistically significant. LncRNA-ANCR was highly expressed in OS tissues and cells RT-PCR was used to detect the expression level of lncRNA-ANCR in OS tissues and tumor-free tissues. Significant high expression level of lncRNA-ANCR was found in the OS tissues than that of the tumor-free tissues (Fig. 1a). In addition, the expression level of lncRNA-ANCR was also detected in a variety of human OS cells lines including MG-63, SW1353, U2OS, Saos2, 143B, Hos, MC3T3-E1, and UMR-106. As shown in Fig.1b, lncRNA-ANCR was highly expressed in OS cell lines in comparison with that of the osteoblast cell line hFOB1.19. These results suggested that lncRNA-ANCR was overexpressed in human OS. MG-63 and UMR-106 cell lines, which showed relatively higher expression levels of lncRNA-ANCR were selected for the following experiments. RNA pull-down assay In vitro transcription of lncRNA-ANCR was performed using T7 RNA polymerase (Ambio Life). The transcrip- tion product was purified using RNeasy Plus Mini Kit (Qiagen) treatment with DNase I (Qiagen). The purified lncRNA-ANCR was then labeled with biotin using biotin RNA Labeling Mix (Ambio Life). MG-63 and UMR-106 cells were harvested and lysed with the RIPA lysis buffer. 50 μl of the lysates were aliquoted as the input, and the remaining supernatant was incubated with biotin-labeled lncRNA-ANCR at 4 °C for 2 h. Afterwards, the M-280 Streptavidin beads (Invitrogen, CA, USA) was added into the supernatant. The mixture was incubated at 4 °C for 2 h. At the same time, beads incubated directly with the supernatant of OS cells in the absence of biotin- labeled lncRNA-ANCR were used as the negative con- trol. Western blot was performed to detect the binding Downregulation of lncRNA-ANCR inhibited the expression of EZH2 and activated p21 and p27 The inhibitory effect of lncRNA-ANCR downregulation on the growth of OS cells promoted us to determine the underlying molecular mechanism. It has been reported that lncRNA-ANCR interacted with EZH2 and regulated osteoblast differentiation [18]. To detect the function of lncRNA-ANCR in OS was associated with the EZH2, the endogenous expression of lncRNA-ANCR was de- pleted (Fig. 3a). We firstly examined the messenger RNA (mRNA) level of EZH2 in MG-63 and UMR-106 cells harboring downregulated lncRNA-ANCR. The result Downregulation of lncRNA-ANCR inhibited the cell growth of OS cells To determine the role of lncRNA-ANCR on the tu- morigenesis of OS cells, endogenous expression of Fig. 1 LncRNA-ANCR is highly expressed in human OS tissues and cell lines. a RT-PCT assay was performed to detect the expression of lncRNA- ANCR in OS patients and tumor-free patients. *P < 0.01. b The expression abundance in OS cell lines and osteoblast cell line hFOB1.19 were detected. P < 0.05 Fig. 1 LncRNA-ANCR is highly expressed in human OS tissues and cell lines. a RT-PCT assay was performed to detect the expression of lncRNA- ANCR in OS patients and tumor-free patients. *P < 0.01. b The expression abundance in OS cell lines and osteoblast cell line hFOB1.19 were detected. P < 0.05 Zhang and Peng Journal of Orthopaedic Surgery and Research (2017) 12:103 Page 4 of 7 Page 4 of 7 lncRNA-ANCR was downregulated by transfecting lncRNA-ANCR-siRNA into OS cells. As shown in Fig. 2a, b, depletion of lncRNA-ANCR significantly inhibited the cell proliferation rate of both MG-63 and UMR-106 cells compared with that of the control cells. In addition, the transwell invasion and migration ability of OS cells harboring downregulated lncRNA-ANCR was also obviously decreased (Fig. 2c, d). Consistent with the inhibitory effect of lncRNA-ANCR depletion on OS cells, both MG-63 and UMR-106 cells expressing lncRNA-ANCR siRNA presented a significantly in- creased cell apoptosis rate (Fig. 2e). The results sug- gested that downregulation of lncRNA-ANCR negatively regulates the growth of OS cells. showed that the mRNA abundance of EZH2 was signifi- cantly decreased in OS cells with the depletion of lncRNA-ANCR (Fig. 3b). Previous studies have reported that overexpression of EZH2 promoted cell proliferation via downregulation of tumor suppressors p21 and p27 [19, 20]. To determine the down-stream effect of the de- creased EZH2 by lncRNA-ANCR knockdown, the mRNA level of both p21 and p27 was detected. As shown in Fig. 3c, d, the mRNA abundance of p21 and p27 was significantly increased in MG-63 and UMR-106 cells with the depletion of lncRNA-ANCR. Western blot analysis also showed enhanced expression level of p21 and p27 in MG-63 cells with the downregulation of lncRNA-ANCR (Fig. 3e). Similar result was also ob- tained in UMR-106 cells (data not shown). These data suggested that downregulation of lncRNA-ANCR inhib- ited the expression of EZH2 and activated the expression of p21 and p27. LncRNA-ANCR interacted with EZH2 and the expression of lncRNA-ANCR is positively correlated with the abundance of EZH2 in OS To further understand the molecular mechanism by which lncRNA-ANCR regulates the expression of EZH2, RNA pull-down assay was performed to detect the inter- action between lncRNA-ANCR with EZH2. As shown in Fig.4a, biotin-labeled lncRNA-ANCR was incubated with Fig. 2 Downregulation of lncRNA-ANCR inhibited the growth of OS cells. a, b The proliferation of MG-63 (a) and UMR-106 cells (b) harboring depleted lncRNA-ANCR or control vector was detected by CCK-8 assay. P < 0.05. c The data of invasion assay of MG-63 and UMR-106 cells with different treatments. P < 0.05. d The migration ability of both MG-63 and UMR-106 cells with downregulation of lncRNA-ANCR or control vector was examined. P < 0.05. e The apoptosis rate of MG-63 and UMR-106 cells with indicated treatments. P < 0.05 Fig. 2 Downregulation of lncRNA-ANCR inhibited the growth of OS cells. a, b The proliferation of MG-63 (a) and UMR-106 cells (b) harboring depleted lncRNA-ANCR or control vector was detected by CCK-8 assay. P < 0.05. c The data of invasion assay of MG-63 and UMR-106 cells with different treatments. P < 0.05. d The migration ability of both MG-63 and UMR-106 cells with downregulation of lncRNA-ANCR or control vector was examined. P < 0.05. e The apoptosis rate of MG-63 and UMR-106 cells with indicated treatments. P < 0.05 Zhang and Peng Journal of Orthopaedic Surgery and Research (2017) 12:103 Page 5 of 7 Fig. 3 Downregulation of lncRNA-ANCR suppressed the expression of EZH2 and activated p21 and p27. a The downregulation of lncRNA-ANCR by siRNA interference was validated by RT-PCR. P < 0.05. b The relative mRNA level of EZH2 in cells with the indicated treatments was measured by RT-PCR. P < 0.05. c, d The mRNA level of p21 (c) and p27 (d) in MG-63 and UMR-106 cells expressing lncRNA-ANCR or control vector was detected. P < 0.05. e The protein abundance of p21 and p27 in OS cells with or without lncRNA-ANCR depletion Fig. 3 Downregulation of lncRNA-ANCR suppressed the expression of EZH2 and activated p21 and p27. a The downregulation of lncRNA-ANCR by siRNA interference was validated by RT-PCR. P < 0.05. b The relative mRNA level of EZH2 in cells with the indicated treatments was measured by RT-PCR. P < 0.05. c, d The mRNA level of p21 (c) and p27 (d) in MG-63 and UMR-106 cells expressing lncRNA-ANCR or control vector was detected. P < 0.05. LncRNA-ANCR interacted with EZH2 and the expression of lncRNA-ANCR is positively correlated with the abundance of EZH2 in OS e The protein abundance of p21 and p27 in OS cells with or without lncRNA-ANCR depletion Fig. 4 LncRNA-ANCR interacted with EZH2 and the expression of lncRNA-ANCR is positively correlated with that of EZH2. a RNA pull-down assay to show the interaction between lncRNA-ANCR and EZH2. b EZH2 is highly expressed in OS patients. P < 0.05. c The expression of lncRNA-ANCR was positively correlated with that of EZH2 Fig. 4 LncRNA-ANCR interacted with EZH2 and the expression of lncRNA-ANCR is positively correlated with that of EZH2. a RNA pull-down assay to show the interaction between lncRNA-ANCR and EZH2. b EZH2 is highly expressed in OS patients. P < 0.05. c The expression of lncRNA-ANCR was positively correlated with that of EZH2 Zhang and Peng Journal of Orthopaedic Surgery and Research (2017) 12:103 Page 6 of 7 Page 6 of 7 the supernatant of OS cell. The binding between lncRNA-ANCR with endogenous EZH2 was detected by western blot with anti-EZH2 antibody. The result showed that lncRNA-ANCR interacted with EZH2 (Fig 4a). We hypothesized that the interaction between lncRNA-ANCR with EZH2 may trigger the regulation of lncRNA-ANCR to EZH2. EZH2 promoted cancer cell proliferation via downregu- lation of tumor suppressors p21 and p27 [19, 20]. In this study, lncRNA-ANCR interacted with EZH2 and deple- tion of lncRNA-ANCR activated the expression of p21 and p27, we hypothesized that downregulation of lncRNA-ANCR may block the recruitment of EZH2 to the promoters of p21 and p27, which attenuates the negative regulation of EZH2 on p21 and p27. This hy- pothesis needs further validation. EZH2 promoted cancer cell proliferation via downregu- lation of tumor suppressors p21 and p27 [19, 20]. In this study, lncRNA-ANCR interacted with EZH2 and deple- tion of lncRNA-ANCR activated the expression of p21 and p27, we hypothesized that downregulation of lncRNA-ANCR may block the recruitment of EZH2 to the promoters of p21 and p27, which attenuates the negative regulation of EZH2 on p21 and p27. This hy- pothesis needs further validation. the supernatant of OS cell. The binding between lncRNA-ANCR with endogenous EZH2 was detected by western blot with anti-EZH2 antibody. The result showed that lncRNA-ANCR interacted with EZH2 (Fig 4a). We hypothesized that the interaction between lncRNA-ANCR with EZH2 may trigger the regulation of lncRNA-ANCR to EZH2. To further confirm the correlation between lncRNA- ANCR and EZH2, the expression level of EZH2 in OS patients was measured. Authors’ contributions Th The manuscript is an original work of ZF and PH. All data, tables, figures, etc. used in the manuscript are prepared by ZF and PH, otherwise the sources are cited and reprint permission is attached. ZF and PH read and approved the final manuscript. Conclusions LncRNA-ANCR was highly expressed in OS tissues and cells. LncRNA-ANCR depletion inhibited the prolifera- tion, invasion, and migration of OS cells. Downregula- tion of lncRNA-ANCR decreased the abundance of EZH2 and activated the expression of both p21 and p27. The interaction between lncRNA-ANCR with EZH2 in- dicated that lncRNA-ANCR might exert its function via binding to EZH2. High expression of lncRNA-ANCR suggested its clinical significance in OS. Availability of data and materials None. Availability of data and materials None. LncRNA-ANCR interacted with EZH2 and the expression of lncRNA-ANCR is positively correlated with the abundance of EZH2 in OS As presented in Fig. 4b, mRNA level of EZH2 was highly expressed in OS patients. The correlation analysis between lncRNA-ANCR and EZH2 showed that the expression of lncRNA-ANCR was posi- tively correlated with that of EZH2 (Fig. 4c, r2 = 0.7541, P < 0.0001). Consistent with the high expression of lncRNA-ANCR in OS patients, overexpressed EZH2 was also found in OS patients. Correlation analyses have shown that the expression of lncRNA-ANCR is positively correlated with that of EZH2. Further investigation is required to explore the upstream regulator that controls the high ex- pression of lncRNA-ANCR and EZH2 in OS. The over- expression of lncRNA-ANCR may be a promising target for the diagnosis and treatment in OS. Discussion As the most common primary bone tumor in both chil- dren and adolescents, OS is a devastating disease with- out accurate early diagnosis and efficient treatment method, which in turn leads to the low long-term sur- vival rate [21]. The increasing morbidity of OS has been observed during the past several decades [22]. Genetic regulation plays pivotal roles in the occurrence and development of cancer. Therefore, identifying the mo- lecular targets involved in the occurrence and develop- ment OS will benefit the diagnosis and treatment of OS. Increasing evidence has demonstrated the involvement of lncRNA in the initiation and development of OS [8]. In our study, we found that lncRNA-ANCR was highly expressed in OS tissues and cell lines. Downregulation of lncRNA-ANCR enhanced the cell apoptosis and inhibited the proliferation, migration, and invasion of OS cells. These data indicated the oncogenetic potential of lncRNA-ANCR in OS. Acknowledgements g Thanks to Wu Han University. Abbreviations CCK-8: Cell counting kit-8; DMEM: Dulbecco’s modified Eagle’s medium; FACS: Fluorescence-activated cell sorting; FBS: Fetal bovine serum; lncRNA: Long non-coding RNAs; miRNAs: MicroRNAs; mRNA: Messenger RNA; OS: Osteosarcoma; PBS: Phosphate-buffered saline; piRNAs: Piwi-interacting RNAs; RT-PCR: Real-time polymerase chain reaction; siRNAs: Short interfering RNAs; snoRNAs: Small nucleolar RNAs Previous studies have showed that EZH2 was over- expressed in a variety of human cancers, which en- hanced tumorigenesis through various signaling pathways [23]. In our study, downregulation of lncRNA- ANCR decreased the expression abundance of EZH2, suggesting that lncRNA-ANCR positively regulates the expression of EZH2. It has been reported that EZH2 promotes the cancer cell proliferation via suppressing the expression level of the cell cycle protein including p21 and p27 [19, 20]. Consistent with these finding, we found that downregulation of lncRNA-ANCR signifi- cantly increased the mRNA and protein expression of both p21 and p27. These results suggested that depletion of lncRNA-ANCR negatively regulated the EZH2-p21/ p27 signaling pathway. Previous studies have shown that LncRNA-ANCR interacted with EZH2, which is import- ant for osteoblast differentiation. Consistently, our data demonstrated the binding of lncRNA-ANCR with EZH2 in OS cells. It has been reported that overexpression of References 1. Link MP. Osteosarcoma in adolescents and young adults: new developments and controversies. Commentary on the use of presurgical chemotherapy. Cancer Treat Res. 1993;62:383–5. 2. Luetke A, Meyers PA, Lewis I, Juergens H. Osteosarcoma treatment—where do we stand? A state of the art review. Cancer Treat Rev. 2014;40:523–32. 2. Luetke A, Meyers PA, Lewis I, Juergens H. Osteosarcoma treatment—where do we stand? A state of the art review. Cancer Treat Rev. 2014;40:523–32. 3. Mirabello L, Troisi RJ, Savage SA. Osteosarcoma incidence and survival rates from 1973 to 2004: data from the Surveillance, Epidemiology, and End Results Program. Cancer. 2009;115:1531–43. 3. Mirabello L, Troisi RJ, Savage SA. Osteosarcoma incidence and survival rates from 1973 to 2004: data from the Surveillance, Epidemiology, and End Results Program. Cancer. 2009;115:1531–43. 4. Farfalli GL, Albergo JI, Lobos PA, Smith DE, Streitenberger PD, Pallotta Rodriguez MG, Aponte-Tinao LA. Osteosarcoma lung metastases. Survival after chemotherapy and surgery. Medicina (B Aires). 2015;75:87–90. 5. Ferguson WS, Goorin AM. Current treatment of osteosarcoma. Cancer Invest. 2001;19:292–315. 6. Jaffe N. Osteosarcoma: review of the past, impact on the future. The American experience. Cancer Treat Res. 2009;152:239–62. 7. Perkel JM. Visiting “noncodarnia”. Biotechniques. 2013;54:301. 303-304. 8. Zhou M, Zhao H, Wang Z, Cheng L, Yang L, Shi H, Yang H, Sun J. Identification and validation of potential prognostic lncRNA biomarkers for predicting survival in patients with multiple myeloma. J Exp Clin Cancer Res. 2015;34:102. 9. Zhang Q, Geng PL, Yin P, Wang XL, Jia JP, Yao J. Down-regulation of long non-coding RNA TUG1 inhibits osteosarcoma cell proliferation and promotes apoptosis. Asian Pac J Cancer Prev. 2013;14:2311–5. 10. Kim KH, Roberts CW. Targeting EZH2 in cancer. Nat Med. 2016;22:128–34. 11. Simon JA, Lange CA. Roles of the EZH2 histone methyltransferase in ca epigenetics. Mutat Res. 2008;647:21–9. 11. Simon JA, Lange CA. Roles of the EZH2 epigenetics. Mutat Res. 2008;647:21–9. epigenetics. Mutat Res. 2008;647:21–9. 12. Chang CJ, Yang JY, Xia W, Chen CT, Xie X, Chao CH, Woodward WA, Hsu JM, Hortobagyi GN, Hung MC. EZH2 promotes expansion of breast tumor initiating cells through activation of RAF1-beta-catenin signaling. Cancer Cell. 2011;19:86–100. 13. Kleer CG, Cao Q, Varambally S, Shen R, Ota I, Tomlins SA, Ghosh D, Sewalt RG, Otte AP, Hayes DF, et al. EZH2 is a marker of aggressive breast cancer and promotes neoplastic transformation of breast epithelial cells. Proc Natl Acad Sci U S A. 2003;100:11606–11. 14. Competing interests The authors declare that they have no competing interests. Consent for publication Permissions from all participants were obtained to publish the data. 20. Mattioli E, Vogiatzi P, Sun A, Abbadessa G, Angeloni G, Ugo DD, Trani D, Gaughan JP, Vecchio FM, Cevenini G, et al. Immunohistochemical analysis of pRb2/p130, VEGF, EZH2, p53, p16(INK4A), p27(KIP1), p21(WAF1), Ki-67 expression patterns in gastric cancer. J Cell Physiol. 2007;210:183–91. 21. Picci P. Osteosarcoma (osteogenic sarcoma). Orphanet J Rare Dis. 2007;2:6. 22. Caudill JS, Arndt CA. Diagnosis and management of bone malignancy in adolescence. Adolesc Med State Art Rev. 2007;18:62–78. IX. 23. Hibino S, Saito Y, Muramatsu T, Otani A, Kasai Y, Kimura M, Saito H. Inhibitors of enhancer of zeste homolog 2 (EZH2) activate tumor-suppressor microRNAs in human cancer cells. Oncogenesis. 2014;3:e104. Authors’ information M.D. Zhang Fei, the first author of this paper, is a clinical doctor of The Third Affiliated Hospital of Inner Mongolia Medical University. He is working under the guidance of Peng Hao in the Ren Min Hospital of Wuhan University. His work focuses on the basic research on orthopaedics. fjogph@sina.com. M.D. Peng Hao, the correspondence author of this paper, is the chief of clinical research lab in the Orthopaedics Department of Ren Min Hospital of Wuhan University. His research focuses on the osteosarcoma. owujnl@sina.com. Page 7 of 7 Page 7 of 7 Zhang and Peng Journal of Orthopaedic Surgery and Research (2017) 12:103 Page 7 of 7 18. Kretz M, Webster DE, Flockhart RJ, Lee CS, Zehnder A, Lopez-Pajares V, Qu K, Zheng GX, Chow J, Kim GE, et al. Suppression of progenitor differentiation requires the long noncoding RNA ANCR. Genes Dev. 2012;26:338–43. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Received: 8 April 2017 Accepted: 17 June 2017 References Varambally S, Dhanasekaran SM, Zhou M, Barrette TR, Kumar-Sinha C, Sanda MG, Ghosh D, Pienta KJ, Sewalt RG, Otte AP, et al. The polycomb group protein EZH2 is involved in progression of prostate cancer. Nature. 2002;419:624–9. 15. Chen YF, Pan KW, Wang PZ, Cao ZY, Wang WB, Wang SY, Hu NG, Xue JH, Li H, Jiang W, et al. HBP1-mediated regulation of p21 protein through the Mdm2/p53 and TCF4/EZH2 pathways and its impact on cell senescence and tumorigenesis. J Biol Chem. 2016;291:12688–705. 16. Kuroki H, Hayashi H, Okabe H, Hashimoto D, Takamori H, Nakahara O, Nakagawa S, Fukushima Y, Chikamoto A, Beppu T, et al. EZH2 is associated with malignant behavior in pancreatic IPMN via p27(Kip1) downregulation. Plos One. 2014;9(8):e100904. Submit your next manuscript to BioMed Central and we will help you at every step: 19. Merola E, Mattioli E, Minimo C, Zuo WN, Rabitti C, Cicala M, Caviglia R, Pollice L, Gabbrielli A, Giordano A, Claudio PP. Immunohistochemical evaluation of pRb2/p130, VEGF, EZH2, p53, p16, p21(waf-1), p27, and PCNA in Barrett’s esophagus. J Cell Physiol. 2006;207:512–9. Submit your next manuscript to BioMed Central and we will help you at every step: Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit and we will help you at every step: 17. Li K, Chen MK, Situ J, Huang WT, Su ZL, He D, Gao X. Role of co-expression of c-Myc, EZH2 and p27 in prognosis of prostate cancer patients after surgery. Chin Med J. 2013;126:82–7. 18. Kretz M, Webster DE, Flockhart RJ, Lee CS, Zehnder A, Lopez-Pajares V, Qu K, Zheng GX, Chow J, Kim GE, et al. Suppression of progenitor differentiation requires the long noncoding RNA ANCR. Genes Dev. 2012;26:338–43. 19. Merola E, Mattioli E, Minimo C, Zuo WN, Rabitti C, Cicala M, Caviglia R, Pollice L, Gabbrielli A, Giordano A, Claudio PP. Immunohistochemical evaluation of pRb2/p130, VEGF, EZH2, p53, p16, p21(waf-1), p27, and PCNA in Barrett’s esophagus. J Cell Physiol. 2006;207:512–9.
https://openalex.org/W3019391325	https://europepmc.org/articles/pmc7240367?pdf=render	English	null	Straightforward Synthetic Protocol to Bio-Based Unsaturated Poly(ester amide)s from Itaconic Acid with Thixotropic Behavior	Polymers	2,020	cc-by	8,222	Received: 30 March 2020; Accepted: 19 April 2020; Published: 22 April 2020 Abstract: In the ﬁeld of polymer chemistry, tremendous eﬀorts have been made over the last decade to replace petrochemical monomers with building blocks from renewable resources. In this respect, itaconic acid has been used as an alternative to acrylic acid or maleic acid in unsaturated polyesters for thermal or UV-curing applications. However, examples of poly(ester amide)s from itaconic acid are scarce. Under standard polycondensation reactions, the presence of free amines leads to aza-Michael addition reactions at the α,β-unsaturated double bond of the itaconic acid and isomerization reactions to mesaconic acid. Both reactions make the resulting materials useless as UV-curing polymer resins. To avoid these undesired side reactions, we herein report the use of preformed, well-deﬁned diols containing internal amide bonds. The resulting unsaturated poly(ester amide) resins were analyzed before and after UV-induced crosslinking. Viscosity measurements revealed a strong thixotropic behavior induced by the amide groups, which is usually not detected in structurally similar polyester resins. Keywords: bio-based polymer; poly(ester amide); itaconic acid; thixotropy; photo-DSC polymers polymers www.mdpi.com/journal/polymers Polymers 2020, 12, 980; doi:10.3390/polym12040980 Article Straightforward Synthetic Protocol to Bio-Based Unsaturated Poly(ester amide)s from Itaconic Acid with Thixotropic Behavior aros Papadopoulos 1 , Marcel Kluge 2,3 , Dimitrios N. Bikiaris 1 and Tobias Robert 2,* Laboratory of Polymer Chemistry and Technology, Department of Chemistry, Aristotle University of Thessaloniki, GR-541 24 Thessaloniki, Greece; lazaros.geo.papadopoulos@gmail.com (L.P.); dbic@chem.auth.gr (D.N.B.) Lazaros Papadopoulos 1 , Marcel Kluge 2,3 , Dimitrios N. Bikiaris 1 and Tobias Robert 2,* p p , g , 1 Laboratory of Polymer Chemistry and Technology, Department of Chemistry, Aristotle University of Thessaloniki, GR-541 24 Thessaloniki, Greece; lazaros.geo.papadopoulos@gmail.com (L.P.); dbic@chem.auth.gr (D.N.B.) 1 Laboratory of Polymer Chemistry and Technology, Department of Chemistry, Aristotle University of Thessaloniki, GR-541 24 Thessaloniki, Greece; lazaros.geo.papadopoulos@gmail.com (L.P.); dbic@chem.auth.gr (D.N.B.) 2 Fraunhofer Institute for Wood Research—Wilhelm-Klauditz-Institut WKI, Bienroder Weg 54E, 38108 Braunschweig, Germany; marcel.kluge@wki.fraunhofer.de 3 Institute for Chemistry of Renewable Resources, Department of Chemistry, University of Natural Resources and Life Sciences Vienna, Konrad-Lorenz-Straße 24, 3430 Tulln, Austria * Correspondence: tobias.robert@wki.fraunhofer.de; Tel.: +49-531-2155-357 * Correspondence: tobias.robert@wki.fraunhofer.de; Tel.: +49-531-2155-357 1. Introduction Over the last years, polymers from renewable resources have attracted considerable attention from both industry and academia in an eﬀort to limit the environmental impact arising from the excessive use of polymeric materials derived from fossil resources [1–4]. However, in order to be able to compete with these established materials, the bio-based counterparts have to be either economically competitive or exhibit unprecedented material properties [5,6]. A viable way to achieve the latter is the utilization of bio-based monomeric building blocks, which are not economically accessible from petrochemical pathways. These can be used to produce novel polymeric structures that in turn can lead to materials with new properties [7–9]. In this context, itaconic acid (IA) has lately drawn considerable attention as it exhibits a promising structure and is available at competitive prices (<2 €/kg) [10]. This monomer is already produced on industrial scale (>80.000 t/a) via the fermentation of sugars with the fungi aspergillus terreus [11–13]. Another interesting feature of IA is its versatility. Since it possesses two carboxylic groups and an exo-double bond, it is a good substrate for diﬀerent chemical transformations. This versatility also applies for the production of polymers. Two main strategies emerge: ﬁrst, the utilization of the Polymers 2020, 12, 980; doi:10.3390/polym12040980 www.mdpi.com/journal/polymers Polymers 2020, 12, 980 2 of 15 Polymers 2020, 12, 980 unsaturated double bonds for the radical polymerization of homo-polymers and co-polymers [14–18] and second, the synthesis of unsaturated polyesters [19–25]. Those materials ﬁnd a number of applications in printing inks, shape memory polymers, and coatings [26–30], while they can also be exploited for post-polymerization reactions [31–34]. Despite the increased research activities in itaconic acid-based materials described above, poly(ester amide)s from itaconic acid have so far received little attention. The main reason for this is the fact that the α,β-unsaturated double bond is prone to a nucleophilic attack of the diamine, leading to the formation of pyrrolidone rings [35,36]. While this eﬀect can be exploited for the synthesis of bio-based polyamides and poly(ester amide)s [37,38], it also results in the complete conversion of the unsaturated double bonds and thus the inability to produce polymers susceptible to radical curing reactions. Recently, we reported two synthetic strategies in order to circumvent these undesired side reactions [39]. The ﬁrst method involved the synthesis of an itaconic acid-based oligoester and an oligo(ester amide), both of which were synthesized independently. 1. Introduction In the next step, these two oligomers were combined in a transesteriﬁcation reaction. The second synthetic pathway involved the in situ formation of a bis-ester amide that was subsequently reacted with IA and further monomers. While both approaches resulted in the successful synthesis of the envisaged structures, the ﬁrst synthetic strategy involved three synthetic steps, which makes the synthesis quite cumbersome. The second strategy suﬀered from a base-catalyzed isomerization of the itaconate moiety to the mesaconate, which was due to some free amines still present after the in situ formation of the bis-ester amide. Another promising and more straightforward approach to this class of unsaturated poly(ester amide)s is the used of well-deﬁned preformed monomers containing amide moieties. In this respect, we recently reported the use of symmetric diamido-α,ω-diols (amido diols) as building blocks for poly(ester amide)s derived from succinic acid [40–42]. By using these monomers, we were able to prevent the formation of succinimide that usually interferes with the synthesis of polyamides or poly(ester amide)s derived from succinic acid. Intrigued by these promising results, we were interested in whether these amido diols could also be used as building blocks for poly(ester amide)s derived from itaconic acid. This would allow for the introduction of amide segments without the occurrence of the undesired aza-Michael additions described above. In this work, two series of unsaturated poly(ester amide)s were synthesized based on itaconic acid and two diﬀerent preformed amido diols. The properties of the polymer resins as well as UV-cured materials were thoroughly characterized, revealing a thixotropic behavior of the resins and considerable inﬂuence of the amide content on the thermal and mechanical properties of the ﬁnal cured materials. 2.2. Measurements FTIR spectra were obtained on a Thermo Scientiﬁc Nicolet iS5 FTIR (Thermo Fisher Scientiﬁc, Waltham, MA, USA) using the ATR technique (32 scans, resolution of 4 cm−1). NMR experiments were conducted on a Bruker Avance III 400-MHz spectrometer (Bruker, Billerica, MA, USA). 1H NMR shifts of polymers are reported in ppm (δ) downﬁeld from tetramethylsilane (TMS) and were determined by reference to the residual solvent peak (chlorofrom-d1, 7.26 ppm for hydrogen atoms). Size-exclusion chromatography (SEC) was performed on a Malvern Viscotek GPCmax (Malvern Instruments, Malvern, United Kingdom) equipped with triple detection, consisting of a Malvern Dual detector and Schambeck RI2012 refractive index detector. The separation was performed by utilizing two PLgel 5 mm MIXED-C, 300 mm columns from Agilent Technologies (Santa Clara, CA, USA) at 35 ◦C. Chloroform was used as the eluent at a ﬂow rate of 0.5 mL min−1. Data acquisition and calculations were performed using Viscotek OmniSec software version 5.0. The samples were ﬁltered over a 0.2 mm PTFE ﬁlter prior to injection. Rheological properties of resins were studied on a Bohlin CVO 100 rheometer (Bohlin Instruments, Cirencester, UK) equipped with cone-plate geometry (CP 4◦, 40 mm). For the table temperature scan, measurements were done with a shear rate of 100 s−1 for 10 s. Five measurements were carried out for each temperature (25 ◦C, 37.5 ◦C, 50 ◦C, 62.5 ◦C, and 75 ◦C), and the average value was calculated for each temperature. For the shear thinning tests and to measure time-dependent behavior, each sample was left on the rheometer for ﬁve minutes prior to measurement to ensure a temperature equilibrium of 25 ◦C. Subsequently, a shear rate of 1 s−1 was applied for three minutes in order to reach the equilibrium state. Then, the shear rate was increased at 100 s−1 for the application of the mechanical load for a duration of two minutes. Finally, a shear rate of 1 s−1 was applied again over a prolonged period of time until the system reached an equilibrium state again. The ability of recovery as a measure of thixotropic behavior for each system was evaluated by comparing the viscosity value of the ﬁrst and the third stage of the measurement. Five samples of each resin were studied, and the average values are given in the results. 2.1. Materials Dimethyl itaconate (DMI, 98%), dimethyl sebacate (DMS, 98%), 1,4-diaminobutane (≥98%), and 1,6-diaminohexane (≥99%) were purchased from Tokyo Chemicals Industry (TCI), Tokyo, Japan. Glycerol (Gly, ≥98%) and chloroform-d1 (99.8% D) + 0.03% TMS v/v were provided by Carl Roth, Karlsruhe, Germany. 2,3-Butanediol (2,3-BD, a mixture of meso-D- and L-form, ≥99%) and 2,6-di-tert-butyl-4-methylphenol (BHT, 99%) were bought from Merck, Darmstadt, Germany. 4-Methoxyphenol (MeHQ, 99%) was purchased from Sigma-Aldrich Chemie, Steinheim, Germany. ε-Caprolactone (99%) was obtained from Alfa Aesar, Kandel, Germany. 1,3-Propanediol (1,3-PD, 99.7%) was kindly provided by DuPont Tate & Lyle Bio Products, Loudon, NH, USA. Isosorbide (Is) was obtained from Ecogreen Oleochemicals, Dessau-Roßlau, Germany. FASCAT 4101 catalyst was provided by PMC Group, Mount Laurel, NJ, USA. Solvents were reagent or analytical grade and were purchased from VWR International, Fontenay-sous-Bois, France. All reagents were used without further puriﬁcation except for isosorbide, which was recrystallized from ethyl acetate. 3 of 15 Polymers 2020, 12, 980 2.2. Measurements 2.4. Polymer Synthesis Six unsaturated poly(ester amide) (PEA) resins as well as a neat polyester, that served as a reference, were prepared via the transesteriﬁcation method, where an excess of 30% hydroxyl groups was targeted (dimethyl ester:diol ratio of 1:1.3). As an example, the synthesis of the neat polyester resin, named PE, will be described. For the synthesis of 60 g of resin, 35.57 g of DMI (224 mmol, 0.8 eq), 12.95 g of DMS (56 mmol, 0.2 eq), 14.71 g of 1,3-PD (193 mmol, 0.75 eq), 5.14 g of Is (35 mmol, 0.125 eq), 3.24 g of Gly (35 mmol, 0.125 eq) and 6.33 g of 2,3-BD (70 mmol, 0.25 eq), along with 0.12 g of FASCAT 4101 (0.2 wt %), 24 mg of BHT (0.08 wt %), and 18 mg of MeHQ (0.06 wt %) were charged into a three-necked round-bottom ﬂask equipped with mechanical stirrer, thermometer, condenser and an immersed thermocouple to control the temperature of the heating mantle. The mixture was heated gradually until homogenization, and then the temperature was set at 180 ◦C for 3 h. Finally, a slight vacuum (20 mbar) was applied for 30 min, and then the system was left to cool down to room temperature. The product was obtained as a yellow-orange resin. For the preparation of the resins containing amide moieties in their backbone, 0.0625, 0.125, and 0.1875 eq of 1,3-PD were substituted by the respective amido diol. 2.2. Measurements Photo-diﬀerential scanning calorimatry (Photo-DSC) measurements were conducted on a DSC 3+ (Mettler-Toledo, Greifensee, Switzerland) equipped with a Lightningcure LC8 UV spot light source (Hamamatsu Photonics, Hamamatsu, Japan) at 70% of its intensity. To get the integration of the heat of reaction, two runs were carried out with a short break between the runs to let the resin cool down. The second run was made once the material was fully cured and the baseline was stable. Then, the second curve was subtracted from the ﬁrst to obtain the curve related to the curing only. Each run is conducted as follows: 30 s at the set temperature (25 ◦C at atmospheric pressure under air) without the lamp; then, the lamp is turned on for 2.5 min. The break between the runs lasts 30 s. Dynamic mechanical analysis (DMA) was carried out on a Tritec 2000 DMA (Triton Technology, Loughborough, UK) in dual cantilever bending mode. Measurements were performed on rectangle bars (80 × 10 × 2 mm) which were prepared using a Teﬂon mold. Irgacure-1173 was added to the resin as photoinitator (5 wt %). Before curing, the mixture was left for 1 h at elevated temperature (50 to 90 ◦C depending on the viscosity of each sample) to reduce the amount of air trapped in the resin that could lead to cracks. Subsequently, it was poured into the mold and cured under UV light using an Aktiprint mini UN50029 UV dryer (Technigraf, Grävenwiesbach, Germany), equipped with a UV 4/120-2 lamp (254 nm, max. 120W cm−1). The conveyor belt was set at 5 m min−1, and ﬁve runs were required to fully cure the specimens. The irradiation energy (UV dose) was measured before and after curing with an average value of 460 mJ cm−2. Samples were studied under nitrogen in a temperature range of −30 to 120 ◦C (5 K min−1, 1 Hz, maximum displacement 0.05 mm). The exact dimensions of specimens were determined before each experiment, and ﬁve test bars were measured for each sample. Thermogravimetric analysis (TGA) of cured resins was performed on a TGA/DSC 1 (Mettler-Toledo, Greifensee, Switzerland) under nitrogen (35 mL min−1). Samples of ca. 10 mg were heated from 25 to 800 ◦C with a heating rate of 10 K min−1. 4 of 15 Polymers 2020, 12, 980 2.3. Synthesis of Amido Diols Two amido diols (AD) with varying alkylene chain lengths were synthesized as described previously [40,42]. In brief, a certain amount of diamine (1,4-diaminobutane or 1,6-diaminohexane) was placed in a three-necked ﬂask equipped with mechanical stirrer and cooler and dissolved in 2-propanol (200 mL mol−1). ε-Caprolactone (2 equivalents) in 2-propanol (50 mL mol−1) was slowly added to the diamine solution at room temperature using a pump (0.6 mL/min). Stirring was continued overnight. The resulting white pasty product was diluted with acetone and then washed several times with the same solvent to remove residual monomers and potentially formed oligomeric by-products by (homo)polymerization of the lactone. It was further puriﬁed by recrystallization from methanol and acetone and then dried under reduced pressure, whereby the respective product was obtained as a white powder. Amido diols are named analogously to polyamides of the AABB type, while the ﬁrst number indicates the number of carbon atoms in the diamine and the second indicates the number of carbon atoms in the lactone. Hence, AD 4.6 was obtained from 1,4-diaminobutane and ε-caprolactone, while AD 6.6 was synthesized from 1,6-diaminohexane. 3.1. Polymer Synthesis One of the main objects of this work was the introduction of amide moieties into the backbone of a bio-based unsaturated polyester resin derived from itaconic acid. As it was already discussed, the presence of free –NH2 moieties leads to the formation of the pyrrolidone lactam ring from itaconic acid, while it also supports the isomerization of itaconic acid to mesaconic acid. For this reason, preformed symmetrical diols that have two internal amide bonds were used for the synthesis of poly(ester amide)s. In order to assure that the amido diol structure would remain intact under the conditions applied for the synthesis, the dimethyl esters of the respective acids were used. Previous studies have shown that the amido diol structure can be lost by an acidolysis-type reaction if free carboxylic acid groups are present during reaction [43–45]. Hence, the dimethyl esters of dicarboxylic acids were utilized to avoid this undesirable side reaction. Another challenge in the synthesis of poly(ester amide) resins is the formation of hydrogen bonds, which should lead to a high increase in viscosity. Indeed, preliminary studies showed very high viscosity values for the produced resins, rendering them nearly non-processable. In an attempt to counter this eﬀect, a range of bio-based diols were incorporated in the polymer backbone to obtain highly irregular polymer chains, which should reduce the intermolecular interaction. Therefore, we anticipated obtaining poly(ester amide) resins with low viscosities. Even though this strategy was 5 of 15 Polymers 2020, 12, 980 Polymers 2020, 12, 980 successful to some extent, the phenomenon could only be mildly suppressed, as the viscosity of the resins increased dramatically even with very small amido diol content. So, in another attempt to reduce the viscosity, the reaction time was set at 3.5 h for all resins, as prolonged reaction times can lead to undesired side reactions and an increased molecular weight that would result in higher viscosities of the resins. Six unsaturated bio-based poly(ester amide) resins containing 5 to 15 mol% of an amido diol (AD 4.6 or 6.6) and a neat polyester, that served as a reference, were successfully synthesized via a transesteriﬁcation method of DMI, DMS, and a variety of sterically demanding diols (Scheme 1). The distance between amide bonds was varied by the choice of the diamine used for the synthesis of amido diols. All polymers were obtained following the same protocol, considering the above-mentioned limitations. 3.1. Polymer Synthesis The monomers and composition used for the synthesis of the resins are given in Table 1. PEA labels include the molar fraction of targeted ester-amide sequences in the resin, which corresponds to the molar percentage of the respective amido diol in the feed. For example, 5 mol% of ester amide moieties were aimed for PEA 4.6-05, corresponding to the equal amount of AD 4.6 in the feed. All building blocks used, but the amido diols, are accessible from renewable resources, resulting in PEA resins with very high bio-based content. Scheme 1. Synthesis route of the unsaturated polyester and poly(ester amide) resins obtained in the course of this study. Scheme 1. Synthesis route of the unsaturated polyester and poly(ester amide) resins obtained in the course of this study. Table 1. Composition of the unsaturated neat polyester and poly(ester amide) resins synthesized. DMI: dimethyl itaconate, DMS: dimethyl sebacate. Sample DMI (eq) DMS (eq) 1,3 PD (eq) 2,3 BD (eq) Is (eq) Gly (eq) Amido Diol (eq) Amido Diol (mol%) PE 0.8 0.2 0.75 0.25 0.125 0.125 - - PEA 4.6-05 0.8 0.2 0.6875 0.25 0.125 0.125 0.0625 5 PEA 4.6-10 0.8 0.2 0.625 0.25 0.125 0.125 0.125 10 PEA 4.6-15 0.8 0.2 0.5625 0.25 0.125 0.125 0.1875 15 PEA 6.6-05 0.8 0.2 0.688 0.25 0.125 0.125 0.0625 5 PEA 6.6-10 0.8 0.2 0.625 0.25 0.125 0.125 0.125 10 PEA 6.6-15 0.8 0.2 0.5625 0.25 0.125 0.125 0.1875 15 3.2. Structural Characterization The ATR-FTIR spectra of synthesized resins are presented in Figure 1. In the spectra of all polymers, the presence of ester bonds is conﬁrmed by the signal at 1720 cm−1, which can be assigned to the C=O stretching vibrations. Peaks at 1640 cm−1 and 810 cm−1, corresponding to the C–C stretch and C–C deformation vibrations of the double bond of itaconic acid, conﬁrm that the double bond did not undergo undesired side reactions during synthesis. A broad peak around 3500 cm−1 can be found 6 of 15 Polymers 2020, 12, 980 in the spectra due to the OH end groups of polymer chains, since diols were used in excess. After the incorporation of an amido diol into the polyester backbone, new signals associated with amide groups can be found in the spectra. The intensity of the signal at 1640 cm−1 increased, since the peak of the double bond of itaconic acid and those associated with the C=O (Amide I) stretching vibrations of the amido diol are overlapping. In addition, a new absorption peak can be found at 1540 cm−1 that is attributed to the N–H (Amide II) bending vibrations of amide bonds. Furthermore, a new signal is observed at 3300 cm−1, which is related to hydrogen-bonded N–H groups. By increasing the amido diol content, the intensity of the peaks associated with amide moieties also increased. Figure 1. FTIR spectra of synthesized resins. C–C double bond vibrations are highlighted in green, vibrations derived from the amide group are in orange. Figure 1. FTIR spectra of synthesized resins. C–C double bond vibrations are highlighted in green, vibrations derived from the amide group are in orange. The chemical structure of the synthesized resins was also investigated by means of 1H NMR spectroscopy. All the spectra of the resins show the signals of the neat polyester resin (PE). The poly(ester amide)s show additional peaks that can be assigned to the ester-amide sequences. In addition, by increasing the amido diol content, the respective peaks increase in intensity. Detailed analysis of the spectra can be found in the Supplementary Materials. Both FTIR and NMR spectra support the successful incorporation of the amido diol building block into the polymer backbone. In addition, the results suggest that aforementioned side reactions, such as ring formation or isomerization, can be suppressed when the preformed amido diols are used. 3.2. Structural Characterization Pyrrolidone lactam ring formation was completely avoided, while the isomerization of itaconic acid was below 3%, which is similar to what is observed when standard polyesters are synthesized from itaconic acid. These results conﬁrm that the copolymerization of amido diols is a viable synthetic strategy for the insertion of amide moieties into unsaturated polyester resins based on itaconic acid. As discussed earlier, the reaction time for the synthesis of the resins in question was limited to obtain PEAs with a viscosity suitable for processing. However, this practice resulted in some methyl ester end groups remaining in the polymer chain, as shown in the 1H NMR spectra (Figure 2). Therefore, rather low molecular weights are assumed for the resins produced. Nevertheless, we consider this practice mandatory, because the increased viscosity makes these resins very diﬃcult to manipulate for the characterizations needed as well as the envisaged UV-curing applications, and a longer reaction would have rendered them non-processable. 7 of 15 7 of 15 Polymers 2020, 12, 980 Figure 2. 1H NMR spectra of the neat polyester resin and PEAs containing diﬀerent amounts of amido diol 4.6. Figure 2. 1H NMR spectra of the neat polyester resin and PEAs containing diﬀerent amounts of amido diol 4.6. 3.3. SEC Measurements SEC results are shown in Figure 3. As expected for the products of polycondensation reactions, the molecular weights of the resins are obtained in gaussian distribution. The absence of any high molecular weight fraction in the elugrams, along with the 1H NMR data, shows that undesired side reactions leading to cross-linking of the polymers were successfully prevented. The molecular weight of the produced resins is rather low, as a result of the synthetic procedure, in the range of 300 to 5000 g/mol. However, the Mn, Mw, and Đ were not calculated, because the calibration of the measurements was limited to 575 g/mol. Nevertheless, they are still suitable for applications in the coatings ﬁeld. The resins derived from AD 6.6 present larger retention volume peaks compared to those based on AD 4.6. This indicates that a higher molecular weight is to be expected when the amide groups are separated by a longer alkylene chain. Figure 3. SEC elugrams of the resins synthesized in this study. Figure 3. SEC elugrams of the resins synthesized in this study. 3.4. Viscosity As anticipated, the incorporation of the amido diol into the polyester backbone led to increased viscosities. However, as a somewhat unexpected feature, the resins also became opaque and almost turned into a waxy solid when allowed to cool overnight in the storage container. At this state, the resins did not ﬂow any more unless an external force was applied. This phenomenon became more pronounced with increasing amido diol content, suggesting that it was derived from intermolecular Polymers 2020, 12, 980 Polymers 2020, 12, 980 8 of 15 hydrogen bonds. Similar claims were made in other works where polyesters containing amide moieties were examined, and it was found via temperature-dependent IR spectroscopy that as the temperature increased, the amide-ester hydrogen bonds were destroyed [46,47]. For the ﬁrst set of measurements, viscosity values were obtained at diﬀerent temperatures under a constant shear rate of 100 s−1. As can be seen in Figure 4, an increase of the temperature resulted in an exponential decrease of the viscosity. In addition to the temperature dependency, we were intrigued to investigate whether the resins also exhibit a non-Newtonian behavior—thus, if the viscosity can be inﬂuenced by a diﬀerence in shear stress and whether there is a time-dependent behavior. This behavior was studied by monitoring the viscosity as a function of the shear rate. In detail, this was done by ﬁrst applying a low shear rate of 1 s−1 until a constant viscosity was obtained. Then, a high shear rate of 100 s−1 was applied for 2 min to break down the interactions between the polymer chains. In a last phase, the shear rate was reduced again to 1 s−1. For both transitions of the shear rates, the eﬀect on the viscosity was examined, as well as a time dependency of the latter, which would indicate a thixotropic behavior. In addition, the ability of the resins to recover to the initial state was examined [48–51]. A strong shear thinning eﬀect could be observed for all resins with amido diols incorporated in the polymer chain (Figure 5). Even at low amido diol contents of 5 mol%, this eﬀect could be observed with AD 6.6 resulting in a more pronounced inﬂuence on the shear thinning behavior (Figure 5b). With increasing ester-amide content, the initial viscosity increased to a point where PEA 6.6–15 could not be measured anymore. 3.4. Viscosity However, for PEA 4.6–15, the viscosity is reduced from 1127 Pa·s at 1 s−1 to 30 Pa·s at 100 s−1, which corresponds to 2.6% of the initial viscosity (Table 2). This shear thinning eﬀect also showed a time-dependent behavior, indicating a thixotropic behavior of the resins. For the neat polyester, no change in viscosity could be observed. The recovery of the initial viscosity was measured in the third phase of the rheology experiment. Here, the thixotropic behavior was more easily observed, as it took several minutes to reach constant viscosities. In addition, it became apparent that resins containing amido diol 6.6 exhibited very high recovery rates that were signiﬁcantly higher than those of the corresponding resins with amido diol 4.6 as a building block (Table 2). This diﬀerence is especially pronounced for the resins with 10 mol% amido diol, where the resin based on AD 4.6 recovers only 69% of the initial viscosity in comparison to 95% in the case of that derived from AD 6.6. These results suggest that in the case of the former amido diol, the initial arrangement of the resin is not achieved within the measurement time, resulting in a lower amount of polymer chain interaction via hydrogen bonds. Most probably, this behavior can be explained by the diﬀerent structure of the amido diols, since the more ﬂexible structure is the one with higher recovery rates. The increased length of the spacer unit between the two amide bonds (1,6-diaminohexane and 1,4-diaminobutane, respectively) gives the chain an extra ﬂexibility that contributes to the faster recreation of the hydrogen bonds after their structure is destroyed by the high shear rate. Figure 4. Temperature dependency of the viscosity of resins synthesized in the course of this study with (a) amido diol 4.6 and (b) amido diol 6.6. Figure 4. Temperature dependency of the viscosity of resins synthesized in the course of this study with (a) amido diol 4.6 and (b) amido diol 6.6. 9 of 15 Polymers 2020, 12, 980 Figure 5. Viscosity experiment conducted to study the thixotropic behavior of the resins containing (a) 5 mol% amido diol and (b) 10 mol% and higher amounts of amido diol. Conditions: 3 min at 1 s−1, then 2 min at 100 s−1, ﬁnally 7 min at 1 s−1. Figure 5. 3.4. Viscosity Viscosity experiment conducted to study the thixotropic behavior of the resins containing (a) 5 mol% amido diol and (b) 10 mol% and higher amounts of amido diol. Conditions: 3 min at 1 s−1, then 2 min at 100 s−1, ﬁnally 7 min at 1 s−1. Table 2. Viscosity values of the polymers at diﬀerent shear rates and percentage of the initial viscosity recovered at the end of the experiment. Sample Starting Viscosity (Pa·s at 1 s−1) Viscosity at High Shear Rate (Pa·s at 100 s−1) % of Starting Viscosity at High Shear Rate Viscosity at Recovery (Pa·s at 1 s−1) % of Recovered Viscosity PE 0.84 0.84 - 0.84 - PEA 4.6-05 3.1 2.75 88.7 2.83 91 PEA 4.6-10 181 7 3.8 123 69 PEA 4.6-15 1127 30 2.6 773 68 PEA 6.6-05 22.9 3.37 14.7 22.3 97 PEA 6.6-10 258 10 3.8 251 95 PEA 6.6-15 Unable to measure at 25 ◦C 3.5. UV-Curing Behavior 3.5. UV-Curing Behavior Photo-DSC measurements were conducted to evaluate the reactivity of the resins toward UV-curing. The double-bond density (DBD) of the resin was calculated from the composition of the resins. In order to obtain the theoretical enthalpy of the UV-induced radical polymerization reaction, the double-bond density was multiplied by the heat of polymerization of dimethyl itaconate (60.67 KJ/mol), which was determined by Dainton et al. [52]. The experimental enthalpy, conversion, and rate of polymerization (ROP) of all resins were calculated from the photo-DSC measurements. The results are all shown in Table 3, and Figure 6 depicts the conversion and ROP over time. The enthalpy, as well as the conversion decreases with increasing ester-amide content. This can be explained by the lower DBD, as well as the increasing viscosity of the resins. The latter compromises the mobility of the polymer chains and therefore the likelihood of radical cross-linking reactions between double bonds. The only exception to the reduction in conversion and enthalpy can be found for PEA 4.6-10, which surpasses the conversion of the neat polyester. The ROP of the polyester is lower than some of the PEAs, but again, a general decreasing trend can be observed with increasing amide content with values between 6.3 and 10.8 × 10−3 s−1. In general, the values are quite low for UV-curing materials, but they are in the same range that have been reported for other materials derived from itaconic acid [39,53]. Polymers 2020, 12, 980 10 of 15 Figure 6. Photo-DSC of the PEAs: Conversion (a) and rate of polymerization (b). Figure 6. Photo-DSC of the PEAs: Conversion (a) and rate of polymerization (b). Table 3. Double-bond density (DBD), theoretical and experimental enthalpy of curing, conversion, and maximum rate of polymerization (ROP) of the resins at 25 ◦C. Table 3. Double-bond density (DBD), theoretical and experimental enthalpy of curing, conversion, and maximum rate of polymerization (ROP) of the resins at 25 ◦C. Table 3. Double-bond density (DBD), theoretical and experimental enthalpy of curing, conversion, and maximum rate of polymerization (ROP) of the resins at 25 ◦C. Sample DBD (mmol/g) ∆Htheo (J/g) ∆H (J/g) C (%) ROP (1000 s−1) PE 3.7 224.5 127 57 8.6 PEA 4.6-05 3.4 206.3 111 54 10.6 PEA 4.6-10 3.2 194.1 112 58 10.8 PEA 4.6-15 3.0 182.0 85 47 6.4 PEA 6.6-05 3.4 206.3 113 55 9.5 PEA 6.6-10 3.2 194.1 92 48 7.7 PEA 6.6-15 3.0 182.0 83. 3.5. UV-Curing Behavior 46 6.3 3.6. DMA Analysis The PEAs were also cast into a Teﬂon mold and cured under UV light. The resulting bars (80 × 10 × 2 mm) were analyzed by means of DMA measurements. Figure 7 shows the storage modulus (E’) as a function of the temperature for the crosslinked polymers. The values for all materials at 25 ◦C are between 11.74 and 2.03 GPa and decrease with increasing temperature, reaching the range of 1.58–0.14 GPa in the rubbery state. The storage modulus E’ is a measurement of material stiﬀness and can be used as providing the information regarding the molecular weight of a polymer and its crosslink density [54]. By introducing the long aliphatic chain of the amido diols into the structure of the polyesters, the spacing between the double bonds increases, leading to a decrease of the crosslinking density in the cured polyesters. The phenomenon gets more pronounced by the introduction of a higher amido diol content into the resins, as well as by the use of AD 6.6, in which the amide bonds are separated by a longer alkylene chain compared to AD 4.6. This statement is supported by the crosslinking density values (νe) i.e., the molar number of elastically eﬀective network chains per cubic centimeter of a sample, which was calculated from the formula: ve = E’/3RT, where E’ is the storage modulus, R is the gas constant, and T is the Kelvin temperature, which are given in Table 4. 11 of 15 11 of 15 Polymers 2020, 12, 980 Figure 7. Storage modulus (E’) and tan δ values of the PEAs after UV curing. PEAs with amido diol 4.6 (A,B) and PEAs with amido diol 6.6 (C,D). Figure 7. Storage modulus (E’) and tan δ values of the PEAs after UV curing. PEAs with amido diol 4.6 (A,B) and PEAs with amido diol 6.6 (C,D). Tan δ graphs are also presented in Figure 7. The tan δ peak corresponds to the Tg of the materials, while its broadening occurs when a less homogenous network is created [23,55]. In all cases, a very broad tan δ peak can be observed. The tan δ peak of the cured neat polyester resin is found at 71 ◦C, and by increasing the amido diol content, a proportional decrease is observed. 3.6. DMA Analysis Simultaneously, the incorporation of the amido diol into the resins resulted in a second peak emerging in the tan δ graphs at temperatures below 0 ◦C. This peak is shifted to higher values and gains in intensity with increasing AD content in the resins. This is an indication that possibly the crosslinked and non-crosslinked regions of the polymer are aﬀected in diﬀerent ways from the addition of the amido diols. As the distance between the double bonds is increasing, a more sparsely crosslinked region is created, and this is reﬂected on the transfer of the peak from 71 ◦C to lower values. In the same time, movement restrictions of the non-crosslinked polymer chains get lifted, and they can interact more with each other, resulting in more hydrogen bonds being formed and thus the second peak shifting to higher temperatures. Table 4. DMA results of the cured resins. Sample E’ at 25 ◦C (GPa) E’ at 115 ◦C (GPa) Crosslink Density (106 × mol × m−3) Tan δ Peaks (◦C) PE 11.74 1.58 0.163 - 71 PEA 4.6-05 11.02 1.19 0.123 −0.4 69 PEA 4.6-10 5.41 0.51 0.053 3.6 63 PEA 4.6-15 2.06 0.22 0.023 6.8 55 PEA 6.6-05 6.70 0.63 0.065 −9 68 PEA 6.6-10 3.01 0.28 0.029 4 61 PEA 6.6-15 2.03 0.14 0.0145 17 58 Table 4. DMA results of the cured resins. Table 4. DMA results of the cured resins. 12 of 15 Polymers 2020, 12, 980 12 of 15 3.7. Thermal Degradation The TGA results of the cured samples are given in Figure 8. The thermal degradation of the crosslinked resins occurs in three main steps as shown by the ﬁrst derivative of the of the TGA curve (dTG graph). In the ﬁrst step, a mass loss of 10%–15% is observed by 250 ◦C, which is assumed to correspond to non-reacted oligomers that have not been incorporated to the crosslinked network. However, the introduction of an amido diol seems to enhance the thermal stability of the material, since an increase of 5 to 16◦C was found in the Td,10% data that are shown in Table 5. In the second step of degradation, polyester segments decompose in the range of 350–400 ◦C, which is very common among polyester resins [55,56]. The incorporation of the amido diol leads to an extra peak in the dTG graph, which originally appeared as a shoulder peak for resin PE and as the amido diol mol% increases, that peak gets separated from the main polyester degradation peak and two clear peaks can be observed around 350 ◦C and 400 ◦C. Although it is unclear why this splitting occurs, it might be associated with the fact that the crosslinking density becomes sparser by introducing higher amido diol mol% in the polymer chains. In the third and ﬁnal step, the amide segments are degrading, as it is shown by the third peak of the dTG graph, around 450 ◦C, the intensity of which increased along with the amido diol content of the resins. Lastly, the resins containing amide moieties presented higher residues at 800 ◦C than the PE resin, while resins containing amido diol 4.6 present slightly higher values for both Td,10% and R800◦C, suggesting better thermal stability. Figure 8. TGA thermograph (a) and dTG (b) of the cured resins. Figure 8. TGA thermograph (a) and dTG (b) of the cured resins. Table 5. TGA results of the cured resins. Sample Td,10% (◦C) R800 ◦C (%) PE 230 11.2 PEA 4.6-05 242 13.3 PEA 4.6-10 245 13.8 PEA 4.6-15 255 12.8 PEA 6.6-05 246 13.1 PEA 6.6-10 235 13.3 PEA 6.6-15 237 11.3 4. Conclusions 4. Conclusions In this work, we report the synthesis of bio-based unsaturated poly(ester amide)s, trying to overcome aza-Michael additions and isomerization reactions that usually take place when itaconic acid and primary diamines are reacted under polycondensation conditions. Our synthetic strategy involved the use of preformed symmetrical amido diols with two internal amide bonds. Evaluation of structural composition veriﬁed the successful synthesis of the desired poly(ester amide)s. In addition, both undesired side reactions were eﬀectively suppressed even at amido diol contents of up to 15 mol%. In this work, we report the synthesis of bio-based unsaturated poly(ester amide)s, trying to overcome aza-Michael additions and isomerization reactions that usually take place when itaconic acid and primary diamines are reacted under polycondensation conditions. Our synthetic strategy involved the use of preformed symmetrical amido diols with two internal amide bonds. Evaluation of structural composition veriﬁed the successful synthesis of the desired poly(ester amide)s. In addition, both undesired side reactions were eﬀectively suppressed even at amido diol contents of up to 15 mol%. 13 of 15 Polymers 2020, 12, 980 The introduction of amide bonds in the polymer structure had a huge impact on the physicochemical properties of the resins e.g., the viscosity and reactivity toward UV-induced crosslinking. The latter was aﬀected, as the conversion of the double bonds along with the rate of polymerization were lowered at higher amido diol content. However, at lower amounts of the amido diol building blocks incorporated into the PEA resins, the curing behavior was not signiﬁcantly aﬀected. For PEA with 10 mol% of the amido diol 4.6, the conversion and ROP was even higher than for the neat PE. In addition, all PEA resins exhibited an intriguing thixotropic behavior. This feature is very interesting, and it could be achieved even with amido diol loadings as low as 5 mol%. Furthermore, despite the lower conversion of some PEA resins, all samples were still suitable for UV-curing applications. The cured test specimen obtained in this study were examined on their thermomechanical properties by means of DMA, with a considerable impact of the amido diol on the properties of the samples. Therefore, the PEAs described in this study could be suitable as additives for certain UV-curing application where thixotropic behavior of the resins is desired. 4. Conclusions Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4360/12/4/980/s1, Figure S1: 1H NMR spectra of the resins containing amido diol 6.6., Figure S2: Close up from 1H NMR spectra. Top row AD 4.6, bottom row AD 6.6. Author Contributions: L.P. and M.K.: Conceptualization, methodology, investigation, writing—original draft preparation; D.N.B.: Conceptualization, supervision, writing—review and editing; T.R.: Conceptualization, methodology, formal analysis, supervision, writing—review and editing. All authors have read and agreed to the published version of this manuscript. Funding: This research was funded by the Projekträger Jülich and the Federal Ministry of Education and Research in Germany. L.P. wants to thank the Erasmus+ program for their support. Acknowledgments: The authors would like to thank K.U. Loos und A.J.J. Woortman for the performance of the SEC measurements. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. References Polymer 2003, 44, 4969–4979. [CrossRef] 16. Retuert, J.; Pedram, M.Y.; Martínez, F.; Jeria, M. Soluble Itaconic Acid–Ethylene Glycol Polyesters. Bull. Chem. Soc. Jpn. 1993, 66, 1707–1708. [CrossRef] 17. Arrighi, V.; McEwen, I.J.; Holmes, P.F. Dielectric Relaxations in Poly(di-n-alkyl itaconate)s. Macromolecules 2004, 37, 6210–6218. [CrossRef] 18. Inciarte, H.; Orozco, M.; Fuenmayor, M.; López-Carrasquero, F.; Oliva, H. Comb-like copolymers of n-alkyl monoitaconates and styrene. e-Polymers 2006, 6. [CrossRef] 19. Robert, T.; Friebel, S. Itaconic acid-a versatile building block for renewable polyesters with enhanced functionality. Green Chem. 2016, 18, 2922–2934. [CrossRef] 20. Pellis, A.; Hanson, P.A.; Comerford, J.W.; Clark, J.H.; Farmer, T.J. Enzymatic synthesis of unsaturated polyesters: Functionalization and reversibility of the aza-Michael addition of pendants. Polym. Chem. 2019, 10, 843–851. [CrossRef] 21. Li, P.; Ma, S.; Dai, J.; Liu, X.; Jiang, Y.; Wang, S.; Wei, J.; Chen, J.; Zhu, J. Itaconic Acid as a Green Alternative to Acrylic Acid for Producing a Soybean Oil-Based Thermoset: Synthesis and Properties. ACS Sustain. Chem. Eng. 2017, 5, 1228–1236. [CrossRef] 22. Dai, J.Y.; Ma, S.Q.; Teng, N.; Dai, X.Y.; Shen, X.B.; Wang, S.; Liu, X.Q.; Zhu, J. 2,5-Furandicarboxylic Acid- and Itaconic Acid-Derived Fully Biobased Unsaturated Polyesters and Their Cross-Linked Networks. Ind. Eng. Chem. Res. 2017, 56, 2650–2657. [CrossRef] 23. Brännström, S.; Malmström, E.; Johansson, M. Biobased UV-curable coatings based on itaconic acid. J. Coat. Technol. Res. 2017, 14, 851–861. [CrossRef] 24. Schoon, I.; Kluge, M.; Eschig, S.; Robert, T. Catalyst inﬂuence on undesired side reactions in the polycondensation of fully bio-based polyester itaconates. Polymers 2017, 9, 693. [CrossRef] [PubMed] 25. Mehta, L.B.; Wadgaonkar, K.K.; Jagtap, R.N. Synthesis and characterization of high bio-based content unsaturated polyester resin for wood coating from itaconic acid: Eﬀect of various reactive diluents as an alternative to styrene. J. Dispers. Sci. Technol. 2019, 40, 756–765. [CrossRef] y 26. Patil, D.M.; Phalak, G.A.; Mhaske, S.T. Design and synthesis of bio-based UV curable PU acrylate resin from itaconic acid for coating applications. Des. Monomers Polym. 2017, 20, 269–282. [CrossRef] [PubMed] 27. Dai, J.; Ma, S.; Wu, Y.; Han, L.; Zhang, L.; Zhu, J.; Liu, X. Polyesters derived from itaconic acid for the properties and bio-based content enhancement of soybean oil-based thermosets. Green Chem. 2015, 17, 2383–2392. [CrossRef] 28. Goerz, O.; Ritter, H. Polymers with shape memory eﬀect from renewable resources: Crosslinking of polyesters based on isosorbide, itaconic acid and succinic acid. Polym. Int. 2013, 62, 709–712. [CrossRef] 29. References 1. Sheldon, R.A. Green and sustainable manufacture of chemicals from biomass: State of the art. Green Chem. 2014, 16, 950–963. [CrossRef] 2. Schneiderman, D.K.; Hillmyer, M.A. 50th Anniversary Perspective: There Is a Great Future in Sustainable Polymers. Macromolecules 2017, 50, 3733–3749. [CrossRef] 3. Bornscheuer, U.T. Feeding on plastic. Science 2016, 351, 1154–1155. [CrossRef] [PubMed] . Zhu, Y.; Romain, C.; Williams, C.K. Sustainable polymers from renewable resources. Nature 2016, 354–362. [CrossRef] [PubMed] 5. Hillmyer, M.A. The promise of plastics from plants. Science 2017, 358, 868–870. [CrossRef] [PubMed] 5. Hillmyer, M.A. The promise of plastics from plants. Science 2017, 358, 868–870. [CrossRef] [PubMed] 6. Iwata, T. Biodegradable and bio-based polymers: Future prospects of eco-friendly plastics. Angew. Chem. Int. Ed. 2015, 54, 3210–3215. [CrossRef] [PubMed] 6. Iwata, T. Biodegradable and bio-based polymers: Future prospects of eco-friendly plastics. Angew. Chem. Int. Ed. 2015, 54, 3210–3215. [CrossRef] [PubMed] 7. Williams, C.K.; Hillmyer, M.A. Polymers from renewable resources: A perspective for a special issue of polymer reviews. Polym. Rev. 2008, 48, 1–10. [CrossRef] 7. Williams, C.K.; Hillmyer, M.A. Polymers from renewable resources: A perspective for a special issue of polymer reviews. Polym. Rev. 2008, 48, 1–10. [CrossRef] 8. Niaounakis, M. Biopolymers: Processing and Products; William Andrew: Oxford, UK; Waltham, MA, USA, 2014. 9. Niaounakis, M. Biopolymers: Reuse, Recycling, and Disposal; William Andrew: Oxford, UK; Waltham, MA, USA, 2013. 9. Niaounakis, M. Biopolymers: Reuse, Recycling, and Disposal; William Andrew: Oxford, UK; Waltham, MA, USA, 2013. 10. Werpy, T.; Petersen, G. Top Value Added Chemicals from Biomass: Volume I—Results of Screening for Potential Candidates from Sugars and Synthesis Gas; U.S. Department of Energy: Washington, DC, USA, 2004; pp. 1–69. [CrossRef] 11. Willke, T.; Vorlop, K.D. Biotechnological production of itaconic acid. Appl. Microbiol. Biotechnol. 2001, 56, 289–295. [CrossRef] 12. Krull, S.; Lünsmann, M.; Prüße, U.; Kuenz, A. Ustilago Rabenhorstiana—An Alternative Natural Itaconic Acid Producer. Fermentation 2020, 6, 4. [CrossRef] 13. Saha, B.C.; Kennedy, G.J. Phosphate limitation alleviates the inhibitory eﬀect of manganese on itaconic acid production by Aspergillus terreus. Biocatal. Agric. Biotechnol. 2019, 18, 101016. [CrossRef] 14 of 15 Polymers 2020, 12, 980 14. Chakraborty, S.; Ju, L.; Galuska, A.A.; Moore, R.B.; Turner, S.R. Suspension polymerization of itaconic acid diesters. J. Appl. Polym. Sci. 2018, 135, 46417. [CrossRef] 15. Lopez-Carrasquero, F.; de Ilarduya, A.M.; Cardenas, M.; Carrillo, M.; Arnal, M.L.; Laredo, E.; Torres, C.; Mendez, B.; Muller, A.J. New comb-like poly(n-alkyl itaconate)s with crystalizable side chains. References [CrossRef] 42. Kluge, M.; Rennhofer, H.; Lichtenegger, H.C.; Liebner, F.W.; Robert, T. Poly(ester amide)s from poly(alkylene succinate)s and rapid crystallizing amido diols: Synthesis, thermal properties and crystallization behavior. Eur. Polym. J. 2020, 129, 109622. [CrossRef] 43. Stapert, H.R.; Bouwens, A.-M.; Dijkstra, P.J.; Feijen, J. Environmentally degradable aliphatic poly(ester-amide)s based on short, symmetrical and uniform bisamide-diol blocks, 1. Synthesis and interchange reactions. Macromol. Chem. Phys. 1999, 200, 1921–1929. [CrossRef] 44. Kotliar, A.M. Interchange Reactions Involving Condensation Polymers. J. Polym. Sci. Macromol. Rev. 1981, 16, 367–395. [CrossRef] 45. Kricheldorf, H.R.; Denchev, Z. Interchange Reactions in Condensation Polymers and Their Analysis by NMR Spectroscopy. In Transreactions in Condensation Polymers; Wiley-VCH Verlag GmbH: Weinheim, Germany, 2007; pp. 1–78. 46. Pillai, C.K.S.; Sandhya, K.Y.; Sudha, J.D.; Saminathan, M. Inﬂuence of hydrogen bonding on the generation and stabilization of liquid crystalline polyesters, poly(esteramide)s and polyacrylates. Pramana 2003, 61, 417–423. [CrossRef] 47. Lips, P. Aliphatic Segmented Poly(Ester amide)s Based on Symmetrical Bisamide-Diols. PhD Thesis, University of Twente, Enschede, The Netherlands, 2005. 48. Shenoy, A. Thermoplastic Melt Rheology and Processing; CRC Press: Boca Raton, FL, USA, 1996. 49. Han, C.D. Rheology and Processing of Polymeric Materials, Volume 1: Polymer Rheology; Oxford University Press: New York, NY, USA, 2007. 50. Shaw, M.T. Introduction to Polymer Rheology; John Wiley & Sons, Inc.: Hoboken, NJ, USA, 2012; Volume 6 1. Kontopoulou, M. Applied Polymer Rheology; John Wiley & Sons, Inc.: Hoboken, NJ, USA, 2011. 52. Dainton, F.S.; Ivin, K.J.; Walmsley, D.A.G. The heats of polymerization of some cyclic and ethylenic compounds. Trans. Faraday Soc. 1960, 56, 1784–1792. [CrossRef] 53. Pérocheau Arnaud, S.; Andreou, E.; Pereira Köster, L.V.G.; Robert, T. Selective Synthesis of Monoesters of Itaconic Acid with Broad Substrate Scope: Biobased Alternatives to Acrylic Acid? ACS Sustain. Chem. Eng. 2020, 8, 1583–1590. [CrossRef] 54. Zhang, Y.; Asif, A.; Shi, W. Highly branched polyurethane acrylates and their waterborne UV curing coating. Prog. Org. Coat. 2011, 71, 295–301. [CrossRef] 55. Panic, V.V.; Seslija, S.I.; Popovic, I.G.; Spasojevic, V.D.; Popovic, A.R.; Nikolic, V.B.; Spasojevic, P.M. Simple One-Pot Synthesis of Fully Biobased Unsaturated Polyester Resins Based on Itaconic Acid. Biomacromolecules 2017, 18, 3881–3891. [CrossRef] 56. Gubbels, E.; Jasinska-Walc, L.; Noordover, B.A.J.; Koning, C.E. Linear and branched polyester resins based on dimethyl-2,5-furandicarboxylate for coating applications. Eur. Polym. J. 2013, 49, 3188–3198. [CrossRef] © 2020 by the authors. Licensee MDPI, Basel, Switzerland. References Guo, B.; Chen, Y.; Lei, Y.; Zhang, L.; Zhou, W.Y.; Rabie, A.B.M.; Zhao, J. Biobased Poly(propylene sebacate) as Shape Memory Polymer with Tunable Switching Temperature for Potential Biomedical Applications. Biomacromolecules 2011, 12, 1312–1321. [CrossRef] [ ] 30. Robert, T.; Eschig, S.; Biemans, T.; Scheiﬂer, F. Bio-based polyester itaconates as binder resins for UV-curing oﬀset printing inks. J. Coat. Technol. Res. 2019, 16, 689–697. [CrossRef] 31. Farmer, T.J.; Clark, J.H.; Macquarrie, D.J.; Ogunjobi, J.K.; Castle, R.L. Post-polymerisation modiﬁcation of bio-derived unsaturated polyester resins via Michael additions of 1,3-dicarbonyls. Polym. Chem. 2016, 7, 1650–1658. [CrossRef] 32. Winkler, M.; Lacerda, T.M.; Mack, F.; Meier, M.A.R. Renewable polymers from itaconic acid by polycondensation and ring-opening-metathesis polymerization. Macromolecules 2015, 48, 1398–1403. [CrossRef] 33. Guarneri, A.; Cutifani, V.; Cespugli, M.; Pellis, A.; Vassallo, R.; Asaro, F.; Ebert, C.; Gardossi, L. Functionalization of Enzymatically Synthesized Rigid Poly(itaconate)s via Post-Polymerization Aza-Michael Addition of Primary Amines. Adv. Synth. Catal. 2019, 361, 2559–2573. [CrossRef] 34. Farmer, T.J.; Macquarrie, D.J.; Comerford, J.W.; Pellis, A.; Clark, J.H. Insights into post-polymerisation modiﬁcation of bio-based unsaturated itaconate and fumarate polyesters via aza-michael addition: Understanding the eﬀects of C=C isomerisation. J. Polym. Sci. Part A Polym. Chem. 2018, 56, 1935–1945. [CrossRef] [PubMed] 15 of 15 Polymers 2020, 12, 980 35. Medway, A.M.; Sperry, J. Heterocycle construction using the biomass-derived building block itaconic acid. Green Chem. 2014, 16, 2084–2101. [CrossRef] 36. Paytash, P.L.; Sparrow, E.; Gathe, J.C. The Reaction of Itaconic Acid with Primary Amines. J. Am. Chem. Soc. 1950, 72, 1415–1416. [CrossRef] 37. Wang, Z.; Wei, T.; Xue, X.; He, M.; Xue, J.; Song, M.; Wu, S.; Kang, H.; Zhang, L.; Jia, Q. Synthesis of fully bio-based polyamides with tunable properties by employing itaconic acid. Polymer 2014, 55, 4846–4856. [CrossRef] 38. Nsengiyumva, O.; Miller, S.A. Synthesis, characterization, and water-degradation of biorenewable polyesters derived from natural camphoric acid. Green Chem. 2019, 21, 973–978. [CrossRef] 39. Ouhichi, R.; Pérocheau Arnaud, S.; Bougarech, A.; Abid, S.; Abid, M.; Robert, T. First Example of Unsaturated Poly(Ester Amide)s Derived From Itaconic Acid and Their Application as Bio-Based UV-Curing Polymers. Appl. Sci. 2020, 10, 2163. [CrossRef] 40. Kluge, M.; Bikiaris, D.N.; Robert, T. Enhancing the properties of poly(propylene succinate) by the incorporation of crystallizable symmetrical amido diols. Eur. Polym. J. 2019, 120, 109195. [CrossRef] 41. Klonos, P.A.; Kluge, M.; Robert, T.; Kyritsis, A.; Bikiaris, D.N. Molecular dynamics, crystallization and hydration study of Poly(Propylene succinate) based Poly(Ester amide)s. Polymer 2020, 186, 122056. References This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W3205545717	https://europepmc.org/articles/pmc8542129?pdf=render	English	null	New approach of prediction of recurrence in thyroid cancer patients using machine learning	Medicine	2,021	cc-by	3,648	1. Introduction This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) which is funded by the Ministry of Science and ICT (2017R1E1A1A03070345). This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) which is funded by the Ministry of Science and ICT (2017R1E1A1A03070345). Well-differentiated thyroid cancer (WDTC) is 1 of the most common types of endocrine malignancy comprising over 90% of all thyroid cancers. Furthermore, it has shown steadily increasing incidence over the last 3 decades.[1,2] Currently, WDTC is the most prevalent cancer in Korea.[2,3] Nonetheless, despite its increasing incidence, the thyroid cancer-related mortality rate remains low.[4] This study was approved by the Institutional Review Board of Gangnam Severance Hospital, Yonsei University College of Medicine, Korea (IRB number 3- 2018-0079). As data were obtained retrospectively, informed consent is not mandatory for retrospective studies in Korea, the institutional review board waived the need for informed consent. The authors have no conﬂicts of interest to disclose. The overall 5-year survival of WDTC is high at 97.9%, and that of low risk patients in stages I and II nearly at 100%.[5] Interestingly, WDTC is unique in that it frequently metastasizes to the lymph nodes. Among the most frequent sites to which it metastasizes are the central lymph nodes. However, a metastasis to the central lymph node has only marginal effects on the long- term survival of patients.[6–8] Supplemental Digital Content is available for this article. The datasets generated during and/or analyzed during the current study are not publicly available, but are available from the corresponding author on reasonable request. a Department of Surgery, Ajou University College of Medicine, Suwon, Korea, b GN Systems Inc., Seoul, Korea, c Department of Surgery, CHA Ilsan Medical Center, Goyang-si, Korea, d Department of Surgery, Thyroid Cancer Center, Gangnam Severance Hospital, Institute of Refractory Thyroid Cancer, Yonsei University College of Medicine, Seoul, Korea, e Department of Mathematics/AI & Data Science, Ajou University, Suwon, Korea, f Department of Industrial Engineering, Ajou University, Suwon, Korea. Although the mortality rate for thyroid cancer is low and 5- year survival rates are high, postoperative recurrence is the primary cause of death in thyroid cancer patients. Reoperations for recurrent thyroid cancer can cause serious complications in the patient’s physical and mental health. Medicine ® Observational Study Abstract bs ac Although papillary thyroid cancers are known to have a relatively low risk of recurrence, several factors are associated with a higher risk of recurrence, such as extrathyroidal extension, nodal metastasis, and BRAF gene mutation. However, predicting disease recurrence and prognosis in patients undergoing thyroidectomy is clinically difﬁcult. To detect new algorithms that predict recurrence, inductive logic programming was used in this study. A total of 785 thyroid cancer patients who underwent bilateral total thyroidectomy and were treated with radioiodine were selected for our study. Of those, 624 (79.5%) cases were used to create algorithms that would detect recurrence. Furthermore, 161 (20.5%) cases were analyzed to validate the created rules. DELMIA Process Rules Discovery was used to conduct the analysis. Of the 624 cases, 43 (6.9%) cases experienced recurrence. Three rules that could predict recurrence were identiﬁed, with postoperative thyroglobulin level being the most powerful variable that correlated with recurrence. The rules identiﬁed in our study, when applied to the 161 cases for validation, were able to predict 71.4% (10 of 14) of the recurrences. Our study highlights that inductive logic programming could have a useful application in predicting recurrence among thyroid patients. Abbreviations: BMI = body mass index, ILP = inductive logic programming, RAI = radioiodine, WDTC = well-differentiated thyroid cancer Our study highlights that inductive logic programming could have a useful application in predicting recurrence among thyroid patients. Our study highlights that inductive logic programming could have a useful application in predicting patients. Abbreviations: BMI = body mass index, ILP = inductive logic programming, RAI = radioiodine, thyroid cancer. Keywords: inductive logic programming, machine learning, recurrence prediction, thyroid cancer, t gic programming, machine learning, recurrence prediction, thyroid cancer, thyroid cancer recurrence Editor: Balaji Thas Moorthy. New approach of prediction of recurrence in thyroid cancer patients using machine learning Soo Young Kim, MD, PhDa, Young-Il Kimb, Hee Jun Kim, MDc, Hojin Chang, MDd, Seok-Mo Kim, MD, PhDd, Yong Sang Lee, MD, PhDd, Soon-Sun Kwon, PhDe, Hyunjung Shin, PhDf, Hang-Seok Chang, MD, PhDd,∗, Cheong Soo Park, MD, PhDc Received: 29 March 2021 / Received in ﬁnal form: 29 August 2021 / Accepted: 23 September 2021 Medicine ® Medicine ® http://dx.doi.org/10.1097/MD.0000000000027493 3.1. Creation of rules Of the total of 785 cases, 624 (79.5%) cases were used for creating rules, whereas 161 cases (20.5%) were used for validation of created models. Among the patients who visited the Thyroid Cancer Clinic at Yonsei University College of Medicine between January, 2009 and June, 2010 as a result of receiving a diagnosis of WDTC, 797 patients who underwent bilateral total thyroidectomy with central compartment lymph node dissection and radioiodine treatment, and were followed up for more than 5years, were included in this study. Of the 797 patients, 12 patients with missing recurrence data were excluded. This study was carried out in accordance with the principles laid out in the World Medical Association’s Declaration of Helsinki, Good Clinical Practice, and associated Korean regulations. This study was approved by the Institutional Review Board of Gangnam Among the 624 cases, there were 43 (6.9%) recurrences, whereas 581 patients (93.1%) were recurrence free (Table 1). Among the 624 cases, there were 43 (6.9%) recurrences, whereas 581 patients (93.1%) were recurrence free (Table 1). In total, 5 rules were identiﬁed that could predict the 581 patients without recurrence, whereas 3 rules were identiﬁed that predict the 43 cases with recurrence (100%) (Fig. 1). In total, 5 rules were identiﬁed that could predict the 581 patients without recurrence, whereas 3 rules were identiﬁed that predict the 43 cases with recurrence (100%) (Fig. 1). Rule 1 predicted that 31 patients had recurrence (72.10%) and represented the sum of the following parameters: BMI (16.65- 29.76kg/m2) AND thyroglobulin level at 1year (0.2-2032(ng/ mL) AND thyroglobulin level at 3years (1.3-611.2ng/mL). 1. Introduction As data were obtained retrospectively, informed consent is not mandatory for retro- spective studies in Korea, the institutional review board waived the need for informed consent. Clinical parameters (age, gender, and body mass index [BMI]), pathological information (cancer size, extrathyroidal extension, multiplicity, central compartment lymph node metastasis, lateral neck lymph node metastasis, and thyroiditis), genetic information (BRAF gene mutation), laboratory parameters (fT4, TSH, thyroglobulin, anti-TPO antibody, anti-thyroglobulin antibody before and after surgery, thyroglobulin levels 1, 2, 3, 4, and 5 years after surgery), and the frequency of radioiodine ablation therapy, radioablation doses, and recurrence were collected for analysis (Table S1, Supplemental Digital Content, http://links. lww.com/MD/G450). Recurrence was diagnosed on the basis of whether it was conﬁrmed through pathological and structural information. Finally, the DELMIA Process Rules Discovery was used for analysis. Inductive logic programming was used to extract rules that represents algorithms to predict recurrence. To create algorithms which detect recurrence, 624 cases (79.5%) were used, whereas 161 cases (20.5%) were analysed for validation of created rules. Brieﬂy, ILP is performed on the basis of the following given information: A background knowledge B represents the knowledge available before learning A set of positive examples E+ and a set of negative examples E The goal is to ﬁnd hypotheses H (set of rules), where: All or almost all positive examples e ∊E+ are covered by H All or almost all positive examples e ∊E+ are covered by H No or few negative examples are covered by H.[14] p p y No or few negative examples are covered by H.[14] The advantages of ILP over propositional learning techniques such as logistic regression are that it can utilize data from relational databases with many tables, discover rules that are based on logic easily understood by humans and computers, and ﬁnally, it can generate rules that can provide meaningful insight about predictive indicators that distinguish the negative examples from positive examples.[12] The objective of this study was to assess rules for prediction of thyroid cancer recurrence from our institutional database using inductive logic programming. 1. Introduction In addition to accurate preoperative assessment and proper treatment, accurate risk stratiﬁcation with close-follow-up to reduce recurrence and detect recurrence early are necessary. ∗Correspondence: Hang-Seok Chang, Department of Surgery, Yonsei University College of Medicine, 211 Eonjuro, Gangnam-gu, Seoul 135-720, Korea (e-mail: surghsc@yuhs.ac). Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc. This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. While WDTC may remain indolent, recurrence rates are reported to be between 12 and 20%,[9,10] with males showing higher recurrence rates than that of females. Larger tumor diameter, lymph node metastasis, and pathological tumor types have been reported to have a higher recurrence rate.[9] The 2015 American Thyroid Associated guideline describes several factors that affect the risk of recurrence, such as extrathyroidal extension, lymph node involvement, multifocality, and BRAF How to cite this article: Kim SY, Kim YI, Kim HJ, Chang H, Kim SM, Lee YS, Kwon SS, Shin H, Chang HS, Park CS. New approach of prediction of recurrence in thyroid cancer patients using machine learning. Medicine 2021;100:42(e27493). Received: 29 March 2021 / Received in ﬁnal form: 29 August 2021 / Accepted: 23 September 2021 http://dx.doi.org/10.1097/MD.0000000000027493 1 Kim et al. Medicine (2021) 100:42 Medicine Table 1 Data set for modelling of rules. No recurrence Recurrence Total Cases (n) 581 42 624 % 93.1 6.9 100 gene mutation status.[11] However, predicting disease recurrence and prognosis in patients undergoing thyroidectomy is clinically difﬁcult. Inductive logic programming (ILP) is a computer programming technique that is particularly helpful in aiding researchers with data mining and the knowledge discovery process.[12] It has evolved from previous research on machine learning, logic programming, and inductive program synthesis.[13] The objective of ILP is to discover a set of if-then rules that predicts the presence or absence of a disease or outcome. To generate rules, the following parameters are necessary: positive and negative examples; background knowledge about given examples; and user-deﬁned constraints about what type of rules may be learned.[12] Severance Hospital, Yonsei University College of Medicine, Korea (IRB number 3-2018-0079). As data were obtained retrospectively, informed consent is not mandatory for retro- spective studies in Korea, the institutional review board waived the need for informed consent. Severance Hospital, Yonsei University College of Medicine, Korea (IRB number 3-2018-0079). 3.1. Creation of rules anti-thyroglobulin antibody, fT4, central and lateral lymph node metastasis, cancer size, and postoperative TSH level. Rule 2 included 11 patients (23.30%) with recurrence who had the following characteristics: an anti-thyroglobulin antibody level of 10 to 194.8IU/mL; a BMI of 19.49 to 25.88kg/m2; a free T4 concentration of 1.3 to 3.2ng/dL; central lymph node metastasis, 0 to 6 in number; lateral lymph node metastasis, 0 to 9 in number; tumor size of 0.5 to 3.8cm; thyroglobulin at 1-year follow-up of 0.1 to 14.2ng/mL; thyroglobulin at 3-year follow- up of 0.2 to 611.2ng/mL; thyroglobulin at 5-year follow-up of 0.1 to 2014ng/mL; thyroglobulin after radioiodine (RAI) of 1.1 to 216.1ng/mL; thyroglobulin before RAI of 1.3 to 4417ng/mL; and a TSH after surgery of 0.02 to 1.8mcIU/mL3. , , p p Although the primary tumor marker for detecting recurrence in cases undergoing total thyroidectomy and radioiodine treatment is the level of thyroid speciﬁc thyroglobulin, in patients with thyroglobulin-antibodies, the value can be over- or under- estimated, which makes it difﬁcult to detect recurrence.[11,15,16] The serum thyroglobulin level is determined by measuring the residual amount of malignant and normal thyroid tissue, degree of injury to thyroid tissue (including ﬁne needle biopsy, operative resection, and RAI), and levels of thyroid-stimulating hor- mone.[17] Although the primary tumor marker for detecting recurrence in cases undergoing total thyroidectomy and radioiodine treatment is the level of thyroid speciﬁc thyroglobulin, in patients with thyroglobulin-antibodies, the value can be over- or under- estimated, which makes it difﬁcult to detect recurrence.[11,15,16] The serum thyroglobulin level is determined by measuring the residual amount of malignant and normal thyroid tissue, degree of injury to thyroid tissue (including ﬁne needle biopsy, operative resection, and RAI), and levels of thyroid-stimulating hor- mone.[17] Rule 3 described 15 patients (34.90%) with recurrence and was deﬁned as the sum of the following parameters: anti- thyroglobulin antibody level of 10to 15.4IU/mL, number of central lymph node metastasis of 0 to 11 regardless of multiplicity, tumor size of 1.3 to 6.2cm, 5-year thyroglobulin level of 0.1 to 2014ng/mL, thyroglobulin after RAI of 31.2 to 5000ng/mL, thyroglobulin level before RAI of 37.2 to 1186ng/ mL, and TSH after surgery of 0.03 to 10.88mcIU/mL. Studies have reported that early postoperative stimulated serum thyroglobulin level is an independent predictor of structural recurrence, and it accurately quantiﬁes the risk of structural disease recurrence. 3.1. Creation of rules All Name Class Purity Size Relative size Age_NewANTI_TG_AB_SHORTBMI BRAF FT4_SHORTYMPH_C_IRRMPH_C_MEYMPH_L_IRRMPH_L_ME MULTI NUMBER SIZE TG_1YEARTG_2YEARTG_3YEAR TG_5YEAR TG_AFTER_RAI1 TG_BEFORE TG_BEFORE_RAI1 TSH_BEFORE TSH_SHORT TYPE BP001 0 100.00% 452 77.80% [ 19.91 ; 67.73 ] [ 0.1 ; 3 ] [ 0.1 ; 0.4 ] [ 0.1 ; 113.4 ] { 1; 3 } BP002 0 100.00% 236 40.60% [ 20.59 ; 77.26 ] [ 0.1 ; 3.1 ] [ 0.1 ; 0.7 ] [ 2.6 ; 110.8 ] [ 0.12 ; 100 ] BP003 0 100.00% 301 51.80% [ 0 ; 50 ] [ 0.1 ; 2.4 ] BP004 0 100.00% 185 31.80% { 1; 9 } [ 0 ; 56 ] [ 0.6 ; 6.5 ] [ 0.1 ; 0.8 ] [ 0.01 ; 0.96 ] BP005 0 100.00% 379 65.20% [ 0.1 ; 2.7 ] [ 0 ; 19 ] [ 0 ; 11 ] [ 0.1 ; 1.7 ] [ 0.1 ; 1.2 ] [ 0.05 ; 98.81 ] RS001 1 100.00% 31 72.10% [ 16.65 ; 29.76 ] [ 0.2 ; 2032 ] [ 1.3 ; 611.2 ] RS002 1 90.90% 11 23.30% [ 10 ; 194.8 ] [ 19.49 ; 25.88 ] [ 1.3 ; 3.2 ] [ 0 ; 6 ] [ 0 ; 9 ] [ 1 ; 1 ] [ 0.5 ; 3.8 ] [ 0.1 ; 14.2 ] [ 0.2 ; 611.2 [ 1.1 ; 216.1 ] [ 1.3 ; 4417 ] [ 0.03 ; 1.83 ] RS003 1 100% 15 34.90% [ 10 ; 15.4 ] [ 0 ; 11 ] { 0; 2 } [ 1.3 ; 6.2 ] [ 0.1 ; 2014 ] [ 31.2 ; 5000 ] [ 37.2 ; 1186 ] [ 0.03 ; 10.88 ] Figure 1. Rules for the prediction of cases with and without recurrence. BP001 to 005 are rules for cases without recurrence; RS001, RS002, and RS003 are rules which predict recur. 2 Kim et al. Medicine (2021) 100:42 www.md-journal.com Table 2 Data set for validation of created rules. No recurrence Recurrence Total Cases 147 14 161 % 91.3 8.7 100 Table 4 Validation of success rates. Actual class distribution No recurrence Recurrence Average Success rate 98% 71.4% 95.7% Failure rate 0% 0% 0% Abstention rate 2% 28.6% 4.3% patients who met the criteria of rule 1 had recurrences, and there were no patients without recurrence who met the above criteria (purity 100%). 3.1. Creation of rules In agreement with these results, our study suggests that early postoperative thyroglobulin could be employed in risk stratiﬁcation using a serum thyroglobulin level of <2ng/mL as a cut-off to guide adjuvant therapy and determine the frequency of surveillance in patients with lower early postoperative thyroglobulin.[18] A total of 5 rules were identiﬁed that described all patients without recurrence. 3.2. Validation of created rules This study is the ﬁrst study that attempted to predict thyroid cancer recurrence using machine learning models. Although the prediction rate was relatively high, the clinical meaning and everyday clinical application should be further clariﬁed. Nevertheless, the results of our study show that ILP with validation can be reliably used to help in the identiﬁcation of novel hypotheses for recurrence in thyroid cancer patients. In the validation group, 11 (7.0%) recurrences were observed among the 159 patients (Table 2). For all created rules, the average prediction success rate was 95.7%. Of the 14 cases with recurrence, only 10 were correctly predicted to be positive for recurrence (success rate 71.4%), whereas 98% of the cases without recurrence were correctly predicted to be negative for recurrence (Tables 3 and 4). 4. Discussion Writing – review & editing: Soo Young Kim, Hang- Cheong Soo Park. [9] Luo XY, Chen AM, Zhou Y, Jiang YQ, Zhang BH, Wu JP. Analysis of risk factors for postoperative recurrence of thyroid cancer. J Buon 2019;24:813–8. Methodology: Soo Young Kim. Methodology: Soo Young Kim. Methodology: Soo Young Kim. Software: Young-Il Kim. Supervision: Soon-Sun Kwon, Hyunjung Shin. [10] Brassard M, Borget I, Edet-Sanson A, et al. Long-term follow-up of patients with papillary and follicular thyroid cancer: a prospective study on 715 patients. J Clin Endocrinol Metab 2011;96:1352–9. Writing – original draft: Soo Young Kim. Writing – review & editing: Soo Young Kim, Hang-Seok Chang, Cheong Soo Park. [11] Haugen BR, Alexander EK, Bible KC, et al. 2015 American Thyroid Association Management Guidelines for Adult Patients with Thyroid Nodules and Differentiated Thyroid Cancer The American Thyroid Association Guidelines Task Force on Thyroid Nodules and Differentiated Thyroid Cancer. Thyroid 2016;26:1–133. 4. Discussion Kim SY made contributions to conception, design of the work, acquisition, analysis, interpretation of data and drafted the work. Kim YI made contributions to analysis and interpretation of data. Kim HJ, Chang H, Kim SM, Lee YS made contributions to the conception, design of work and acquisition. Kim SY made contributions to conception, design of the work, acquisition, analysis, interpretation of data and drafted the work. Kim YI made contributions to analysis and interpretation of data. Ki HJ Ch H Ki SM L YS d t ib ti t th In our study, we identiﬁed 3 rules that described all patients with recurrence in the model creating group and could correctly predict 71.40% of the recurrences. The most important parameters included in the model were thyroglobulin levels at 1, 2, 3, 4, and 5years after onset and thyroglobulin levels before and after surgery. Other factors included in the rules were BMI, Kim HJ, Chang H, Kim SM, Lee YS made contributions to the conception, design of work and acquisition. Kwon SS and Shin H made contributions to analysis and interpretation of data. Kwon SS and Shin H made contributions to analysis and interpretation of data. Chang HS and Park CS substantially revised the manuscript. Chang HS and Park CS substantially revised the manuscript. Table 3 Validation of created rules to predict recurrence. Actual class distribution No recurrence Recurrence Total Prediction No recurrence 144 0 144 Recurrence 0 10 10 Abstention 3 4 7 Total 147 14 161 Chang HS and Park CS substantially revised the manuscript. All authors have approved the submitted version and have agreed both to be personally accountable for the author’s own contributions and to ensure that questions related to the accuracy or integrity of any part of the work. Conceptualization: Soo Young Kim, Hee Jun Kim, Hojin Chang, Seok-Mo Kim, Yong Sang Lee. Conceptualization: Soo Young Kim, Hee Jun Kim, Hojin Chang, Seok-Mo Kim, Yong Sang Lee. Data curation: Soo Young Kim, Hee Jun Kim, Hojin Chang, Seok-Mo Kim, Yong Sang Lee. Data curation: Soo Young Kim, Hee Jun Kim, Hojin Chang, Seok-Mo Kim, Yong Sang Lee. Formal analysis: Soo Young Kim, Young-Il Kim, Soon-Sun Kwon, Hyunjung Shin. Formal analysis: Soo Young Kim, Young-Il Kim, Soon-Sun Kwon, Hyunjung Shin. 3 Kim et al. Medicine (2021) 100:42 Medicine Methodology: Soo Young Kim. Software: Young-Il Kim. Supervision: Soon-Sun Kwon, Hyunjung Shin. Writing – original draft: Soo Young Kim. References [1] Greenlee RT, Hill-Harmon MB, Murray T, Thun M. Cancer statistics, 2001. CA Cancer J Clin 2001;51:15–36. [12] Dzeroski S. From inductive logic programming to relational data mining. Lect Notes Artif Int 2006;4160:1–14. Lect Notes Artif Int 2006;4160:1–14. [2] Pellegriti G, Frasca F, Regalbuto C, Squatrito S, Vigneri R. Worldwide increasing incidence of thyroid cancer: update on epidemiology and risk factors. J Cancer Epidemiol 2013;2013:965212. Epub Ahead of Print. [13] Bergadano FGD. Inductive Logic ProgrammingFrom Machine Learning to Software Engineering. Cambridge, MA: Massachusetts Institute of Technology; 1996. [3] Jung KW, Won YJ, Kong HJ, Lee ES. Cancer statistics in Korea: incidence, mortality, survival, and prevalence in 2016. Cancer Res Treat 2019;51:417–30. gy; [14] S.M. Inductive logic programming. New Gener Comput 1991;8: 295–318. [15] Ringel MD, Nabhan F. Approach to follow-up of the patient with differentiated thyroid cancer and positive anti-thyroglobulin antibodies. J Clin Endocrinol Metab 2013;98:3104–10. [4] Ahn HS, Kim HJ, Welch HG. Korea’s thyroid-cancer “epidemic”– screening and overdiagnosis. N Engl J Med 2014;371:1765–7. [5] Tsang TW, Brierley JD, Simpson WJ, Panzarella T, Gospodarowicz MK, Sutcliffe SB. The effects of surgery, radioiodine, and external radiation therapy on the clinical outcome of patients with differentiated thyroid carcinoma. Cancer 1998;82:375–88. [16] de Meer SGA, Vorselaars W, Kist JW, et al. Follow-up of patients with thyroglobulin-antibodies: rising Tg-Ab trend is a risk factor for recurrence of differentiated thyroid cancer. Endocr Res 2017;42: 302–10. ; [6] Shaha AR. Management of the neck in thyroid cancer. Otolaryngol Clin North Am 1998;31:823–31. [17] Spencer CA, LoPresti JS. Technology insight: measuring thyroglobulin and thyroglobulin autoantibody in patients with differentiated thyroid cancer. Nat Clin Pract Endoc 2008;4:223–33. [7] Noguchi S, Noguchi A, Murakami N. Papillary carcinoma of the thyroid. II. Value of prophylactic lymph node excision. Cancer 1970;26:1061–4. alue of prophylactic lymph node excision. Cancer 1970;26:10 [18] Jayasekara J, Jonker P, Lin JF, et al. Early postoperative stimulated serum thyroglobulin quantiﬁes risk of recurrence in papillary thyroid cancer. Surgery 2020;167:40–5. [8] Gimm O, Rath FW, Dralle H. Pattern of lymph node metastases in papillary thyroid carcinoma. Br J Surg 1998;85:252–4. 4 4
https://openalex.org/W4360999140	https://www.nature.com/articles/s41561-023-01159-6.pdf	English	null	A solar wind-derived water reservoir on the Moon hosted by impact glass beads	Nature geoscience	2,023	cc-by	13,731	Article https://doi.org/10.1038/s41561-023-01159-6 A solar wind-derived water reservoir on the Moon hosted by impact glass beads Huicun He 1,2, Jianglong Ji 1,2, Yue Zhang3, Sen Hu 1,2 , Yangting Lin 1,2, Hejiu Hui 3,4 , Jialong Hao 1, Ruiying Li 1, Wei Yang 1,2, Hengci Tian 1,2, Chi Zhang 1,2, Mahesh Anand 5,6, Romain Tartèse 7, Lixin Gu 1, Jinhua Li 1,2, Di Zhang 8, Qian Mao8, Lihui Jia 8, Xiaoguang Li 8, Yi Chen 8, Li Zhang9,4, Huaiwei Ni9,4, Shitou Wu 8, Hao Wang 8, Qiuli Li 8, Huaiyu He8, Xianhua Li 8 & Fuyuan Wu 8 Received: 9 June 2022 Accepted: 24 February 2023 Published online: 27 March 2023 Check for updates The past two decades of lunar exploration have seen the detection of substantial quantities of water on the Moon’s surface. It has been proposed that a hydrated layer exists at depth in lunar soils, buffering a water cycle on the Moon globally. However, a reservoir has yet to be identified for this hydrated layer. Here we report the abundance, hydrogen isotope composition and core-to-rim variations of water measured in impact glass beads extracted from lunar soils returned by the Chang’e-5 mission. The impact glass beads preserve hydration signatures and display water abundance profiles consistent with the inward diffusion of solar wind-derived water. Diffusion modelling estimates diffusion timescales of less than 15 years at a temperature of 360 K. Such short diffusion timescales suggest an efficient water recharge mechanism that could sustain the lunar surface water cycle. We estimate that the amount of water hosted by impact glass beads in lunar soils may reach up to 2.7 × 1014 kg. Our direct measurements of this surface reservoir of lunar water show that impact glass beads can store substantial quantities of solar wind-derived water on the Moon and suggest that impact glass may be water reservoirs on other airless bodies. It has long been argued that there could be water and other volatile species at the surface of the Moon1–3. Renewed lunar exploration and advances in remote-sensing measurements in the late 1990s allowed the neutron spectrometer on board the Lunar Prospector mission to confirm the existence of water ice at the lunar poles4. Following this, the Moon mineralogy mapper instrument on board the Chandrayaan-1 spacecraft detected the diagnostic absorption bands of hydroxyl and/or water at 2.8–3.0 μm on the surface of the Moon5. 1Key Laboratory of Earth and Planetary Physics, Chinese Academy of Sciences, Beijing, China. 2University of Chinese Academy of Sciences, Beijing, China. 3State Key Laboratory for Mineral Deposits Research and Lunar and Planetary Science Institute, School of the Earth Sciences and Engineering, Nanjing University, Nanjing, China. 4CAS Center for Excellence in Comparative Planetology, Hefei, China. 5School of Physical Sciences, The Open University, Milton Keynes, UK. 6Department of Earth Sciences, The Natural History Museum, London, UK. 7Department of Earth and Environmental Sciences, The University of Manchester, Manchester, UK. 8State Key Laboratory of Lithospheric Evolution, Chinese Academy of Sciences, Beijing, China. 9School of Earth and Space Sciences, University of Science and Technology of China, Hefei, China. e-mail: husen@mail.iggcas.ac.cn; hhui@nju.edu.cn nature geoscience nature geoscience nature geoscience A solar wind-derived water reservoir on the Moon hosted by impact glass beads Blue squares indicate locations of NanoSIMS in the first analytical session. Each NanoSIMS analysis pit is 7 × 7 μm2. 50 µm c 3 5 7 9 11 13 14 15 17 19 21 27 29 23 1 2 25 Profile 2 Profile 1 b c 25 µm d 4 3 5 6 7 8 9 1 2 10 Profile 4 d f e f e 20 µm Fig. 1 \| Representative back-scattered electron images of CE5 impact glass beads after nanoSIMS analyses. a, CE5#33,002. b, CE5#33,003. c, CE5#33,036. d, CE5#33,046. e, CE5#33,052. f, CE5#33,076. These impact glass beads have spherical shapes and have homogeneous chemical compositions (Supplementary Table 2). Beads CE5#33,003 and CE5#33,036 are coated by finer agglutinates. Six profiles carried out on five impact glass beads in the second session are outlined in red. Blue squares indicate locations of NanoSIMS in the first analytical session. Each NanoSIMS analysis pit is 7 × 7 μm2. surface of the Moon13. To sustain a water cycle at the surface of the Moon, there should be a hydrated layer (reservoir) at depth in lunar soils13. However, finding this water reservoir has remained elusive, despite several studies having investigated the water inventory of fine mineral grains in lunar soils (for example, refs. 34,35), impact-produced agglutinates27, volcanic rocks (for example, ref. 26) and pyroclastic glass beads21,36. Consequently, there must be a yet-unidentified water reservoir in lunar soils that has the capacity to buffer a lunar surface water cycle. shadowed regions within Cabeus crater11. Elevated water-ice abun- dance in lunar polar regions was further supported by the neutron flux measurements performed by the Lunar Exploration Neutron Detector on board the Lunar Reconnaissance Orbiter spacecraft12. Recently, the neutral mass spectrometer on the Lunar Atmosphere and Dust Environment Explorer detected exospheric water liberated by meteoroid impacts13, and ground-based telescope observations detected molecular water on the lunar surface14. Today, there is little doubt that most of the Moon’s surface harbours water in one form or another. However, the origin(s) of this lunar surface water and its spatial distribution and evolution during regolith gardening remain largely unknown, despite key implications for future lunar surface exploration and for better understanding the (sub)surface water reservoir and processing on Solar System airless bodies. The lunar soils returned by the Apollo, Luna and Chang’e-5 (CE5) missions comprise mainly rock and mineral fragments, pyroclastic glass beads, impact-produced agglutinates and impact glass beads37,38. A solar wind-derived water reservoir on the Moon hosted by impact glass beads Detailed analyses indicated that hydroxyl/water could be present all over the Moon’s surface, which also displayed temporal and spatial variations, with equivalent water abundances ranging between ~10 and 1,000 μg g−1 (refs. 6–10). Furthermore, the Lunar Crater Observation and Sensing Satellite impact experiment carried out in 2009 provided direct evi- dence for high water-ice abundances (5.6 ± 2.9 wt%) in permanently It has long been argued that there could be water and other volatile species at the surface of the Moon1–3. Renewed lunar exploration and advances in remote-sensing measurements in the late 1990s allowed the neutron spectrometer on board the Lunar Prospector mission to confirm the existence of water ice at the lunar poles4. Following this, the Moon mineralogy mapper instrument on board the Chandrayaan-1 spacecraft detected the diagnostic absorption bands of hydroxyl and/or water at 2.8–3.0 μm on the surface of the Moon5. Detailed analyses indicated that hydroxyl/water could be present all over the Moon’s surface, which also displayed temporal and spatial variations, with equivalent water abundances ranging between ~10 and 1,000 μg g−1 (refs. 6–10). Furthermore, the Lunar Crater Observation and Sensing Satellite impact experiment carried out in 2009 provided direct evi- dence for high water-ice abundances (5.6 ± 2.9 wt%) in permanently Nature Geoscience \| Volume 16 \| April 2023 \| 294–300 Nature Geoscience \| Volume 16 \| April 2023 \| 294–300 294 Article https://doi.org/10.1038/s41561-023-01159-6 15 µm 25 µm 50 µm 25 µm 20 µm 20 µm a b c d e f 4 3 5 6 7 8 Profile 3 9 10 11 3 5 7 9 11 13 14 15 17 19 21 27 29 4 3 5 6 7 8 9 3 4 5 7 8 9 10 14 3 4 5 6 7 8 9 10 11 12 13 1 2 23 1 2 1 2 1 2 1 2 1 2 6 25 10 Profile 2 Profile 1 Profile 6 Profile 5 Profile 4 Fig. 1 \| Representative back-scattered electron images of CE5 impact glass beads after nanoSIMS analyses. a, CE5#33,002. b, CE5#33,003. c, CE5#33,036. d, CE5#33,046. e, CE5#33,052. f, CE5#33,076. These impact glass beads have spherical shapes and have homogeneous chemical compositions (Supplementary Table 2). Beads CE5#33,003 and CE5#33,036 are coated by finer agglutinates. Six profiles carried out on five impact glass beads in the second session are outlined in red. Nature Geoscience \| Volume 16 \| April 2023 \| 294–300 Solar wind origin of water The rims of CE5 impact glass beads are generally characterized by higher water abundances (up to ~2,000 μg g−1) and lower δD values (~−990‰) compared with pyroclastic glasses, impact-produced agglu- tinates and melt inclusions reported in previous studies (Fig. 2 and Supplementary Table 4). The extremely low δD values measured at the rims of CE5 impact glasses (Fig. 3) are consistent with the H iso- tope composition of solar wind40,41. These low δD values are distinct from other potential lunar water reservoirs, such as indigenous water outgassed during lunar volcanism (δD ≈ −300 to 1,200‰) (refs. 22,36,42), water-rich carbonaceous chondrites (δD ≈ −200 to 800‰) (refs. 43,44) and comets (δD ≈0 to 2,500‰) (refs. 45,46), indicating that the water retained in CE5 impact glasses is of a solar wind origin. endmember and a water-rich but extremely low D/H endmember, reproduces well the range of water abundances and δD values meas- ured in CE5 impact glasses (Fig. 2). Modelling requires that the initial water abundances of CE5 impact glass beads are less than 50 μg g−1 before the addition of solar wind-derived water (Fig. 2). This require- ment is consistent with the water abundances measured at the cores of CE5 impact glasses, which are comparable to the instrument H2O background (~10–30 μg g−1; Methods), indicating that any water poten- tially present in the precursor materials was lost during formation of the CE5 impact glass beads, before the addition of solar wind-derived water. Most important, the hydration profiles in CE5 impact glass beads show that solar wind-derived water has diffused inwards into the glasses (Fig. 3), unambiguously demonstrating that the addition of solar wind-derived water post-dated the formation of the impact glass beads. There are two potential mechanisms, de-gassing and two- endmember mixing, that could explain the negative correlation between water abundances and δD values defined by CE5 impact glasses (Fig. 2). Because the initial D/H ratio is so low (D/H = 3.1 × 10−6 for the analysis with the highest H2O abundance), the H2 de-gassing modelling indicates that D/H fractionation due to H2 loss would yield an increase in δD values from about −980‰ up to only −926‰ for 99.9% loss, starting with an initial water abundance in CE5 impact glasses of ~2,000 μg g−1 (Fig. 2 and Methods). https://doi.org/10.1038/s41561-023-01159-6 Article 0 500 1,000 1,500 2,000 –1,000 –500 0 500 1,000 0 20 40 60 80 100 –1,000 –500 H2O0 = 50 µg g –1 H2O0 = 5 µg g –1 H2O0 = 50 µg g –1 0 500 1,000 CE5 impact glasses First session Second session Literature Melt inclusions Pyroclastic glasses Agglutinates Modelling Binary mixing Degassing Water abundance (µg g–1) δDSMOW (‰) Fig. 2 \| Water abundances and δD values measured in CE5 impact glass beads. All glass bead analyses define a negative correlation between the water abundances and δD values, which is consistent with a two-endmember mixing model (red lines) that indicates addition of solar wind-derived water with δD value of −990‰ into a low water abundance (H2O0 = 5–50 μg g−1) CE5 impact glass with δD value of +500‰ (ref. 50). De-gassing of H2 from CE5 impact glasses cannot be responsible for the large observed H2O–δD variations (brown line). The inset diagram is the zoom-in view of water abundance range of 0–100 μg g−1. For more details, see Methods. The literature data for melt inclusions, pyroclastic glasses and impact-produced agglutinates are also plotted for comparison, and the datasets are listed in Supplementary Table 4. All error bars correspond to 2 s.d. analytical uncertainties. SMOW, standard mean ocean water. H2O0 is the water abundance of impact glass beads prior to the inward diffusion of solar wind- derived water. Solar wind origin of water Therefore, H2 loss from CE5 impact glasses via de-gassing is not a viable mechanism to account for the observed δD variations from around −990‰ to +522‰ (Fig. 2). More important, the high water content at the rim but low water content in the core of the glass beads is not consistent with the de-gassing scenario. Water abundance and hydrogen isotope composition of impact glasses All glass bead analyses define a negative correlation between the water abundances and δD values, which is consistent with a two-endmember mixing model (red lines) that indicates addition of solar wind-derived water with δD value of −990‰ into a low water abundance (H2O0 = 5–50 μg g−1) CE5 impact glass with δD value of +500‰ (ref. 50). De-gassing of H2 from CE5 impact glasses cannot be responsible for the large observed H2O–δD variations (brown line). The inset diagram is the zoom-in view of water abundance range of 0–100 μg g−1. For more details, see Methods. The literature data for melt inclusions, pyroclastic glasses and impact-produced agglutinates are also plotted for comparison, and the datasets are listed in Supplementary Table 4. All error bars correspond to 2 s.d. analytical uncertainties. SMOW, standard mean ocean water. H2O0 is the water abundance of impact glass beads prior to the inward diffusion of solar wind- derived water. Fig. 2 \| Water abundances and δD values measured in CE5 impact glass beads. All glass bead analyses define a negative correlation between the water abundances and δD values, which is consistent with a two-endmember mixing model (red lines) that indicates addition of solar wind-derived water with δD value of −990‰ into a low water abundance (H2O0 = 5–50 μg g−1) CE5 impact glass with δD value of +500‰ (ref. 50). De-gassing of H2 from CE5 impact glasses cannot be responsible for the large observed H2O–δD variations (brown line). The inset diagram is the zoom-in view of water abundance range of 0–100 μg g−1. For more details, see Methods. The literature data for melt inclusions, pyroclastic glasses and impact-produced agglutinates are also plotted for comparison, and the datasets are listed in Supplementary Table 4. All error bars correspond to 2 s.d. analytical uncertainties. SMOW, standard mean ocean water. H2O0 is the water abundance of impact glass beads prior to the inward diffusion of solar wind- derived water. A solar wind-derived water reservoir on the Moon hosted by impact glass beads Among these components, the water inventory of impact glass beads has not yet been investigated in detail, despite these glassy beads being potential candidates for playing a significant role in a lunar surface water cycle. To investigate this possibility, we carried out a systematic characterization of the petrography, major element composition, Raman characteristics, water abundance and hydrogen isotope com- position on the impact glass beads returned by the CE5 mission, aiming to identify and characterize the missing water reservoir on the Moon’s surface. A recent geochronological study of CE5 impact glass beads has shown that they formed more or less continuously for the past 2 Gyr, with prominent peaks in formation ages at ~575 million years ago (Ma), 380 Ma, 68 Ma and 35 Ma, originating predominantly from a few 1- to 5-km-diameter impact craters in the Em4 (Eratosthenian-aged mare) basaltic unit where CE5 landed39. There are several potential sources and processes that could have contributed to the water inventory at the surface of the Moon15, such as (1) solar wind implantation16–20, (2) outgassing of volatiles dur- ing lunar volcanism21–23, (3) deposition of volatile-bearing pyroclastic deposits24 and minerals25 and (4) delivery by impacts of comets and asteroids26–29. It is generally thought that solar wind hydrogen-ion implantation could react with surface minerals to produce hydroxyl or water in lunar soils30,31. The surface water produced and/or delivered on the Moon’s equatorial regions may migrate to polar regions, driven by temperature oscillations16,19,32,33, and could also be partially released to space13. Hence, a scenario of lunar water cycle was further proposed to describe the retention, release and replenishment of water on the 295 https://doi.org/10.1038/s41561-023-01159-6 Nature Geoscience \| Volume 16 \| April 2023 \| 294–300 Water abundance and hydrogen isotope composition of impact glasses 0 500 1,000 1,500 2,000 –1,000 –500 0 500 1,000 0 20 40 60 80 100 –1,000 –500 H2O0 = 50 µg g –1 H2O0 = 5 µg g –1 H2O0 = 50 µg g –1 0 500 1,000 CE5 impact glasses First session Second session Literature Melt inclusions Pyroclastic glasses Agglutinates Modelling Binary mixing Degassing Water abundance (µg g–1) δDSMOW (‰) p p g The studied impact glass beads were hand picked from a lunar soil sample (sample CE5C0100YJFM00103; ~1 g) scooped by the CE5 lander robotic arm. A total of 117 individual spherical glass beads were characterized using field-emission scanning electron microscopy (SEM), electron probe microanalyser (EPMA) and Raman spectro scopy (Fig. 1, Supplementary Figs. 1 and 2, Supplementary Table 1 and Extended Data Fig. 1). Among them, 32 impact glass beads charac terized by smooth exposed surfaces and chemical compositions consistent with those of bulk CE5 basalts were chosen for in situ water abundance and H isotope analysis using a nanoscale secondary ion mass spectrometer (NanoSIMS) (Fig. 1, Extended Data Figs. 2 and 3 and Supplementary Table 1). The CE5 impact glass beads con- tain 0–1,909 μg g−1 equivalent H2O, with H isotope compositions (given in delta notation, δD = 1000 × (D/Hsample/D/HSMOW−1), where D/Hsample is the measured D/H ratio measured in the sample and D/ HSMOW is the D/H ratio (1.5576 × 10−4) of the standard mean ocean water) ranging from −990 ± 6‰ to 522 ± 440‰ and which are negatively correlated with the water abundances (Fig. 2). We also analysed water abundances and H isotope compositions along six transects in five glass beads (CE5#33,003, CE5#33,036, CE5#33,046, CE5#33,052 and CE5#33,076; Fig. 1), which show elevated water abundances at the rims that gradually decrease towards the cores while δD values decrease from cores to rims (Fig. 3 and Extended Data Figs. 4 and 5). Three glass beads display sharp decreases in water content from the rims towards the cores, while the other two glass beads display a smoother water abundance variation (Fig. 3 and Extended Data Figs. 4 and 5). δDSMOW (‰) , Water abundance (µg g–1) Fig. 2 \| Water abundances and δD values measured in CE5 impact glass Fig. 2 \| Water abundances and δD values measured in CE5 impact glass beads. Impact glasses as the driver for the lunar surface water cycle The apparent distances along the profiles were measured from the rim on a side (Fig. 1c). A water-diffusion coefficient of 20.84 μm2 yr−1 at T = 360 K was used48. The maximum diffusion durations for Profile 1 and Profile 2 are 9 and 15 yr at T = 360 K, respectively. Profile 2 was further modelled by a post-diffusion de-gassing on the basis of the bell shape of water abundance on the right side. More modelling details can be found in Methods. All error bars correspond to 2 s.d. analytical uncertainties. 0 20 40 60 80 0 500 1,000 1,500 2,000 a H2O abundance (µg g–1) Distance (µm) T = 360 K t = 3 yr t = 9 yr Profile 1 –1,000 –800 –600 –400 –200 0 b T = 360 K t = 3 yr t = 9 yr Profile 1 δDSMOW (‰) 0 20 40 60 80 Distance (µm) b a T = 360 K t = 5 yr t = 15 yr Diffusion+degassing Profile 2 c 0 500 1,000 1,500 2,000 H2O abundance (µg g–1) 0 20 40 60 100 80 Distance (µm) d –1,000 –800 –600 –400 –200 0 T = 360 K t = 5 yr t = 15 yr Profile 2 δDSMOW (‰) 0 20 40 60 100 80 Distance (µm) d c Fig. 3 \| Two profiles across CE5 impact glass bead CE5#33,036. a,b, Water used48. The maximum diffusion durations for Profile 1 and Profile 2 are 9 and 15 yr at T = 360 K, respectively. Profile 2 was further modelled by a post-diffusion de-gassing on the basis of the bell shape of water abundance on the right side. More modelling details can be found in Methods. All error bars correspond to 2 s.d. analytical uncertainties. g p p g abundance (a) and δD values (b) measured along Profile 1, as outlined in Fig. 1c. c,d, Water abundance (c) and δD values (d) measured along Profile 2, as outlined in Fig. 1c. The apparent distances along the profiles were measured from the rim on a side (Fig. 1c). A water-diffusion coefficient of 20.84 μm2 yr−1 at T = 360 K was Some of the CE5 impact glass rims contain higher water abun- dance (up to ~2,000 μg g−1) than that reported in Apollo agglutinates (<600 μg g−1) (Fig. Impact glasses as the driver for the lunar surface water cycle The negative correlation between water abundances and δD values, coupled with the hydration profiles observed for CE5 impact glass beads, reveal that solar wind-derived water can diffuse into and be stored in lunar impact glasses on the Moon’s surface, probably via post-implantation diffusion19,47. To model the diffusion timescale, we used Raman spectroscopy to confirm that water hosted in CE5 impact glass beads is in the form of hydroxyl/molecular water (Extended Data Fig. 6). The diffusion coefficient of hydroxyl/water in glasses48 was used for modelling the duration timescale necessary for forming the observed hydration profiles of CE5 impact glass beads (Methods). Diffusion modelling results indicate that the diffusion time needed In the other scenario, the CE5 impact glasses could have lost most of the original water inventory of their precursors during their formation at high temperature, following which solar wind-derived water started diffusing inwards into these glass beads on the Moon’s surface. This scenario is consistent with the notable lower abun- dances of volatile elements such as Na2O (most <0.05 wt%) and K2O (<0.06 wt%) in CE5 impact glasses compared with CE5 mare basalt fragments (Na2O ≈ 0.6 wt% and K2O ≈ 0.1–0.2 wt%, on average)39. This binary mixing model, between a water-poor but relatively high D/H Nature Geoscience \| Volume 16 \| April 2023 \| 294–300 296 https://doi.org/10.1038/s41561-023-01159-6 Article T = 360 K t = 5 yr t = 15 yr Diffusion+degassing Profile 2 0 20 40 60 80 0 500 1,000 1,500 2,000 –1,000 –800 –600 –400 –200 0 a b c d H2O abundance (µg g–1) Distance (µm) T = 360 K t = 3 yr t = 9 yr Profile 1 T = 360 K t = 3 yr t = 9 yr Profile 1 δDSMOW (‰) 0 20 40 60 80 Distance (µm) 0 500 1,000 1,500 2,000 H2O abundance (µg g–1) 0 20 40 60 100 80 Distance (µm) –1,000 –800 –600 –400 –200 0 T = 360 K t = 5 yr t = 15 yr Profile 2 δDSMOW (‰) 0 20 40 60 100 80 Distance (µm) Fig. 3 \| Two profiles across CE5 impact glass bead CE5#33,036. a,b, Water abundance (a) and δD values (b) measured along Profile 1, as outlined in Fig. 1c. c,d, Water abundance (c) and δD values (d) measured along Profile 2, as outlined in Fig. 1c. Impact glasses as the driver for the lunar surface water cycle 2); however, they may still not be water saturated compared with terrestrial basaltic glasses of similar chemical compo- sitions that can contain up to 2.43 wt% H2O (ref. 49). This could be the result of dynamic diffusion and release of water in the impact glass beads controlled by the time-of-the-day temperature oscillations10. The dynamic ingress and egress of water in impact glass beads could have acted as a buffer to explain the global and daily variations of water abundance on the lunar surface and in the lunar exosphere7,8,10. to produce the observed hydration profiles varies from 1 year to 15 years at a peak temperature (T) of 360 K, relevant for the landing site of CE532 (Fig. 3, Extended Data Figs. 4 and 5 and Methods). This short diffusion time indicates that the solar wind-derived water can be rapidly accumulated and stored in lunar impact glass beads. It is inter- esting to note that one profile on bead CE5#33,036 displays notable post-diffusion water loss at the very rim (Fig. 3, and Methods), suggest- ing that CE5 impact glass beads have the capacity of both incorporat- ing solar wind-derived water and releasing it to the lunar exosphere if T increases. Therefore, we propose that impact glass beads in lunar soils are a prime water reservoir candidate able to drive the lunar surface water cycle (Fig. 4). Nature Geoscience \| Volume 16 \| April 2023 \| 294–300 Inventory of water in lunar soils The impact glass beads at the surface would still be ab for e is est of 3– of 3– 132– Solar wind implantation and diffusion Irradiation Release Solar wind implantation and diffusion Irradiation Impact glass beads: water reservoir on the Moon Stage 1 a b c Stage 2 Stage 3 Impact glass beads: water reservoir on the Moon 2.7 × 1014 kg water Solar wind implantation and diffusion Irradiation Solar wind i and di Impact glass beads: water reservoir on the Moon Stage 1 a b Stage 2 Release implantation iffusion Irradiation c Stage 3 Impact glass beads: water reservoir on the Moon 2.7 × 1014 kg water a Release implantation iffusion Irradiation c Stage 3 Impact glass beads: water reservoir on the Moon 2.7 × 1014 kg water c Irradiation Solar wind implantation and diffusion Rele Irradiation Solar wind implantation and diffusion Impact glass beads: water reservoir on the Moon Stage 1 Stage 2 Stage 3 Fig. 4 \| Schematic diagram of the lunar surface water cycle associated with be able to release water into the lunar exosphere, due to meteoroid impact, for example13. The amount of water held by impact glass beads in lunar soils is estimated to be 0.03–27.00 × 1013 kg, assuming a global depth of lunar soils of 3–12 m (refs. 56,57), a modal abundance of impact glass beads in lunar soils of 3–5 vol.% (ref. 51) and impact glass beads having a bulk H2O abundance of 132–1,570 μg g−1. g \| g y impact glass beads. a, Because of the high temperature of formation of impact glass beads, most water present in precursor materials would have been lost. b, After their deposition at the surface, solar wind-derived water would diffuse into impact glass beads after solar wind H+ implantation. c, Gardening would transfer the impact glass beads deeper into the soils, creating a water reservoir at the subsurface of the Moon. The impact glass beads at the surface would still References 1. Watson, K., Murray, B. & Brown, H. On the possible presence of ice on the Moon. J. Geophys. Res. 66, 1598–1600 (1961). 1. Watson, K., Murray, B. & Brown, H. On the possible presence of ice on the Moon. J. Geophys. Res. 66, 1598–1600 (1961). 2. Watson, K., Murray, B. C. & Brown, H. The behavior of volatiles on the lunar surface. J. Geophys. Res. 66, 3033–3045 (1961). 2. Watson, K., Murray, B. C. & Brown, H. The behavior of volatiles on the lunar surface. J. Geophys. Res. 66, 3033–3045 (1961). 3. Zeller, E. J., Ronca, L. B. & Levy, P. W. Proton-induced hydroxyl formation on the lunar surface. J. Geophys. Res. 71, 4855–4860 (1966). 4. Feldman, W. C. et al. Fluxes of fast and epithermal neutrons from Lunar Prospector: evidence for water ice at the lunar poles. Science 281, 1496–1500 (1998). 5. Pieters, C. M. et al. Character and spatial distribution of OH/H2O on the surface of the Moon seen by M3 on Chandrayaan-1. Science 326, 568–572 (2009). 5. Pieters, C. M. et al. Character and spatial distribution of OH/H2O on the surface of the Moon seen by M3 on Chandrayaan-1. Science 326, 568–572 (2009). 6. Clark, R. N. Detection of adsorbed water and hydroxyl on the Moon. Science 326, 562–564 (2009). 6. Clark, R. N. Detection of adsorbed water and hydroxyl on the Moon. Science 326, 562–564 (2009). Online content on the Moon’s surface is not high enough to drive hydroxyl out from the apatite structure32,55, indicating that impact glass beads are probably the dominant reservoir involved in the lunar surface water cycle, except for the space-weathered rims and possibly water ice in polar regions. Any methods, additional references, Nature Portfolio reporting sum- maries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author con- tributions and competing interests; and statements of data and code availability are available at https://doi.org/10.1038/s41561-023-01159-6. The global lunar surface is covered by lunar soils with a thickness ranging from about 3 m to 12 m (refs. 56,57). The amount of water in lunar soils contributed by impact glass beads varies from 3.0 × 1011 kg to 2.7 × 1014 kg, based on the bulk water abundance of 132–1,570 μg g−1 in impact glass beads and a modal abundance of ~3–5 vol.% (ref. 51) (Fig. 4). Importantly, this estimate provides a minimum quantity of water avail- able at the lunar subsurface as it does not take into account the water contribution from permanently shadowed regions4–6,11, indigenous lunar rocks (for example, ref. 26), delivery by meteoritic impacts13,27 and solar wind-derived water preserved in the space-weathered rims of lunar soils34,35. These findings indicate that the lunar soils contain a much higher amount of solar wind-derived water than previously thought, which could be a water reservoir for in situ utilization in future lunar exploration. Indeed, this water entrapped in impact glass beads appears to be quite easy to extract as shown by the post- diffusion de-gassing profile for bead CE5#33,036 (Fig. 3 and Methods). Inventory of water in lunar soils Linking a lunar surface water cycle with impact glass beads implies three major processes: (1) formation of impact glass beads, (2) solar wind-derived water implantation and diffusion into the beads and (3) water release to the lunar exosphere during T increase, possibly due to sunlight irradiation and/or meteoroid impacts (Fig. 4). Impact glass beads are produced during melting of lunar soil and regolith targets when impactors hit the Moon. Most of the water present in the precursor materials would have probably been lost during high-temperature melting that led to the formation of the impact glass beads. Solar wind hydrogen ions would then be continually implanted into lunar soils and impact glass beads and would combine with oxygen atoms to form the hydroxyl/water that diffused into the glass interiors and is stored in glass beads. This water stored in impact glass beads could then be released to the lunar exosphere due to sunlight irradiation19 and/or meteoroid impacts13. Using the water abundance measured in CE5 impact glass beads and mare basalts50, we can estimate the relative contribution of solar wind implanted in impact glasses and basalts to the global water inventory in the CE5 regolith. Considering the zoning features of water in CE5 impact glass beads, we estimate that these beads have a bulk water abundance of 132–1,570 μg g−1, which is notably higher than that of the bulk CE5 mare basalts (7 ± 3 μg g−1) (ref. 50). The modal abundance of impact glass beads in lunar soils is in the region of 3–5 vol.% estimated from Apollo lunar soils51. Using this modal abundance yields a contribution by the CE5 impact glass beads to bulk soils of 4–78 μg g−1 equivalent water, which is consistent with bulk water abundance estimates derived from reflectance spectroscopy measurements carried out from the CE5 lander52. Importantly, the major water-bearing phase in CE5 basalts is apatite in which water occurs as hydroxyl occupying the X site in apatite crystal structure53,54. The daily temperature variation of 93 K to 423 K 297 Article https://doi.org/10.1038/s41561-023-01159-6 Solar wind implantation and diffusion Irradiation Release Solar wind implantation and diffusion Irradiation Impact glass beads: water reservoir on the Moon Stage 1 a b c Stage 2 Stage 3 Impact glass beads: water reservoir on the Moon 2.7 × 1014 kg water Fig. 4 \| Schematic diagram of the lunar surface water cycle associated with impact glass beads. Nature Geoscience \| Volume 16 \| April 2023 \| 294–300 Inventory of water in lunar soils a, Because of the high temperature of formation of impact glass beads, most water present in precursor materials would have been lost. b, After their deposition at the surface, solar wind-derived water would diffuse into impact glass beads after solar wind H+ implantation. c, Gardening would transfer the impact glass beads deeper into the soils, creating a water reservoir at the subsurface of the Moon. The impact glass beads at the surface would still be able to release water into the lunar exosphere, due to meteoroid impact, for example13. The amount of water held by impact glass beads in lunar soils is estimated to be 0.03–27.00 × 1013 kg, assuming a global depth of lunar soils of 3–12 m (refs. 56,57), a modal abundance of impact glass beads in lunar soils of 3–5 vol.% (ref. 51) and impact glass beads having a bulk H2O abundance of 132–1,570 μg g−1. Solar wind implantation and diffusion Irradiation Impact glass beads: w reservoir on the Mo Stage 1 a b Stage 2 Fig. 4 \| Schematic diagram of the lunar surface water cycle associated with impact glass beads. a, Because of the high temperature of formation of impact glass beads, most water present in precursor materials would have been lost. b, After their deposition at the surface, solar wind-derived water would diffuse into impact glass beads after solar wind H+ implantation. c, Gardening would transfer the impact glass beads deeper into the soils, creating a water reservoir at the subsurface of the Moon. Implications for water on airless bodies Lunar soil hydration constrained by exospheric water liberated by meteoroid impacts. Nat. Geosci. 12, 333–338 (2019). 34. Zhou, C. et al. Chang’e-5 samples reveal high water content in lunar minerals. Nat. Commun. 13, 5336 (2022). 14. Honniball, C. I. et al. Molecular water detected on the sunlit Moon by SOFIA. Nat. Astron. 5, 121–127 (2021). 35. Xu, Y. et al. High abundance of solar wind-derived water in lunar soils from the middle latitude. Proc. Natl Acad. Sci. USA 119, e2214395119 (2022). 15. McCord, T. B. et al. Sources and physical processes responsible for OH/H2O in the lunar soil as revealed by the Moon Mineralogy Mapper (M3). J. Geophys. Res. 116, E00G05 (2011). 36. Saal, A. E., Hauri, E. H., Van Orman, J. A. & Rutherford, M. J. Hydrogen isotopes in lunar volcanic glasses and melt inclusions reveal a carbonaceous chondrite heritage. Science 340, 1317–1320 (2013). 16. Schaible, M. J. & Baragiola, R. A. Hydrogen implantation in silicates: the role of solar wind in SiOH bond formation on the surfaces of airless bodies in space. J. Geophys. Res. Planets 119, 2017–2028 (2014). 37. Morris, R. V. Handbook of Lunar Soils (Lyndon B. Johnson Space Center, 1983). 17. Schörghofer, N. Migration calculations for water in the exosphere of the Moon: dusk–dawn asymmetry, heterogeneous trapping, and D/H fractionation. Geophys. Res. Lett. 41, 4888–4893 (2014). 38. Li, C. et al. Characteristics of the lunar samples returned by the Chang’e-5 mission. Natl Sci. Rev. 9, nwab188 (2022). 18. Farrell, W. M., Hurley, D. M. & Zimmerman, M. I. Solar wind implantation into lunar regolith: hydrogen retention in a surface with defects. Icarus 255, 116–126 (2015). 39. Long, T. et al. Constraining the formation and transport of lunar impact glasses using the ages and chemical compositions of Chang’e-5 glass beads. Sci. Adv. 8, eabq2542 (2022). 19. Jones, B. M., Aleksandrov, A., Hibbitts, K., Dyar, M. D. & Orlando, T. M. Solar wind-induced water cycle on the Moon. Geophys. Res. Lett. 45, 10959–10967 (2018). 40. Hashizume, K., Chaussidon, M., Marty, B. & Robert, F. Solar wind record on the moon: deciphering presolar from planetary nitrogen. Science 290, 1142–1145 (2000). 41. Epstein, S. & Taylor, H. P. Jr. The isotopic composition and concentration of water, hydrogen, and carbon in some Apollo 15 and 16 soils and in the Apollo 17 orange soil. Proc. Lunar Sci. Conf. 4, 1559–1575 (1973). 20. Tucker, O. J., Farrell, W. M., Killen, R. Implications for water on airless bodies 7. Sunshine, J. M. et al. Temporal and spatial variability of lunar hydration as observed by the Deep Impact spacecraft. Science 326, 565–568 (2009). Asteroid and comet impacts are the major exogenous processes that reshape the surface morphologies of airless bodies, as evidenced by the widespread presence of impact craters on the Moon58, Mercury59 and asteroids60,61. These impacts probably create impact glasses and glass beads on any airless bodies, as evidenced by the studies of lunar soils62 and howardites63. This study demonstrates that impact glass beads have the capacity to store significant quantities of solar wind-derived water at the surface of airless bodies, in addition to the possible presence of water ice trapped in permanently shadowed areas in polar regions. The presence of water, stored in impact glass beads, is consistent with the remote detection of water at lower-latitude regions of the Moon5–12, 4 Vesta64 and Mercury65,66. Our findings indicate that the impact glasses on the surface of Solar System airless bodies are capable of storing solar wind-derived water and releasing it to space. 7. Sunshine, J. M. et al. Temporal and spatial variability of lunar hydration as observed by the Deep Impact spacecraft. Science 326, 565–568 (2009). 8. Li, S. & Milliken, R. E. Water on the surface of the Moon as seen by the Moon Mineralogy Mapper: distribution, abundance, and origins. Sci. Adv. 3, e1701471 (2017). 9. Bandfield, J. L., Poston, M. J., Klima, R. L. & Edwards, C. S. Widespread distribution of OH/H2O on the lunar surface inferred from spectral data. Nat. Geosci. 11, 173–177 (2018). 10. Wohler, C., Grumpe, A., Berezhnoy, A. A. & Shevchenko, V. V. Time-of-day-dependent global distribution of lunar surficial water/hydroxyl. Sci. Adv. 3, e1701286 (2017). 11. Colaprete, A. et al. Detection of water in the LCROSS ejecta plume. Science 330, 463–468 (2010). Nature Geoscience \| Volume 16 \| April 2023 \| 294–300 298 Article https://doi.org/10.1038/s41561-023-01159-6 spectra and Diviner temperatures. J. Geophys. Res. Planet. 121, 2081–2107 (2016). 12. Mitrofanov, I. G. et al. Hydrogen mapping of the lunar south pole using the LRO neutron detector experiment LEND. Science 330, 483–486 (2010).f 33. Jones, B. M., Aleksandrov, A., Hibbitts, C. A. & Orlando, T. M. Thermal evolution of water and hydrogen from Apollo lunar regolith grains. Earth Planet. Sci. Lett. 571, 117107 (2021). 13. Benna, M., Hurley, D. M., Stubbs, T. J., Mahaffy, P. R. & Elphic, R. C. Implications for water on airless bodies M. & Hurley, D. M. Solar wind implantation into the lunar regolith: Monte Carlo simulations of H retention in a surface with defects and the H2 exosphere. J. Geophys. Res. Planets 124, 278–293 (2019). 21. Saal, A. E. et al. Volatile content of lunar volcanic glasses and the presence of water in the Moon’s interior. Nature 454, 192–195 (2008). 42. Tartèse, R. et al. Apatites in lunar KREEP basalts: the missing link to understanding the H isotope systematics of the Moon. Geology 42, 363–366 (2014). 43. Robert, F., Gautier, D. & Dubrulle, B. The solar system D/H ratio: observations and theories. Space Sci. Rev. 92, 201–224 (2000). 22. Tartèse, R. et al. The abundance, distribution, and isotopic composition of hydrogen in the Moon as revealed by basaltic lunar samples: implications for the volatile inventory of the Moon. Geochim. Cosmochim. Acta 122, 58–74 (2013). 44. Alexander, C. M. et al. The provenances of asteroids, and their contributions to the volatile inventories of the terrestrial planets. Science 337, 721–723 (2012). 23. Hui, H. J. et al. Cooling rates of lunar orange glass beads. Earth Planet. Sci. Lett. 503, 88–94 (2018). 24. Milliken, R. E. & Li, S. Remote detection of widespread indigenous water in lunar pyroclastic deposits. Nat. Geosci. 10, 561–565 (2017). 45. Altwegg, K. et al. Cometary science. 67P/Churyumov-Gerasimenko, a Jupiter family comet with a high D/H ratio. Science 347, 1261952 (2015). 46. Hartogh, P. et al. Ocean-like water in the Jupiter-family comet 103P/Hartley 2. Nature 478, 218–220 (2011). 25. Hui, H. J., Peslier, A. H., Zhang, Y. X. & Neal, C. R. Water in lunar anorthosites and evidence for a wet early Moon. Nat. Geosci. 6, 177–180 (2013). 47. Jones, B. M., Aleksandrov, A., Dyar, M. D., Hibbitts, C. A. & Orlando, T. M. Investigation of water interactions with Apollo lunar regolith grains. J. Geophys. Res. Planet. 125, e06147 (2020).f 26. Greenwood, J. P. et al. Hydrogen isotope ratios in lunar rocks indicate delivery of cometary water to the Moon. Nat. Geosci. 4, 79–82 (2011). 27. Liu, Y. et al. Direct measurement of hydroxyl in the lunar regolith and the origin of lunar surface water. Nat. Geosci. 5, 779–782 (2012). 48. Zouine, A., Dersch, O., Walter, G. & Rauch, F. Diffusivity and solubility of water in silica glass in the temperature range 23–200 °C. Phys. Chem. Glasses B 48, 85–91 (2007). 28. Füri, E., Deloule, E., Gurenko, A. https://doi.org/10.1038/s41561-023-01159-6 https://doi.org/10.1038/s41561-023-01159-6 65. Harmon, J. K. & Slade, M. A. Radar mapping of Mercury: full-disk images and polar anomalies. Science 258, 640–643 (1992). 56. Zhang, J. et al. Volcanic history of the Imbrium basin: a close-up view from the lunar rover Yutu. Proc. Natl Acad. Sci. USA 112, 5342–5347 (2015). 65. Harmon, J. K. & Slade, M. A. Radar mapping of Mercury: full-disk images and polar anomalies. Science 258, 640–643 (1992). 66. Lawrence, D. J. et al. Evidence for water ice near Mercury’s north pole from MESSENGER neutron spectrometer measurements. Science 339, 292–296 (2013). 66. Lawrence, D. J. et al. Evidence for water ice near Mercury’s north pole from MESSENGER neutron spectrometer measurements. Science 339, 292–296 (2013). 57. Zhang, J. et al. Lunar regolith and substructure at Chang’e-4 landing site in south pole–Aitken basin. Nat. Astron. 5, 25–30 (2020). Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 58. Head, J. W. 3rd et al. Global distribution of large lunar craters: implications for resurfacing and impactor populations. Science 329, 1504–1507 (2010). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. 59. Strom, R. G., Chapman, C. R., Merline, W. J., Solomon, S. C. & Head, J. W. 3rd Mercury cratering record viewed from MESSENGER’s first flyby. Science 321, 79–81 (2008). 60. Jaumann, R. et al. Vesta’s shape and morphology. Science 336, 687–690 (2012). 61. Abe, S. et al. Mass and local topography measurements of Itokawa by Hayabusa. Science 312, 1344–1347 (2006). 62. Taylor, G. J. et al. Modal analyses of lunar soils by quantitative X-ray diffraction analysis. Geochim. Cosmochim. Acta 266, 17–28 (2019). 63. Singerling, S. A., McSween, H. Y. Jr. Nature Geoscience \| Volume 16 \| April 2023 \| 294–300 Implications for water on airless bodies & Marty, B. New evidence for chondritic lunar water from combined D/H and noble gas analyses of single Apollo 17 volcanic glasses. Icarus 229, 109–120 (2014). 49. Hauri, E. et al. SIMS analysis of volatiles in silicate glasses. Chem. Geol. 183, 99–114 (2002). 50. Hu, S. et al. A dry lunar mantle reservoir for young mare basalts of Chang’e-5. Nature 600, 49–53 (2021). 29. Joy, K. H. et al. The isotopic composition of volatiles in the unique Bench Crater carbonaceous chondrite impactor found in the Apollo 12 regolith. Earth Planet. Sci. Lett. 540, 116265 (2020). 51. McKay, D. S. et al. The lunar regolith in Lunar sourcebook Ch. 7, 285–356 (Cambridge Univesity Press, 1991). 52. Lin, H. et al. In situ detection of water on the Moon by the Chang’E-5 lander. Sci. Adv. 8, eabl9174 (2022). 30. Housley, R., Grant, R. & Paton, N. Origin and characteristics of excess Fe metal in lunar glass welded aggregates. Proc. Lunar Sci. Conf. 4, 2737–2749 (1973). 53. Boyce, J. W., Tomlinson, S. M., McCubbin, F. M., Greenwood, J. P. & Treiman, A. H. The lunar apatite paradox. Science 344, 400–402 (2014). 31. Managadze, G. G., Cherepin, V. T., Shkuratov, Y. G., Kolesnik, V. N. & Chumikov, A. E. Simulating OH/H2O formation by solar wind at the lunar surface. Icarus 215, 449–451 (2011). 54. McCubbin et al. Experimental investigation of F, Cl, and OH partitioning between apatite and Fe-rich basaltic melt at 1.0–1.2 GPa and 950–1000 °C. Am. Mineral. 4779, 83–89 (2015).f 55. Ingrin, J. & Blanchard, M. Diffusion of hydrogen in minerals. Rev. Mineral. Geochem. 62, 291–320 (2006). 32. Li, S. & Milliken, R. E. An empirical thermal correction model for Moon Mineralogy Mapper data constrained by laboratory Nature Geoscience \| Volume 16 \| April 2023 \| 294–300 299 Article https://doi.org/10.1038/s41561-023-01159-6 & Taylor, L. A. Glasses in howardites: impact melts or pyroclasts? Meteorit. Planet. Sci. 48, 715–729 (2013). 64. Prettyman, T. H. et al. Elemental mapping by Dawn reveals exogenic H in Vesta’s regolith. Science 338, 242–246 (2012). © The Author(s) 2023 300 Article https://doi.org/10.1038/s41561-023-01159-6 Raman spectroscopy h f The Raman spectra of impact glasses were collected using a WITec alpha 300 R confocal Raman system and carried out after the NanoSIMS measurements at the IGGCAS. An optical microscope with an objective of 0.9 NA and ×50 magnification was used to focus the Ar+ laser beam with a 484 nm excitation wavelength on the target phases. Raman spectra were acquired with a total integration time of 500 s, a 9.2 mW laser power and a beam size of 5 μm. Three CE5 impact glass beads CE5#33,076 with water abundances zoning from 1,909 μg g−1 at the rims to <10 μg g−1 in the cores were chosen for Raman analyses (Extended Data Fig. 6). Along with the CE5 impact glass beads, terrestrial MORB glasses, MRN-G1 (H2O = 1.5 wt%) (ref. 73) and EPR-G3 (H2O = 0.22 wt%)73, were also measured to demonstrate the variation of Raman peak inten- sity of water and/or hydroxyl at ~3,300 cm−1 to 3,700 cm−1 with their water abundance (Extended Data Fig. 6). The sample and the standard prepared in different mounts were loaded in the same sample holder of NanoSIMS and were baked over- night at ~60 °C in the NanoSIMS airlock. The holders were then stored in the NanoSIMS sample chamber to improve the vacuum quality and minimize the H background49,67. The vacuum pressure in the anal- ysis chamber was 1.5 × 10−10 to 1.9 × 10−10 Torr during analysis. Each 7 μm × 7 μm analysis area was pre-sputtered for 2 min with a Cs+ ion beam current of 2 nA to remove the surface coating and potential contamination. During analysis, the secondary anions 1H−, 2D−, 12C− and 18O− were simultaneously counted by electron multipliers (EMs) from the central 5 μm × 5 μm areas using the NanoSIMS electronic gate technique (49% blanking). A 44 ns dead time was corrected for all EMs, while the EM noise (<10−2 cps) was ignored. We used a primary ion In situ water abundance and hydrogen isotope analyses Two analytical sessions were designed to measure the water abun- dances and hydrogen isotope compositions of CE5 impact glass beads using a CAMECA NanoSIMS 50L at the IGGCAS. In the first session, measurements at the cores and rims of CE5 impact glass beads were carried out to investigate the potential water zonation features in the glasses. In the second session, more-detailed profile measure- ments on five individual CE5 impact glasses were acquired, as well as several parallel measurements on other glass beads for cross checking. A liquid nitrogen cold trap was used to further improve the vacuum quality in the second session. Electron probe microanalysis Before the NanoSIMS analyses, we used the CAMECA SXFive EPMA at IGGCAS to quantify the major and minor elemental abundances in CE5 impact glass beads. The operating accelerating voltage was 20 kV and the beam current was 10 nA, with a 10 μm beam diameter. The total counting time was 10 min for each analysis. One basalt glass (VG-2) and one komatiite glass (MPI-DING-GOR128) were used to monitor the analysis accuracy and precision68. The detection limits were (3σ) 0.02 wt% for Na, Ni, K, S and P, 0.04 wt% for Ca, 0.05 wt% for Al, Mg and Cr, 0.06 wt% for Si and Ti and 0.09 wt% for Mn and Fe. The EPMA data obtained for impact glass beads are listed in Supplementary Table 2. Methods Sample preparation beam current of ~0.5 nA for analysis, corresponding to a beam size of ~500 nm in diameter. The charging effect on the sample surfaces was compensated by an electron gun during analysis. The CE5 lunar soil allocated by the China National Space Administration and used in this study is CE5C0100YJFM00103, weighing ~1 g. The CE5 lunar soils were scooped by the robotic arm on board the CE5 lander. A total of 150 individual grains, varying in size from ~50 μm to ~1 mm in diameter, were hand picked under a binocular microscope in the ultraclean room at the Institute of Geology and Geophysics, Chinese Academy of Sciences (IGGCAS). The hand-picked grains were mounted in epoxy and prepared as a double polished section with a thickness of about 100 μm (Supplementary Fig. 1 and Supplementary Table 1). The surrounding epoxy was removed to improve the vacuum quality for the water abundance measurements using NanoSIMS67. The prepared section was cleaned using anhydrous ethanol before drying at 60 °C in a baking oven overnight. A chip of the nominally anhydrous San Carlos olivine reference, with a reported water content of 1.4 μg g−1 (ref. 69), was used for instrument H2O background corrections, following the relationship H/Obg = (Hcounts – Hbg)/Ocounts and D/Hmeasured = (1 – f) × D/Htrue + f × D/Hbg, where f is the proportion of H emitted from the instrumental back- ground70. In the first session, D/Hbg was 2.83 (±1.98) × 10−4 and Hbg = 815 ± 457 cps (2 s.d., N = 16, corresponding to an instrument H2O background abundance of 29.8 ± 14.8 μg g−1 (2 s.d.)). In the second session, the measured H counts on the central areas of CE5 impact glass beads are even lower than that of San Carlos olivine. Therefore, the extremely low H/O ratio measurements on CE5 impact glass beads were treated as reference (Hbg = 215 ± 40 cps, corresponding to an instrument H2O background of 11.7 ± 2 μg g−1) for background correction. SEM observation After an instrument H2O background subtraction, the water abundances of CE5 impact glass beads were calculated from the background-subtracted H/O ratios multiplied by the slope of the cali- bration lines (Extended Data Fig. 2), which were determined by meas- uring two apatite standards, Durango apatite (H2O = 0.0478 wt% and δD = −120 ± 5‰) (refs. 26,71) and Kovdor apatite (H2O = 0.98 ± 0.07 wt% and δD = −66 ± 21‰) (ref. 72), the SWIFT mid-ocean-ridge basalt (MORB) glass (H2O = 0.258 wt% and δD = −73 ± 2‰) and two basaltic glasses, 519-4-1 (H2O = 0.17 wt%) (ref. 49) and 1833-11 (H2O = 1.2 wt%) (ref. 49) (Supplementary Table 3). Corrections for instrumental mass frac- tionation (IMF) on H isotopic compositions were carried out using repeated analyses of the SWIFT MORB glass standard and monitored by analysing both the Durango apatite and Kovdor apatite standards during the whole analytical session (Extended Data Fig. 3). The matrix effects on water abundance and IMF on hydrogen isotope composi- tion are the same between apatite and silicate glass within analytical uncertainties67. Hydrogen isotopic compositions are given using the delta notation, δD = ((D/H)sample/(D/H)SMOW) − 1) × 1,000‰, where SMOW is the standard mean ocean water with a D/H ratio of 1.5576 × 10−4. More technical details can be found in ref. 67. All data are reported with their 2 s.d. analytical uncertainties, which include reproducibility of D/H measurements on the reference materials, uncertainty of H2O background subtraction and internal precision on each analysis (Supplementary Tables 3 and 4). The raw measured D/H ratios were corrected for the background, followed by correction for IMF. Petrographic observations were carried out using field-emission SEM using FEI Nova NanoSEM 450 and Thermofisher Apreo instruments at the IGGCAS, using electron beam currents of 2.0–6.4 nA and an acceleration voltage of 15 kV. The prepared sections were initially coated with carbon for petrographic observations and analyses of mineral chemistry and then recoated with Au for in situ NanoSIMS measurement of water content and hydrogen isotopes. After NanoSIMS analyses, Au coating was removed, and the samples were recoated with carbon and observed by SEM to confirm the positions of the NanoSIMS spots. Petrography and chemistry of CE5 impact glass beads Considering the scooped samples were collected from less than 3 cm deep38, diffusion simulations were performed using a peak tem- perature of 360 K, relevant for a latitude of 45° N on the Moon32, because the peak temperature is the main controlling factor for the diffusion time35. The H diffusion coefficients of water/hydroxyl (DH) determined on silica glass48, DH = 20.84 μm2 yr−1 at 360 K, were used in our simula- tions. It is not possible to reconstruct the orientation of the exposed surface of the studied impact glass beads on the lunar surface. Thus, we carried out a simplistic diffusion modelling based on the measured profiles, assuming a constant Cs at the rims for each bead based on their diverse Pb–Pb ages39. The diffusion duration is roughly a maximum constraint for qualitatively estimating the efficiency of solar wind- derived OH/H2O recharged in impact glass beads. The water abun- dances at the rims (Cs) of the six CE5 impact glass bead profiles are extrapolated from the measured profiles using 2,000 μg g−1, 2,000 μg g−1, 1,600 μg g−1, 500 μg g−1, 2,600 μg g−1 and 3,000 μg g−1 and a C0 of 10 μg g−1 for Profiles 1–6, respectively. The diffusional durations vary from 3.0 to 9.0 years, 5.0 to 15.0 years, 1.0 to 4.5 years, 2.0 to 6.0 years, 0.5 to 4.0 years and 2.0 to 14.0 years at T = 360 K for Profiles 1–6, respectively, based on the apparent distance (Fig. 1) and a fixed Cs at the rims for each profile. Petrography. A total of 150 impact glass fragments were mounted in CE5#33 mount (Supplementary Fig. 1 and Supplementary Table 1). There are 117 glass fragments displaying ovoid to spherical shapes, which are mainly composed mainly of glass, 1 glass rod, 3 olivine and plagioclase monomict clasts, 1 basaltic fragment and 28 glass frag- ments showing irregular shapes, which are composed mainly of glass, mafic mineral fragments and large bubbles (Supplementary Fig. 1 and Supplementary Table 1). The ovoid to spherical fragments are referred to as glass beads in this work and further classified into two textural types, homogeneous and heterogeneous glass beads (Fig. 1, Supplementary Fig. 2 and Supplementary Table 1). The homogeneous glass beads display smooth surfaces and homogeneous contrast in back-scattered electron images while the heterogeneous glass beads usually contain some vesicles, pyroxene and plagioclase clasts and micrometre-sized sulfide spheres (Fig. Two-endmember mixing modelling In the two-endmember mixing model, the solar wind endmember is set to have a δD value of −980‰ (ref. 41) and a water abundance of 2,000 μg g−1, which corresponds to the maximum measured value in this work (Fig. 2). The initial water component in CE5 impact glass beads could have a minimum δD value of ~500‰ based on the measure- ments on CE5 apatite50, yielding a best fit of initial water abundance of 5–50 μg g−1 before the addition of solar wind-derived water (Fig. 2). On the basis of the following mass balance (equations (2) and (3)), we can model the relationship between the measured water abundances and δD values: H2O0 + H2Osolar = H2Omeasured (2) (2) Correction of water abundances and D/H ratios for spallation effects The measured D/H ratios have been corrected for the potential effects of spallation by cosmic ray, using a D production rate of 2.17 × 10−12 mol g–1 Myr–1 (ref. 74) for impact glasses. The correction errors induced by D spallation are around 5% on D/H ratios and negligible Nature Geoscience https://doi.org/10.1038/s41561-023-01159-6 Article (Fig. 3). The water abundance on the right side modelled in Stage 1 was taken as the initial condition for modelling of Stage 2. The de-gassing rate is assumed to be proportional to the surface water abundance along the profile, where transmission capacity of a boundary was formulated as an additional boundary condition for the diffusion equation (see ref. 35 for more details). The corresponding δD profiles are calculated by a binary mixing model (Two-endmember mixing modelling) considering a limited diffusional fractionation for H iso- tope78 compared with the large difference in δD values for the two endmembers (δD ≈ −980‰, D/H ≈ 3.0 × 10−6 for solar wind-derived water and δD ≈ 500‰, D/H ≈ 230 × 10−6 for the initial water in impact glass beads). on water content36. Because no cosmic-ray exposure age is yet avail- able for the CE5 impact glass beads, a cosmic-ray exposure age of 50 Ma estimated by Hu et al.50 was used for correction of spallation in this study (Supplementary Table 3). Another D production rate, of 9.2 × 10−13 mol g–1 Myr–1 (ref. 75), was also used for correction of spalla- tion effects to account for the suggestion that the D production rate of 2.17 × 10−12 mol g–1 Myr–1 (ref. 74) may not be appropriate for impact glasses. As the D production rate in Ref. 75 is slower, corrections for spallation-produced D are even smaller, not making any notable dif- ference overall for our analytical results (Supplementary Table 4). De-gassing modelling The hydrogen isotope fractionation during volatile loss into a vacuum is given by α2 = M1/M2, where M1 and M2 are the masses of the volatile phase isotopologues. The change of the isotopic composition of H during volatile loss by Rayleigh fractionation is given by R = R0 × f(α − 1), where R0 and R are the initial and final D/H ratios for a fraction f of remaining hydrogen76. De-gassing of H2 (M1 = 2 for H2 and M2 = 3 for HD) yields an α value of ~0.8165 (ref. 76) (Fig. 2). The de-gassing modelling assumes that the CE5 impact glass beads have an initial water abundance of 2,000 μg g−1, an initial D/H ratio of 3.1 × 10−6 (corresponding to that of the analysis with the highest H2O abundance) and de-gas in the form of H2, yielding a δD value increase from −980‰ at f = 1 to −926‰ at f = 0.001. Diffusion modelling The five sharp hydration profiles of CE5 impact glass beads were simulated by a simplified diffusion process that follows Fick’s second law as equation (1) (ref. 77). (3) H2O0 × D/H0 + H2Osolar × D/Hsolar = H2Omeasured × D/Hmeasured (3) where H2O0 and D/H0 are the initial water abundance and D/H ratio of CE5 impact glass beads before the addition of solar wind-derived water, respectively, H2Osolar is the water abundance contributed by solar wind-derived water, D/Hsolar is the hydrogen isotopic composi- tion of solar wind-derived water, and H2Omeasured and D/Hmeasured are the water abundance and D/H ratio measured from CE5 impact glass beads, respectively. C (x) = (Cs −C0) erfc ( x 2√Dt ) + C0 (1) (1) In equation 1, D is the diffusion coefficient that depends on tempera- ture, x is the distance to the rim, C0 is the initial water abundance of the CE5 impact glass beads, C(x) is the water abundance at distance x, Cs is the maximum water abundance at the rims, t is duration of diffusion and erfc is the complementary error function. Nature Geoscience https://doi.org/10.1038/s41561-023-01159-6 CE5 impact glass beads are composed mainly of SiO2 (31.2–44.5 wt%), TiO2 (3.95–6.54 wt%), Al2O3 (10.4–17.3 wt%), FeO (17.6–23.5 wt%), MgO (5.94–10.9 wt%) and CaO (10.1–15.4 wt%), with minor Cr2O3 (0.12–0.28 wt%), MnO (0.22–0.31 wt%), NiO (0.15–0.61 wt%), Na2O (<0.17 wt%, most <0.05 wt%) and K2O (<0.06 wt%) (Supplementary Table 2). Each single glass bead is homogeneous in terms of its major element chemical composition (Supplementary Table 2). Most CE5 homogeneous impact glass beads have compositions similar to the bulk CE5 basalt compositions reported by refs. 38,81, indicating that they could have been formed locally rather than ejected from other regions. This prediction is consistent with the similar geochemical characteris- tics between CE5 impact glass beads and CE5 bulk basalts82. The Pb–Pb dating of the CE5 impact glass beads indicates that they were formed in the range of 4–2,000 Ma by various local meteoritic impacts39. CE5 impact glass beads are composed mainly of SiO2 (31.2–44.5 wt%), TiO2 (3.95–6.54 wt%), Al2O3 (10.4–17.3 wt%), FeO (17.6–23.5 wt%), MgO (5.94–10.9 wt%) and CaO (10.1–15.4 wt%), with minor Cr2O3 (0.12–0.28 wt%), MnO (0.22–0.31 wt%), NiO (0.15–0.61 wt%), Na2O (<0.17 wt%, most <0.05 wt%) and K2O (<0.06 wt%) (Supplementary Table 2). Each single glass bead is homogeneous in terms of its major element chemical composition (Supplementary Table 2). Most CE5 homogeneous impact glass beads have compositions similar to the bulk CE5 basalt compositions reported by refs. 38,81, indicating that they could have been formed locally rather than ejected from other regions. This prediction is consistent with the similar geochemical characteris- tics between CE5 impact glass beads and CE5 bulk basalts82. The Pb–Pb dating of the CE5 impact glass beads indicates that they were formed in the range of 4–2,000 Ma by various local meteoritic impacts39. 70. Tartèse, R., Anand, M. & Franchi, I. A. H and Cl isotope characteristics of indigenous and late hydrothermal fluids on the differentiated asteroidal parent body of Grave Nunataks 06128. Geochim. Cosmochim. Acta 266, 529–543 (2019). 71. Greenwood, J. P., Itoh, S., Sakamoto, N., Vicenzi, E. P. & Yurimoto, H. Hydrogen isotope evidence for loss of water from Mars through time. Geophys. Res. Lett. 35, L05203 (2008). 72. Nadeau, S. L., Epstein, S. & Stolper, E. Hydrogen and carbon abundances and isotopic ratios in apatite from alkaline intrusive complexes, with a focus on carbonatites. Geochim. Cosmochim. Acta 63, 1837–1851 (1999). 73. Shimizu, K. et al. Water abundances and hydrogen isotope compositions of the CE5 impact glass beads p g A total of 32 CE5 impact glass beads with smooth exposed surface (Fig. 1 and Supplementary Fig. 2) and a mare origin (Extended Data Fig. 2) were selected for the NanoSIMS measurements of water abundances and hydrogen isotopic compositions. In the first analytical session, the objective was to investigate the homogeneity of water abundance within each glass bead; therefore, two analyses of the cores and the rims on relatively large glass beads were carried out to identify the hydration and or de-gassing candidate profiles. In the second session, six more-detailed profile analyses were carried out on five CE5 impact glass beads (Fig. 1), as well as several duplicate measurements on other glass beads (Supplementary Fig. 2). Overall, the analytical water abundances in the first session are in the range of the second session due to the heterogeneous distribution of water in the CE5 impact glass beads (Fig. 2 and Supplementary Table 4). The water abundances of the analysed CE5 impact glass beads vary from ~0 to 1,909 μg g−1 and are negatively correlated with their δD values (−990 ± 6‰ to +522 ± 440‰) (Fig. 2 and Supplementary Table 4). Water abundances measured in all CE5 impact glass beads display higher values at the rims and decrease towards the glass bead cores (Fig. 3 and Extended Data Figs. 4 and 5). The δD values display the reverse trends (Fig. 3 and Extended Data Figs. 4 and 5). Three profile measurements (Profile 3, Profile 4 and Profile 5) show a sharp decrease in water abundance from the rim towards the core (Fig. 3 and Extended Data Figs. 4 and 5), while the other three (Profile 1, Profile 2 and Profile 6) display a modest decrease of water abundance towards the cores (Fig. 3 and Extended Data Figs. 4 and 5). It is also noticeable that the latter profiles have higher water abundances (1,055–1,909 μg g−1) at the rim than do the former profiles (176–1,423 μg g−1) (Fig. 3, Extended Data Figs. 4 and 5 and Supplementary Table 4). All of the profile measurements acquired on the central areas of CE5 impact glass beads have water abundances comparable to the instrument H2O background (Supplementary Tables 3 and 4). 75. Merlivat, L., Leiu, M., Neif, G. & Roth, E. Spallation deuterium in rock 70215. In 7th Proc. Lunar Sci. Conf, 649–658 (1976). 76. Sharp, Z. D., McCubbin, F. M. Water abundances and hydrogen isotope compositions of the CE5 impact glass beads & Shearer, C. K. A hydrogen- based oxidation mechanism relevant to planetary formation. Earth Planet. Sci. Lett. 380, 88–97 (2013).f 77. Crank, J. The Mathematics of Diffusion (Oxford Univ. Press, 1975). . Crank, J. The Mathematics of Diffusion (Oxford Univ. P 78. Graham, C. M. Experimental hydrogen isotope studies III: diffusion of hydrogen in hydrous minerals, and stable isotope exchange in metamorphic rocks. Contrib. Mineral. Petrol. 76, 216–228 (1981). 79. Delano, J. W. Pristine lunar glasses: criteria, data, and implications. Proc. Lunar Sci. Conf. 91, D201–D213 (1986). 80. Naney, M., Crowl, D. & Papike, J. The Apollo 16 drill core: statistical analysis of glass chemistry and the characterization of a high alumina-silica poor (HASP) glass. In 7th Proc. Lunar Sci. Conf., 155–184 (1976). 81. Tian, H. C. et al. Non-KREEP origin for Chang’e-5 basalts in the Procellarum KREEP Terrane. Nature 600, 59–63 (2021). 82. Yang, W. et al. Geochemistry of impact glasses in the Chang’e-5 regolith: constraints on impact melting and the petrogenesis of local basalt. Geochim. Cosmochim. Acta 335, 183–196 (2022). 83. Hu, S. et al. NanoSIMS analyses of apatite and melt inclusions in the GRV 020090 Martian meteorite: hydrogen isotope evidence for recent past underground hydrothermal activity on Mars. Geochim. Cosmochim. Acta 140, 321–333 (2014). https://doi.org/10.1038/s41561-023-01159-6 H2O, CO2, F, S, Cl, and P2O5 analyses of silicate glasses using SIMS: report of volatile standard glasses. Geochem. J. 51, 299–313 (2017). 74. Füri, E., Deloule, E. & Trappitsch, R. The production rate of cosmo genic deuterium at the Moon’s surface. Earth Planet. Sci. Lett. 474, 76–82 (2017). Data availability All geochemical data generated in this study are included in Supplementary Tables 1–5 and are available via Zenodo (https://doi.org/ 10.5281/zenodo.7660603). Acknowledgements We thank D. Chew for providing Durango and Kovdor apatite, E. Hauri for providing basaltic glass 519-4-1 and 1833-11, R. Francis for providing SWIFT MORB glass, K. Shimizu for providing MRN-G1 and EPR-G3 MORB glasses, and H. Ma and Q. Guo for hand picking fragments in CE5 soils. This study was funded by the National Natural Science Foundation of China (42241104 and 41973062 to S.H.), the Strategic Priority Research Program of Chinese Academy of Sciences (XDB 41000000 to W.Y.), the key research programme of Chinese Academy of Sciences (ZDBS-SSW-JSC007-15 to W.Y.), the key research programme of the Institute of Geology and Geophysics, Chinese Academy of Sciences (IGGCAS-202101 to W.Y. and 201904 and 202204 to S.H.) and Pre-research project on Civil Aerospace Technologies by CNSA (D020201, D020203, and D020205 to S.H., W.Y., and H. Hui., respectively). M.A. and R.T. acknowledge funding from the UK Science and Technology Facilities Council (grant #ST/P000657/1 and #ST/P005225/1, respectively). The CE5 samples were allocated by the China National Space Administration. Petrography and chemistry of CE5 impact glass beads 1 and Supplementary Fig. 2). The textural differences between homogeneous and heterogeneous glass beads could reflect a higher formation temperature for the homogeneous glass beads (Fig. 1 and Supplementary Fig. 2). Chemistry. A total of 32 homogeneous impact glass beads were selected for EPMA analyses. The analytical results are listed in Supplementary Table 2. The analysed glass beads have a mare origin, except for two glass beads plotting in the highlands popula- tion and another two distributed in pyroclastic areas judging from their CaO/Al2O3 and MgO/Al2O3 ratios (Extended Data Fig. 1)79,80. These glasses plotting in non-mare fields are not discussed further in this work because their origin is currently uncertain. The homogeneous For the notable post-diffusion de-gassing feature of Profile 2, a two-stage modelling of water diffusion (Stage 1) and post-diffusion de-gassing (Stage 2) was carried out to fit the measured water abun- dance profile. The water-diffusion details in Stage 1 have been described in the preceding. A post-diffusion de-gassing of water (Stage 2) was modelled to fit the bell shape of water abundance on the right side Nature Geoscience Article https://doi.org/10.1038/s41561-023-01159-6 References 67. Hu, S. et al. Measurements of water content and D/H ratio in apatite and silicate glasses using a NanoSIMS 50L. J. Anal. At. Spectrom. 30, 967–978 (2015). 67. Hu, S. et al. Measurements of water content and D/H ratio in apatite and silicate glasses using a NanoSIMS 50L. J. Anal. At. Spectrom. 30, 967–978 (2015). 68. Zhang, D. et al. High-precision measurement of trace level Na, K, P, S, Cr, and Ni in lunar glass using electron probe microanalysis. At. Spectrosc. 43, 28–41 (2022). 68. Zhang, D. et al. High-precision measurement of trace level Na, K, P, S, Cr, and Ni in lunar glass using electron probe microanalysis. At. Spectrosc. 43, 28–41 (2022). Author contributions 69. Zhang, W. F. et al. Optimization of SIMS analytical parameters for water content measurement of olivine. Surf. Interface Anal. 52, 224–233 (2019). S.H. and H. Hui designed this research. Huicun He, J.J., Y.Z., H.T., C.Z., L.G., J.L., D.Z., Q.M., L.J., Xiaoguang Li, Y.C., S.W., H.W., Q.L. and Nature Geoscience Article https://doi.org/10.1038/s41561-023-01159-6 S.H. prepared the sample and characterized the petrography and mineral chemistry of CE5 basalts. Huicun He, J.H., R.L., J.J., W.Y. and S.H. conducted the NanoSIMS measurements. C.Z., L.Z., H.N. and S.H. carried out the diffusion modelling. S.H., Y.L., H. Hui, Huicun He, Huaiyu He, Xianhua Li, F.W., M.A. and R.T. wrote the manuscript. All authors contributed to the preparation of the manuscript. Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41561-023-01159-6. Correspondence and requests for materials should be addressed to Sen Hu or Hejiu Hui. Peer review information Nature Geoscience thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editor: Tamara Goldin, in collaboration with the Nature Geoscience team. Competing interests g The authors declare no competing interests. Reprints and permissions information is available at www.nature.com/reprints. Extended data is available for this paper at h //d i / 0 038/ 6 023 0 9 6 Additional information Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at Extended data is available for this paper at https://doi.org/10.1038/s41561-023-01159-6. Extended data is available for this paper at https://doi.org/10.1038/s41561-023-01159-6. https://doi.org/10.1038/s41561-023-01159-6. Nature Geoscience Article https://doi.org/10.1038/s41561-023-01159-6 0.4 0.6 0.8 1.0 1.2 1.4 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Pyroclastic Mare Impact glass CE5 Apollo Luna LMs Pyroclastic glass Apollo MgO/Al2O3 CaO/Al2O3 Highlands CE5 bulk Extended Data Fig. 1 \| MgO/Al2O3 versus CaO/Al2O3 for the studied CE5 impact glass beads. Most smooth and homogeneous CE5 glass beads plot in the mare domain (Supplementary Table 2). Literature data for lunar impact and pyroclastic glasses in Apollo samples, Luna samples, and lunar meteorites (LMs) are from MoonDB (http://search.moondb.org) and are shown for comparison. Compositions of the CE5 bulk basalts and bulk soils (shaded yellow square) are from Tian et al.81 and Li et al.38. Classification of lunar glasses (dashed lines) is from Refs. 79,80. 0.4 1.4 are from MoonDB (http://search.moondb.org) and are shown for comparison. Compositions of the CE5 bulk basalts and bulk soils (shaded yellow square) are from Tian et al.81 and Li et al.38. Classification of lunar glasses (dashed lines) is from Refs. 79,80. Extended Data Fig. 1 \| MgO/Al2O3 versus CaO/Al2O3 for the studied CE5 impact glass beads. Most smooth and homogeneous CE5 glass beads plot in the mare domain (Supplementary Table 2). Literature data for lunar impact and pyroclastic glasses in Apollo samples, Luna samples, and lunar meteorites (LMs) Nature Geoscience Nature Geoscience Article https://doi.org/10.1038/s41561-023-01159-6 0.0 0.3 0.6 0.9 0.0 0.3 0.6 0.9 1.2 y = (1.41 ± 0.014)x (R2 = 0.9994) SCOL MORB 1833-11 The 1st session standards linear fit The 2nd session standards linear fit H2O (wt %) 1H/18O y = (1.43 ± 0.012)x (R2 = 0.9996) KOV DAP 519-4-1 Water abundance calibration lines Extended Data Fig. 2 \| The water abundance calibration lines established on standards. The linear slopes of the two analytical sessions are comparable and consistent with the slope of the instrument’s long-term stability50,67,83. The analytical uncertainty is 1.7% (2σ) for the 1st session and 2.0% (2σ) for the 2nd session. The error bars are 2σ. KOV: Kovdor apatite, DAP: Durango apatite, SCOL: San Carlos olivine. analytical uncertainty is 1.7% (2σ) for the 1st session and 2.0% (2σ) for the 2nd session. The error bars are 2σ. KOV: Kovdor apatite, DAP: Durango apatite, SCOL: San Carlos olivine. Extended Data Fig. 2 \| The water abundance calibration lines established on standards. Reprints and permissions information is available at The linear slopes of the two analytical sessions are comparable and consistent with the slope of the instrument’s long-term stability50,67,83. The Nature Geoscience Nature Geoscience Article https://doi.org/10.1038/s41561-023-01159-6 0 100 200 300 -1000 -500 0 500 1000 1st session 2nd session #Sequence Standards MORB 1833-11 519-4-1 KOV DAP CE5 impact glass beads 1st session 2nd session SMOW Extended Data Fig. 3 \| Hydrogen isotope analysis sequences for the two analytical sessions. Analysis sequences are shown to monitor the reproducibility of hydrogen isotope analysis of the standards. δD values of Kovdor apatite (KOV), Durango apatite (DAP), SWIFT MORB (MORB), 1833-11, and 519-4-1 have been corrected for instrumental mass fractionation. And the fractionation factor (αIMF) was established by SWIFT MORB glass. The reproducibility of δD analysis throughout the whole analytical session-1 and −2 are 58 ‰ (N = 38) and 117 ‰ (N = 11) (2σ), estimated by repeated analyses of the SWIFT MORB glass standard. The average δD values calculated for the Kovdor and Durango apatite are −39 ± 37 ‰ and −80 ± 134 ‰ (Supplementary Table 3), respectively, consistent with their recommended values within analytical errors26,71. The average δD values measured for the basaltic glass standards 1833-11 and 519-4-1 are −54 ± 42 ‰ and −80 ± 28 ‰ (Supplementary Table 3), respectively, which is consistent with the δD values reported in our previous work50. The error bars are 2 SD. 0 100 200 300 -1000 -500 0 500 1000 1st session 2nd session #Sequence Standards MORB 1833-11 519-4-1 KOV DAP CE5 impact glass beads 1st session 2nd session SMOW #Sequence Extended Data Fig. 3 \| Hydrogen isotope analysis sequences for the two analytical sessions. Analysis sequences are shown to monitor the reproducibility of hydrogen isotope analysis of the standards. δD values of Kovdor apatite (KOV), Durango apatite (DAP), SWIFT MORB (MORB), 1833-11, and 519-4-1 have been corrected for instrumental mass fractionation. And the fractionation factor (αIMF) was established by SWIFT MORB glass. The reproducibility of δD analysis throughout the whole analytical session-1 and −2 are 58 ‰ (N = 38) and 117 ‰ (N = 11) (2σ), estimated by repeated analyses of the d Data Fig. 3 \| Hydrogen isotope analysis sequences for the SWIFT MORB glass standard. Reprints and permissions information is available at The average δD values calculated for the Kovdor and Durango apatite are −39 ± 37 ‰ and −80 ± 134 ‰ (Supplementary Table 3), respectively, consistent with their recommended values within analytical errors26,71. The average δD values measured for the basaltic glass standards 1833-11 and 519-4-1 are −54 ± 42 ‰ and −80 ± 28 ‰ (Supplementary Table 3), respectively, which is consistent with the δD values reported in our previous work50. The error bars are 2 SD. Nature Geoscience Nature Geoscience https://doi.org/10.1038/s41561-023-01159-6 Article 0 20 40 60 0 400 800 1200 1600 T=360 K t=1 year t=4.5 year profile-3 0 20 40 60 -1000 -800 -600 -400 -200 0 0 20 40 60 80 100 0 100 200 300 400 500 0 20 40 60 80 100 -1000 -800 -600 -400 -200 0 T=360 K t=1 years t=4.5 years profile-3 H2 -1) a. CE5#33,003, water abundance profile δDSMOW b. CE5#33,003, δD profile c. CE5#33,046, water abundance profile T=360 K t=2 years t=6 years profile-4 H2 -1) d. CE5#33,046, δD profile T=360 K t=2 year t=6 year profile-4 δDSMOW Extended Data Fig. 4 \| Two zoning profiles of water abundances and δD values for CE5 impact glass beads (profile-3 on CE5#33,003 and profile-4 on CE5#33,046). a. water abundance profile on CE5#33,003. b. δD profile on CE5#33,003. c. water abundance profile on CE5#33,046. d. δD profile on CE5#33,046. The analytical locations of profile-3 on CE5#33,003 and profile-4 on CE5#33,046 are shown in Fig. 1. Hydroxyl/water diffusion modelling results at 360 K are shown in black lines. The diffusional durations vary from 1 to 4.5 years an 2 to 6 years for profile-3 and −4, respectively. For modelling details see Methods. The error bars are 2 SD. 0 20 40 60 0 400 800 1200 1600 T=360 K t=1 years t=4.5 years profile-3 H2 -1) a. CE5#33,003, water abundance profile T=360 K t=1 year t=4.5 year profile-3 0 20 40 60 -1000 -800 -600 -400 -200 0 δDSMOW b. CE5#33,003, δD profile δDSMOW 0 20 40 60 80 100 0 100 200 300 400 500 c. CE5#33,046, water abundance profile T=360 K t=2 years t=6 years profile-4 H2 -1) 0 20 40 60 80 100 -1000 -800 -600 -400 -200 0 d. CE5#33,046, δD profile T=360 K t=2 year t=6 year profile-4 δDSMOW on CE5#33,046 are shown in Fig. 1. Hydroxyl/water diffusion modelling results at 360 K are shown in black lines. Reprints and permissions information is available at The diffusional durations vary from 1 to 4.5 years an 2 to 6 years for profile-3 and −4, respectively. For modelling details see Methods. The error bars are 2 SD. Extended Data Fig. 4 \| Two zoning profiles of water abundances and δD values for CE5 impact glass beads (profile-3 on CE5#33,003 and profile-4 on CE5#33,046). a. water abundance profile on CE5#33,003. b. δD profile on CE5#33,003. c. water abundance profile on CE5#33,046. d. δD profile on CE5#33,046. The analytical locations of profile-3 on CE5#33,003 and profile-4 Nature Geoscience Nature Geoscience https://doi.org/10.1038/s41561-023-01159-6 Article Extended Data Fig. 5 \| Another two zoning profiles of water abundances and δD values for two CE5 impact glass beads (profile-5 on CE5#33,052 and profile-6 on CE5#33,076). a. water abundance profile on CE5#33,052. b. δD profile on CE5#33,052. c. water abundance profile on CE5#33,076. d. δD profile on CE5#33,076. The analytical locations of profile-3 and profile-4 are shown in Fig. 1. The diffusional durations vary from 0.5 to 4 years and 2 to 14 years at 360 K for profile-5 and −6, respectively. For modelling details see Methods. The error bars are 2 SD. 0 20 40 60 0 600 1200 1800 2400 T 360 K t=0.5 years t=4 years profile-5 0 20 40 60 -1000 -800 -600 -400 -200 0 0 20 40 60 80 -1000 -800 -600 -400 -200 0 0 20 40 60 80 0 1000 2000 3000 T=360 K t=0.5 years t=4 years profile-5 H2 -1) a. CE5#33,052, water abundance profile δDSMOW b. CE5#33,052, δD profile T=360 K t=2 years t=14 years profile-6 δDSMOW d. CE5#33,076, δD profile c. CE#33,076, water abundance profile T=360 K t=2 years t=14 years profile-6 H2 -1) 0 20 40 60 0 600 1200 1800 2400 T=360 K t=0.5 years t=4 years profile-5 H2 -1) a. CE5#33,052, water abundance profile T 360 K t=0.5 years t=4 years profile-5 0 20 40 60 -1000 -800 -600 -400 -200 0 δDSMOW b. CE5#33,052, δD profile H2 -1) 40 0 20 40 60 80 0 1000 2000 3000 c. CE#33,076, water abundance profile T=360 K t=2 years t=14 years profile-6 H2 -1) 0 20 40 60 80 -1000 -800 -600 -400 -200 0 T=360 K t=2 years t=14 years profile-6 δDSMOW d. CE5#33,076, δD profile δDSMOW on CE5#33,076. The analytical locations of profile-3 and profile-4 are shown in Fig. 1. Reprints and permissions information is available at The diffusional durations vary from 0.5 to 4 years and 2 to 14 years at 360 K for profile-5 and −6, respectively. For modelling details see Methods. The error bars are 2 SD. Extended Data Fig. 5 \| Another two zoning profiles of water abundances and δD values for two CE5 impact glass beads (profile-5 on CE5#33,052 and profile-6 on CE5#33,076). a. water abundance profile on CE5#33,052. b. δD profile on CE5#33,052. c. water abundance profile on CE5#33,076. d. δD profile Nature Geoscience Nature Geoscience Article https://doi.org/10.1038/s41561-023-01159-6 Extended Data Fig. 6 \| Raman spectra. a,b, MORB glasses MRN-G1 (a) and EPR-G3 (b). c, CE5 impact glass bead, CE5#33,076. Raman peak at ~3500 cm−1 is ascribed to hydroxyl/molecular water. The grey lines are apparent baseline of the Raman peak between ~3300 and 3700 cm−1. A notable H2O zoning CE5 impact glass bead (CE5#33, 076) measured by NanoSIMS was chosen for Raman profile analysis. The Raman analytical positions of CE5 impact glass beads are outlined in Supplementary Fig. 2. 3000 3300 3600 3900 4200 a. MRN-G1 (H2O = 1.5 wt%) MRN-G b. EPR-G3 (H2O = 0.22 wt%) rim c. CE5#033,076 core Intensity (a.u.) Raman shift (cm-1) Extended Data Fig. 6 \| Raman spectra. a,b, MORB glasses MRN-G1 (a) and EPR-G3 (b). c, CE5 impact glass bead, CE5#33,076. Raman peak at ~3500 cm−1 is ascribed to hydroxyl/molecular water. The grey lines are apparent baseline of theRamanpeakbetween 3300and3700 cm−1 AnotableH OzoningCE5impact glass bead (CE5#33, 076) measured by NanoSIMS was chosen for Raman profile analysis. The Raman analytical positions of CE5 impact glass beads are outlined in Supplementary Fig. 2. 3000 3300 3600 3900 4200 a. MRN-G1 (H2O = 1.5 wt%) MRN-G b. EPR-G3 (H2O = 0.22 wt%) rim c. CE5#033,076 core Intensity (a.u.) Raman shift (cm-1) Extended Data Fig. 6 \| Raman spectra. a,b, MORB glasses MRN-G1 (a) and EPR-G3 (b). c, CE5 impact glass bead, CE5#33,076. Raman peak at ~3500 cm−1 is ascribed to hydroxyl/molecular water. The grey lines are apparent baseline of the Raman peak between ~3300 and 3700 cm−1. A notable H2O zoning CE5 impact glass bead (CE5#33, 076) measured by NanoSIMS was chosen for Raman profile analysis. The Raman analytical positions of CE5 impact glass beads are outlined in Supplementary Fig. 2. Nature Geoscience Nature Geoscience
https://openalex.org/W2560219982	https://europepmc.org/articles/pmc5191059?pdf=render	English	null	Application of Fast Dynamic Allan Variance for the Characterization of FOGs-Based Measurement While Drilling	Sensors	2,016	cc-by	13,578	Application of Fast Dynamic Allan Variance for the Characterization of FOGs-Based Measurement While Drilling Lu Wang 1, Chunxi Zhang 1, Shuang Gao 1,, Tao Wang 2, Tie Lin 1 and Xianmu Li 1 Lu Wang 1, Chunxi Zhang 1, Shuang Gao 1,, Tao Wang 2, Tie Lin 1 and Xianmu Li 1 1 Key Laboratory of Inertial Technology, Institute of Opto-electronics Technology, School of Instrument Science and Opto-electronics Engineering, Beihang University, Beijing 100191, China; wanglu@buaa.edu.cn (L.W.) zhangchunxi@buaa.edu.cn (C.Z.); opticlin@163.com (T.L.); lixianmu@buaa.edu.cn (X.L.) 2 Beijing Aerospace Times Laser Inertial Technology Company, Ltd., Beijing 100094, China; wtwdqa@163.com * Correspondence: gaoshuang@buaa.edu.cn; Tel.: +86-138-1002-5968; Fax: +86-10-8231-6547 (ext. 818) A d i Edit Vitt i M N P 1 Key Laboratory of Inertial Technology, Institute of Opto-electronics Technology, School of Instrument Science and Opto-electronics Engineering, Beihang University, Beijing 100191, China; wanglu@buaa.edu.cn (L.W.) zhangchunxi@buaa.edu.cn (C.Z.); opticlin@163.com (T.L.); lixianmu@buaa.edu.cn (X.L.) 2 Beijing Aerospace Times Laser Inertial Technology Company, Ltd., Beijing 100094, China; wtwdqa@163.com * Correspondence: gaoshuang@buaa.edu.cn; Tel.: +86-138-1002-5968; Fax: +86-10-8231-6547 (ext. 818) A d i Edi Vi i M N P Received: 12 August 2016; Accepted: 10 November 2016; Published: 7 December 2016 Received: 12 August 2016; Accepted: 10 November 2016; Published: 7 December 2016 Abstract: The stability of a ﬁber optic gyroscope (FOG) in measurement while drilling (MWD) could vary with time because of changing temperature, high vibration, and sudden power failure. Abstract: The stability of a ﬁber optic gyroscope (FOG) in measurement while drilling (MWD) could vary with time because of changing temperature, high vibration, and sudden power failure. The dynamic Allan variance (DAVAR) is a sliding version of the Allan variance. It is a practical tool that could represent the non-stationary behavior of the gyroscope signal. Since the normal DAVAR takes too long to deal with long time series, a fast DAVAR algorithm has been developed to accelerate the computation speed. However, both the normal DAVAR algorithm and the fast algorithm become invalid for discontinuous time series. What is worse, the FOG-based MWD underground often keeps working for several days; the gyro data collected aboveground is not only very time-consuming, but also sometimes discontinuous in the timeline. In this article, on the basis of the fast algorithm for DAVAR, we make a further advance in the fast algorithm (improved fast DAVAR) to extend the fast DAVAR to discontinuous time series. The improved fast DAVAR and the normal DAVAR are used to responsively characterize two sets of simulation data. Application of Fast Dynamic Allan Variance for the Characterization of FOGs-Based Measurement While Drilling The simulation results show that when the length of the time series is short, the improved fast DAVAR saves 78.93% of calculation time. When the length of the time series is long (6 × 105 samples), the improved fast DAVAR reduces calculation time by 97.09%. Another set of simulation data with missing data is characterized by the improved fast DAVAR. Its simulation results prove that the improved fast DAVAR could successfully deal with discontinuous data. In the end, a vibration experiment with FOGs-based MWD has been implemented to validate the good performance of the improved fast DAVAR. The results of the experience testify that the improved fast DAVAR not only shortens computation time, but could also analyze discontinuous time series. Keywords: dynamic stability; FOGs-MWD; dynamic Allan variance; fast algorithm; discontinuous data sensors sensors sensors 1. Introduction Gyroscopes are sensors that are appropriate for a wide variety of applications in the inertial navigation scope. The ﬁber optic gyroscope (FOG), which has become mainstream in inertial navigation systems, is utilized to determine altitude for satellites and missiles [1]. The Ring Laser gyroscope is the ideal angular sensor for high-precision and long-endurance inertial navigation systems [2]. The MEMS (Micro-electromechanical systems) gyroscope is widely used in cheap and small applications, such as unmanned aerial vehicles [3]. The measurement stability of gyroscopes can vary over time due to several factors, such as temperature, humidity, radiation, and ageing [4]. A slight instability in the gyroscopes will lead to an increase in measurement error. FOGs in measurement while drilling (MWD) Sensors 2016, 16, 2078; doi:10.3390/s16122078 www.mdpi.com/journal/sensors 2 of 18 Sensors 2016, 16, 2078 systems work even in poor conditions. Their temperature increases as the drilling depth increases [5]. Additionally, FOGs withstand shock when drilling the oil hole. Therefore, it is fundamental to evaluate how the measurement stability of gyroscopes behaves over time. Allan variance is a common and standard method to analyze gyroscopes [6], but it cannot describe the dynamic characteristic. In 2003, in order to track and reveal the anomaly in the atom clock behavior, Galleani and Tavella developed Dynamic Allan Variance (DAVAR) [7]. Although it is an extension of Allan variance, DAVAR is a new method that can track and describe the non-stationary characteristics of time series [8–13]. Li, Zhang, and Wei extended the DAVAR to diagnose the non-stationary of gyroscope [14–18]. Since the DAVAR requires instant calculation of Allan variance, its calculation burden is a critical shortcoming [19–22]. Wang and Zhang developed a fast algorithm for DAVAR to simply the DAVAR algorithm and accelerate the calculation speed [23]. An MWD system keeps working for several days underground. The output signal of the gyroscopes in the MWD system would be interrupted due to error in the mud pulse communication or sudden power outages. In this case, both the classical DAVAR and the fast DAVAR algorithms are unable to deal with discontinuous time series. Hence, in order to deal with the discontinuous gyroscope data, a further improvement is made on the basis of the fast DAVAR. The recursion characteristic of the fast DAVAR will judge whether the data contains unreadable code or not. It makes the fast DAVAR available to deal with discontinuous gyroscope data. 1. Introduction Thus, the improved fast algorithm of the DAVAR is more valuable in engineering applications. This article is organized as follows. In Section 2, the structure of the FOG-based MWD and its working characteristics are introduced. In Section 3, the theory of Allan variance is brieﬂy presented. In Section 4, we sum up the calculation process of the classical DAVAR. In addition, a 2D diagram illustrating the noise characteristics of FOGs is reported. In Section 5, the fast algorithm of the DAVAR is derived step by step. In Section 6, a further improvement has been made and the improved fast DAVAR is extended to discontinuous data. In Section 7, in order to test that the improved fast DAVAR is superior to the classical method, three sets of simulation data have been analyzed by the two methods. In Section 8, a set of discontinuous time series collected from a vibration experiment with the FOGs-based MWD is analyzed by the fast DAVAR. The conclusions are presented in Section 9. 2. Structure of the FOG-Based MWD The MWD prototype utilizes a FOG-based IMU, as shown in Figure 1. MWD is composed of three-axis FOGs sharing one laser light source, three quartz ﬂexible accelerometers with one A/D converter circuit, a navigation computer, and a mechanical bracket to support the above units [24]. All the components are orthogonally mounted along the lengthwise axis of the MWD so that the diameter can be minimized to satisfy the demands of a borehole environment. The FOGs and accelerometers are arranged in three mutually orthogonal directions, as shown in Figure 2. The three-axis FOGs measure the angular velocity of the carrier, while three-axis accelerometers provide the 3D acceleration measurements of the body. The navigation computer is mainly utilized to collect all sensor data, pre-process the data, and run the navigation algorithm. 3 of 18 3 of 18 Sensors 2016, 16, 2078 Sensors 2016, 16, 2078 Figure 1. 3D graphic model of the MWD. xf yf zf y  z  x  Figure 2. Sensors of IMU. Due to the particularity of the working environment in oil wells, the FOGs-based MWD working manner and characteristics are different from other application fields such as aerospace, Figure 1. 3D graphic model of the MWD. Figure 1. 3D graphic model of the MWD. xf yf zf y  z  x  Figure 2. Sensors of IMU. Figure 2. Sensors of IMU. Figure 1. 3D graphic model of the MWD. Figure 1. 3D graphic model of the MWD. Figure 1. 3D graphic model of the MWD. Figure 1. 3D graphic model of the MWD. Figure 1. 3D graphic model of the MWD. Figure 1. 3D graphic model of the MWD. Figure 1. 3D graphic model of the MWD. Figure 1. 3D graphic model of the MWD. Figure 1. 3D graphic model of the MWD. xf yf zf y  z  x  Figure 2. Sensors of IMU. ularity of the working environment in oil wells, haracteristics are different from other application f xf yf zf y  z  x  Figure 2. Sensors of IMU. Figure 2. Sensors of IMU. e working environment in s are different from other a Figure 2. Sensors of IMU. Figure 2. Sensors of IMU. aviation, and land navigation. 2. Structure of the FOG-Based MWD Comprehensive analysis shows that the detection and location of oil wells have the following characteristics: (1) Long working hours: the FOGs-based MWD system keeps working underground for more than 200 h. Putting the MWD system down into the well and pulling it up to the ground costs too much time and money [25]. Due to the particularity of the working environment in oil wells, the FOGs-based MWD working manner and characteristics are different from other application fields such as aerospace, aviation, and land navigation. Comprehensive analysis shows that the detection and location of oil wells have the following characteristics: Due to the particularity of the working environment in oil wells, the FOGs-based MWD working manner and characteristics are different from other application ﬁelds such as aerospace, aviation, and land navigation. Comprehensive analysis shows that the detection and location of oil wells have the following characteristics: (2) Strong vibration: FOGs in MWD system hold up to strong shocks due to the obstruction of the underground stone. (3) High rotation speed. FOGs in MWD systems work with a high-speed rotation up to 300 rounds per second (1) Long working hours: the FOGs-based MWD system keeps working underground for more than 200 h. Putting the MWD system down into the well and pulling it up to the ground costs too much time and money [25]. (2) Strong vibration: FOGs in MWD system hold up to strong shocks due to the obstruction of the (1) Long working hours: the FOGs-based MWD system keeps working underground for more than 200 h. Putting the MWD system down into the well and pulling it up to the ground costs too much time and money [25]. per second. (4) High Temperature. The temperature of the working environment increases with the increase in the drilling depth at a rate of 30 °C/km. (2) Strong vibration: FOGs in MWD system hold up to strong shocks due to the obstruction of the underground stone. (3) High rotation speed. FOGs in MWD systems work with a high-speed rotation up to 300 rounds (2) Strong vibration: FOGs in MWD system hold up to strong shocks due to the obstruction of the underground stone. (5) Data loss. The data transmission method of the MWD system is mud pulse. 2. Structure of the FOG-Based MWD When drilling the oil well, the mud pulse transmits the data collected downhole to the monitoring equipment on th d It t f t i l It bit t i hi h d th li bilit i l (3) High rotation speed. FOGs in MWD systems work with a high speed rotation up to 300 rounds per second. (4) High Temperature. The temperature of the working environment increases with the increase in (3) High rotation speed. FOGs in MWD systems work with a high-speed rotation up to 300 rounds per second. the ground. Its transfer rate is low. Its bit error rate is high and the reliability is very low. (6) Eclectic battery-powered. A battery provides power to support the MWD system working underground. ( ) g p p g the drilling depth at a rate of 30 °C/km. (5) Data loss. The data transmission method of the MWD system is mud pulse. When drilling the (4) High Temperature. The temperature of the working environment increases with the increase in the drilling depth at a rate of 30 ◦C/km. underground. When drilling the oil well, FOGs’ signal in MWD system is non-stationary, since the FOGs can be easily influenced by the increasing temperature, humidity, radiation, and sudden power failure. Occasionally the data received aboveground appears to be discontinuous Even a slight variation in y p g oil well, the mud pulse transmits the data collected downhole to the monitoring equipment on the ground. Its transfer rate is low. Its bit error rate is high and the reliability is very low. (6) Eclectic battery-powered. A battery provides power to support the MWD system working (5) Data loss. The data transmission method of the MWD system is mud pulse. When drilling the oil well, the mud pulse transmits the data collected downhole to the monitoring equipment on the ground. Its transfer rate is low. Its bit error rate is high and the reliability is very low. Occasionally, the data received aboveground appears to be discontinuous. Even a slight variation in the gyroscope stability can generate significant measurement error. Accordingly, it is crucial to identify the dynamic instability of the FOGs in MWD. ( ) y p y p p pp y g underground. When drilling the oil well FOGs’ signal in MWD system is non-stationary since the FOGs can (6) Eclectic battery-powered. 3. Allan Variance FOGs sense the angle velocity. Their output signal is the angle velocity ω(t). Allan variance [26] has commonly been used to evaluate the stability of FOG signal ω(t). The standard concept of the Allan variance is: σ2 ω(τ) = 1 2 D (ω(t + τ) −ω(t))2E , (1) (1) where τ is the observation interval and ⟨⟩indicates a time averaging. The average of ϖ(t) is given by where τ is the observation interval and ⟨⟩indicates a time averaging. The average of ϖ(t) is given by ω(t) = 1 τ w t+τ t ω(u)du, (2) (2) where u is the integral variable. Equation (1) is the deﬁnition of Allan variance for continuous data. If the Allan variance is to be estimated on discrete samples ω [n] whose total number is N, t is sampled as where u is the integral variable. Equation (1) is the deﬁnition of Allan variance for continuous data. If the Allan variance is to be estimated on discrete samples ω [n] whose total number is N, t is sampled as t = nτ0, (n = 1, 2, · · · N), (3) (3) where τ0 is the sampling interval. Consequently, the observation interval τ is discretized is τ = kτ0, (k = 1, 2, · · · N 2 ). (4) (4) For the discrete data, the Allan variance becomes For the discrete data, the Allan variance becomes σ2 ω(k) = 1 2k2τ2 0 1 N −2k N−2k−1 ∑ n=0 (ω[n + k] −ω[n])2). (5) (5) The average of ϖ[n] is given by The average of ϖ[n] is given by The average of ϖ[n] is given by ω[n] = 1 k n+k−1 ∑ i=n ω[i]. (6) (6) Therefore, according to the different observation interval k, the corresponding Allan variance can be obtained. Therefore, according to the different observation interval k, the corresponding Allan variance can be obtained. Ng [26] shows that a unique relationship existing between σ2 ω(τ) and the power spectral density (PSD) of the intrinsic random processes. This relationship is σ2 ω(τ) = 4 w ∞ 0 SΩ( f )sin4 (π f τ) (π f τ)2 du, (7) (7) where SΩ( f ) is the PSD of the random process Ω(t), namely the instantaneous output rate of the g Equation (7) is the focal point of the Allan variance technique. 2. Structure of the FOG-Based MWD A battery provides power to support the MWD system working underground. be easily influenced by the increasing temperature, humidity, radiation, and sudden power failure. Occasionally, the data received aboveground appears to be discontinuous. Even a slight variation in the gyroscope stability can generate significant measurement error. Accordingly, it is crucial to identify the dynamic instability of the FOGs in MWD. When drilling the oil well, FOGs’ signal in MWD system is non-stationary, since the FOGs can be easily inﬂuenced by the increasing temperature, humidity, radiation, and sudden power failure. Occasionally, the data received aboveground appears to be discontinuous. Even a slight variation in the gyroscope stability can generate signiﬁcant measurement error. Accordingly, it is crucial to identify the dynamic instability of the FOGs in MWD. Sensors 2016, 16, 2078 4 of 18 3. Allan Variance Noise Terms Noise Coefﬁcient SΩ(v) PSD of the Random Process σ2(τ) Slope of logσ(τ) −logτ the quantization noise Q SΩ( f ) =        4Q2 τ sin2 (π f τ) f ≥1 2τ (2π f )2τQ2 f < 1 2τ 3Q2 τ2 −1 angular random walk N SΩ( f ) = N2 N2 τ −1/2 bias instability B SΩ( f ) =        ( B2 2π ) 1 f f ≤f0 0 f > f0 2B2 π ln2 0 rate random walk K SΩ( f ) = ( K2 2π ) 1 f 2 K2τ 3 1/2 the rate slope R SΩ( f ) = R2 (2π f )3 R2τ2 2 1 Noise Terms Noise Coefficient S ( ) v  PSD of the Random Process 2( )   Slope of log ( ) log    - the quantization noise Q 2 2 2 2 1 4 sin ( ) 2 S ( )= 1 < (2 ) 2 f Q f f f f Q            2 2 3Q  −1 angular random walk N 2 S ( ) = N f  2 N  −1/2 bias instability B 2 0 0 1 ( ) S ( )= 2 > 0 B f f f f f f       2 2 ln 2 B  0 rate random walk K 2 2 1 S ( ) = ( ) 2 K f f   2 3 K  1/2 the rate slope R 2 3 S ( ) = (2 ) R f f   2 2 2 R  1 Figure 3. Sample plot of Allan variance analysis results. Figure 3. Sample plot of Allan variance analysis results. Figure 3. Sample plot of Allan variance analysis results. Figure 3. Sample plot of Allan variance analysis results. Figure 3. Sample plot of Allan variance analysis results. Figure 3. Sample plot of Allan variance analysis results. In general, any number of the random processes discussed above can be present in the data. Thus, a typical Allan variance plot looks like the one shown in Figure 3. Different noise terms appear in different regions of . 3. Allan Variance The PSD of any physically meaningful random process can be substituted in the integral and an expression for the Allan variance σ2 ω(τ) as a function of cluster length τ is identiﬁed [26,27]. Equation (7) states that the Allan variance is proportional to the total power output of the random process when passed through a ﬁlter with the transfer function sin4 (π f τ) (π f τ)2 . This particular transfer function is the result of the method used to create and operate on the cluster. It is seen from Equation (7) that the ﬁlter bandpass depends on τ. This suggests that different types of random processes can be examined by adjusting the ﬁlter bandwidth, namely by varying τ [28]. C l i 2 ( ) i bl i l f 2 ( ) id di the transfer function sin4 (π f τ) (π f τ)2 . This particular transfer function is the result of the method used to create and operate on the cluster. It is seen from Equation (7) that the ﬁlter bandpass depends on τ. This suggests that different types of random processes can be examined by adjusting the ﬁlter bandwidth, namely by varying τ [28]. ( f ) to create and operate on the cluster. It is seen from Equation (7) that the ﬁlter bandpass depends on τ. This suggests that different types of random processes can be examined by adjusting the ﬁlter bandwidth, namely by varying τ [28]. Consequently, since σ2 ω(τ) is a measurable quantity, a plot of σ2 ω(τ) versus τ provides a direct indication of the types of random processes that exist in the gyro data. A log-log plot of the square root of the Allan variance σω(τ) −τ provides a means of identifying and quantifying various noise terms 5 of 18 ntifying Sensors 2016, 16, 2078 square root of th that exist in the gyro sensor data, as Figure 3 shows. Table 1 gives a representation of noise terms using Allan variance. gy g g representation of noise terms using Allan variance. Table 1 Representation of noise terms using Allan variance Table 1. Representation of noise terms using Allan variance. Table 1. Representation of noise terms using Allan variance. Noise 2 Slope of Table 1. Representation of noise terms using Allan variance. 3. Allan Variance This allows for easy identification of the various random processes that exist in the gyro data. If it can be assumed that the existing random processes are all statistically In general, any number of the random processes discussed above can be present in the data. Thus, a typical Allan variance plot looks like the one shown in Figure 3. Different noise terms appear in different regions of τ. This allows for easy identiﬁcation of the various random processes that exist in the gyro data. If it can be assumed that the existing random processes are all statistically independent, then it can be shown that the Allan variance at any given τ is the sum of the Allan variances due to the individual random processes at the same τ [27]. In other words, σ2(τ) = σ2 Q(τ) + σ2 N(τ) + σ2 B(τ) + σ2 K(τ) + σ2 R(τ) = 3Q2 τ2 + N2 τ + 2B2 π ln2 + K2τ 3 + R2τ2 2 , (8) (8) where σ2 Q(τ), σ2 N(τ), σ2 B(τ), σ2 K(τ), σ2 R(τ) represent Allan variance due to the individual random processes. Then the noise coefﬁcient of the ﬁve noise terms can be obtained by a data ﬁtting algorithm. Sensors 2016, 16, 2078 6 of 18 When the dimension of FOG output data is in degrees per hour (◦/h), the different noise terms are as follows: N = p C−1 60 (◦/h 1 2 ) K = 60√3C1(◦/h 3 2 ) B = √C0 0.664(◦/h) Q = 106π√C−2 180 × 3600 × √ 3(′′) R = 3600√2C2(◦/h2) . (9) (9) 4.1. Computation Process of Dynamic Allan Variance 4.1. Computation Process of Dynamic Allan Variance The dynamic Allan variance (DAVAR) is an extension of the Allan variance. Firstly, truncate the data with a rectangular window centered at a given time point. Secondly, calculate the Allan variance of the truncated data. Then repeat the above two steps at every time epoch. In the end, by collecting all the variances obtained at every epoch and plotting the results in a signal 3D graph [8], we can obtain the DAVAR. The detailed computation process can be found in [9]. For continuous-time signals, the DAVAR could be obtained using Equation (10): σ2 ω(t, τ) = 1 2τ2(Nw −2τ) w t+ Nw 2 −τ t−Nw 2 +τ (ϖ(u + τ) −ϖ(u))2du, (10) (10) where Nw is the length of the truncation window, t is the analysis time point, τ is the observation interval, and σ2 ω(t, τ) is the DAVAR. The DAVAR given in Equation (10) is continuous both in t and τ. For discrete time signals, by sampling the integral variable u and the observation interval τ as follows, u = mτ0, (m = NW 2 , 2, · · · N −NW 2 ), τ = kτ0, (k = 1, 2, · · · NW 2 ), (11) (11) we can obtain the standard notation of discrete-time signals. we can obtain the standard notation of discrete-time signals. σ2 ω[n, k] = 1 2k2τ2 0 1 Nw −2k × n+Nw/2−2k−1 ∑ m=n−Nw/2 (ϖ[m + k] −ϖ[k])2, (12) (12) where τ0 is the sampling interval, kτ0 is the observation interval of the discrete time, and N is the available number of ω(n). Nw is the length of the analysis window. Nw is assumed to be even. 4. Dynamic Allan Variance 4.1. Computation Process of Dynamic Allan Variance 4.2. 2D Description of FOG Noise Terms 4.2. 2D Description of FOG Noise Terms The DAVAR is simply obtained by sliding the Allan variances on the time series. At each given time t, Equation (8) still holds true. Therefore, the Allan variance at any given time can be written as Equation (13): σ2(t, τ) = σ2 Q(t, τ) + σ2 N(t, τ) + σ2 B(t, τ) + σ2 K(t, τ) + σ2 R(t, τ). (13) (13) At a given time t, there is a σ(t, τ) −τ curve. Correspondingly, we can obtain the coefﬁcients Q(t), N(t), B(t), K(t), and R(t) of the ﬁve noise terms. Therefore, coefﬁcients of the noise terms for all t could be obtained. In the end, by plotting Q(t), N(t), B(t), K(t), and R(t) according to the chronological order, At a given time t, there is a σ(t, τ) −τ curve. Correspondingly, we can obtain the coefﬁcients Q(t), N(t), B(t), K(t), and R(t) of the ﬁve noise terms. Therefore, coefﬁcients of the noise terms for all t could be obtained. In the end, by plotting Q(t), N(t), B(t), K(t), and R(t) according to the chronological order, 7 of 18 efficients s for all Sensors 2016, 16, 2078 At a given Q( ) N( ) B( ) K the time-varying coefﬁcients of FOGs noise terms can be characterized in a 2D diagram. Figure 4 gives a ﬂowchart of the computation process of the DAVAR. could be obtained. In the end, by plotting Q(t), N(t), B(t), K(t), and R(t) according to the chronologica order, the time-varying coefficients of FOGs noise terms can be characterized in a 2D diagram Figure 4 gives a flowchart of the computation process of the DAVAR. 2 0 ( , ), = in k    log 2( , ) in   W N ( ) T in  ( ) T in  in in in in in ( ) n  ( ) n  in in 2 log ( , ) in   1 = in n 0 k 2 0 ( , ) in k   0 0 2 W N k   Figure 4. Flow chart of dynamic Allan variance. Figure 4. Flow chart of dynamic Allan variance. in in in in in Figure 4. Flow chart of dynamic Allan variance Figure 4. Flow chart of dynamic Allan variance. 5. Fast Dynamic Allan Variance Figure 4 shows that the DAVAR is obtained by computing the Allan variance at each analysis time epoch t. With the length of ω(t) increasing, it can result in a large computational burden. Therefore it is necessary to develop a fast algorithm for DAVAR. On the basis of the recursive characteristic of Allan variance, the recursive property of the DAVAR was found [29]. With this special characteristic, we can develop a recursive algorithm for the normal DAVAR, i.e., the fast DAVAR. Sensors 2016, 16, 2078 8 of 18 First deﬁne another time-series θ(t) connected to ω(t). The relationship between them is ω(t) = dθ(t) dt (14) (14) θ(n) denotes the angle, for which the dimension is given in degrees. Both ω(t) and θ(n) are random quantities. Then, substituting Equations (2) and (14) into Equation (10), Equation (10), which is the normal DAVAR, can be rewritten as σ2 ω(t, τ) = 1 2τ2(Nw −2τ) w t+ Nw 2 −τ t−Nw 2 +τ (θ(u + 2τ) −2θ(u + τ) + θ(u))2du. (15) (15) Using Equation (11), we can obtain the DAVAR for the discrete time gyro signal θ(n) as in Equation (16): Using Equation (11), we can obtain the DAVAR for the discrete time gyro signal θ(n) as in Equation (16): σ2 ω[n, k] = 1 2k2τ2 0 1 Nw −2k n+Nw/2−2k−1 ∑ m=n−Nw/2 (θ[m + 2k] −2θ[m + k] + θ[m])2. (16) (16) In order to describe the derivation simply, we name ∆k[m] = θ[m + 2k] −2θ[m + k] + θ[m] as the discrete second order difference. Therefore Equation (16) can be rewritten as Equation (17): In order to describe the derivation simply, we name ∆k[m] = θ[m + 2k] −2θ[m + k] + θ[m] as the discrete second order difference. Therefore Equation (16) can be rewritten as Equation (17): σ2 ω[n, k] = 1 2k2τ2 0 1 Nw −2k n+Nw/2−2k−1 ∑ m=n−Nw/2 ∆2 k[m]. (17) (17) Using Equation (17), we can obtain the DAVAR at time point n + 1 as follows: σ2 ω[n + 1, k] = 1 2k2τ2 0 1 Nw −2k n+Nw/2−2k ∑ m=n+1−Nw/2 ∆2 k[m]. (18) (18) σ2 ω[n + 1, k] can also be written with σ2 ω[n, k], as Equation (19) shows. 5. Fast Dynamic Allan Variance σ2 ω[n + 1, k] = 1 2k2τ2 0 1 Nw −2k n+Nw/2−2k−1 ∑ m=n−Nw/2 ∆2 k[m] + 1 2k2τ2 0 1 Nw −2k(∆2 k[n + Nw 2 −2k] −∆2 k[n −Nw 2 ]) = σ2 ω[n, k] + 1 2k2τ2 0 1 Nw −2k(∆2 k[n + Nw 2 −2k] −∆2 k[n −Nw 2 ]) (19) (19) At time n, the limit of m in discrete second-order difference ∆k[m] lies in [n −Nw 2 , n + Nw 2 −2k −1]. 2 2 At time n + 1, the limit of m lies in [n + 1 −Nw 2 , n + Nw 2 −2k]. Therefore, when we compute the σ2 ω[n + 1, k] based on the σ2 ω[n, k], we just need to add the discrete second-order difference ∆2 k[n + Nw/2 −2k] and subtract the discrete second-order difference ∆2 k[n −Nw/2]. Equation (19) shows that the DAVAR at time n + 1 can be written as a function of the DAVAR at time n. Since Equation (19) is a recursive algorithm for DAVAR, the initial value is required before the computation. On the assumption that the analysis starts at time n1, the starting value is as in Equation (20): σ2 ω[n1, k] = 1 2k2τ2 0 1 Nw −2k n1+Nw/2−2k−1 ∑ m=n1−Nw/2 ∆2 k[m]. (20) (20) In conclusion, with the recursive formula, it is not necessary to compute the Allan variance at every time epoch. The new DAVAR can be obtained based on the previous DAVAR. This can reduce the computation time dramatically. We named the recursive algorithm fast DAVAR and the ordinary one classical DAVAR. Figure 5 gives a ﬂowchart of fast DAVAR. 5. Fast Dynamic Allan Variance 9 of 18 and the Sensors 2016, 16, 2078 the computation di l y g g 2[ / 2 2 ] k i w n N k    W N ( ) n  ( ) n  2 0 ( , ), =1 =1,2 2 W i N n k i k    ， 2 -1 0 ( , ) in k   0 0 =( +1) k k   0 0 2 W N k   2[ / 2] k i w n N   2 -1 0 ( , ) in k   2 0 ( , ), = in k    log 2( , ) in   in in in in in 2 log ( , ) in   in 0 0 = k  Figure 5. Flowchart of fast dynamic Allan variance. Figure 5. Flowchart of fast dynamic Allan variance. 2( , ) in   in in in in in Figure 5. Flowchart of fast dynamic Allan variance Figure 5. Flowchart of fast dynamic Allan variance. 7. Models and Simulations In order to testify the proposal algorithm, two sets of simulation data are created. The model of the simulation data is shown as follows: (23) x[n] = σ[n] f [n], (23) where f [n] is the white Gaussian noise [5], σ[n] is the standard deviation of f [n], and x[n] is simulating data of gyroscopes whose unit is ◦/h. The sampling interval of the two sets of simulation data is 0.01 s. The ﬁrst simulation dataset x1[n] is stationary white Gaussian noise, whose standard deviation σ[n] is always equal to 1. The number of x1[n] is L1 = 6 × 103 samples. So the time length of x1[n] is 60 s. The second one x2[n] is also white Gaussian noise, whose variance σ[n] increases with time linearly as Equation (24) shows. The number of x2[n] is L2 = 6 × 105 samples. Thus the time length of x2[n] is 6000 s. where f [n] is the white Gaussian noise [5], σ[n] is the standard deviation of f [n], and x[n] is simulating data of gyroscopes whose unit is ◦/h. The sampling interval of the two sets of simulation data is 0.01 s. The ﬁrst simulation dataset x1[n] is stationary white Gaussian noise, whose standard deviation σ[n] is always equal to 1. The number of x1[n] is L1 = 6 × 103 samples. So the time length of x1[n] is 60 s. The second one x2[n] is also white Gaussian noise, whose variance σ[n] increases with time linearly as Equation (24) shows. The number of x2[n] is L2 = 6 × 105 samples. Thus the time length of x2[n] is 6000 s. σ[n] = 1 + 10 L2 × n (24) (24) Two sets of simulation data are represented in Figure 6a,c. x1[n] is stationary. The amplitude of x2[n] increases with time. Two sets of simulation data are represented in Figure 6a,c. x1[n] is stationary. The amplitude of x2[n] increases with time. Figure 6b,d exhibits the Allan variance of the two sets of simulated data. Because x1[n] is stationary white Gaussian noise, its slope is approximately −1/2 for all τ [30]. x2[n] is non-stationary white noise, but the shapes and slopes of Allan variance are the same as for the stationary white noise x1[n]. Obviously, the Allan variance cannot track and reveal the non-stationary characteristics. Then the classical DAVAR and the improved fast DAVAR are applied to analyze the simulation data. 6. Extension to Discontinuous Time Series 6. Extension to Discontinuous Time Series At present, when drilling oil wells, the MWD system utilizes the mud pulse to send the data from underground to the monitoring equipment on the ground. Its transfer rate and reliability are low while its bit error rate is high. Consequently, the data received on the ground often appears to be abnormal, such as with unreadable words or showing a loss of data in a certain period of time. So the data are discontinuous in the timeline. However, when meeting the discontinuous gyroscope data, both the classical DAVAR and the fast DAVAR are invalid. It is critical to further improve the algorithm of the DAVAR to deal with discontinuous time series. Based on the recursive character of the fast DAVAR algorithm, we make a further advance to extend the fast DAVAR to discontinuous time series. Sensors 2016, 16, 2078 10 of 18 Before computing σ2 ω[n + 1, k], we need to compute the discrete second-order difference ∆2 k[n + Nw/2 −2k] and ∆2 k[n −Nw/2]. For the given n and k, ∆2 k[n + Nw/2 −2k] and ∆2 k[n −Nw/2] can be obtained by Equations (21) and (22): ∆2 k[n + Nw 2 −2k] = (θ[n + Nw 2 ] −2θ[n + Nw 2 −k] + θ[n + Nw 2 −2k]) 2 (21) ∆2 k[n −Nw 2 ] = (θ[n −Nw 2 + 2k] −2θ[n −Nw 2 + k] + θ[n −Nw 2 ]) 2 . (22) (21) ∆2 k[n −Nw 2 ] = (θ[n −Nw 2 + 2k] −2θ[n −Nw 2 + k] + θ[n −Nw 2 ]) 2 . (22) (22) At this time, we can judge whether θ[n + Nw 2 ], θ[n + Nw 2 −k] and θ[n + Nw 2 −2k] are unreadable words or not. If one of them is an unreadable word, we set ∆2 k[n + Nw/2 −2k] to zero and then continue the computation. ∆2 k[n −Nw/2] could be obtained in the same way. The recursion characteristic of the fast DAVAR will judge whether the data contains unreadable code or not. So the fast DAVAR could be extended to deal with discontinuous time series. We call this modiﬁed algorithm improved fast DAVAR. 7. Models and Simulations The analysis results of the classical DAVAR Figure 6a,c and the improved fast DAVAR Figure 6b,d are shown in Figure 7. 11 of 18 Sensors 2016, 16, 2078 -20 X (a) (b) (c) (d) Figure 6. Two sets of simulation data. (a) white Gaussian noise 1[ ] x n ; (b) Allan variance of 1[ ] x n ; (c) white Gaussian noise with “increase” 2[ ] x n ; (d) Allan variance of 2[ ] x n . 0 20 40 60 -4 -2 0 2 4 t/s X1(t)°/h 10 -2 10 -1 10 0 10 1 10 -2 10 -1 10 0 10 1 Allan variance and curve-fitting τ(s) σ(τ)(。/h) allan deviation fitting curve 0 2000 4000 6000 -40 -20 0 20 40 t/s X3(t)°/h 10 -2 10 0 10 2 10 4 10 -3 10 -2 10 -1 10 0 10 1 Allan variance and curve-fitting τ(s) σ(τ)(。/h) allan deviation fitting curve Figure 6. Two sets of simulation data. (a) white Gaussian noise x1[n]; (b) Allan variance of x1[n]; (c) white Gaussian noise with “increase” x2[n]; (d) Allan variance of x2[n]. (c) (d) Figure 6. Two sets of simulation data. (a) white Gaussian noise 1[ ] x n ; (b) Allan variance of 1[ ] x n ; (c) white Gaussian noise with “increase” 2[ ] x n ; (d) Allan variance of 2[ ] x n . Figure 6b,d exhibits the Allan variance of the two sets of simulated data. Because 1[ ] x n tionary white Gaussian noise, its slope is approximately −1/2 for all [30]. 2[ ] x n n-stationary white noise, but the shapes and slopes of Allan variance are the same as for th tionary white noise 1[ ] x n . Obviously, the Allan variance cannot track and reveal th n-stationary characteristics. Then the classical DAVAR and the improved fast DAVAR are applied to analyze the simulatio ta. The analysis results of the classical DAVAR Figure 6a,c and the improved fast DAVA gure 6b,d are shown in Figure 7. With a rectangular window of L = 1000 samples and a step whose width is 30 samples, th alysis results of 1[ ] x n are obtained. As can be seen in Figure 7a, both DAVAR methods a nstant over time and have a typical white noise slope. 7. Models and Simulations 2x opes of Allan variance are the same as 10 -2 10 0 10 2 10 10 3 τ(s) (b) (d) 10 -2 10 0 10 2 10 4 10 -3 10 -2 10 -1 10 0 10 1 Allan variance and curve-fitting τ(s) σ(τ)(。/h) allan deviation fitting curve p Allan variance cannot track and revea t DAVAR are applied to analyze the simu R Figure 6a,c and the improved fast DA es and a step whose width is 30 sample seen in Figure 7a, both DAVAR metho ope. The value of the improved fast DAV .The analysis results of 2[ ] x n are obtain ples and a step whose width is 300 sam (d) whose (c) num Figure 6. Two sets of simulation data. (a) white Gaussian noise 1[ ] x n ; (b) Allan variance of 1[ ] x n ; (c) white Gaussian noise with “increase” 2[ ] x n ; (d) Allan variance of 2[ ] x n . Figure 6. Two sets of simulation data. (a) white Gaussian noise x1[n]; (b) Allan variance of x1[n]; (c) white Gaussian noise with “increase” x2[n]; (d) Allan variance of x2[n]. igure 7b, the improved fast DAVAR and classical DAVAR reveal that noise increases linea h time. Figure 6b,d exhibits the Allan variance of the two sets of simulated data. Because 1[ ] x n is stationary white Gaussian noise, its slope is approximately −1/2 for all [30]. 2[ ] x n is non-stationary white noise, but the shapes and slopes of Allan variance are the same as for the stationary white noise 1[ ] x n . Obviously, the Allan variance cannot track and reveal the non-stationary characteristics. Then the classical DAVAR and the improved fast DAVAR are applied to analyze the simulation data. The analysis results of the classical DAVAR Figure 6a,c and the improved fast DAVAR Figure 6b,d are shown in Figure 7. With a rectangular window of L = 1000 samples and a step whose width is 30 samples, the analysis results of 1[ ] x n are obtained. As can be seen in Figure 7a, both DAVAR methods are constant over time and have a typical white noise slope. 7. Models and Simulations The value of the improved fast DAVAR is similar to that of the classical DAVAR at fixed t and .The analysis results of 2[ ] x n are obtained by a rectangular window of number W N = 2000 samples and a step whose width is 300 samples. In Figure 7b, the improved fast DAVAR and classical DAVAR reveal that noise increases linearly with time. (a) (b) (a) (b) nsors 2016, 16, 2078 12 of 18 (c) (d) Figure 7. The classical DAVAR and the improved fast DAVAR. (a) DAVAR of white Gaussian noise 1[ ] x n ; (b) The improved fast DAVAR of 1[ ] x n ; (c) DAVAR of white noise with “increase” 2[ ] x n ; (d) The improved fast DAVAR of 2[ ] x n . Figure 7. The classical DAVAR and the improved fast DAVAR. (a) DAVAR of white Gaussian noise x1[n]; (b) The improved fast DAVAR of x1[n]; (c) DAVAR of white noise with “increase” x2[n]; (d) The improved fast DAVAR of x2[n]. he two sets of simulated data. Because 1[ ] x n is approximately −1/2 for all [30]. 2[ ] x n is lopes of Allan variance are the same as for the Allan variance cannot track and reveal the ast DAVAR are applied to analyze the simulation AR Figure 6a,c and the improved fast DAVAR ples and a step whose width is 30 samples, the e seen in Figure 7a, both DAVAR methods are slope. The value of the improved fast DAVAR is (b) 12 of Figure 6b,d exhibits the Allan varianc stationary white Gaussian noise, its slo non-stationary white noise, but the shapes stationary white noise 1[ ] x n . Obviously non-stationary characteristics. Then the classical DAVAR and the impr data. The analysis results of the classical Figure 6b,d are shown in Figure 7. With a rectangular window of L = 100 analysis results of 1[ ] x n are obtained. As constant over time and have a typical white (a) ors 2016, 16, 2078 1[ e and (a) impro (b) ope e a ue o e i p o e a A A i d .The analysis results of 2[ ] x n are obtained by amples and a step whose width is 300 samples. 7. Models and Simulations The value of the improved fast DAVAR milar to that of the classical DAVAR at fixed t and .The analysis results of 2[ ] x n are obtained b rectangular window of number W N = 2000 samples and a step whose width is 300 sample Figure 7b, the improved fast DAVAR and classical DAVAR reveal that noise increases linear th time. 0 2000 4000 6000 -40 t/s 10 -2 10 0 10 2 10 4 10 -3 τ(s) (b) (d) 10 -2 10 -1 10 0 10 1 10 -2 10 -1 10 0 10 1 Allan variance and curve-fitting τ(s) σ(τ)(。/h) allan deviation fitting curve 10 -2 10 0 10 2 10 4 10 -3 10 -2 10 -1 10 0 10 1 Allan variance and curve-fitting τ(s) σ(τ)(。/h) allan deviation fitting curve (d) aussian noise 1[ ] x n ; (b) Allan variance of 1[ ] x n ; Allan variance of 2[ ] x n . e two sets of simulated data. Because 1[ x n approximately −1/2 for all [30]. 2[ x n opes of Allan variance are the same as fo Allan variance cannot track and reveal st DAVAR are applied to analyze the simula R Figure 6a,c and the improved fast DA les and a step whose width is 30 samples seen in Figure 7a, both DAVAR method lope. The value of the improved fast DAVA .The analysis results of 2[ ] x n are obtaine mples and a step whose width is 300 sam 10 -2 10 0 10 2 10 4 10 3 τ(s) (b) 10 -2 10 -1 10 0 10 1 10 -2 10 -1 10 0 10 1 Allan variance and curve-fitting τ(s) σ(τ)(。/h) allan deviation fitting curve (d) aussian noise 1[ ] x n ; (b) Allan variance of 1[ x n Allan variance of 2[ ] x n . two sets of simulated data. Because x approximately −1/2 for all [30]. 7. Models and Simulations sical DAVAR reveal that noise increases linearly ( ) (d) similar to that of the classical DAVAR at fixed t a rectangular window of number W N = 2000 In Figure 7b, the improved fast DAVAR and c with time. (c) (d) (c) (a) (b) Figure 7. The classical DAVAR and the improved fast DAVAR. (a) DAVAR of white Gaussian noise 1[ ] x n ; (b) The improved fast DAVAR of 1[ ] x n ; (c) DAVAR of white noise with “increase” 2[ ] x n ; (d) The improved fast DAVAR of 2[ ] x n . Figure 7. The classical DAVAR and the improved fast DAVAR. (a) DAVAR of white Gaussian noise x1[n]; (b) The improved fast DAVAR of x1[n]; (c) DAVAR of white noise with “increase” x2[n]; (d) The improved fast DAVAR of x2[n]. Sensors 2016, 16, 2078 12 of 18 12 of 18 With a rectangular window of L = 1000 samples and a step whose width is 30 samples, the analysis results of x1[n] are obtained. As can be seen in Figure 7a, both DAVAR methods are constant over time and have a typical white noise slope. The value of the improved fast DAVAR is similar to that of the classical DAVAR at ﬁxed t and τ. The analysis results of x2[n] are obtained by a rectangular window of number NW = 2000 samples and a step whose width is 300 samples. In Figure 7b, the improved fast DAVAR and classical DAVAR reveal that noise increases linearly with time. (c) (d) Figure 7. The classical DAVAR and the improved fast DAVAR. (a) DAVAR of white Gaussian noise 1[ ] x n ; (b) The improved fast DAVAR of 1[ ] x n ; (c) DAVAR of white noise with “increase” 2[ ] x n ; (d) The improved fast DAVAR of 2[ ] x n . The coefficient of the angle random walk (N) denotes the magnitude of the white noise [30]. The coefﬁcient of the angle random walk (N) denotes the magnitude of the white noise [30]. According to the 2D description method of noise terms mentioned in Section 4.2, the coefﬁcient N(t) of the Angle random walk can be acquired. Figure 8 shows the time-varying parameter N(t) of the two sets’ simulation data. 7. Models and Simulations Figure 8a,c shows the results of the classical DAVAR while Figure 8b,d shows the results of the improved fast DAVAR. The coefﬁcient N(t) of x1[n] ﬂuctuates around a constant while x2[n] increases linearly with time. The results prove that the improved fast DAVAR is correct. According to the 2D description method of noise terms mentioned in Section 4.2, the coefficient N(t) of the Angle random walk can be acquired. Figure 8 shows the time-varying parameter N(t) of the two sets’ simulation data. Figure 8a,c shows the results of the classical DAVAR while Figure 8b,d shows the results of the improved fast DAVAR. The coefficient N(t) of 1[ ] x n fluctuates around a constant while 2[ ] x n increases linearly with time. The results prove that the improved fast DAVAR is correct. (a) (b) (c) (d) Figure 8. Angle random walk of the simulation data. (a,b) Angle random walk of 1[ ] x n ; (c,d) angle random walk of 2[ ] x n . 10 20 30 40 50 60 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5x 10 -3 times(s) N (°/sqrt(h) 0 20 40 60 2.1 2.15 2.2 2.25x 10 -3 times(s) N (°/sqrt(h) 0 1000 2000 3000 4000 5000 6000 2 2.5 3 3.5 4 4.5 5 5.5 6 x 10 -3 times(s) N (°/sqrt(h) 0 2000 4000 6000 2 3 4 5 6 x 10 -3 times(s) N (°/sqrt(h) Figure 8. Angle random walk of the simulation data. (a,b) Angle random walk of x1[n]; (c,d) angle random walk of x2[n]. (b) 0 20 40 60 2.1 2.15 2.2 2.25x 10 -3 times(s) N (°/sqrt(h) (a) 10 20 30 40 50 60 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5x 10 -3 times(s) N (°/sqrt(h) (a) (b) (b) (d) 0 2000 4000 6000 2 3 4 5 6 x 10 -3 times(s) N (°/sqrt(h) ( ) (c) 0 1000 2000 3000 4000 5000 6000 2 2.5 3 3.5 4 4.5 5 5.5 6 x 10 -3 times(s) N (°/sqrt(h) (d) (c) Figure 8. Angle random walk of the simulation data. (a,b) Angle random walk of 1[ ] x n ; (c,d) angle random walk of 2[ ] x n . Figure 8. Angle random walk of the simulation data. (a,b) Angle random walk of x1[n]; (c,d) angle random walk of x2[n]. 7. Models and Simulations With the time series increasing, the classical DAVAR needs to calculate the Allan variance more and more times while the improved fast DAVAR just needs to compute it one time (the initial value of the DAVAR). Thus, the improved fast DAVAR could shorten the calculation time. When the time series is long, the improved fast DAVAR is meaningful and significant. Now we extend the improved fast DAVAR to discontinuous time series. On the basis of the simulation data x1[n], we created simulation dataset x3[n] using the same noise model as x1[n], but it is longer than x1[n]. Two pieces of data in x3[n] are deleted. One piece of data is between 200 s and 220 s and the other piece of data is between 400 s and 500 s. The discontinuous simulation data is represented in Figure 9. Now we extend the improved fast DAVAR to discontinuous time series. On the basis of the simulation data 1[ ] x n , we created simulation dataset 3[ ] x n using the same noise model as 1[ ] x n , but it is longer than 1[ ] x n . Two pieces of data in 3[ ] x n are deleted. One piece of data is between 200 s and 220 s and the other piece of data is between 400 s and 500 s. The discontinuous simulation data is represented in Figure 9. Figure 9. The discontinuous series time. 0 100 200 300 400 500 600 -5 0 5 time(s) X3(t)°/h Figure 9. The discontinuous series time. Figure 9. The discontinuous series time. Figure 9. The discontinuous series time. Then the classical DAVAR and the improved fast DAVAR are applied to analyzing the simulation data. The classical DAVAR is forced to stop because the program could not identify the NaN. However, the improved fast DAVAR could successfully analyze the data. Figure 10a represents fast DAVAR obtained with a window of 1000 W N  samples and a step whose width is 30 samples. When t < 200 s, the DAVAR is essentially stationary. The slope of the DAVAR surface correctly indicates the presence of white phase noise. In the time interval 200 s < t < 220 s, the DAVAR shows a little canyon corresponding to a few areas of missing data. 7. Models and Simulations Both the results are obtained by the Matlab program on an Intel(R) Core(TM) i7-3770 CPU with a 3.4 GHz clock. Table 2 shows a computational comparison of the classical DAVAR and the improved fast DAVAR. In the second column, the number of simulation data points is reported. The third column shows the length of the truncation window and the fourth column shows the step width. The ﬁfth column represents the computational time. The last column indicates the calculation times of the Allan variance in the whole calculation process. 13 of 18 s of the Sensors 2016, 16, 2078 column represents Alla a ia e i t Table 2. The computational comparison. Table 2. The computational comparison. Data Numbers NW Step Width Time (s) Computation Times of the Allan Variance DAVAR Fast DAVAR DAVAR Fast DAVAR x1[n] 6 × 103 1000 30 3.656864 0.770367 166 1 x2[n] 6 × 105 2000 300 960.422362 27.92727 1993 1 Data Numbers W N Step Width Time (s) Computation Times of the Allan Variance DAVAR Fast DAVAR DAVAR Fast DAVAR x1[n] 3 6 10  1000 30 3.656864 0.770367 166 1 x2[n] 5 6 10  2000 300 960.422362 27.92727 1993 1 When the length of the time series is short (6 × 103 samples), the improved fast DAVAR saves 78.93% of computing time. When the length of the time series is long (6 × 105 samples), the improved fast DAVAR costs only 27.92727 s while the classical DAVAR costs 960.422362 s. The improved fast DAVAR reduces 97.09% of computing time. The last column reports the reason. With the time series increasing, the classical DAVAR needs to calculate the Allan variance more and more times while the improved fast DAVAR just needs to compute it one time (the initial value of the DAVAR). Thus, the improved fast DAVAR could shorten the calculation time. When the time series is long, the improved fast DAVAR is meaningful and signiﬁcant. When the length of the time series is short ( 3 6 10  samples), the improved fast DAVAR saves 78.93% of computing time. When the length of the time series is long ( 5 6 10  samples), the improved fast DAVAR costs only 27.92727 s while the classical DAVAR costs 960.422362 s. The improved fast DAVAR reduces 97.09% of computing time. The last column reports the reason. 7. Models and Simulations In the region of the large gap 400 s < t < 500 s, the surface of the DAVAR exhibits a large canyon corresponding to a greater amount of missing data. Figure 10b shows the coefficient of the angle random walk noise. The Then the classical DAVAR and the improved fast DAVAR are applied to analyzing the simulation data. The classical DAVAR is forced to stop because the program could not identify the NaN. However, the improved fast DAVAR could successfully analyze the data. Figure 10a represents fast DAVAR obtained with a window of NW = 1000 samples and a step whose width is 30 samples. When t < 200 s, the DAVAR is essentially stationary. The slope of the DAVAR surface correctly indicates the presence of white phase noise. In the time interval 200 s < t < 220 s, the DAVAR shows a little canyon corresponding to a few areas of missing data. In the region of the large gap 400 s < t < 500 s, the surface of the DAVAR exhibits a large canyon corresponding to a greater amount of missing data. Figure 10b shows the coefﬁcient of the angle random walk noise. The coefﬁcient N(t) of x3[n] is similar to Figure 8a. Moreover, it has the same order of magnitude and the same shape as that of the continuous data besides the two canyons. With this geometrical representation, the fast DAVAR clearly describes the dynamic instability of a gyroscope with missing data. 14 of 18 cope with pe with Sensors 2016, 16, 2078 representation, representation, th (a) (a) (a) (b) Figure 10. The fast DAVAR of the discontinuous data. (a) The improved fast DAVAR of 3[ ] x n ; (b) Angle random walk of 3[ ] x n . 0 100 200 300 400 500 600 2.1 2.15 2.2 2.25 2.3x 10 -3 times(s) N (°/sqrt(h) Figure 10. The fast DAVAR of the discontinuous data. (a) The improved fast DAVAR of x3[n]; (b) Angle random walk of x3[n]. (a) (b) Figure 10. The fast DAVAR of the discontinuous data. (a) The improved fast DAVAR of 3[ ] x n ; (b) Angle random walk of 3[ ] x n . 8. Experiment 8. Experiments 8. Experiments Aiming to further verify the proposal algorithm, a vibration experiment was implemented with a ready-made FOGs-based MWD. The accuracy of FOGs in MWD system is 0.03 /h. The vibration experiment is carried out at the high temperature of 65 °C. The x-axis FOG is mounted along with the vibration direction of the vibration platform. The PSD of the vibration is reported in Figure 11. It vibration level is high, which simulates the drilling vibration underground. When the vibration frequency is between 700 Hz and 800 Hz, the PSD of the vibration is up to 1 g2/Hz. Th root-mean-square value of the whole vibration is 13.39 g. Aiming to further verify the proposal algorithm, a vibration experiment was implemented with a ready-made FOGs-based MWD. The accuracy of FOGs in MWD system is 0.03 ◦/h. The vibration experiment is carried out at the high temperature of 65 ◦C. The x-axis FOG is mounted along with the vibration direction of the vibration platform. The PSD of the vibration is reported in Figure 11. Its vibration level is high, which simulates the drilling vibration underground. When the vibration frequency is between 700 Hz and 800 Hz, the PSD of the vibration is up to 1 g2/Hz. The root-mean-square value of the whole vibration is 13.39 g. Aiming to further verify the proposal algorithm, a vibration experiment was implemented with a ready-made FOGs-based MWD. The accuracy of FOGs in MWD system is 0.03 /h. The vibration experiment is carried out at the high temperature of 65 °C. The x-axis FOG is mounted along with the vibration direction of the vibration platform. The PSD of the vibration is reported in Figure 11. Its vibration level is high, which simulates the drilling vibration underground. When the vibration frequency is between 700 Hz and 800 Hz, the PSD of the vibration is up to 1 g2/Hz. The root-mean-square value of the whole vibration is 13.39 g. Figure 11. PSD of the random vibration. Figure 11. PSD of the random vibration. Figure 11. PSD of the random vibration. Figure 11. PSD of the random vibration Figure 11. PSD of the random vibration. Figure 11. PSD of the random vibration. The vibration experiment was conducted via the following steps. First, the vibration platform was kept static (0–330 s). Secondly, the vibration platform began to vibrate and kept vibrating fo 5 min (330 s–630 s). 7. Models and Simulations 0 100 200 300 400 500 600 2.1 2.15 2.2 2.25 2.3x 10 -3 times(s) N (°/sqrt(h) (b) 0 100 200 300 400 500 600 2.1 2.15 2.2 2.25 2.3x 10 -3 times(s) N (°/sqrt(h) (b) 0 100 200 300 400 500 600 2.1 2.15 2.2 2.25 2.3x 10 -3 times(s) N (°/sqrt(h) (b) b) (a) a) Figure 10. The fast DAVAR of the discontinuous data. (a) The improved fast DAVAR of 3[ ] x n (b) Angle random walk of 3[ ] x n . Figure 10. The fast DAVAR of the discontinuous data. (a) The improved fast DAVAR of x3[n]; (b) Angle random walk of x3[n]. Figure 10. The fast DAVAR of the discontinuous data. (a) The improved fast DAVAR of 3[ ] x n ; (b) Angle random walk of 3[ ] x n . 8. Experiment 8. Experiments 8. Experiments Output signal of x-axis FOG. Figure 12. Output signal of x-axis FOG. Figure 12. Output signal of x-axis FOG. t/s Then the improved fast DAVAR has been applied to analyzing this discontinuous vibration data. Its result is obtained with a truncation window of length 2000 W N  samples, and a step width of 1000 samples. The Matlab program calculation is carried out on an Intel (R) Core (TM) i7-3770 CPU with a 3.4 GHz clock. The improved fast DAVAR costs only 28.616520 s to deal with this long time series. Figure 13 is the improved fast DAVAR result It can be seen that the DAVAR surface is Then the improved fast DAVAR has been applied to analyzing this discontinuous vibration data. Its result is obtained with a truncation window of length NW = 2000 samples, and a step width of 1000 samples. The Matlab program calculation is carried out on an Intel (R) Core (TM) i7-3770 CPU with a 3.4 GHz clock. The improved fast DAVAR costs only 28.616520 s to deal with this long time series. Then the improved fast DAVAR has been applied to analyzing this discontinuous vibration data. Its result is obtained with a truncation window of length 2000 W N  samples, and a step width of 1000 samples. The Matlab program calculation is carried out on an Intel (R) Core (TM) i7-3770 CPU with a 3.4 GHz clock. The improved fast DAVAR costs only 28.616520 s to deal with this long time series Figure 13 is the improved fast DAVAR result. It can be seen that the DAVAR surface is stationary at the beginning. Then there appears a large crest that starts at t = 330 s and stops at t = 630 s. In the end it goes back to being stationary. Obviously, the canyon (430 s < t < 450 s) is the graphical representation of the missing data in the time series. The changing process of the DAVAR surface is consistent with Figure 12. In conclusion, the improved fast DAVAR could track and reveal the non-stationary characteristics in a clear way. Figure 13 is the improved fast DAVAR result. It can be seen that the DAVAR surface is stationary at the beginning. Then there appears a large crest that starts at t = 330 s and stops at t = 630 s. In the end it goes back to being stationary. 8. Experiment 8. Experiments 8. Experiments Obviously, the canyon (430 s < t < 450 s) is the graphical representation of the missing data in the time series. The changing process of the DAVAR surface is consistent with Figure 12. In conclusion, the improved fast DAVAR could track and reveal the non-stationary characteristics in a clear way. long time series. Figure 13 is the improved fast DAVAR result. It can be seen that the DAVAR surface is stationary at the beginning. Then there appears a large crest that starts at t = 330 s and stops at t = 630 s. In the end it goes back to being stationary. Obviously, the canyon (430 s < t < 450 s) is the graphical representation of the missing data in the time series. The changing process of the DAVAR surface is consistent with Figure 12. In conclusion, the improved fast DAVAR could track and reveal the non-stationary characteristics in a clear way. Figure 13. The improved fast DAVAR of the vibration data. g the double logarithmic curve 2( ,t    )- at any given time t, the tim Figure 13. The improved fast DAVAR of the vibration data. Figure 13. The improved fast DAVAR of the vibration data. le logarithmic curve 2(t    )- at any given time Figure 13. The improved fast DAVAR of the vibration data. Figure 13. The improved fast DAVAR of the vibration data. coefficients of noise terms can be obtained [27,28]. Figure 14 shows the changing coefficients of each noise terms evaluated by the improved fast DAVAR. Aside from the canyon at t = 432 s, it is easy to distinguish the change in the noise. Before b ( ) h OG h ff f h ll h d Fitting the double logarithmic curve 2( ,t    )- at any given time t, the time-varying coefficients of noise terms can be obtained [27,28]. Figure 14 shows the changing coefficients of each noise terms evaluated by the improved fast DAVAR. A id f th t t 432 it i t di ti i h th h i th i B f Fitting the double logarithmic curve σ2(t, τ) −τ at any given time t, the time-varying coefﬁcients of noise terms can be obtained [27,28]. Figure 14 shows the changing coefﬁcients of each noise terms evaluated by the improved fast DAVAR. 8. Experiment 8. Experiments 8. Experiments Finally the vibration platform returned to static state (630 s–930 s). Th movement of the MWD system was in accordance with the movement of the vibration platform. W collected the output signal of the x-axis FOG in the MWD system. The sampling interval is 2.5 ms Hence, the total value of this gyro data is 4 37.2 10  . The primary signal is shown in Figure 12 The vibration experiment was conducted via the following steps. First, the vibration platform was kept static (0–330 s). Secondly, the vibration platform began to vibrate and kept vibrating for 5 min (330 s–630 s). Finally the vibration platform returned to static state (630 s–930 s). The movement of the MWD system was in accordance with the movement of the vibration platform. We collected the output signal of the x-axis FOG in the MWD system. The sampling interval is 2.5 ms. Hence, the total value of this gyro data is 4 37.2 10  . The primary signal is shown in Figure 12. The vibration experiment was conducted via the following steps. First, the vibration platform was kept static (0–330 s). Secondly, the vibration platform began to vibrate and kept vibrating for 5 min (330 s–630 s). Finally the vibration platform returned to static state (630 s–930 s). The movement of the MWD system was in accordance with the movement of the vibration platform. We collected the output signal of the x-axis FOG in the MWD system. The sampling interval is 2.5 ms. Hence, the total value of this gyro data is 37.2 × 104. The primary signal is shown in Figure 12. It shows that the platform began to vibrate at t = 330 s and stopped at t = 630 s. In addition, we must pay attention to a piece of lost data when 432 s < t < 450 s. 15 of 18 we must we must Sensors 2016, 16, 2078 It shows that the p tt ti t It shows that the p Figure 12. Output signal of x-axis FOG. 0 200 400 600 800 -6 -4 -2 0 2 4 6 t/s y(t)°/s Figure 12. Output signal of x-axis FOG. e of lost data when 432 s < t < 450 s. Figure 12. Output signal of x-axis FOG. 0 200 400 600 800 -6 -4 -2 0 2 4 6 t/s y(t)°/s Figure 12. 9. Conclusions 9. Conclusions The working environment of FOGs-based MWD is hostile: the vibration is very strong and the temperature is very high. The gyroscope is heavily influenced by factors such as temperature, vibration, aging, and sudden breakdowns. A slight variation of the gyroscope stability can turn into a measurement error. Hence, it is important to monitor the behavior of gyroscopes through the use of DAVAR. Th DAVAR i t ti f th ti i t bilit f th Th f t DAVAR The working environment of FOGs-based MWD is hostile: the vibration is very strong and the temperature is very high. The gyroscope is heavily inﬂuenced by factors such as temperature, vibration, aging, and sudden breakdowns. A slight variation of the gyroscope stability can turn into a measurement error. Hence, it is important to monitor the behavior of gyroscopes through the use of DAVAR. The DAVAR is a representative of the time-varying stability of the gyroscope. The fast DAVAR is a fast algorithm for the classical DAVAR. However, both the fast DAVAR and the classical DAVAR could not analyze the discontinuous gyroscope data. What is worse, in many applications, the gyroscope often gives discontinuous data, for example in a FOGs-based MWD system. In this paper utilizing the recursive characteristic of the fast DAVAR we make a further The DAVAR is a representative of the time-varying stability of the gyroscope. The fast DAVAR is a fast algorithm for the classical DAVAR. However, both the fast DAVAR and the classical DAVAR could not analyze the discontinuous gyroscope data. What is worse, in many applications, the gyroscope often gives discontinuous data, for example in a FOGs-based MWD system. In this paper, utilizing the recursive characteristic of the fast DAVAR, we make a further advance on the fast algorithm to extend the fast DAVAR to discontinuous gyroscope data. This not only dramatically reduces the computation time, but could also allow us to analyze the discontinuous gyroscope data. Both the simulation results and the experimental results show that the improved fast DAVAR could not only save more than 90% of the computational time, but also deal successfully with discontinuous data. In this paper, utilizing the recursive characteristic of the fast DAVAR, we make a further advance on the fast algorithm to extend the fast DAVAR to discontinuous gyroscope data. This not only dramatically reduces the computation time, but could also allow us to analyze the discontinuous gyroscope data. 8. Experiment 8. Experiments 8. Experiments vibration (t < 330 s), since the FOG is static, the coefficient of each noise is small without evident change. When the platform starts to vibrate (t = 432 s), the noise terms of the FOG change dramatically, especially the Rate Random walk, which is affected by the vibration. Meanwhile, the coefficient of the quantization noise increases when the FOG is vibrating. The data acquisition circuit Aside from the canyon at t = 432 s, it is easy to distinguish the change in the noise. Before vibration (t < 330 s), since the FOG is static, the coefficient of each noise is small without evident change. When the platform starts to vibrate (t = 432 s), the noise terms of the FOG change dramatically, especially the Rate Random walk, which is affected by the vibration. Meanwhile, the coefficient of the quanti ation noise increases when the FOG is ibrating The data acquisition circuit Aside from the canyon at t = 432 s, it is easy to distinguish the change in the noise. Before vibration (t < 330 s), since the FOG is static, the coefﬁcient of each noise is small without evident change. When the platform starts to vibrate (t = 432 s), the noise terms of the FOG change dramatically, especially the Rate Random walk, which is affected by the vibration. Meanwhile, the coefﬁcient of the quantization 16 of 18 16 of 18 Sensors 2016, 16, 2078 noise increases when the FOG is vibrating. The data acquisition circuit of FOG has better aseismic performance. After vibration, all conﬁdents of noise terms are back to the previous state. We can make a conclusion that the FOG could endure the drilling vibration. In conclusion, the improved fast algorithm of the DAVAR could successfully analyze the discontinuous gyroscope data. The improved fast DAVAR identiﬁes and reveals the highly dynamic instability in the FOG’s discontinuous time series. of FOG has better aseismic performance. After vibration, all confidents of noise terms are back to the previous state. We can make a conclusion that the FOG could endure the drilling vibration. In conclusion, the improved fast algorithm of the DAVAR could successfully analyze the discontinuous gyroscope data. The improved fast DAVAR identifies and reveals the highly dynamic instability in the FOG’s discontinuous time series. Figure 14. The coefficients of each noise term. 8. Experiment 8. Experiments 8. Experiments 0 100 200 300 400 500 600 700 800 0 0.1 0.2 Q(＇＇) 0 100 200 300 400 500 600 700 800 0 0.05 0.1 N (°/sqrt(h) 0 100 200 300 400 500 600 700 800 0 1 2 B(°/h) 0 100 200 300 400 500 600 700 800 0 20 40 K(°/h(3/2) 0 100 200 300 400 500 600 700 800 0 200 400 time(s) R(°/h2) Figure 14. The coefﬁcients of each noise term. Figure 14. The coefficients of each noise term. Figure 14. The coefﬁcients of each noise term. References In Proceedings of the 2003 IEEE International Frequency Control Symposium and PDA Exhibition Jointly with the 17th European Frequency and Time Forum, Tampa, FL, USA, 4–8 May 2003; pp. 239–244. , ; , y In Proceedings of the 2003 IEEE International Frequency Control Symposium and PDA Exhibition Jointly with the 17th European Frequency and Time Forum Tampa FL USA 4–8 May 2003; pp 239–244 8. Galleani, L.; Tavella, P. Tracking Nonstationarities in Clock Noises Using the Dynamic Allan Variance. In Proceedings of the IEEE International Frequency Control Symposium and Exposition, Vancouver, BC, Canada, 29–31 August 2005; pp. 392–396. 9. Galleani, L.; Tavella, P. Interpretation of the Dynamic Allan Variance of Nonstationary Clock Data. In Proceedings of the IEEE International Frequency Control Symposium, 2007 Joint with the 21st European Frequency and Time Forum, Geneva, Switzerland, 29 May–1 June 2007; pp. 992–997. 10. Nunzi, E.; Galleani, L.; Tavella, P. Detection of Anomalies in the Behavior of Atomic Clocks. IEEE Trans. Instrum. Meas. 2007, 56, 523–528. [CrossRef] 11. Sesia, I.; Galleani, L.; Tavella, P. Implementation of the Dynamic Allan Variance for the Galileo System Test Bed V2. In Proceedings of the IEEE International Frequency Control Symposium, 2007 Joint with the 21st European Frequency and Time Forum, Geneva, Switzerland, 29 May–1 June 2007; pp. 946–949. 12. Galleani, L.; Tavella, P. The Dynamic Allan Variance. IEEE Trans. Ultrason. Ferroelectr. Freq. Control 2009, 56, 450–464. [CrossRef] [PubMed] 13. Sesia, I.; Galleani, L.; Tavella, P. Application of the Dynamic Allan Varinacne for the Characterization of Space Clock Behavior. IEEE Trans. Aerosp. Electron. Syst. 2011, 47, 884–895. [CrossRef] 14. Li, Y.; Chen, X.; Song, S. Dynamic Allan Variance Analysis for the Drift Error of Fiber Optical Gyroscope. J. Optoelectron. Laser 2008, 19, 183–186. 15. Wei, G.; Long, X. Research on Stochastic Errors of Dithered Ring Laser Gyroscope Based on Dynamic Allan Variance. Chin. J. Lasers 2010, 37, 2975–2979. 16. Li, X.; Zhang, N. Analysis of Dynamic Characteristics of a Fiber Optic Gyroscope Based on Dynamic Allan Variance. J. Harbin Eng. Univ. 2011, 32, 183–187. 17. Zhang, N.; Li, X. Research on Theoretical Improvement of Dynamic Allan Variance and Its Application. Acta Opt. Sin. 2011, 31, 1–6. [CrossRef] 18. Zhang, C.; Wang, L.; Gao, S.; Li, H.; Lin, T.; Li, X.; Wang, T. Dynamic Allan Variance Analysis for Stochastic Errors of Fiber Optic Gyroscope. Infrared Laser Eng. 2014, 43, 3081–3088. 19. Galleani, L.; Tavella, P. References 1. Sun, J.; Xu, X.; Liu, Y.; Zhang, T.; Li, Y. FOG Random Drift Signal Denoising Based on the Improved AR Model and Modiﬁed Sage-Huas Adaptive Kalman Filter. Sensors 2016, 16, 1073. [CrossRef] [PubMed] 1. Sun, J.; Xu, X.; Liu, Y.; Zhang, T.; Li, Y. FOG Random Drift Signal Denoising Based on the Improved AR Model and Modiﬁed Sage-Huas Adaptive Kalman Filter. Sensors 2016, 16, 1073. [CrossRef] [PubMed] 2. Yu, H.; Wu, W.; Wu, M.; Feng, G.; Hao, M. Systematic Angle Random Walk E Biased Ring Laser Gyro. Sensors 2013, 13, 2750–2762. [CrossRef] [PubMed] 2. Yu, H.; Wu, W.; Wu, M.; Feng, G.; Hao, M. Systematic Angle Random Walk Estimation of the Constant Rate Biased Ring Laser Gyro. Sensors 2013, 13, 2750–2762. [CrossRef] [PubMed] 3. Quinchia, A.G.; Falco, G.; Falletti, E.; Dovis, F.; Ferrer, C. A Comparison between Different Error Modeling of MEMS Applied to GPS/INS Integrated Systems. Sensors 2013, 13, 9549–9588. [CrossRef] [PubMed] 3. Quinchia, A.G.; Falco, G.; Falletti, E.; Dovis, F.; Ferrer, C. A Comparison between Different Error Modeling of MEMS Applied to GPS/INS Integrated Systems. Sensors 2013, 13, 9549–9588. [CrossRef] [PubMed] Miao, Z.; Shen, F.; Xu, D.; He, K.; Tian, C. Online Estimation of Allan Variance Coefﬁcients Based on a Neural-Extended Kalman Filter Sensors 2015 15 2496–2524 [CrossRef] [PubMed] 4. Miao, Z.; Shen, F.; Xu, D.; He, K.; Tian, C. Online Estimation of Allan Variance Coefﬁc Neural-Extended Kalman Filter. Sensors 2015, 15, 2496–2524. [CrossRef] [PubMed] 5. Wang, L.; Zhang, C.; Lin, T.; Li, X.; Wang, T. Characterization of a Fiber Optic Gyroscope in a Measurement While Drilling System with the Dynamic Allan Variance Measurement 2015 75 263 272 [CrossRef] Wang, L.; Zhang, C.; Lin, T.; Li, X.; Wang, T. Characterization of a Fiber Optic Gyroscope in a Measuremen While Drilling System with the Dynamic Allan Variance. Measurement 2015, 75, 263–272. [CrossRef] 6. Allan, D.W. Statistics of Atomic Frequency Standards. Proc. IEEE 1966, 54, 221–230. [CrossRef] 7. Galleani, L.; Tavella, P. The Characterization of Clock Behavior with the Dynamic Allan Variance. In Proceedings of the 2003 IEEE International Frequency Control Symposium and PDA Exhibition Jointly with the 17th European Frequency and Time Forum, Tampa, FL, USA, 4–8 May 2003; pp. 239–244. 7. Galleani, L.; Tavella, P. The Characterization of Clock Behavior with the Dynamic Allan Variance. 9. Conclusions 9. Conclusions Both the simulation results and the experimental results show that the improved fast DAVAR could not only save more than 90% of the computational time, but also deal successfully with discontinuous data. Acknowledgments: The authors would like to thank Huipeng Li for his helpful discussions and comme This research was supported by the National Science Foundation of China (Grant No. 60207002). Acknowledgments: The authors would like to thank Huipeng Li for his helpful discussions and comm This research was supported by the National Science Foundation of China (Grant No. 60207002). Author Contributions: All the authors made contributions to this work. The idea was originally from Chunxi Zhang and Shuang Gao; Lu Wang proposed the scheme, developed the algorithms, completed the simulation, and wrote the manuscript; Tao Wang developed the primary algorithms; Tie Lin and Xianmu Li processed the simulation and critically reviewed the paper Author Contributions: All the authors made contributions to this work. The idea was originally from Chunxi Zhang and Shuang Gao; Lu Wang proposed the scheme, developed the algorithms, completed the simulation, and wrote the manuscript; Tao Wang developed the primary algorithms; Tie Lin and Xianmu Li processed the simulation and critically reviewed the paper. processed the simulation and critically reviewed the paper. Conflicts of Interest: The authors declare no conflict of interest Conﬂicts of Interest: The authors declare no conﬂict of interest. 17 of 18 17 of 18 Sensors 2016, 16, 2078 References Fast Computation of the Dynamic Allan Variance. In Proceedings of the 2009 Joint Conference of the IEEE International Frequency Control Symposium and European Frequency and Time Forum (IEEE FCS-EFTF), Belfast, France, 20–24 April 2009; pp. 685–687. 20. Galleani, L. The Dynamic Allan Variance II: A Fast Computational Algorithm. IEEE Trans. Ultrason. Ferroelectr. Freq. Control 2009, 57, 182–188. [CrossRef] [PubMed] 21. Galleani, L. The Dynamic Allan Variance III: Conﬁdence and Detection Surfaces. IEEE Trans. Ultrason. Ferroelectr. Freq. Control 2011, 58, 1550–1558. [CrossRef] [PubMed] 22. Galleani, L.; Tavella, P. Characterization of Atomic Clock Anomalies in the Dynamic Allan Variance Domain. In Proceedings of the 2013 Joint European Frequency and Time Forum & International Frequency Control Symposium, Prague, Czech, 21–25 July 2013. 23. Wang, L.; Zhang, C. Fast Algorithm of the Dynamic Allan Variance for FOG. Optik 2016, 127, 2413–2418. [CrossRef] 18 of 18 Sensors 2016, 16, 2078 24. Lin, T.; Zhang, C.; Gao, S. A Surveying method based on motion features for a dual FOGs-based MWD. In Proceeding of the 4th International Conference on Electronics Communications and Networks, Beijing, China, 12–15 December 2014; pp. 764–769. 25. Zhang, C.; Lin, T. A Long-term Performance Enhancement Method for FOG-based Measuremen Drilling. Sensors 2016, 16, 1186. [CrossRef] [PubMed] 26. Ng, L.C.; Pines, D.J. Characterization of Ring Laser Gyro performance Using the Allan variance Method. J. Guid. Control Dyn. 1997, 20, 211–214. [CrossRef] 27. Tehrani, M.M. Ring Laser Gyro Data Analysis with Cluster Sampling Technique. Proc. SPIE 1983, 0412. [CrossRef] 28. IEEE Standard Speciﬁcation Format Guide and Test Procedure of Single Axis Interferometric Fiber Optic Gyros; IEEE Std 952-1997; The Institute of Electrical and Electronics Engineers, Inc.: New York, NY, USA, 16 September 1997. 29. Hou, H. Modeling Inertial Sensors Errors Using Allan Variance; University of Calgary: Calgary, AB, Canada, 2004. 30. Xu, B. Fiber Optic Gyro Signal Random Drift Testing and Noise Error Analysis. In Proceedings of the 2010 3rd Conference on Computer Science and Information Technology, Chengdu, China, 9–11 July 2010; pp. 189–192. © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W2092020720	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0019394&type=printable	English	null	Zebrafish Her8a Is Activated by Su(H)-Dependent Notch Signaling and Is Essential for the Inhibition of Neurogenesis	PloS one	2,011	cc-by	10,583	Introduction the transcription of target genes, Hairy and Enhancer-of-split [Hairy/ E(spl)]. Hairy/E(spl) related proteins are transcription factors belong to the bHLH superfamily and are structurally related to the Drosophila Hairy and Enhancer-of-split proteins. It is generally believed that Hairy/E(spl) homologues act as potential targets for Notch mediated signals that suppress the expression of proneural genes such as Neurogenin1 and Achaete-scute homolog 1, resulting in inhibition of neurogenesis [1]. However, some Hairy/E(spl) genes have been identified that do not respond to Notch signaling ([3], and reference therein) and a recent study showed that a zebrafish hairy/E(spl) homologue, her3, is repressed rather than induced by Notch signaling [4]. It therefore appears that not all the Hairy/ E(spl) genes respond to Notch signaling in the same way, and the regulation of these genes’ expression may vary in different developmental stages and different cell populations. In addition to the variable response to Notch signaling, the function of Hairy/E(spl) proteins themselves is also controversial. Most of the Hairy/E(spl) homologues studied so far act as transcriptional repressors for the proneural bHLH genes, and thus inhibit neurogenesis. However, one of the mouse homologues, HES6, has been shown to promote rather than inhibit neurogenesis [5,6]. Hence, our understanding of the roles of Hairy/E(spl) in neurogenesis and their response to Notch signaling remains incomplete. The nervous system consists of a diverse collection of neural cells that arise during a sequence of developmental events including neural induction, cell proliferation, differentiation, migration, process formation, and synapse formation. These events require numerous gene regulatory and signaling processes. One of the key regulators in these processes is Notch signaling which is conserved throughout evolution and components of this pathway have been characterized both in Drosophila and vertebrates [1]. In the developing nervous system, Notch signaling plays fundamental roles in neuronal progenitor maintenance and the decision between neuronal and glial lineages. It also influences aspects of the behavior of terminally differentiated neurons and is important in patterning cellular fields during brain morphogenesis. In addition, Notch signaling also participates in the pathogenesis of several human cancers and genetic disorders [2]. The Notch receptor is a single transmembrane protein that acts as both a sensor for ligand-activation and a mediator of the resulting signal to the cell nucleus. Notch signaling regulates neural fate commitment upon interaction with its canonical ligands Delta or Serrate (JAGGED in mammals). Abstract nding: This work was supported by Chang Gung Memorial Hospital (CMRPD34019) and the National Science Council of Taiwan, Rep 11-B-182-001). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manus supported by Chang Gung Memorial Hospital (CMRPD34019) and the National Science Council of Taiwan, Republic of China (NSC96- ders 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: yccheng@mail.cgu.edu.tw Zebrafish Her8a Is Activated by Su(H)-Dependent Notch Signaling and Is Essential for the Inhibition of Neurogenesis Pei-Chen Chung1, Wen-Shiuan Lin1, Paul J. Scotting2, Fu-Yu Hsieh1, Hui-Lan Wu1, Yi-Chuan Cheng1* 1 School of Medicine, Graduate Institute of Biomedical Sciences, Chang-Gung University, Taoyuan, Taiwan, 2 Children’s Brain Tumour Research Centre, Centre for Genetics and Genomics, Queen’s Medical Centre, University of Nottingham, Nottingham, United Kingdom Abstract Understanding how diversity of neural cells is generated is one of the main tasks of developmental biology. The Hairy/E(spl) family members are potential targets of Notch signaling, which has been shown to be fundamental to neural cell maintenance, cell fate decisions, and compartment boundary formation. However, their response to Notch signaling and their roles in neurogenesis are still not fully understood. In the present study, we isolated a zebrafish homologue of hairy/ E(spl), her8a, and showed this gene is specifically expressed in the developing nervous system. her8a is positively regulated by Su(H)-dependent Notch signaling as revealed by a Notch-defective mutant and injection of variants of the Notch intracellular regulator, Su(H). Morpholino knockdown of Her8a resulted in upregulation of proneural and post-mitotic neuronal markers, indicating that Her8a is essential for the inhibition of neurogenesis. In addition, markers for glial precursors and mature glial cells were down-regulated in Her8a morphants, suggesting Her8a is required for gliogenesis. The role of Her8a and its response to Notch signaling is thus similar to mammalian HES1, however this is the converse of what is seen for the more closely related mammalian family member, HES6. This study not only provides further understanding of how the fundamental signaling pathway, Notch signaling, and its downstream genes mediate neural development and differentiation, but also reveals evolutionary diversity in the role of H/E(spl) genes. Citation: Chung P-C, Lin W-S, Scotting PJ, Hsieh F-Y, Wu H-L, et al. (2011) Zebrafish Her8a Is Activated by Su(H)-Dependent Notch Signaling and Is Essential for the Inhibition of Neurogenesis. PLoS ONE 6(4): e19394. doi:10.1371/journal.pone.0019394 Editor: Domingos Henrique, Instituto de Medicina Molecular, Portugal Citation: Chung P-C, Lin W-S, Scotting PJ, Hsieh F-Y, Wu H-L, et al. (2011) Zebrafish Her8a Is Activated by Su(H)-Dependent Notch Signaling and Is Essential for the Inhibition of Neurogenesis. PLoS ONE 6(4): e19394. doi:10.1371/journal.pone.0019394 Received October 19, 2010; Accepted April 4, 2011; Published April 26, 2011 October 19, 2010; Accepted April 4, 2011; Published April 26, 20 ung et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits tion, and reproduction in any medium, provided the original author and source are credited. Copyright: 2011 Chung et al. This is an open-access article distributed under the terms of the Creative Commons Attributi unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. PLoS ONE \| www.plosone.org Characterization of zebrafish her8a Primers were designed according to the zebrafish genome database (www.ensembl.org, version Zv7) to amplify the open reading frame of her8a (also known as her8.1 in [7] and [16]) (GenBank accession number AY007990 and NM_199624). The product of the polymerase chain reaction comprised 663 bp encoding a 221 residue peptide identical to her8a. Alignment with the genomic sequence showed the open reading frame was contained within four exons which correspond to exon 1-4 in comparison to the prediction of the Ensembl database (reference number ENSDARG00000016363). The amino acid sequence of Her8a shows structural features of H/E(spl) family members that contains a bHLH domain, a two-helix orange domain and a C- terminal WRPW motif, implying that it may act as a transcrip- tional repressor (Fig. 1A). Her8a can be further grouped into E(spl) subfamily due to lack of the HC domain present in other Hairy subfamilies [17]. The most similar paralogues of the zebrafish h/ E(spl) subfamily are Her8.2, Her13.1.and Her13.2 which show 43%, 32% and 35% identity to Her8a, respectively. Compared to other orthologues of the E(spl) family, Her8a showed the highest degree of similarity to chicken HES6-2 with 50% identical and 66% conserved amino acids [18]and slightly weaker homology with Xenopus hes6.1 (previously named hes6 in [6]; 34% identical and 54% conserved amino acids) and mouse HES6 (34% identical and 52% conserved amino acids) (Fig. 1). Within the bHLH domain the identity increases to 71% for chicken HES6-2, 55% for Xenopus hes6.1 and 54% for mouse HES6. The three introns interrupt the Her8a protein coding sequence in an identical position in chicken HES6-2 and in a very similar position in mammalian homologues (Fig. 1A). The conservation of intron/ exon structure within the Hes6 genes is strong evidence for evolution from a common ancestor gene specific to this subgroup. y p The expression of her8a was also compared to all identified notch homologues (notch1a, notch1b, notch2 and notch3) (Fig. S1). The results showed her8a expression most resembled that of notch1a, for which significant expression was detected in the brain and anterior spinal cord and also exhibited a segmental pattern in the developing hindbrain from the 9-somite stage (Fig. 2; Fig. S1C1 and D1). A region of weak expression of both genes was also observed in the forebrain–midbrain boundary (Fig. 2; Fig. S1C1 and D1). her8a is expressed in the developing nervous system her8a is expressed in the developing nervous system her8a expression was analyzed by whole mount in situ hybridization. Transcripts first appeared in the developing nervous system at bud stage in the primodia of telencephalon, hypothalamus, midbrain and hindbrain (Fig. 2A). From the 3-somite stage onwards, strong her8a expression was retained in the telencephalon, midbrain, hindbrain, and started to be expressed in the spinal cord (Fig. 2B–G). The expression in the spinal cord was retained until the latest stage analyzed (48 hpf, Fig. 2F–G). Clear gaps of weak expression could be found in the forebrain–midbrain boundary from the 9-somite stage to 24 hpf (Fig. 2C–E). It was notable that dynamic expression was observed in the developing hindbrain with all rhombomeres expressing her8a at different time points and at different levels. During hindbrain segmentation, her8a transcripts were initially detected broadly in all rhombomeres (3-somite stage, Fig. 2B), became clearly segmented at 9-somite stage (Fig. 2C) and was later strongly expressed in rhombomere 3, 5, and 7, and comparatively weaker in the other hindbrain segments (Fig. 2D), suggesting Her8a may play a role in hindbrain patterning. This segmental expression pattern gradually disappeared after 24 hpf, instead the expression became restricted to the midline and hindbrain neurons (Fig. 2E). In conjunction with previous studies showing that Her4 and Her9 regulate the specification of midline precursor cells [21,22], our data suggest that Her8a may also be involved in midline formation. In the developing eyes, transient expression in the lens and the adjacent ventral retinal neuroepithelial cells was detected at 24 hpf but lost by 36 hpf (Fig. 2E). During these stages, the lens epithelium proliferates and differentiates into crystalline fiber cells [23], and the first postmitotic retinal cells appear forming ganglion cells in the ventral retina [24]. Therefore, Her8a may also play a crucial role in retinal neuron proliferation and differentiation. In general, the dynamic expression of her8a in neural tissues suggests its importance in central nervous system development. Here we present the isolation of a previously uncharacterized Hairy/E(Spl) homologue, her8a, in zebrafish and show that its expression is restricted in the developing nervous system. her8a can be grouped into the Hes6 subgroup by sequence similarity. However, unlike mammalian Hes6, her8a is regulated by Su(H)- dependent Notch signaling and it is essential for the inhibition of neurogenesis. We also found Her8a to be required for gliogenesis. her8a is expressed in the developing nervous system Our data provide further insights into the diverse roles of Hairy/ E(Spl) homologues in neural development and their response to Notch signaling. Her8a Is Essential to Inhibit Neurogenesis Her8a Is Essential to Inhibit Neurogenesis The zebrafish, Danio rerio, has emerged as an excellent vertebrate model, with advantages over other organisms for the study of many aspects of developmental biology. In particular, the morpholino approach provides efficient and economic phenocop- ies of gene mutations. To date, more than 20 zebrafish Hairy/ E(spl) homologues, named Her [zebrafish homologue of Hairy/ E(spl) related] and Hey [Hairy/E(spl) related with YxxW motifs], have been identified [7]. Nine genes have been reported to exhibit expression in the developing nervous system (her3: [4]; her4: [8]; her5: [9]; her6: [10]; her9: [11]; her11: [7]; her12 and her15:[7]; hey2: [12]) and six (her3, her4, her5, her6, her9, her11) have been functionally analyzed. Among those, Her3, Her4, Her5, Her9 and Her11 have been shown to repress proneural or neuronal genes, and expression of her3 and her5 is repressed by Notch activation whereas her4 and her6 are induced by Notch signaling, and her9 is independent of Notch activation ([4,9,13,14,15]). Outside of the nervous system, some of the her genes are also expressed in the presomitic mesoderm and are essential for somite segmentation [3]. In comparison to the well-characterized sequence of mouse HES6, Her8a does not contain the negatively charged acidic residues which are responsible for heterodimerization [19], but it contains an identical LNHLL motif which was shown to be important for inhibition of astrocyte differentiation [20] (Fig. 1A). Conserved SPXXSP motifs were also found at the C-terminus of HES6 and Her8a which are putative MAPK phosphorylation sites [19]. Introduction These trigger proteolytic cleavages that release the Notch intracellular domain (NICD) to enter the nucleus. NICD then interacts with the DNA binding protein CSL [CBF/RBP-J, Su(H), LAG-1], leading to April 2011 \| Volume 6 \| Issue 4 \| e19394 PLoS ONE \| www.plosone.org 1 Characterization of zebrafish her8a Nonetheless, notch1a was expressed strongly in the posterior spinal cord and tail bud from bud to 9-somite stage where no her8a expression could be detected. notch1b and notch2 were ubiquitously expressed in the developing central nervous system, and notch3 expression was predominantly in tissues outside of nervous system sharing little in common with her8a expression (Fig. S1). The results suggest that her8a is most likely to be regulated by Notch1a. her8a expression was next compared to those her genes which are known Notch targets (her3 and her4) as well as the well-established target of notch signaling, neurogenin1, in the developing nervous system (Fig. S1). During early stages of neurogenesis, her3 was expressed in the inter-proneuronal domains where it represses proneural genes (Fig. S1) [13,4] and Her3 also negatively regulates neurogenin1 in rhombomeres 2 and 4 [4]. In comparison, the expression of her4 was similar to neurogenin1, expressed in the PLoS ONE \| www.plosone.org April 2011 \| Volume 6 \| Issue 4 \| e19394 PLoS ONE \| www.plosone.org 2 Her8a Is Essential to Inhibit Neurogenesis Figure 1. Alignment and phylogenetic analysis of Her8a and the H/E(spl) family. (A) Amino acid alignment of human, mouse, Chicken, Xenopus laevis, and zebrafish Hes6 related sequences. Residues identical in all proteins are marked in black boxes and partial conservation is shown in grey boxes. The basic helix-loop-helix domain, orange domain and C terminal WRPW motif are indicated, and LNHLL and SPXXSP motifs are underlined. (B) Phylogenetic tree for the H/E(spl) protein family. The full coding protein sequence was used for each family member. Trees were calculated with a bootstrap support of 100 replicates. The phylogram demonstrates only sequence relationships, but does not absolutely imply sequence ancestry since no ancestral relationship is assumed in initial alignments. The initial letter ‘‘h’’ denotes human, ‘‘m’’ mouse, ‘‘c’’ chicken, ‘‘x’’ Xenopus laevis and ‘‘z’’ zebrafish. doi:10.1371/journal.pone.0019394.g001 Figure 1. Alignment and phylogenetic analysis of Her8a and the H/E(spl) family. (A) Amino acid alignment of human, mouse, Chicken, Xenopus laevis, and zebrafish Hes6 related sequences. Residues identical in all proteins are marked in black boxes and partial conservation is shown in grey boxes. The basic helix-loop-helix domain, orange domain and C terminal WRPW motif are indicated, and LNHLL and SPXXSP motifs are underlined. (B) Phylogenetic tree for the H/E(spl) protein family. The full coding protein sequence was used for each family member. her8a is regulated by the canonical Notch signaling In the developing nervous system, Hairy/E(Spl) homologues can suppress or promote neurogenesis acting in either a Notch- dependent or independent manner (reviewed in [25] and [3]). To examine the response of her8a to Notch signaling, we analyzed its expression in mind bomb mutant embryos (mibta52b) that have a strong Notch pathway deficiency [26] due to mutation of a ubiquitin ligase required for Delta ligand activity [27]. We found that in mibta52b embryos, her8a was significantly downregulated compared to wild-type embryos (100%, N = 30; Fig. 3A–E), suggesting that her8a is activated by Notch signaling. Nonetheless, low level expression could be detected in the midbrain and hindbrain boundaries which may due to residual Notch activity in Characterization of zebrafish her8a Trees were calculated with a bootstrap support of 100 replicates. The phylogram demonstrates only sequence relationships, but does not absolutely imply sequence ancestry since no ancestral relationship is assumed in initial alignments. The initial letter ‘‘h’’ denotes human, ‘‘m’’ mouse, ‘‘c’’ chicken, ‘‘x’’ Xenopus laevis and ‘‘z’’ zebrafish. doi:10.1371/journal.pone.0019394.g001 the mibta52b mutants (Fig. 3A–D). An alternative explanation is that her8b is not dependent upon Notch and could be activated by other signaling pathways in these regions (see Discussion). proneuronal domains complementary to the inter-proneuronal regions (Fig. S1) [8]. A previous study showed that her4 can be induced by Notch activation to suppress neurogenin1 in the proneuronal domains [8]. We found the expression of her8a in the spinal cord was more restricted in the middle region which did not resemble her3 expression but partially overlapped with expression of her4. In addition, strong her8a expression in the telecephalon was more similar to her4 expression where no her3 transcript could be detected (Fig. 2; Fig. S1). Notch signaling can be mediated via Su(H)-dependent (canon- ical) and Su(H)-independent (non-canonical) pathways ([28]. A putative Su(H) binding sequence GTGGGAA [16] was identified at positon 2203 to 2197 upstream of the her8a translation start codon. To test whether her8a is activated through the Su(H)- dependent Notch pathway, we expressed RNAs encoding a constitutive-active form (ANK) or dominant-negative form of Su(H) (DBM) which have been shown to activate or suppress Notch signaling target genes, respectively [29]. The Su(H) variants were injected at the 1–2 blastomere stage and harvested at the 3- somite stage for in situ hybridization with a her8a riboprobe (Fig. 3F–H). The results showed her8a transcripts were upregulated in constitutive-active Su(H) injected embryos (70%, N = 50; Fig. 3F) and downregulated after dominant-negative Su(H) injection (80%, N = 55; Fig. 3H), indicating that her8a is activated by Su(H)-dependent Notch signaling. Loss of Her8a is sufficient to cause a neurogenic phenotype doi:10.1371/journal.pone.0019394.g002 for sequencing, which confirmed exon 3 deletion and further showed a predicted premature stop codon in exon 4 that would severely truncate the encoded protein (Fig. 4C). its expression. An antisense morpholino (MO1) was synthesized to target the translation start site of her8a mRNA to block protein production. Comparison to available database sequences predicted binding of MO1 to her8a with no other similar sequence in the zebrafish genome. In order to confirm the specifity of MO1, a second morpholino (MO2) was designed that targets the intron2- exon3 boundary resulting in a truncated product (Fig. 4A). Comparison of MO2 sequence with available zebrafish genomic sequences again predicted that MO2 would specifically binds only to her8a. Four other regions with lower identity to MO2 were found, which show less than 20 bp identity compare to the 25 bp MO2 sequence, and these fragments do not correspond to any 59 UTR or exon-intron splicing site of predicted or characterized genes, suggesting that MO2 would also act specifically on her8a. RT-PCR with primers that spanned the MO2 binding sequence was employed and a fragment corresponding to an mRNA lacking the 79 bp of exon 3 was detected in the morpholino injected embryo extract (Fig. 4B). The mis-spliced product was subjected its expression. An antisense morpholino (MO1) was synthesized to target the translation start site of her8a mRNA to block protein production. Comparison to available database sequences predicted binding of MO1 to her8a with no other similar sequence in the zebrafish genome. In order to confirm the specifity of MO1, a second morpholino (MO2) was designed that targets the intron2- exon3 boundary resulting in a truncated product (Fig. 4A). Comparison of MO2 sequence with available zebrafish genomic sequences again predicted that MO2 would specifically binds only to her8a. Four other regions with lower identity to MO2 were found, which show less than 20 bp identity compare to the 25 bp MO2 sequence, and these fragments do not correspond to any 59 UTR or exon-intron splicing site of predicted or characterized genes, suggesting that MO2 would also act specifically on her8a. RT-PCR with primers that spanned the MO2 binding sequence was employed and a fragment corresponding to an mRNA lacking the 79 bp of exon 3 was detected in the morpholino injected embryo extract (Fig. 4B). Loss of Her8a is sufficient to cause a neurogenic phenotype To delineate the role of Her8a in neurodevelopment, the morpholino (MO) knockdown approach was used to interfere with April 2011 \| Volume 6 \| Issue 4 \| e19394 PLoS ONE \| www.plosone.org 3 Her8a Is Essential to Inhibit Neurogenesis Figure 2. her8a expression in the developing zebrafish embryos. her8a expression is restricted in the developing nervous system during zebrafish embryogenesis analyzed by in situ hybridization. Stages of embryos shown in bottom left corner of each panel. Yolks were removed and embryos were flat-mounted, dorsal view except F and G lateral. her8a expression first appears in the developing brain at bud stage (A) and later becomes restricted to specific brain areas (B–E). The transcripts can be detected in the spinal cord from the 3-somite stage (3ss) (B) and retained until the latest stage analyzed (48 hpf) (C–G). Dashed circles in D mark the otic vesicles. Insert panel in D is an enlargement of the hindbrain, and in E is an enlargement of the eye. Arrowheads in D and E indicate her8a expressing cells at the midline. No signal was detected using sense riboprobe (C’). (H) her4 expression at 48 hpf . fmb, forebrain–midbrain boundary; hb, hindbrain; hp, hypothalamus; le, lens; mb, midbrain; r1-r7, rhombomere 1–7; rn, retinal neuroepithelial cells; sc, spinal cord; tel, telencephalon. doi:10.1371/journal.pone.0019394.g002 Figure 2. her8a expression in the developing zebrafish embryos. her8a expression is restricted in the developing nervous system during zebrafish embryogenesis analyzed by in situ hybridization. Stages of embryos shown in bottom left corner of each panel. Yolks were removed and embryos were flat-mounted, dorsal view except F and G lateral. her8a expression first appears in the developing brain at bud stage (A) and later becomes restricted to specific brain areas (B–E). The transcripts can be detected in the spinal cord from the 3-somite stage (3ss) (B) and retained until the latest stage analyzed (48 hpf) (C–G). Dashed circles in D mark the otic vesicles. Insert panel in D is an enlargement of the hindbrain, and in E is an enlargement of the eye. Arrowheads in D and E indicate her8a expressing cells at the midline. No signal was detected using sense riboprobe (C’). (H) her4 expression at 48 hpf . fmb, forebrain–midbrain boundary; hb, hindbrain; hp, hypothalamus; le, lens; mb, midbrain; r1-r7, rhombomere 1–7; rn, retinal neuroepithelial cells; sc, spinal cord; tel, telencephalon. Loss of Her8a is sufficient to cause a neurogenic phenotype The mis-spliced product was subjected Embryos injected with MO1 or MO2 were analyzed at 24 hpf, 3 days post fertilization (dpf) and 5 dpf for morphological defects. 6 ng of MO1 or 4 ng of MO2 injection caused an identical phenotype exhibiting pericardial edema at 5 dpf (Fig. 4K; Fig. S2). In comparison, higher dosage (12 ng MO1 or 8 ng of MO2) injection resulted in more severe defects including brain malformation and a bent trunk from 24 hpf (Fig. 4D–L) and edemas in eyes, pericardial sac and the abdominal cavity from 3 dpf (Fig. 4G–L; Fig. S2). The phenotypes caused by morpholino injection could be rescued by concomitant injection of her8a mRNA, indicating that the morpholino-induced defect was due to loss of Her8a function (92%, N = 39; Fig. 4M). Many of the Hairy/E(Spl) proteins act as transcriptional repressors to inhibit genes responsible for neurogenesis. In order to address the role of Her8a during neurodevelopment, we PLoS ONE \| www.plosone.org PLoS ONE \| www.plosone.org April 2011 \| Volume 6 \| Issue 4 \| e19394 April 2011 \| Volume 6 \| Issue 4 \| e19394 4 Her8a Is Essential to Inhibit Neurogenesis Figure 3. her8a is regulated by the Su(H)-dependent Notch signaling pathway. her8a is expressed at lower levels in the Notch activity- deficient mind bomb mutant embryos (C and D) in comparison to wild-type siblings (A and B). B and D are higher power views of hindbrain regions in A and C, respectively. Note the remnants of her8a transcripts in mind bomb mutants at the midbrain and hindbrain rhombomere boundaries (arrowheads in D). (E) Quantification of her8a expression analyzed by qPCR showing significantly reduced level of her8a in mind bomb mutants. (F–H) RNA encoding constitutively-active Su(H) (ANK) (G) or dominant-negative Su(H) (DBM) (H) was injected at the one or two-cell stage and analyzed at the 3-somite stage. (F) Control embryos injected with GFP mRNA; (G) her8a expression is upregulated by constitutive-active Su(H) injection; (H) her8a expression is downregulated in dominant-negative Su(H) injected embryos. (I) The levels of her8a expression in Su(H) variant injected embryos were further quantified by qPCR, showing significantly increased her8a expression in ANK injected embryos and decreased expression in DBM over- expressed embryos. ANK, constitutive-active Su(H); DBM, dominant-negative Su(H), mib: mind bomb mutants. doi:10.1371/journal.pone.0019394.g003 Figure 3. her8a is regulated by the Su(H)-dependent Notch signaling pathway. Loss of Her8a is sufficient to cause a neurogenic phenotype Embryos injected with 6 ng of MO1 or 4 ng of MO2 exhibited upregulation of HuC/D expression (67%, N = 48; Fig. 5B and 5E; Fig. S3). The phenotype appeared more severe when injected with higher dosage of MO1 (12 ng; 77%, N = 52) or MO2 (8 ng; 84%, N = 51; Fig. 5C and 5F). To examine whether the increased HuC/D expression was due to cell number changes or increased expression in individual cells, a z- stack of fluorescence images was acquired by confocal microscopy and the HuC/D positive-cells were counted. The results showed that the number of HuC/D-positive neurons was increased by approximately 2-fold and 4-fold in low dose and high dose morpholino injected embryos, respectively. Western blot analysis also showed a 2.3 fold increase of HuC/D expression in Her8a morphants (Fig. 5J). Co-injection with her8a mRNA attenuated the effect of morpholino (8 ng; 78%, N = 50; Fig. 5I) indicating that the neuronal defects in MO1 and MO2 morphants were the result of specific inhibition of Her8a function. analyzed the effect of Her8a knockdown using post-mitotic neuronal marker, HuC/D. Embryos injected with 6 ng of MO1 or 4 ng of MO2 exhibited upregulation of HuC/D expression (67%, N = 48; Fig. 5B and 5E; Fig. S3). The phenotype appeared more severe when injected with higher dosage of MO1 (12 ng; 77%, N = 52) or MO2 (8 ng; 84%, N = 51; Fig. 5C and 5F). To examine whether the increased HuC/D expression was due to cell number changes or increased expression in individual cells, a z- stack of fluorescence images was acquired by confocal microscopy and the HuC/D positive-cells were counted. The results showed that the number of HuC/D-positive neurons was increased by approximately 2-fold and 4-fold in low dose and high dose morpholino injected embryos, respectively. Western blot analysis also showed a 2.3 fold increase of HuC/D expression in Her8a morphants (Fig. 5J). Co-injection with her8a mRNA attenuated the effect of morpholino (8 ng; 78%, N = 50; Fig. 5I) indicating that the neuronal defects in MO1 and MO2 morphants were the result of specific inhibition of Her8a function. inhibition and consequently caused premature differentiation of neurons [30]. Since neuronal differentiation is initiated by proneural genes, we next examined whether knockdown of Her8a also affect the expression of proneural markers, neurogenin1 and zash1. Loss of Her8a is sufficient to cause a neurogenic phenotype her8a is expressed at lower levels in the Notch activity- deficient mind bomb mutant embryos (C and D) in comparison to wild-type siblings (A and B). B and D are higher power views of hindbrain regions in A and C, respectively. Note the remnants of her8a transcripts in mind bomb mutants at the midbrain and hindbrain rhombomere boundaries (arrowheads in D). (E) Quantification of her8a expression analyzed by qPCR showing significantly reduced level of her8a in mind bomb mutants. (F–H) RNA encoding constitutively-active Su(H) (ANK) (G) or dominant-negative Su(H) (DBM) (H) was injected at the one or two-cell stage and analyzed at the 3-somite stage. (F) Control embryos injected with GFP mRNA; (G) her8a expression is upregulated by constitutive-active Su(H) injection; (H) her8a expression is downregulated in dominant-negative Su(H) injected embryos. (I) The levels of her8a expression in Su(H) variant injected embryos were further quantified by qPCR, showing significantly increased her8a expression in ANK injected embryos and decreased expression in DBM over- expressed embryos. ANK, constitutive-active Su(H); DBM, dominant-negative Su(H), mib: mind bomb mutants. doi:10.1371/journal.pone.0019394.g003 inhibition and consequently caused premature differentiation of neurons [30]. Since neuronal differentiation is initiated by proneural genes, we next examined whether knockdown of Her8a also affect the expression of proneural markers, neurogenin1 and zash1. The result showed proneural genes were upregulated in the morpholino injected embryos (neurogenin1: 74%, N = 68; zash1a: 71%, N = 56), and the phenotype could be rescued by her8a mRNA injection (neurogenin1: 80%, N = 56; zash1a: 95%, N = 40) (Fig. 6A–F). Quantitative real time PCR (qPCR) analysis also showed a 2.5 fold and 4.4 fold increase of neurogenin1 and zash1a expression in Her8a morphants, respectively (Fig. 6G). On the contrary, widespread over-expression of her8a mRNA alone did not alter the expression of proneural and pan-neuronal markers (Fig. S4), indicating that Her8a alone was not sufficient to inhibit neurogenesis. Subsequently, we analyzed the effect of Her8a in neural stem cells. Sox2 is expressed in neural stem cells where it maintains the stemness identity and inhibits neurogenesis [31]. We found that sox2 expression remained intact after Her8a knock down (Fig. S5). To confirm, we used sox3 as another neural progenitor maker ([32]; [33]) and found the expression of sox3 also remained intact in Her8a morphants (Fig. S5C and S5D). Since analyzed the effect of Her8a knockdown using post-mitotic neuronal marker, HuC/D. PLoS ONE \| www.plosone.org Loss of Her8a is sufficient to cause a neurogenic phenotype The result showed proneural genes were upregulated in the morpholino injected embryos (neurogenin1: 74%, N = 68; zash1a: 71%, N = 56), and the phenotype could be rescued by her8a mRNA injection (neurogenin1: 80%, N = 56; zash1a: 95%, N = 40) (Fig. 6A–F). Quantitative real time PCR (qPCR) analysis also showed a 2.5 fold and 4.4 fold increase of neurogenin1 and zash1a expression in Her8a morphants, respectively (Fig. 6G). On the contrary, widespread over-expression of her8a mRNA alone did not alter the expression of proneural and pan-neuronal markers (Fig. S4), indicating that Her8a alone was not sufficient to inhibit neurogenesis. Subsequently, we analyzed the effect of Her8a in neural stem cells. Sox2 is expressed in neural stem cells where it maintains the stemness identity and inhibits neurogenesis [31]. We found that sox2 expression remained intact after Her8a knock down (Fig. S5). To confirm, we used sox3 as another neural progenitor maker ([32]; [33]) and found the expression of sox3 also remained intact in Her8a morphants (Fig. S5C and S5D). Since The increased number of neurons resulting from Her8a knockdown resembles the effect observed in Notch deficient embryos which have been shown to be the result of lost of lateral April 2011 \| Volume 6 \| Issue 4 \| e19394 5 Her8a Is Essential to Inhibit Neurogenesis Her8a Is Essential to Inhibit Neurog Figure 4. Knockdown of Her8a by specific morpholino antisense oligonucleotides causes developmental abnormalities. (A) Schematic representation shows the genomic organization of the her8a gene. Regions targeted by translational-blocking (MO1) and splice-blocking (MO2) morpholinos are shown. (B) The efficacy of MO2 was validated by RT-PCR using primers as indicated in A. Wild-type her8a mRNA produces a 666 bp PCR product while alternatively spliced transcripts from morphant embryos yield a 587 bp fragment. (C) The mis-spliced event resulted in loss of exon 3 confirmed by sequencing the 587 bp PCR product in B (left panel). The mis-splicing event resulted in a premature stop codon in exon 4 (boxed, right panel). (D–M) Representative images of morpholino injected embryos. (D, G and J) Control morpholino; (E, H and K) ‘low dose’ indicates 6 ng of MO1 or 4 ng of MO2 injection, resulting in indistinguishable phonotype; (F, I and L) ‘high dose’ represents 12 ng MO1 or 8 ng of MO2 injection, which resulted in identical morphological defects. Loss of Her8a is sufficient to cause a neurogenic phenotype Arrows denote bent body characteristics, and arrowheads indicate edemas in eyes, pericardial sac and abdominal cavity. Malformation of brains is marked by asterisks. (M) These morphological phenotypes can be rescued by concomitant injection of 2 ng her8a mRNA. doi:10 1371/journal pone 0019394 g004 Figure 4. Knockdown of Her8a by specific morpholino antisense oligonucleotides causes developmental abnormalities. (A) Schematic representation shows the genomic organization of the her8a gene. Regions targeted by translational-blocking (MO1) and splice-blocking (MO2) morpholinos are shown. (B) The efficacy of MO2 was validated by RT-PCR using primers as indicated in A. Wild-type her8a mRNA produces a 666 bp PCR product while alternatively spliced transcripts from morphant embryos yield a 587 bp fragment. (C) The mis-spliced event resulted in loss of exon 3 confirmed by sequencing the 587 bp PCR product in B (left panel). The mis-splicing event resulted in a premature stop codon in exon 4 (boxed, right panel). (D–M) Representative images of morpholino injected embryos. (D, G and J) Control morpholino; (E, H and K) ‘low dose’ indicates 6 ng of MO1 or 4 ng of MO2 injection, resulting in indistinguishable phonotype; (F, I and L) ‘high dose’ represents 12 ng MO1 or 8 ng of MO2 injection, which resulted in identical morphological defects. Arrows denote bent body characteristics, and arrowheads indicate edemas in eyes, pericardial sac and abdominal cavity. Malformation of brains is marked by asterisks. (M) These morphological phenotypes can be rescued by concomitant injection of 2 ng her8a mRNA. doi:10.1371/journal.pone.0019394.g004 of neurogenesis, and loss of Her8a results in premature neural differentiation. Notch signaling-initiated lateral inhibition comes into play after the Sox2/3-positive neural progenitor regions is determined, these data are consistent with the expectation that these stem cell markers would not be altered in Her8a deficient embryos. Taken together, these results suggest that Her8a is required for inhibition Previous studies have shown that H/E(spl) transcription factors not only repress the expression of proneural genes during neurogenesis, but also promote the differentiation of many glial PLoS ONE \| www.plosone.org April 2011 \| Volume 6 \| Issue 4 \| e19394 April 2011 \| Volume 6 \| Issue 4 \| e19394 6 Figure 5. Her8a morphants exhibit upregulation of HuC/D expression. HuC/D expression is upregulated in Her8a morpholino injected embryos analyzed by immunohistochemistry with anti-Hu antibody at 24 hpf. The black-and-white fluorescent signals were inverted to negative film for a better presentation. Loss of Her8a is sufficient to cause a neurogenic phenotype (B and E) 6 ng of MO1 or 4 ng of MO2 injection (low dose) resulted in indistinguishable phonotype and therefore only embryos injected with 4 ng of MO2 are shown (see Fig. S2 for MO1). (C and F) Injection of 12 ng MO1 or 8 ng MO2 (high dose) resulted in identical morphological defects and only MO2 injected embryos are shown, which shows more dramatic upregulation of HuC/D expression. (A–C) Brain regions; (D-I) 3-somite to 9-somite level of the spinal cord. (A–F) Hu-positve cells were significantly increased in the morpholino injected embryos. (G–I) The phenotype can be rescued by co-injection of morpholino with her8a mRNA. (J) Western blot analysis confirming the levels of HuC/D expression in Her8a morphants were up-regulated in comparison to the control. * p,0.05. doi:10.1371/journal.pone.0019394.g005 and we did not observe any detectable deviation in Her8a morphants with or without p53 MO (Fig. S6). Since the phenotypes caused by her8a morpholino injection could also be rescued by concomitant injection of her8a mRNA as shown above, we suggest that the phenotypes in Her8a morphants were the result of specific inhibition of Her8a function independent of p53 activity. subtypes (reviewed in [1]). In contrast, mouse HES6 inhibits astroglia differentiation in vitro [20]. Consistent with this, we found that the early glial marker, slc1a3a (Glast in mammals, [34,35]) was reduced in Her8a knockdown embryos (83%, N = 12, Fig. 7A–C, J), suggesting that Her8a is required for gliogenesis. To further confirm the reduction of slc1a3a in Her8a morphants was not due to increased glial differentiation, we analyzed the expression of mature radial glial cell markers (zrf-1 antibody for radial glial fibers and gfap riboprobe for radial glial cell body) and found the expression were downregulated in Her8a knockdown embryos (Fig. 7 D–J)), supporting our notion that Her8a is required for gliogensis. However, the possibility that the reduction of glial markers was due the bias of neuronal differentiation cannot be ruled out. Loss of Her8a is sufficient to cause a neurogenic phenotype (A and D) embryos injected with control morpholino. (B and E) 6 ng of MO1 or 4 ng of MO2 injection (low dose) resulted in indistinguishable phonotype and therefore only embryos injected with 4 ng of MO2 are shown (see Fig. S2 for MO1). (C and F) Injection of 12 ng MO1 or 8 ng MO2 (high dose) resulted in identical morphological defects and only MO2 injected embryos are shown, which shows more dramatic upregulation of HuC/D expression. (A–C) Brain regions; (D-I) 3-somite to 9-somite level of the spinal cord. (A–F) Hu-positve cells were significantly increased in the morpholino injected embryos. (G–I) The phenotype can be rescued by co-injection of morpholino with her8a mRNA. (J) Western blot analysis confirming the levels of HuC/D expression in Her8a morphants were up-regulated in comparison to the control. * p,0.05. doi:10.1371/journal.pone.0019394.g005 Her8a Is Essential to Inhibit Neurogenesis Her8a Is Essential to Inhibit Neurogenesis Figure 5. Her8a morphants exhibit upregulation of HuC/D expression. HuC/D expression is upregulated in Her8a morpholino injected embryos analyzed by immunohistochemistry with anti-Hu antibody at 24 hpf. The black-and-white fluorescent signals were inverted to negative film for a better presentation. (A and D) embryos injected with control morpholino. (B and E) 6 ng of MO1 or 4 ng of MO2 injection (low dose) resulted in indistinguishable phonotype and therefore only embryos injected with 4 ng of MO2 are shown (see Fig. S2 for MO1). (C and F) Injection of 12 ng MO1 or 8 ng MO2 (high dose) resulted in identical morphological defects and only MO2 injected embryos are shown, which shows more dramatic upregulation of HuC/D expression. (A–C) Brain regions; (D-I) 3-somite to 9-somite level of the spinal cord. (A–F) Hu-positve cells were significantly increased in the morpholino injected embryos. (G–I) The phenotype can be rescued by co-injection of morpholino with her8a mRNA. (J) Western blot analysis confirming the levels of HuC/D expression in Her8a morphants were up-regulated in comparison to the control. * p,0.05. doi:10.1371/journal.pone.0019394.g005 Figure 5. Her8a morphants exhibit upregulation of HuC/D expression. HuC/D expression is upregulated in Her8a morpholino injected embryos analyzed by immunohistochemistry with anti-Hu antibody at 24 hpf. The black-and-white fluorescent signals were inverted to negative film for a better presentation. (A and D) embryos injected with control morpholino. PLoS ONE \| www.plosone.org Discussion H/E(spl) family members efficiently inhibit neurogenesis by forming homodimers or heterodimers with other H/E(spl) factors. These then bind to target sequences to actively repress the transcription of target genes, or form non-DNA binding heterodimers with bHLH activators such as E47 and inhibit transcriptional activation [3]. We have isolated her8a, a member of the E(spl) family in zebrafish. Intriguingly, although we found that knockdown of Her8a resulted in premature neurogenesis and this Previous studies have shown that morpholinos can cause off- target apoptosis mediated by p53 activation [36]. To rule out the possibility that the effect of her8a morpholinos was mediated by off- targeted apoptosis, all her8a MOs were co-injected with a p53 MO April 2011 \| Volume 6 \| Issue 4 \| e19394 PLoS ONE \| www.plosone.org April 2011 \| Volume 6 \| Issue 4 \| e19394 7 Her8a Is Essential to Inhibit Neurogenesis could be rescued by concomitant over-expression of Her8a mRNA, According to the sequence similarity and conserved intron/ Figure 6. Increased neuronal precursors in Her8a knockdown embryos. Proneural genes were upregulated in the morpholino injected embryos and this effect was rescued by co-injection with her8a mRNA. Proneuronal markers neurogenin1 (A–C) and zash1 (D–F) were analyzed by in situ hybridization. Embryos were injected with control morpholino (A and D), 8 ng of MO2 (B and E), or co-injection of 8 ng of MO2 with 480 pg her8a mRNA (C and F). The most dramatic phenotypes are indicated by arrows. Stages of embryos are 6-somite stage (A-C) and 16-somite stage (D–F). (G) qPCR analysis showing the levels of neurogenin1 and zash1 are both significantly increased in embryos injected with her8a morpholino. * p,0.05. doi:10.1371/journal.pone.0019394.g006 Her8a Is Essential to Inhibit Neurogenesis could be rescued by concomitant over-expression of Her8a mRNA, over-expressing Her8a alone failed to efficiently inhibit the formation of post-mitotic neurons. This suggests that Her8a may According to the s exon structure her8a Strikingly, we found th Figure 6. Increased neuronal precursors in Her8a knockdown embryos. Proneural genes embryos and this effect was rescued by co-injection with her8a mRNA. Proneuronal markers neuroge situ hybridization. Embryos were injected with control morpholino (A and D), 8 ng of MO2 (B and E), o mRNA (C and F). The most dramatic phenotypes are indicated by arrows. Stages of embryos are 6-so qPCR analysis showing the levels of neurogenin1 and zash1 are both significantly increased in embr doi:10.1371/journal.pone.0019394.g006 Figure 6. Discussion Increased neuronal precursors in Her8a knockdown embryos. Proneural genes were upregulated in the morpholino injected embryos and this effect was rescued by co-injection with her8a mRNA. Proneuronal markers neurogenin1 (A–C) and zash1 (D–F) were analyzed by in situ hybridization. Embryos were injected with control morpholino (A and D), 8 ng of MO2 (B and E), or co-injection of 8 ng of MO2 with 480 pg her8a mRNA (C and F). The most dramatic phenotypes are indicated by arrows. Stages of embryos are 6-somite stage (A-C) and 16-somite stage (D–F). (G) qPCR analysis showing the levels of neurogenin1 and zash1 are both significantly increased in embryos injected with her8a morpholino. * p,0.05. doi:10.1371/journal.pone.0019394.g006 According to the sequence similarity and conserved intron/ exon structure her8a is a member of the HES6 subfamily. Strikingly, we found that her8a was activated by Su(H)-dependent Notch signaling, in contrast to chick HES6-2 which has been shown to be repressed by Notch signaling [18] and Xenopus hes6.1 which has been shown not to respond to Notch activation [6] in the developing nervous system. However, it is noteworthy that although her8a was significantly downregulated in mibta52b embryos could be rescued by concomitant over-expression of Her8a mRNA, over-expressing Her8a alone failed to efficiently inhibit the formation of post-mitotic neurons. This suggests that Her8a may need to form a heterodimer with other H/E(spl) factors to inhibit neuronal differentiation. In support of this, we did find several Her homologues could interact with Her8a by yeast two-hybrid analysis (data not shown), but whether they can operate together in vivo to inhibit neurogenesis remains to be tested. PLoS ONE \| www.plosone.org PLoS ONE \| www.plosone April 2011 \| Volume 6 \| Issue 4 \| e19394 PLoS ONE \| www.plosone.org 8 Figure 7. Glial precursors and mature glial cells were reduced in Her8a knockdown embryos. Her8a morpholino injection downregulates the expression of the glial precursor marker slc1a3a (A, B) and radial glial markers (gfap for radial glial cell body (D, E) and zrf-1 for glial fibers (G, H)), and this effect was rescued by co-injection with her8a mRNA (C, F, I). (A, D, G) Control morpholino, (B, E, H) 8 ng of MO2, (C, F, I) Co-injection of 8 ng of MO2 with 0.5 ng her8a mRNA. Embryos were harvested at the 18-somite stage (A–C) and 36 hpf (D–I). Discussion (A–F) in situ hybridization; (G–I) immunohistochemistry with zrf-1 antibody, fluorescent signals were inverted to negative film for a better presentation. (J) qPCR analysis showing the levels of slc1a3a and zash1 and zrf-1 are significantly decreased in embryos injected with her8a morpholino. * p,0.05. doi:10.1371/journal.pone.0019394.g007 Her8a Is Essential to Inhibit Neurogenesis Her8a Is Essential to Inhibit Neurogenesis Figure 7. Glial precursors and mature glial cells were reduced in Her8a knockdown embryos. Her8a morpholino injection downregulates the expression of the glial precursor marker slc1a3a (A, B) and radial glial markers (gfap for radial glial cell body (D, E) and zrf-1 for glial fibers (G, H)), and this effect was rescued by co-injection with her8a mRNA (C, F, I). (A, D, G) Control morpholino, (B, E, H) 8 ng of MO2, (C, F, I) Co-injection of 8 ng of MO2 with 0.5 ng her8a mRNA. Embryos were harvested at the 18-somite stage (A–C) and 36 hpf (D–I). (A–F) in situ hybridization; (G–I) immunohistochemistry with zrf-1 antibody, fluorescent signals were inverted to negative film for a better presentation. (J) qPCR analysis showing the levels of slc1a3a and zash1 and zrf-1 are significantly decreased in embryos injected with her8a morpholino. * p,0.05. doi:10.1371/journal.pone.0019394.g007 The roles of the H/E(spl) family members are generally evolutionarily conserved in the developing nervous system where they have been demonstrated to repress neurogenesis. In contrast, The Hes6 subfamily have a role distinct from other H/E(spl) members, which is to promote instead of to inhibit neurogenesis. And all Hes6 homologues characterized to date showed a similar function. For example, chicken HES6-2 represses HES5 and cooperates with proneural genes to promote neuronal differenti- ation [18], mouse HES6 promotes neuronal cell differentiation by suppressing HES1 [13], and Xenopus hes6.1 also promotes neuronal differentiation [6]. Interestingly, we found that zebrafish Her8a is essential for the inhibition of neurogenesis, the converse of the earlier demonstrated roles for Hes6 homologues. Previously, another Hes6 homologue named her13.2 was identified in suggesting that her8a is activated by Notch signaling, residual expression of her8a was maintained at the midbrain and the hindbrain rhombomere boundaries in Notch deficient mutants, suggesting that her8a may also response to other singling pathways in these regions. Fish Maintenance and Mutants 52b Tu¨ (wild type) and mibta52b mutant zebrafish embryos were purchased from the Zebrafish International Resource Center (ZIRC, Oregon, USA) and were raised, maintained and paired under standard conditions. The embryos were staged according to the number of somites, hours post fertilization and days post fertilization [40]. Quantitative analysis For quantitative real time PCR (qPCR), embryos were homogenized in TRIzol reagent (Invitrogen) and total RNA was extracted using a standard method. cDNA was synthesized from total RNA with random hexamer priming using RevertAid First Strand cDNA Synthesis Kit (Fermentas). qPCR was performed on an ABI StepOneTM Real-Time PCR System (Applied Biosystems) with SYBR green fluorescent label (Fermentas). Primers for ngn1 (F: 59-CGCACACGGATGATGAAGACTCGCG-39; R: 59-CG- GTTCTTCTTCACGACGTGCACAGTGG-39), zash1 (F:59-AC- CGGGTGAAGCTTGTGAAC-39; R: 59-CGTCATGCYCG RNA and Morpholino Injection Capped RNA encoding the full coding sequence of Her8a, constitutive-active Su(H), and dominant-negative Su(H) [29] was prepared as described previously [41]. The Su(H) constructs were kindly provided by Chris Kintner. Antisense morpholino oligonu- cleotides were purchased from Gene Tools, LLC (Oregon, USA). Two morpholinos against her8a were used: MO1 (CTTAT- TACTGCCGGAGGTTGTACCC) that overlaps the ATG start codon, and MO2 (TATTAAACTTAAGGGTGTCGTTAGA) that corresponds to intron2-exon3 boundary region sequence. A p53 morpholino with the sequence GACCTCCTCTCCAC- TAAACTACGAT (Gene Tools, LLC) was used to rule out the possibility that the effect of her8a MOs was mediated through an off-targeted p53 activation. All injections were performed at the one to two-cell stage and mRNAs or morpholinos were introduced into blastomeres. Sequence comparisons and phylogeny q p p y g y Amino acid sequences were aligned and displayed using the Vector NTI (Invitrogen). Phylogenetic tree calculation was performed with ClustalX [39]. The GenBank accession numbers of the compared proteins are as follows: zebrafish Her1 (NM_131078), Her2 (NM_131089), Her3 (NM_131080), Her4.1 (NM_001161408), Her4.2 (NM_131090), Her5 (NM_131077), Her6 (NM_131079), Her7 (NM_131609), Her8a (NM_199624), Her8.2 (NM_001166166), Her9 (NM_131873), Her11 (NM_ 001003886), Her12 (NM_205619), Her13.1 (NM_001017901), Her13.2 (NM_194400), Her15.1 (NM_182875); chicken Hes1 isoform 1 (c-hairy1A, NM_001005848), Hes1 isoform 2 (c- hairy1B, NM_204472), Hes5 (NM_001012695), Hes6-2 (XP_422641); Xenopus laevis hes1-a (NM_001087927), hes1-b (NM_001085917), hes2 (NM_001122882), hes3.1 (NM_ 001088503.1), hes3.3 (NM_001088694), hes4-a (NM_ 001089105), hes4-b (NM_001088692), hes5.1 (NM_001095627), hes5.2-a (NM_001088505), hes5.2-b (NM_001095626), hes6.1 (BC130161), hes7.1 (NM_001088706), hes7.2 (AAD42783), hes9.1-a (NM_001088237), hes9.1-b (NM_001095628.1); mouse HES1 (NM_008235.2), HES2 (NM_008236.4), HES3 (NM_008237), HES5 (NM_010419.4), HES6 (NM_019479.3), HES7 (NM_033041); human HES1 (NM_005524), HES2 (NM_019089), HES3 (NM_001024598), HES4 (NM_ 001142467), HES5 (NM_001010926), HES6 (NM_018645), HES7 (NM_001165967). Her8a Is Essential to Inhibit Neurogenesis pCS2+ MT vector. PCR amplifications were performed with the high fidelity Pfu polymerase (Promega) and constructs were sequenced to check for the absence of mutations. zebrafish that is required for somite segmentation, but its role in the developing nervous system has not been analyzed [38]. The sequences of two other predicted genes, named her8.2 and her13.1, have been described in zebrafish that also show high similarity to mammalian Hes6, but their roles have not yet been studied [7]. Therefore, it is possible that at least one of the above genes could share the functional role of the mammalian HES6 protein to promote neurogenesis. Taken together, our results suggest her8a was created by duplication events which allowed functional diversity to develop within the paralogues. However, within this group Her8a is essential to repress neuronal differentiation. Since her8a expression is regulated by Notch signaling, these data provide further understanding of how Notch signaling and downstream genes mediate neural cell fate determination. RT-PCR Total RNA was extracted from zebrafish embryos using standard protocol (TRIzol; Invitrogen) and resuspended in nuclease-free water. The concentration and purity of RNA were measured with a spectrophotometer (NanoDrop Technologies), and contaminating genomic DNA was removed using DNase I (Ambion). Reverse transcription was performed using the Thermoscript RT-PCR system (Invitrogen) priming with random hexamers. Synthesized cDNA was used with the primers (forward, 59- AGAATTCATGACGGCCTCCAACATGGGC -39; reverse, 59- GGAATTCCTCACCAGGGCCTCCACATG -39) spanning the MO2 binding sequence in standard PCR conditions. Histological Analysis All experiments were performed in strict accordance to standard guidelines for zebrafish work and approved by the Institutional Animal Care and Use Committee of Chang Gung University (IACUC approval number: CGU04-57 and CGU08-86). y Digoxigenin-UTP labeled riboprobes to detect her8a, her3, her4, her5, her6, notch1a, notch1b, notch2, notch3, deltaA, neurog1, zash1, sox2, sox3, and slc1a3a transcripts were synthesized according to manufacturer’s instructions (Roche), and in situ hybridizations were performed as described previously [30]. The color reaction was carried out using NBT/BCIP substrate (Roche). For immunohistochemistry, the embryos were blocked in 5% goat serum and anti-HuC/HuD neuronal protein monoclonal 16A11 antibody (1/500 dilution, Invitrogen) or zrf-1 radial glial fibers antibody (1/500 dilution, Zebrafish International Resource Center) was applied. Fluorochrome conjugated antibodies Alexa Fluor 488 (or 594) goat anti-mouse IgG (Invitrogen) was used to detect the primary antibodies. Embryos were mounted with Vectashield mouting medium with DAPI (Vector Laboratories, Inc.). Confocal Microscopy was performed using a Zeiss LSM 510 microscope. Discussion In support of this, a previous study in chick embryos showed that H/E(spl) genes can respond to FGF and TGFb signaling at the rhombomere boundaries [37], and other work showed that H/E(spl) could be regulated by BMP, TGFb, JAK-STAT and Ras signaling cascades in several tissue systems (review in [25]). Therefore, her8a is positively-regulated by Notch signaling to repress neuronal differentiation, but it may also respond to other signaling pathways in the midbrain and the rhombomere boundaries where the role of Her8a is currently not clear and needs to be further analyzed. suggesting that her8a is activated by Notch signaling, residual expression of her8a was maintained at the midbrain and the hindbrain rhombomere boundaries in Notch deficient mutants, suggesting that her8a may also response to other singling pathways in these regions. In support of this, a previous study in chick embryos showed that H/E(spl) genes can respond to FGF and TGFb signaling at the rhombomere boundaries [37], and other work showed that H/E(spl) could be regulated by BMP, TGFb, JAK-STAT and Ras signaling cascades in several tissue systems (review in [25]). Therefore, her8a is positively-regulated by Notch signaling to repress neuronal differentiation, but it may also respond to other signaling pathways in the midbrain and the rhombomere boundaries where the role of Her8a is currently not clear and needs to be further analyzed. PLoS ONE \| www.plosone.org PLoS ONE \| www.plosone.org April 2011 \| Volume 6 \| Issue 4 \| e19394 9 Her8a Is Essential to Inhibit Neurogenesis Supporting Information Figure S1 Expression comparison of notch homologues, her3, her4 and neurogenin1. Expression of notch homologues and known Notch target, her3 and her4, and neurogenenin1 analyzed by in situ hybridization. Name of the gene analyzed shown on the top row and stages of embryos shown on the left column. Embryos were flat-mounted, dorsal view. (TIF) Figure S6 The phenotypes in Her8a morphants were not caused by none specific p53 activation. Embryos co- injected her8a MOs with p53 MO (C, F, I, L, O, R) were compared to Her8a morphants (B, E, H, K, N, Q) and did not cause any detectable deviation analyzed with all markers tested. (A, D, G, J, M, P) Embryos injected with control morpholino. (TIF) Figure S2 Embryos injected with MO1 or MO2 resulted in very similar morphological phenotypes. (A, B) Embryos injected with control morpholino analyzed at 5 days post fertilization. (C, D) Injection of 6 ng of MO1 or 4 ng of MO2 (lower dosage) caused very similar phenotype exhibiting pericar- dial edema. (E, F) Embryos injected with 12 ng MO1 or 8 ng of MO2 (higher dosage) show an identical phenotype including brain malformation and edemas in eyes, pericardial sac and the abdominal cavity. Acknowledgments We thank Haiwei Pi and Yaa-Jyuhn J. Meir for discussions, Han-Ting Lee and Chu-Li Tsao for technical assistance, and Chris Kintner for constructs. Figure S3 Embryos injected with MO1 exhibit upregu- lation of HuC/D expression. (A, B) Embryos injected with Figure S3 Embryos injected with MO1 exhibit upregu- lation of HuC/D expression. (A, B) Embryos injected with Constructs Generation The open reading frame of her8a was PCR amplified with the primers 59-GAATTCGCCACCATGACGGCCTCCAACATG- GGC-39and 59-GGAATTCCTCACCAGGGCCTCCACATG- 39, which introduce EcoRI restriction sites suitable for cloning. The PCR product was digested with EcoRI and cloned into the PLoS ONE \| www.plosone.org April 2011 \| Volume 6 \| Issue 4 \| e19394 10 Her8a Is Essential to Inhibit Neurogenesis TCCAGAAGTT-39), slc1a3 (F: 59-GTAACGGGGAGACGC- GTCTGCAGCG-39; R: 59-GATTATTCCCACGATGACGG- CGGCG-39), gfap(F: 59-ACTGAGGAGTGGTATCGCTCAAA- 39; R: 59-AGACCCACGGAGAGATTCCA-39)and gapdh (F: 59- ACCCGTGCTGCTTTCTTGAC-39; R: 59-GACCAGTTT- GCCGCCTTCT-39) were used. Gene expression levels were normalized to gapdh and assessed using the comparative CT (40 cycles) according to the manufacturer’s instructions (Applied Biosystems). control morpholino. (C, D) Injection of 6 ng of MO1 (low dose) resulted in upregulation of HuC/D expression (E, F) 12 ng of MO1 injection (high dose) displayed more dramatic upregulation of HuC/D. (TIF) Figure S4 Over-expression of her8a mRNA did not alter the expression of proneural and pan-neuronal markers. Embryos were analyzed by immunohistochemistry with HuC/D antibody (A, B) and in situ hybridization with neurogenin1 (C, D) or zash1 (E, F). (A, C, E) Embryos were injected with GFP mRNA as control. (B, D, F) Injection of her8a mRNA revealed no significant deviation. For Western blot analysis, embryos were homogenized in SDS lysis buffer. 60ug were loaded on 12% SDS polyacrylamide gel and transferred to a PVDF membrane and detected with anti- HuC/HuD monoclonal antibody (1:1000, Invitrogen). After washes, membranes were incubated with goat anti-Mouse HRP- conjugated secondary Ab (Chemicon) and developed with ECL(Millipore). Band intensities were quantified using Multi Gaugre analysis software. Figure S5 No significant alteration can be detected in sox2 or Sox3 expressing neural progenitors in Her8a morphants. Embryo injection with control morpholino (A and C) or 8 ng of MO2 (B and D) analyzed with sox2 (A and B) or sox3 (C and D) riboprobes. sox2 and sox3 were expressed in the neural precursors located within the neuroectodermal region (arrow- heads). 75% epiboly; dorsal view, animal pole toward the top. (TIF) Statistical analysis was performed by student’s t-test using Microsoft ExcelH 2007. The significance level was set at P,0.05. All Reaction was performed in triplicate for each sample. Statistical analysis was performed by student’s t-test using Microsoft ExcelH 2007. The significance level was set at P,0.05. All Reaction was performed in triplicate for each sample. References 1. Louvi A, Artavanis-Tsakonas S (2006) Notch signalling in vertebrate neural development. Nat Rev Neurosci 7: 93–102. 10. Pasini A, Henrique D, Wilkinson DG (2001) The zebrafish Hairy/Enhancer-of- split-related gene her6 is segmentally expressed during the early development of hindbrain and somites. Mech Dev 100: 317–321. p 2. Roy M, Pear WS, Aster JC (2007) The multifaceted role of Notch in cancer. Curr Opin Genet Dev 17: 52–59. 11. Leve C, Gajewski M, Rohr KB, Tautz D (2001) Homologues of c-hairy1 (her9) and lunatic fringe in zebrafish are expressed in the developing central nervous system, but not in the presomitic mesoderm. Dev Genes Evol 211: 493–500. 3. Kageyama R, Ohtsuka T, Kobayashi T (2007) The Hes gene family: repressors and oscillators that orchestrate embryogenesis. Development 134: 1243–1251. 12. Zhong TP, Rosenberg M, Mohideen MA, Weinstein B, Fishman MC (2000) gridlock, an HLH gene required for assembly of the aorta in zebrafish. Science 287: 1820–1824. 4. Hans S, Scheer N, Riedl I, v Weizsacker E, Blader P, et al. (2004) her3, a zebrafish member of the hairy-E(spl) family, is repressed by Notch signalling. Development 131: 2957–2969. 13. Bae YK, Shimizu T, Hibi M (2005) Patterning of proneuronal and inter- proneuronal domains by hairy- and enhancer of split-related genes in zebrafish neuroectoderm. Development 132: 1375–1385. 5. Bae S, Bessho Y, Hojo M, Kageyama R (2000) The bHLH gene Hes6, an inhibitor of Hes1, promotes neuronal differentiation. Development 127: 2933–2943. 14. Takke C, Campos-Ortega JA (1999) her1, a zebrafish pair-rule like gene, acts downstream of notch signalling to control somite development. Development 126: 3005–3014. 6. Koyano-Nakagawa N, Kim J, Anderson D, Kintner C (2000) Hes6 acts in a positive feedback loop with the neurogenins to promote neuronal differentiation. Development 127: 4203–4216. 15. Ninkovic J, Tallafuss A, Leucht C, Topczewski J, Tannhauser B, et al. (2005) Inhibition of neurogenesis at the zebrafish midbrain-hindbrain boundary by the combined and dose-dependent activity of a new hairy/E(spl) gene pair. Development 132: 75–88. 7. Sieger D, Tautz D, Gajewski M (2004) her11 is involved in the somitogenesis clock in zebrafish. Dev Genes Evol 214: 393–406. 8. Takke C, Dornseifer P, v Weizsacker E, Campos-Ortega JA (1999) her4, a zebrafish homologue of the Drosophila neurogenic gene E(spl), is a target of NOTCH signalling. Development 126: 1811–1821. p 16. Gajewski M, Voolstra C (2002) Comparative analysis of somitogenesis related genes of the hairy/Enhancer of split class in Fugu and zebrafish. Author Contributions Conceived and designed the experiments: YCC. Performed the experi- ments: PCC WSL FYH HLW YCC. Analyzed the data: YCC. Contributed reagents/materials/analysis tools: PJS YCC. Wrote the paper: PJS YCC. Her8a Is Essential to Inhibit Neurogenesis 18. Fior R, Henrique D (2005) A novel hes5/hes6 circuitry of negative regulation controls Notch activity during neurogenesis. Dev Biol 281: 318–333. 31. Lefebvre V, Dumitriu B, Penzo-Mendez A, Han Y, Pallavi B (2007) Control of cell fate and differentiation by Sry-related high-mobility-group box (Sox) transcription factors. Int J Biochem Cell Biol 39: 2195–2214. controls Notch activity during neurogenesis. Dev Biol 281: 318– y g g 19. Belanger-Jasmin S, Llamosas E, Tang Y, Joachim K, Osiceanu AM, et al. (2007) Inhibition of cortical astrocyte differentiation by Hes6 requires amino- and carboxy-terminal motifs important for dimerization and phosphorylation. J Neurochem 103: 2022–2034. 32. Okuda Y, Yoda H, Uchikawa M, Furutani-Seiki M, Takeda H, et al. (2006) Comparative genomic and expression analysis of group B1 sox genes in zebrafish indicates their diversification during vertebrate evolution. Dev Dyn 235: 811–825. 20. Jhas S, Ciura S, Belanger-Jasmin S, Dong Z, Llamosas E, et al. (2006) Hes6 inhibits astrocyte differentiation and promotes neurogenesis through different mechanisms. J Neurosci 26: 11061–11071. 33. Shih YH, Kuo CL, Hirst CS, Dee CT, Liu YR, et al. (2010) SoxB1 transcription factors restrict organizer gene expression by repressing multiple events downstream of Wnt signalling. Development 137: 2671–2681. J 21. Latimer AJ, Dong X, Markov Y, Appel B (2002) Delta-Notch signaling induces hypochord development in zebrafish. Development 129: 2555–2563. 34. Storck T, Schulte S, Hofmann K, Stoffel W (1992) Structure, expression, and functional analysis of a Na(+)-dependent glutamate/aspartate transporter from rat brain. Proc Natl Acad Sci U S A 89: 10955–10959. 22. Latimer AJ, Shin J, Appel B (2005) her9 promotes floor plate development in zebrafish. Dev Dyn 232: 1098–1104. 23. Fadool JM, Dowling JE (2008) Zebrafish: a model system for the study of eye genetics. Prog Retin Eye Res 27: 89–110. 35. Shibata T, Yamada K, Watanabe M, Ikenaka K, Wada K, et al. (1997) Glutamate transporter GLAST is expressed in the radial glia-astrocyte lineage of developing mouse spinal cord. J Neurosci 17: 9212–9219. 24. Hu M, Easter SS (1999) Retinal neurogenesis: the formation of the initial central patch of postmitotic cells. Dev Biol 207: 309–321. patch of postmitotic cells. Dev Biol 207: 309–321. p g p J 36. Robu ME, Larson JD, Nasevicius A, Beiraghi S, Brenner C, et al. (2007) p53 activation by knockdown technologies. PLoS Genet 3: e78. 25. Fischer A, Gessler M (2007) Delta-Notch—and then? References BMC Genomics 3: 21. p 16. Gajewski M, Voolstra C (2002) Comparative analysis of somitogenesis related genes of the hairy/Enhancer of split class in Fugu and zebrafish. BMC Genomics 3: 21. 9. Geling A, Plessy C, Rastegar S, Strahle U, Bally-Cuif L (2004) Her5 acts as a prepattern factor that blocks neurogenin1 and coe2 expression upstream of Notch to inhibit neurogenesis at the midbrain-hindbrain boundary. Develop- ment 131: 1993–2006. 9. Geling A, Plessy C, Rastegar S, Strahle U, Bally-Cuif L (2004) Her5 acts as a prepattern factor that blocks neurogenin1 and coe2 expression upstream of Notch to inhibit neurogenesis at the midbrain-hindbrain boundary. Develop- ment 131: 1993–2006. 17. Davis RL, Turner DL (2001) Vertebrate hairy and Enhancer of split related proteins: transcriptional repressors regulating cellular differentiation and embryonic patterning. Oncogene 20: 8342–8357. 17. Davis RL, Turner DL (2001) Vertebrate hairy and Enhancer of split related proteins: transcriptional repressors regulating cellular differentiation and embryonic patterning. Oncogene 20: 8342–8357. PLoS ONE \| www.plosone.org 11 April 2011 \| Volume 6 \| Issue 4 \| e19394 Her8a Is Essential to Inhibit Neurogenesis Protein interactions and proposed modes of repression by Hes and Hey bHLH factors. Nucleic Acids Res 35: 4583–4596. activation by knockdown technologies. PLoS Genet 3: e78. 37. Sela-Donenfeld D, Kayam G, Wilkinson DG (2009) Boundary cells regulate a switch in the expression of FGF3 in hindbrain rhombomeres. BMC Dev Biol 9: 16. 26. Jiang YJ, Brand M, Heisenberg CP, Beuchle D, Furutani-Seiki M, et al. (1996) Mutations affecting neurogenesis and brain morphology in the zebrafish, Danio rerio. Development 123: 205–216. 38. Kawamura A, Koshida S, Hijikata H, Sakaguchi T, Kondoh H, et al. (2005) Zebrafish hairy/enhancer of split protein links FGF signaling to cyclic gene expression in the periodic segmentation of somites. Genes Dev 19: 1156–1161. p 27. Itoh M, Kim CH, Palardy G, Oda T, Jiang YJ, et al. (2003) Mind bomb is a ubiquitin ligase that is essential for efficient activation of Notch signaling by Delta. Dev Cell 4: 67–82. 39. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25: 4876–4882. 28. Ramain P, Khechumian K, Seugnet L, Arbogast N, Ackermann C, et al. (2001) Novel Notch alleles reveal a Deltex-dependent pathway repressing neural fate. Curr Biol 11: 1729–1738. 40. Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF (1995) Stages of embryonic development of the zebrafish. Dev Dyn 203: 253–310. 29. Wettstein DA, Turner DL, Kintner C (1997) The Xenopus homolog of Drosophila Suppressor of Hairless mediates Notch signaling during primary neurogenesis. Development 124: 693–702. 41. Xu Q, Alldus G, Holder N, Wilkinson DG (1995) Expression of truncated Sek-1 receptor tyrosine kinase disrupts the segmental restriction of gene expression in the Xenopus and zebrafish hindbrain. Development 121: 4005–4016. 30. Cheng YC, Amoyel M, Qiu X, Jiang YJ, Xu Q, et al. (2004) Notch activation regulates the segregation and differentiation of rhombomere boundary cells in the zebrafish hindbrain. Dev Cell 6: 539–550. 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https://openalex.org/W2132774628	https://europepmc.org/articles/pmc3401951?pdf=render	English	null	Development of visual working memory precision in childhood	Developmental science	2,012	cc-by	10,321	Abstract Visual working memory (VWM) is the facility to hold in mind visual information for brief periods of time. Developmental studies have suggested an increase during childhood in the maximum number of complete items that can simultaneously be stored in VWM. Here, we exploit a recent theoretical and empirical innovation to investigate instead the precision with which items are stored in VWM, where precision is a continuous measure reflecting VWM resolution. Ninety boys aged 7 to 13 years completed one-item and three-item VWM tasks in which stimuli were coloured bars varying in orientation. On each trial, participants used a rotating dial to reproduce the probed stimulus from memory. Results show linear age-related improvement in recall precision for both one-item and three-item VWM tasks. However, even the youngest age group stored a significant amount of information about all three items on the difficult 3-item VWM task. Importantly, the development of VWM precision was not accounted for by development on a sensorimotor control task. Whereas storage of a single complete item was previously thought to be well within the capacity limitations of the current age range, these results suggest protracted development during childhood and early adolescence in the resolution with which single and multiple items are stored in VWM. Probabilistic modelling of response distribution data suggests that improvement in VWM performance is attributable to a specific decrease in variability of stored feature representations, rather than to a decrease in misbinding or random noise. As such, we highlight a novel, potentially developmentally plausible mechanism that may underlie developmental improvement in VWM performance, independent of any alterations in the maximum number of complete items which can be stored. Development of visual working memory precision in childhood Stephanie Burnett Heyes,1,2 Nahid Zokaei,2 Irene van der Staaij,2,3 Paul M. Bays2 and Masud Husain2 1. Department of Experimental Psychology, University of Oxford, UK 2. UCL Institute of Cognitive Neuroscience and UCL Institute of Neurology, UK 3. Department of Psychology, Vrije Universiteit, The Netherlands Developmental Science 15:4 (2012), pp 528–539 DOI: 10.1111/j.1467-7687.2012.01148.x Developmental Science 15:4 (2012), pp 528–539 DOI: 10.1111/j.1467-7687.2012.01148.x Developmental Science 15:4 (2012), pp 528–539 Address for correspondence: Stephanie Burnett Heyes, Department of Experimental Psychology, University of Oxford, South Parks Road, Oxford OX1 3UD, UK; e-mail: burstephanie@gmail.com Introduction is held in mind during a delay. These well-validated par- adigms have demonstrated, within large datasets, robust age trajectories and evidence for developmental stability in the relationship of VWM to other cognitive compo- nents (Gathercole et al., 2004). Visual working memory (VWM) or visual short-term memory,1 is the facility to hold in mind visual informa- tion for brief periods of time (Luck & Vogel, 1997). This ability is considered to be a fundamental cognitive pro- cess, essential for complex reasoning, decision-making and goal-directed action (Baddeley, 2003). Developmen- tal studies have shown that performance on well-estab- lished neuropsychological tests of VWM improves linearly during childhood (Alloway, Gathercole & Pic- kering, 2006; Gathercole, Pickering, Ambridge & Wear- ing, 2004). Typically in these paradigms, participants view a static visual array (e.g. coloured shapes) or spatiotem- poral sequence of visual events (e.g. block tapping) which However, the component mechanisms that underlie the development of VWM during childhood remain to be established (Astle & Scerif, 2011). What actually improves through development of VWM? One major focus of research has been on improvements in the capacity of working memory, i.e. the number of items that can be held in VWM, using visual change detection paradigms (Luck & Vogel, 1997). In such tasks, participants judge whether a test array of visual items differs (‘change’) or does not differ (‘no change’) from a comparison array presented following a brief retention period. Test and comparison arrays are either identical, or differ by a feature of one item (e.g. colour, location). Proportion correct judgments, sensi- tivity (d’) and estimates of the maximum number of items encoded (e.g. Cowan’s K) increase with age during 1 Some authors use the term visual short-term memory to refer to a passive store while visual working memory refers to active manipulation of items in memory. We make no such distinction here and, like some other investigators, we use visual working memory to include storage processes. Introduction The findings are consistent with the view that the mature visual system can maintain a maximum of 3–4 complete items in working memory ‘slots’ at any one time (Luck & Vogel, 1997). We applied a previously validated probabilistic model to characterize sources of error in VWM across age in the three-item task (Bays et al., 2009; Zhang & Luck, 2008). This enabled us to test whether effects of age on VWM precision are due to a change in the variability of stored features, a change in the number of items which completely fail to be stored, or some other factor, for example a change in the frequency of misbinding (Cowan et al., 2006a). Recently, an alternative theoretical and empirical approach to VWM has been developed in studies of adult participants. This approach investigates working memory precision, where precision is a continuous measure reflecting the resolution of items held in VWM (Bays & Husain, 2008; Bays, Wu & Husain, 2011b; Fougnie, Asplund & Marois, 2010; Gorgoraptis, Catalao, Bays & Husain, 2011; Wilken & Ma, 2004; Zhang & Luck, 2008). In precision paradigms, participants typically view either a simultaneous or a sequential array of items. Following a short delay, they are prompted to reproduce a given fea- ture of one of the items (e.g. bar orientation) using the method of adjustment (Stevens, 1958). Precision is calcu- lated as 1⁄standard deviation (SD) of error in response. Because there is evidence for continuing maturation of fine motor precision and sensorimotor co-ordination across the age range of our sample (Pehoski, Henderson & Tickle-Degnen, 1997), we administered a sensorimotor control task to correct for any such factors as potential confounding effects on VWM precision estimates. Finally, to investigate the construct validity of our VWM precision measure, we investigated its relationship to indices of intelligence (IQ). Crucially, VWM precision shows a robust dependency on the total number of items presented (Bays & Husain, 2008; Bays, Catalao & Husain, 2009; Gorgoraptis et al., 2011). Thus, with increasing memory load, each feature is stored with decreasing precision, but importantly the number of items that can be stored need not be limited: With larger set sizes, each item is stored with more var- iance. Introduction : Stephanie Burnett Heyes, Department of Experimental Psychology, University of Oxford, South Parks Road, Oxford stephanie@gmail.com Address for correspondence: Stephanie Burnett Heyes, Department of Experimental Psychology, University of Oxford, South Parks Road, Oxford OX1 3UD, UK; e-mail: burstephanie@gmail.com Re-use of this article is permitted in accordance with the Terms and Conditions set out at http://wileyonlinelibrary.com/onlineopen#OnlineOpen_ Terms mitted in accordance with the Terms and Conditions set out at http://wileyonlinelibrary.com/onlineopen#OnlineOpen_ Re-use of this article is permitted in accordance with the Terms and Conditions set out at http://wileyonlinelibrary.com/on Terms 2012 Blackwell Publishing Ltd, 9600 Garsington Road, Oxford OX4 2DQ, UK and 350 Main Street, Malden, MA 02148, USA. Blackwell Publishing Ltd, 9600 Garsington Road, Oxford OX4 2DQ, UK and 350 Main Street, Malden, MA 02148, USA Development of visual working memory precision 529 childhood (Cowan, Fristoe, Elliott, Brunner & Saults, 2006a; Cowan, Morey, AuBuchon, Zwilling & Gilchrist, 2010; Cowan, Naveh-Benjamin, Kilb & Saults, 2006b; Riggs, McTaggart, Simpson & Freeman, 2006; Riggs, Simpson & Potts, 2011). (Cowan et al., 2006a; Riggs et al., 2011). To our knowl- edge, no previous study has shown continuing develop- ment within the current age range in VWM for a single item. In the current study, an age-associated increase in precision on the one-item VWM task would therefore provide novel evidence for the development of compo- nents other than estimated maximum capacity for com- plete items held in VWM. Such a result would potentially highlight a distinct developmental mechanism underlying observed improvements in VWM performance with age. (Cowan et al., 2006a; Riggs et al., 2011). To our knowl- edge, no previous study has shown continuing develop- ment within the current age range in VWM for a single item. In the current study, an age-associated increase in precision on the one-item VWM task would therefore provide novel evidence for the development of compo- nents other than estimated maximum capacity for com- plete items held in VWM. Such a result would potentially highlight a distinct developmental mechanism underlying observed improvements in VWM performance with age. These results suggest that improvement during child- hood could be due at least in part to a discrete increase in VWM capacity, that is, the maximum number of com- plete items that can be held in VWM (Cowan, Elliott, Saults, Morey, Mattox, Hismjatullina & Conway, 2005; Riggs et al., 2006). Introduction These findings have led to the proposal that VWM might best be considered a limited resource that can be distributed flexibly among memoranda, but without any limit to the number of complete items that can be stored (Bays & Husain, 2008; Wilken & Ma, 2004). 2012 Blackwell Publishing Ltd. Colour naming task Colour naming task Figure 1 Precision tasks: (a) Sensorimotor precision control task (25 trials). A rotating dial is used to match the orientation of the probe bar (above, in circle) to that of the target bar (below). The probe bar remains on screen. (b) One-item visual working memory task (30 trials). Following a delay, the par- ticipant uses a rotating dial to match the orientation of the probe bar (below, in circle) to the remembered orientation of the target bar (above). Dotted lines denote a blank intervening delay (500 ms). (c) Three-item visual working memory task (90 trials). Following a delay, the participant uses a rotating dial to match the orientation of the probe bar (bottom, in circle) to that of the remembered orientation of the equivalently coloured target bar presented in sequence. Dotted lines denote blank intervening delays (500 ms). Figure 1 Precision tasks: (a) Sensorimotor precision control task (25 trials). A rotating dial is used to match the orientation of the probe bar (above, in circle) to that of the target bar (below). The probe bar remains on screen. (b) One-item visual working memory task (30 trials). Following a delay, the par- ticipant uses a rotating dial to match the orientation of the probe bar (below, in circle) to the remembered orientation of the target bar (above). Dotted lines denote a blank intervening delay (500 ms). (c) Three-item visual working memory task (90 trials). Following a delay, the participant uses a rotating dial to match the orientation of the probe bar (bottom, in circle) to that of the remembered orientation of the equivalently coloured target bar presented in sequence. Dotted lines denote blank intervening delays (500 ms). A colour naming task was administered at the start of the experimental session. Participants were shown five screenshots from the VWM task, each containing a bar in one of five stimulus colours: red, yellow, green, blue and pink. Participants named aloud each of the five colours. Any participant who did not give 5⁄5 correct responses was excluded from all analyses. Participants Following a delay, the participant uses a rotating dial to match the orientation of the probe bar (bottom, in circle) to that of the remembered orientation of the equivalently coloured target bar presented in sequence. Dotted lines denote blank intervening delays (500 ms). Sensorimotor precision task 1-item visual working memory task 3-item visual working memory task (a) (b) (c) Figure 1 Precision tasks: (a) Sensorimotor precision control task (25 trials). A rotating dial is used to match the orientation of the probe bar (above, in circle) to that of the target bar (below). The probe bar remains on screen. (b) One-item visual working memory task (30 trials). Following a delay, the par- ticipant uses a rotating dial to match the orientation of the probe bar (below, in circle) to the remembered orientation of the target bar (above). Dotted lines denote a blank intervening delay (500 ms). (c) Three-item visual working memory task (90 trials). Following a delay, the participant uses a rotating dial to match the orientation of the probe bar (bottom, in circle) to that of the remembered orientation of the equivalently coloured target bar presented in sequence. Dotted lines denote blank intervening delays (500 ms). 1-item visual working memory task (b) Sensorimotor precision task 1-item visual working memory task (a) (b) Table 1 Participant information School year (grade) N Age range (years) Mean (SD) age Mean (SD) FSIQe 3 2 7.9–8.1 8.00 (.141) 4 14 8.8–10.3 9.30 (.440) 110.72 (13.73) 5 17 9.5–10.7 10.22 (.341) 110.16 (13.89) 6 18 9.9–11.7 11.24 (.430) 111.72 (11.70) 7 18 11.5–12.8 12.17 (.341) 115.54 (10.47) 8 18 12.8–13.6 13.22 (.260) 120.24 (11.66) Due to small N we collapsed years 3 and 4. Sensorimotor precision task (a) Table 1 Participant information (c) (a) Due to small N we collapsed years 3 and 4. Sensorimotor control task Participants completed 25 trials of a sensorimotor con- trol task (Figure 1a), directly after completion of the colour naming task. On each trial, a coloured oriented target bar (approx. 2 · 0.3 of visual angle) appeared on a grey background. After a 500 ms delay, a probe bar of the same colour appeared above the target bar, sur- rounded by a dark grey circle. Participants used a rotating dial (Griffin Powermate; Griffin Technology, Nashville, USA) to adjust the probe bar orientation to match that of the target, and when they were satisfied with the match they clicked the dial to proceed to the next trial. Response time was unconstrained. Note that in this task, the target bar stayed on-screen throughout the trial. motor control task. The task was identical to the sen- sorimotor control task, with the exception that a 500 ms delay (blank screen) intervened between the target and the probe stimulus, and the target and probe appeared at the same, central location. Thus, participants now rotated the probe stimulus to match the remembered orientation of the target. Participants were prompted every 15 trials by the task programme to take a short break. During this break participants were encouraged to focus on a far point in the room, to minimize ocular fatigue. Bar colour was drawn at random on each trial from the possible colour set (red, yellow, green, blue, pink). Target and probe orientation were independently ran- domized across p rad on each trial. The inter-trial interval (ITI) was 500 ms. Materials and measures A colour naming task, sensorimotor control task and two visual working memory (VWM) tasks were admin- istered on a laptop computer with the screen at a viewing distance of 60 cm. Participants were tested individually by one of two experimenters in one of two quiet rooms in school, during school hours. The duration of the exper- imental session was 35 minutes per participant, timed to correspond to a single 35-minute school lesson (period). 2012 Blackwell Publishing Ltd. Participants Ninety participants were recruited from a single-sex (male) preparatory school. Boys were selected randomly from each school year by a teacher, with the caveat that the developmental disorders dyspraxia and attention- deficit⁄hyperactivity disorder be excluded. Parental consent was given for each participant. The study was approved by the local ethics committee. We excluded three boys with suspected colour blind- ness who could not perform a colour-naming control task or commented that ‘two bars were the same colour’ in the three-item VWM task. The final sample consisted of 87 participants aged 7.9–13.6 (mean 11.26 years, SD 1.48; see Table 1). What happens to VWM precision during childhood? In the context of data showing discrete increases in the capacity to store complete items, it is possible that mean precision per successfully encoded item simply remains constant across age. Alternatively, precision might improve, indicating changes in the resource that can be deployed to maintain individual visual items. In the current study, we tested these competing hypotheses in a cross-sectional sample of 90 children aged 7–13 years, using a variant of a computerized, sequential VWM task that has been validated in adults (Gorgoraptis et al., 2011). In this paradigm, coloured bars of varying orien- tation were presented one at a time at a central location. Standardized yearly test scores (CAT-3; http://www. gl-assessment.co.uk) were provided by the school for all but one participant. We used these scores to estimate full- scale (FS) IQ, as per Wright, Strand and Wonders (2005): FSIQe ¼ 1:1 CAT-Av 12:0 ð1Þ ð1Þ where FSIQe is estimated FSIQ and CAT-Av is average CAT-3 score calculated by combining standardized scores on verbal, non-verbal and quantitative reasoning subtests (Wright et al., 2005). 2 where FSIQe is estimated FSIQ and CAT-Av is average CAT-3 score calculated by combining standardized scores on verbal, non-verbal and quantitative reasoning subtests (Wright et al., 2005). 2 Crucially, we administered both one-item and three- item versions of the task. Developmental studies using change detection paradigms estimate that during middle childhood (e.g. age 10 years) the maximum capacity of VWM is limited to between two and three complete items FSIQe was correlated with age (r2 = .069, p = .014). Therefore, we used FSIQe as a covariate in subsequent analyses. 530 Stephanie Burnett Heyes et al. Participants Materials and measures A colour naming task, sensorimotor control task and two visual working memory (VWM) tasks were admin- istered on a laptop computer with the screen at a viewing distance of 60 cm. Participants were tested individually by one of two experimenters in one of two quiet rooms in school, during school hours. The duration of the exper- imental session was 35 minutes per participant, timed to correspond to a single 35-minute school lesson (period). Colour naming task A colour naming task was administered at the start of the experimental session. Participants were shown five screenshots from the VWM task, each containing a bar in one of five stimulus colours: red, yellow, green, blue and pink. Participants named aloud each of the five colours. Any participant who did not give 5⁄5 correct responses was excluded from all analyses. Sensorimotor control task Participants completed 25 trials of a sensorimotor con- trol task (Figure 1a), directly after completion of the colour naming task. On each trial, a coloured oriented target bar (approx. 2 · 0.3 of visual angle) appeared on b k d Aft 500 d l b b f t t l t k Th t k id ti l t th Table 1 Participant information School year (grade) N Age range (years) Mean (SD) age Mean (SD) FSIQe 3 2 7.9–8.1 8.00 (.141) 4 14 8.8–10.3 9.30 (.440) 110.72 (13.73) 5 17 9.5–10.7 10.22 (.341) 110.16 (13.89) 6 18 9.9–11.7 11.24 (.430) 111.72 (11.70) 7 18 11.5–12.8 12.17 (.341) 115.54 (10.47) 8 18 12.8–13.6 13.22 (.260) 120.24 (11.66) *Due to small N we collapsed years 3 and 4. Sensorimotor precision task 1-item visual working memory task 3-item visual working memory task (a) (b) (c) Figure 1 Precision tasks: (a) Sensorimotor precision control task (25 trials). A rotating dial is used to match the orientation of the probe bar (above, in circle) to that of the target bar (below). The probe bar remains on screen. (b) One-item visual working memory task (30 trials). Following a delay, the par- ticipant uses a rotating dial to match the orientation of the probe bar (below, in circle) to the remembered orientation of the target bar (above). Dotted lines denote a blank intervening delay (500 ms). (c) Three-item visual working memory task (90 trials). Development of visual working memory precision Development of visual working memory precision 531 blank inter-stimulus interval. Sequential rather than simultaneous presentation was used, in order to mini- mize the effect of potential age differences in the ability to efficiently distribute spatial attention among items in a scene (Enns & Girgus, 1985; Lane & Pearson, 1983). Bar colour was drawn at random without replacement on each trial from the possible colour set used in the colour naming task (comprising five colours). Bar orientation (stimuli, probe) was independently randomized across p rad on each trial, with the constraint that the three stimulus bars must be at a minimum of 0.175 rad sepa- ration. Following a blank 500 ms duration after the last test bar, a probe bar of the same colour as one of the test bars appeared. Participants rotated the probe bar to match the remembered orientation of the target, and the remaining stimuli were not probed. All items in the sequence were probed with equal probability. sonable to assume additivity of variance, i.e. that sen- sorimotor and recall errors from individual and multiple items contribute independently to response variability. However, variance is not distributed normally in our tasks, so we describe below methods implemented to compare age effects across task and serial position, i.e. interactions. At the outset we tested whether precision in the sen- sorimotor control task was correlated with age. A posi- tive result entailed correction for sensorimotor precision of raw precision values in the one-item and three-item VWM tasks. Assuming that sensorimotor errors and recall errors contribute independently to response vari- ability on VWM trials, recall precision can be estimated from the difference between VWM error variance (i.e. SD2) and sensorimotor error variance, i.e. corrected VWM precision = 1⁄(SDVWM 2 – SDSM 2). This correc- tion is implemented throughout. Participants were encouraged every 15 trials to take a break. Four participants did not complete all 90 trials of the three-item task due to lack of time. Mean one-item and three-item VWM precision values, corrected for sensorimotor performance, were interro- gated for effects of participant age using linear regression and partial correlation controlling for FSIQe. For the three-item VWM task, mean precision was calculated by averaging variance across serial positions of the target. We predicted effects of age on precision in the VWM tasks that were not entirely attributable to developmental improvement in sensorimotor precision. Development of visual working memory precision To guide inter- pretation of any age effects, we used one-sample t-tests to evaluate whether performance was above chance in each year group and task (in particular, younger year groups and the three-item VWM task). 2012 Blackwell Publishing Ltd. Analysis We investigated our dependent variables (see below) for effects of participant age and in some cases school year group (i.e. grade). Year groups 3 and 4 were collapsed due to low N in year group 3 (see Table 1). The pattern of results was the same when students were grouped according to age quintiles (data available on request). Outliers > 2.5 SD from the sample mean of each dependent variable were excluded for that variable only (for further details, see footnotes. If it is not specified that outliers were excluded, there were no outliers). Statistical significance is p < .05 two-tailed unless otherwise speci- fied. To compare age effects on VWM precision across tasks, we calculated precision differences. This was implemented by subtracting raw one-item error variance from raw three-item error variance, and recalculat- ing precision accordingly, i.e. precision difference = 1⁄ (SDVWM(3) 2 – SDVWM(1) 2). A significant correlation between age and this dependent variable would imply a specific effect of age on storage or recall of multiple items in VWM, i.e. an age effect on the degree of independence of error in the recall of multiple items (e.g. age-sensitive constant attention or decay cost). An absence of associ- ation would constitute no evidence for differential rates of development between single- and multiple-item tasks. Visual working memory task: three-item condition Participants completed 90 trials of a three-item VWM task (see Figure 1c), subsequent to completion of the one-item task. The task had a similar format to that of the one-item VWM task. On each trial, three coloured oriented test bars appeared sequentially on the screen for a duration of 500 ms per stimulus, separated by a 500 ms Visual working memory task: one-item condition Participants completed 30 trials of a one-item VWM task (Figure 1b), subsequent to completion of the sensori- 2012 Blackwell Publishing Ltd. Precision For each trial of the sensorimotor, one-item VWM and three-item VWM tasks, we calculated the angular devi- ation between the response orientation and the original orientation of the target, i.e. the angular error. Precision was then calculated as the reciprocal of the standard deviation of error across trials (1⁄SD). Since the parameter space for orientation is circular, we used Fisher’s definition of SD for circular data (Fisher, 1996), subtracting the value expected for chance, so that a precision value of zero corresponds to responding at random. This method has been described previously (Bays & Husain, 2008; Bays et al., 2009; Bays et al., 2011b; Gorgoraptis et al., 2011), and provides a simple and intuitive measure of the fidelity with which the target orientation was reproduced. Note that it would not be valid to interpret an inter- action resulting from an ANOVA on precision across age group and task in the usual way. This is because ANOVA assumes additivity (i.e. linearity) of the dependent vari- able across conditions. However, precision does not be- have in this way. As such, a significant interaction in repeated measures ANOVA would merely be consistent with our understanding that precision does not behave linearly; it would not necessarily indicate an interesting result. Instead, it is justified to assume additivity of variance (1⁄precision2), i.e. independent contribution to response variability of VWM errors from individual and multiple items. However, since response variability is not distributed normally, this violates the assumptions of Note that it is not justified to test for a group · task (or group · serial position) interaction in precision using repeated measures ANOVA, since precision is not expected to show additive effects across group and task (or group and serial position). Rather, it is more rea- 532 32 Stephanie Burnett Heyes et al. ANOVA. Hence, our alternative analysis described above, the output of which is conceptually equivalent to the output of a repeated measures ANOVA on group and task. non-target orientation (the orientation of a bar observed in a sequence, but different from the colour of the probed bar) would constitute a misbinding error (Bays et al., 2009). The model is described as follows: To evaluate serial order effects, we calculated mean precision for each of the three serial positions (SP) of the three-item VWM task, i.e. first (SP1), second (SP2) and third item in sequence (SP3). Relationship to FSIQe We investigated the relationship between FSIQe and mean three-item VWM precision, corrected for sensori- motor precision and standardized within each year group, using linear regression. However, in order to investigate more formally the distribution of responses, and thence to identify potential mechanisms underlying age-related improvement in VWM performance, we fit a probabilistic model (Bays et al., 2009) to each participant dataset. This model has the potential to offer insights into which aspect of VWM performance might be altering with development. Four parameters (see Figure 5a) were extracted for each par- ticipant: j (kappa), a concentration parameter encapsu- lating Gaussian variability in memory for target orientations, and parameters representing the probability of reporting the target orientation, a non-target orienta- tion and a random orientation. Note that reporting a Distribution of responses Precision gives an indication of the overall variability in responses. However, we wanted to determine how this variability was distributed across feature space. This could give some indication of sources of error in VWM. Therefore, to visualize these data we plotted the fre- quency of responses at each of nine arbitrary orientation bins spaced evenly across p rad of response space in the three-item VWM task, first relative to the target orien- tation and then relative to the non-target (unprobed) stimuli in a sequence. Precision Effects across the sample of the serial position of the probed item were evaluated using one-way ANOVA (within-subjects factor: SP). We expected to observe a recency effect, i.e. an advantage for the item presented last (Gorgoraptis et al., 2011; Hitch, Halliday, Schaafstal & Schraagen, 1988). To test for a differential effect of age on the recency effect, we calcu- lated precision differences between the final (SP3) and preceding (SP1⁄2) items using the method outlined above. A significant result would imply age differences in the degree of independence of error in recall of multiple, serially presented items (e.g. age-sensitive constant attention or decay cost). pð^hÞ ¼ a/kð^h hÞ þ b 1 m X m i /kð^h /iÞ þ c 1 2p ð2Þ ð2Þ h is the true orientation of the target item, ^h the orientation reported by the participant and /k is the von Mises distribution (the circular analogue of the Gaussian distribution) with mean zero and concentration param- eter j (kappa). The probability of reporting the target item, p(T) is given by a, the probability of reporting a non-target item and p(NT) is given by b. {u1, u2 … um} are the orientations of the m non-target items, and the probability of responding at random, p(U) is given by c = 1 – a - b. Maximum likelihood estimates (Myung, 2003) of the parameters j, a, b and c were obtained separately for each participant for the three-item VWM task (mean across serial positions of target), using an expectation- maximization algorithm (MATLAB code available at http://www.sobell.ion.ucl.ac.uk/pbays/code/JV10/). Since the four parameters are non-independent, we tested for effects of age on each parameter separately, using lin- ear regression and partial correlation covarying out FSIQe. 2012 Blackwell Publishing Ltd. Results Sensorimotor precision improves with age Recency and serial position effects on the three-item task Recency and serial position effects on the three-item task Precision improved with age for each SP of the three-item VWM task4 (SP1: r2 adj = .120, p = .001; SP2: r2 adj = .112, p = .001; SP3: r2 adj = .124, p = .001). This improvement remained significant after covarying out FSIQe (SP1: q = .324, p = .003; SP2: q = .310, p = .005; SP3: q = .327, p = .003). Remarkably, VWM precision at each SP was above chance in all age groups (all p < .001; Working memory precision improves with age on the three-item task Working memory precision improves with age on the three-item task Mean precision across the three-item VWM task,2 i.e. calculated by averaging variance across serial position, improved significantly with age (r2 adj = .156, p < .001). This improvement remained significant after covarying out FSIQe (q = .333, p = .002). Precision was signifi- cantly above chance in all year groups (all p < .001; Table 2, Figure 2) indicating that even the youngest participants encoded information in this more difficult task. Figure 2 Mean precision in one-item and three-item VWM tasks, corrected for sensorimotor performance, increase linearly with age. Year group data (with mean ages) are shown for the purposes of illustration; inferential statistics were conducted using age as a continuous variable (see text). Improvement with age is greater on the three-item VWM task Sensorimotor precision improves with age Mean precision in the sensorimotor control task (see Table 2) improved significantly with age (r2 adj = .179, p < .001). We therefore factor this out in all subsequent analyses of VWM precision. Sensorimotor precision (1⁄SD error) was consistently higher than VWM preci- sion (see Table 2). Table 2 Mean (SD) precision values (rad)1) in each task by year group (mean age shown) and condition, text). All VWM precision values are corrected for sensorimotor precision values (rad)1) in each task by year group (mean age shown) and condition, excluding outliers (see are corrected for sensorimotor precision Table 2 Mean (SD) precision values (rad)1) in each task by year group (mean age shown) and condition, excluding outliers (see text). All VWM precision values are corrected for sensorimotor precision Age 9 Age 10 Age 11 Age 12 Age 13 Sensorimotor 8.08 (3.25) 8.44 (2.57) 9.00 (3.06) 12.17 (3.28) 11.93 (3.06) One-item VWM 2.08 (1.06) 2.70 (1.37) 3.14 (1.56) 3.67 (1.70) 3.87 (1.46) Three-item VWM .41 (.26) .50 (.25) .93 (.64) .79 (.51) 1.15 (.71) SP1 .39 (.24) .62 (.43) .85 (.54) .87 (.55) 1.10 (.81) SP2 .52 (.33) .60 (.49) .98 (.63) .76 (.46) 1.21 (.78) SP3 1.58 (1.23) 1.40 (.95) 2.61 (1.61) 2.94 (1.62) 2.98 (1.67) Development of visual working memory precision 533 Figure 2 Mean precision in one-item and three-item VWM tasks, corrected for sensorimotor performance, increase linearly with age. Year group data (with mean ages) are shown for the purposes of illustration; inferential statistics were conducted using age as a continuous variable (see text). Working memory precision improves with age on the one-item task Mean precision on the one-item VWM task improved significantly with age (r2 adj = .167, p < .001). This improve- ment remained significant after covarying out FSIQe (q = .339, p = .002). That is, VWM precision for a singly encoded item continues to develop between age 7 and 13 years. Precision was significantly above chance in all year groups (all p < .002; see Table 2, Figure 2). 2 Three outliers > 2.5 SD > mean excluded. 3 Three outliers > 2.5 SD > mean excluded. 4 SP1: two outliers > 2.5 SD > mean excluded; SP2: one outlier > 2.5 SD > mean excluded; SP3: four outliers > 2.5 SD > mean excluded. Improvement with age is greater on the three-item VWM task The precision difference between one-item and three-item VWM tasks3 showed an effect of age (r2 adj = .054, p = .019). This relationship remained significant after covarying out FSIQe (q = .187, p = .046 one-tailed). Thus, whereas we demonstrate significant improvement with age on both the one-item and three-item VWM tasks, the relative magnitude of improvement in VWM performance with age is greater for multiply encoded items. Table 2, Figure 3). That is, even though the youngest participants show substantial immaturity in precision for single items, they are able to store some information about each of the three sequentially presented items. There was an effect of target serial position (SP) on the precision of recall (F1,99 = 94.08, p < .001; DoF reduced to correct for non-sphericity; see Figure 3). Mean preci- sion was higher for stimuli presented in SP3 than in SP2 (t82 = 11.54, p < .001) and SP1 (t80 = 11.58, p < .001); items presented in SP1 and SP2 did not differ significantly in precision from one another (p > .05). Thus, the last item in a sequence was remembered with greater precision than previous items, as expected. In other words, the contribution to VWM variability of recall errors from multiple items becomes more inde- pendent with age, although this effect is modest. This implies a specific effect of age on storage or recall of multiple items in VWM. This could arise, for example, due to an age-sensitive constant attention or decay cost for each item. The difference in precision between SP3 and SP1⁄2 mean showed a significant effect of age (r2 adj = .083, p = .003; covarying out FSIQe: q = .266, p = .017). 2012 Blackwell Publishing Ltd. 2 Three outliers > 2.5 SD > mean excluded. 3 3 Three outliers > 2.5 SD > mean excluded. 4 Distribution of responses To visualize response distributions in the three-item VWM task we plotted the frequency of responses at each of nine orientation bins spaced evenly across p rad of response space with reference to target and non-target orientations. As shown in Figure 4, the variability of responses around the target alters with age (note width at half-maximum height). However, the likelihood of responding to a non-target is extremely low, and con- sistent across age groups (flat response distribution; not shown). 2012 Blackwell Publishing Ltd. 534 Stephanie Burnett Heyes et al. 534 To evaluate more formally these observations, we fit a probabilistic model (Figure 5a) to each participant (r2 adj = .023, b = .456, p = .045 one-tailed), but again this was not the case for the remaining model parameters (all p > .05). Serial position Mean precision (1/radian) [corrected for sensorimotor precision] Mean age 13 12 9 10 11 Figure 3 Mean precision improves with age for items pre- sented at each serial position. Precision is highest for the most recent item in a sequence, and this effect becomes more pro- nounced with age. Year group data (with mean ages) are shown for the purposes of illustration; inferential statistics were conducted using age as a continuous variable (see text). Mean age 13 12 9 10 11 Proportion of responses Distribution of responses around target (rad) –1.5 –1 –0.5 0 0.5 1 1.5 0 5 10 15 20 25 30 35 Figure 4 Distribution of responses with respect to target narrows with age: With increasing age, there is a decrease in variability of responses around the target orientation (three- item VWM task). This decrease in variability with age is cap- tured within our model by the concentration parameter, kappa. Mean age 13 12 9 10 11 Proportion of responses Distribution of responses around target (rad) –1.5 –1 –0.5 0 0.5 1 1.5 0 5 10 15 20 25 30 35 Figure 4 Distribution of responses with respect to target narrows with age: With increasing age, there is a decrease in variability of responses around the target orientation (three- item VWM task). This decrease in variability with age is cap- tured within our model by the concentration parameter, kappa. Distribution of responses Serial position Mean precision (1/radian) [corrected for sensorimotor precision] Mean age 13 12 9 10 11 Mean age 13 12 9 10 11 Proportion of responses Distribution of responses around target (rad) –1.5 –1 –0.5 0 0.5 1 1.5 0 5 10 15 20 25 30 35 Proportion of responses Figure 4 Distribution of responses with respect to target narrows with age: With increasing age, there is a decrease in variability of responses around the target orientation (three- item VWM task). This decrease in variability with age is cap- tured within our model by the concentration parameter, kappa. Figure 3 Mean precision improves with age for items pre- sented at each serial position. Precision is highest for the most recent item in a sequence, and this effect becomes more pro- nounced with age. Year group data (with mean ages) are shown for the purposes of illustration; inferential statistics were conducted using age as a continuous variable (see text). (r2 adj = .023, b = .456, p = .045 one-tailed), but again this was not the case for the remaining model parameters (all p > .05). To evaluate more formally these observations, we fit a probabilistic model (Figure 5a) to each participant dataset5 and evaluated age effects on each of the four parameters. This showed that kappa or the ‘concentra- tion’ parameter increased significantly with participant age (r2 adj = .133, p < .001; covarying out FSIQe: q = .324, p = .003; for year group mean parameter values, see Figure 5b). Since kappa is inversely related to variance, this indicates an age-associated decrease in Gaussian VWM variance for target recall. Discussion The current study was conducted to investigate the development of precision in VWM during middle child- hood and early adolescence. Results show that the pre- cision with which items are recalled from VWM increases with age in a cross-sectional sample aged 7–13 years. This was the only significant effect of age on the four model parameters. Thus there was no significant effect of age on the probability of responding to targets, responding to non-targets or making random guesses (all p > .05). Importantly, this improvement in precision was observed for items presented individually (one-item VWM task), as well as in sequences of three (three-item VWM task). These effects withstood correction for the effects of age on a control task requiring fine hand–eye co-ordination, so are not readily explicable on the basis of improvement in sensorimotor factors. Thus, regardless of any putative increase in the maximum number of items that can be stored, we have shown an age-associated increase in the precision with which items – and even a single item – are stored. This is consistent with an increase in resolution of a continuous, dynamic memory resource. 2012 Blackwell Publishing Ltd. Development of working memory precision Parameter value Previous studies have shown improvement across child- hood in performance on standard tests of VWM (Alloway et al., 2006; Gathercole et al., 2004). This improvement has been attributed to a discrete increase in capacity, that is, an increase in the maximum number of complete items that can be held in working memory (Cowan et al., 2005). In the current study we have shown evidence for an age-related increase in the resolution of items in VWM, whether presented individually or in sequences of three (Table 2; Figure 2). This represents a fundamen- tally different approach than estimating the maximum number of complete items encoded. Furthermore, to our knowledge, previous studies have not demonstrated development up to the age of 13 years in recall of items presented individually. Measuring precision, rather than estimating the maximum number of complete items stored, appears to be a particularly sensitive method for investigating VWM performance across age. Figure 5 Model shows decrease in concentration parameter kappa with age: (a) Schematic showing model parameters. Kappa, the ‘concentration’ parameter corresponds to variabil- Figure 5 Model shows decrease in concentration parameter kappa with age: (a) Schematic showing model parameters. Kappa, the ‘concentration’ parameter corresponds to variabil- ity in feature representations: in this case orientation. p(T), p(NT) and p(U) represent the probability of responding with the recalled target, non-target or a random orientation, respec- tively. (b) Model parameters in each year group. The concen- tration parameter, kappa increases with age. No other parameter shows an effect of age. Year group data (with mean ages) are shown for the purposes of illustration; inferential statistics were conducted using age as a continuous variable (see text). Figure 5 Model shows decrease in concentration parameter kappa with age: (a) Schematic showing model parameters. The effect of age on precision in the three-item VWM task amounted to a substantial decrease in SD error between the ages of 7 and 13 years (see Table 2). How- ever, at age 13, mean performance across serial positions of the three-item VWM task (1.15 rad)1) remained somewhat lower than that observed previously in adults, using a similar paradigm (see Figure 3 in Gorgoraptis et al., 2011: specifically, solid black line, y 1.5 rad)1 at x = 3 items). This raises the intriguing possibility that VWM precision may continue to develop beyond the upper limit of the age range in the current study. Relationship between precision and IQ Mean precision on the three-item VWM task, corrected for sensorimotor precision and standardized within each year group, accounted for a significant proportion of variance in FSIQe (r2 adj = .126, p = .001; Figure 6). Year-group ranked kappa was correlated with FSIQe Applying a probabilistic model (Bays et al., 2009; Gorgoraptis et al., 2011) to the data confirmed that the age-related increase in VWM precision was driven by a significant decrease in the variability of feature repre- sentations in working memory, rather than changes in the frequency of random guessing or misbinding errors. 5 Subsequent to model fitting, individually outlying parameter values were excluded: three outliers > 2.5 SD > mean for kappa and p(U); three outliers > 2.5 SD < mean for p(T); four outliers > 2.5 SD > mean for p(NT). Development of visual working memory precision 535 13 12 9 10 11 Response orientation Target orientation Non-target orientation Uniform error Response probability α1/k Mean age Parameter Parameter value n.s n.s n.s p<.001 α1/k (a) (b) Figure 5 Model shows decrease in concentration parameter kappa with age: (a) Schematic showing model parameters. Kappa, the ‘concentration’ parameter corresponds to variabil- ity in feature representations: in this case orientation. p(T), p(NT) and p(U) represent the probability of responding with the recalled target, non-target or a random orientation, respec- tively. (b) Model parameters in each year group. The concen- tration parameter, kappa increases with age. No other parameter shows an effect of age. Year group data (with mean ages) are shown for the purposes of illustration; inferential statistics were conducted using age as a continuous variable Estimated FSIQ 3-item VWM precision: year group Z-score –2 80 90 100 110 120 130 140 –1 0 1 2 Figure 6 Relationship between precision and IQ: Mean pre- cision in the three-item visual working memory task, corrected for sensorimotor performance and standardized within year groups, is correlated with FSIQe. Response Uniform error Response probability α1/k α1/k (a) Estimated FSIQ 3-item VWM precision: year group Z-score –2 80 90 100 110 120 130 140 –1 0 1 2 Response probability Figure 6 Relationship between precision and IQ: Mean pre- cision in the three-item visual working memory task, corrected for sensorimotor performance and standardized within year groups, is correlated with FSIQe. 2012 Blackwell Publishing Ltd. Relationship between precision and IQ (b) 13 12 9 10 11 Mean age Parameter Parameter value n.s n.s n.s p<.001 more plausible than positing a step change in maximum VWM capacity. Modelling the distribution of responses Previous developmental studies have shown linear improvement, during middle childhood, in standard tests of the ability to store items in VWM (Alloway et al., 2006; Gathercole et al., 2004). While our results are in agreement with these existing findings, our modelling analysis sheds new light on potential mechanisms that may underlie improvements in VWM performance. More broadly, our findings lend weight to the notion that working memory precision offers a complementary and potentially more sensitive metric for characterizing childhood VWM performance than do traditional mea- sures of capacity. Whilst our working memory precision findings are not directly comparable with capacity esti- mates (e.g. it is not possible to recover ‘total capacity’ by adding up precision per item), they complement earlier findings by providing evidence within a distinctly dif- ferent empirical and conceptual framework to that of capacity, including recent reformulations of slot models (Zhang & Luck, 2008). We applied the probabilistic model described by Bays et al. (2009) following a related proposal by Zhang and Luck (2008), and subsequently applied within a sequen- tial VWM task in adults by Gorgoraptis et al. (2011), to decompose sources of error in working memory. This analysis confirmed that the age-associated improvement in VWM performance was attributable specifically to variability in the representation of targets, and was not due to changes in the frequency of random or misbinding errors. This variability was encapsulated in our model (Figure 5a) by kappa, the ‘concentration’ parameter. The age-related increase in kappa (see Figure 5b for year group average) corresponds closely to the raw response data shown in Figure 4 (note width at half-maximum height for each year group). As a next step, potential effects of target presentation time on precision in each age group might be investi- gated. It is possible that younger children were detri- mentally affected by the short presentation times (500 ms) of each stimulus. Whereas evidence from change detection paradigms suggests comparable child- hood performance when viewing objects for 500 ms vs. 1000 ms (Cowan, AuBuchon, Gilchrist, Ricker & Saults, 2011; Cowan et al., 2010), recent evidence shows that continuous measures of VWM performance may be more sensitive to differences in encoding time (Bays, Gorgoraptis, Wee, Marshall & Husain, 2011a). Based on this finding, one important implication of our findings for brain mechanisms is that the variability of feature representations stored in VWM decreases during childhood and early adolescence. Development of working memory precision The extent to which this may be due to developmental This finding reveals an important potential mechanism underlying developmental improvement in VWM per- formance, and one which in developmental terms may be 6 Stephanie Burnett Heyes et al. 536 Serial position effects changes in attention, VWM decay or metacognition is an empirical question. Importantly, the age effects on VWM precision observed here were not accounted for by concurrent improvement on a sensorimotor control task. As in previous studies investigating VWM in children and adults (Gorgoraptis et al., 2011; Hitch et al., 1988; Neath, 1993; Wright, Santiago, Sands, Kendrick & Cook, 1985), there was an effect of serial position on recall performance (Figure 3). Specifically, the final item in a sequence was recalled with higher precision than the preceding items (i.e. a recency effect), which did not differ in precision from one another. This effect became more marked with age. However, precision at each serial position improved significantly with age, which indicates that age effects were not solely due to an increase in precision for the most recent item. Our results are consistent with findings from change detection paradigms, which show continuing develop- ment during middle childhood in estimates of the max- imum number of items that may be held in VWM (Cowan et al., 2005; Riggs et al., 2006). However, our findings go beyond those from traditional measures of capacity by demonstrating protracted development throughout middle childhood and early adolescence in VWM precision for individual items, whether presented individually or in sequence. An implication of this find- ing is that childhood VWM development is best char- acterized not as a discrete increase in the capacity to store complete items, but by an increase in the precision with which these items are encoded, stored or retrieved. 2012 Blackwell Publishing Ltd. Acknowledgements Whether VWM capacity is discrete (slot models) or continuous (dynamic resource models) is debated (Bays & Husain, 2009; Cowan & Rouder, 2009). Intermediate, hybrid models propose that a dynamic resource is dis- tributed continuously among slots (Zhang & Luck, 2008, 2009). The current data are inconsistent with a fully discrete model. Two empirical points support our inter- pretation of a developmental increase in precision of a dynamic resource. First, above-chance performance at each serial position of the three-item task suggests that even the youngest children in our sample (aged 7– 9 years) succeed at encoding information about each of the three items. Consistent with this interpretation, there was no effect of age on the model parameter p(U), which indicates a lack of evidence that younger children were more likely than older children to respond at random on the difficult three-item task. Note that if younger chil- dren were able to store < 3 discrete items, whereas older children were able to store ‡ 3 items, for example, then this component would be expected to show an effect of age (since un-encoded items would give rise to uniformly distributed response errors). This research was supported by the Wellcome Trust and the NIHR BRC at UCL⁄UCLH. Relationship between precision and IQ The results presented here demonstrate a positive cor- relation between VWM precision and full-scale IQ esti- mated from CAT-3 scores (FSIQe; Figure 6). It has been suggested that, during development, improvements in the efficiency of complex working memory (i.e. the ability to manipulate information held in memory) underpin developmental improvements in tasks that measure the capacity of working memory (Fry & Hale, 2000; Gath- ercole & Baddeley, 1993). Therefore, it is possible that the association between FSIQe and precision observed here is underpinned by correlated improvement in higher-level processing capabilities. Development of visual working memory precision Development of visual working memory precision 537 (Blakemore, 2008; Gonzalez-Burgos, Kroene, Zaitsev, Povysheva, Krimer, Barrionuevo & Lewis, 2008). Such mechanisms, therefore, could potentially underlie the developmental increase in VWM precision observed in the current study. (Blakemore, 2008; Gonzalez-Burgos, Kroene, Zaitsev, Povysheva, Krimer, Barrionuevo & Lewis, 2008). Such mechanisms, therefore, could potentially underlie the developmental increase in VWM precision observed in the current study. (Blakemore, 2008; Gonzalez-Burgos, Kroene, Zaitsev, Povysheva, Krimer, Barrionuevo & Lewis, 2008). Such mechanisms, therefore, could potentially underlie the developmental increase in VWM precision observed in the current study. tion is not stored in a fully discrete, all-or-none manner. Independent of any putative increase in the maximum number of complete items that can be stored, we have shown an age-associated increase in the precision with which even a single item is stored, consistent with an increase in resolution of a dynamic resource. A recent non-human primate study has linked response properties of delay neurons in DLPFC to VWM performance decline between young adulthood and healthy seniority (Wang, Gamo, Yang, Jin, Wang, Laubach, Mazer, Lee & Arnste, 2011). Potentially, using more sensitive precision measures in such experiments will offer important insights into developmental neuro- cognitive mechanisms of working memory. Conclusions Visual working memory is the facility to hold in mind visual objects for brief periods of time. In this study, we have shown evidence that the precision of VWM develops throughout middle childhood and early ado- lescence. This development is attributable to an increase in the resolution of feature representations held in memory, without the need to invoke alterations in the capacity to store discrete items. As such, these results demonstrate protracted development in VWM perfor- mance, and shed new light on the potential mechanisms that may underlie this development. Measuring preci- sion, rather than estimating the maximum number of complete items that can be stored in VWM, may pro- vide a more sensitive metric for assessing childhood VWM development. Questions remain as to whether this development reflects an increase in the ‘amount’ of VWM resource, or an increase in the extent to which VWM can be flexibly distributed across targets (Astle & Scerif, 2011). 2012 Blackwell Publishing Ltd. Modelling the distribution of responses According to prominent computational neuroscience models, feature representations are implemented in the brain by popu- lation coding across neurons with tuned response prop- erties (Pouget, Dayan & Zemel, 2000; Seung & Sompolinsky, 1993). In the case of working memory, there is evidence that this information is maintained during a delay by local recurrent excitatory networks, for example in dorsolateral prefrontal cortex (DLPFC) (Compte, Brunel, Goldman-Rakic & Wang, 2000). Pro- gressive sharpening of these networks during develop- ment is thought to result in more precise feature representations (Munakata, 2004; Rolls & Deco, 2011; Schutte, Spencer & Schçner, 2003). There is evidence that such sharpening may be effected in DLPFC during childhood ⁄adolescence by excitatory synaptic pruning Of interest, our analyses showed an age-related in- crease in the precision difference between one-item and three-item VWM tasks, conceptually equivalent to an age by task interaction. Thus, whereas we demonstrate significant improvement with age on both one-item and three-item VWM tasks, consistent with age-associated development in the resolution of VWM, the relative age- associated improvement on the three-item task was more substantial. Further empirical studies are needed to identify the cognitive mechanism underlying this result. Possibilities include serial order effects (see below), emerging metacognitive capability, and the development of attention (Astle, Nobre & Scerif, 2010): for example, each additional item to be stored may incur a constant attentional cost, which is greater at younger ages. References Visual working memory in young chil- dren. Memory & Cognition, 16, 120–132. doi:10.3758/ BF03213479 Blakemore, S.-J. (2008). The social brain in adolescence. Nature Reviews Neuroscience, 9 (4), 267–277. doi:10.1038/nrn2353 Compte, A., Brunel, N., Goldman-Rakic, P.S., & Wang, X.J. (2000). Synaptic mechanisms and network dynamics under- lying spatial working memory in a cortical network model. Cerebral Cortex, 10 (9), 910–923. Lane, D.M., & Pearson, D.A. (1983). Attending to spatial locations: a developmental study. Child Development, 54 (1), 98–104. doi:10.2307/1129866 Cowan, N., AuBuchon, A.M., Gilchrist, A.L., Ricker, T.J., & Saults, J.S. (2011). Age differences in visual working memory capacity: not based on encoding limitations. Developmental Science, 14 (5), 1066–1074. doi:10.1111/j.1467-7687.2011. 01060.x Luck, S.J., & Vogel, E.K. (1997). The capacity of visual working memory for features and conjunctions. Nature, 390 (6657), 279–281. doi:10.1038/36846 Munakata, Y. (2004). Computational cognitive neuroscience of early memory development. Developmental Review, 24 (1), 133–153. doi:10.1016/j.dr.2003.09.008 Cowan, N., Elliott, E.M., Saults, J.S., Morey, C.C., Mattox, S., Hismjatullina, A., & Conway, A.R A. (2005). On the capacity of attention: its estimation and its role in working memory and cognitive aptitudes. Cognitive Psychology, 51 (1), 42–100. doi:10.1016/j.cogpsych.2004.12.001 Myung, I.J. (2003). Tutorial on maximum likelihood estima- tion. Journal of Mathematical Psychology, 47 (1), 90–100. doi:10.1016/S0022-2496(02)00028-7 Neath, I. (1993). Distinctiveness and serial position effects in recognition. Memory & Cognition, 21 (5), 689–698. Cowan, N., Fristoe, N.M., Elliott, E.M., Brunner, R.P., & Saults, J.S. (2006a). Scope of attention, control of attention, and intelligence in children and adults. Memory & Cognition, 34 (8), 1754–1768. Pehoski, C., Henderson, A., & Tickle-Degnen, L. (1997). In-hand manipulation in young children: rotation of an ob- ject in the fingers. American Journal of Occupational Therapy, 51 (7), 544–552. Cowan, N., Morey, C.C., AuBuchon, A.M., Zwilling, C.E., & Gilchrist, A.L. (2010). Seven-year-olds allocate attention like adults unless working memory is overloaded. Develop- mental Science, 13 (1), 120–133. doi:10.1111/j.1467-7687. 2009.00864.x Pouget, A., Dayan, P., & Zemel, R. (2000). Information pro- cessing with population codes. Nature Reviews Neuroscience, 1 (2), 125–132. doi:10.1038/35039062 Riggs, K.J., McTaggart, J., Simpson, A., & Freeman, R.P.J. (2006). Changes in the capacity of visual working memory in 5- to 10-year-olds. Journal of Experimental Child Psychology, 95 (1), 18–26. doi:10.1016/j.jecp.2006.03.009 Cowan, N., Naveh-Benjamin, M., Kilb, A., & Saults, J.S. (2006b). Life-span development of visual working memory: when is feature binding difficult? Developmental Psychology, 42 (6), 1089–1102. doi:10.1037/0012-1649.42.6.1089 Riggs, K.J., Simpson, A., & Potts, T. (2011). References Alloway, T.P., Gathercole, S.E., & Pickering, S.J. (2006). Ver- bal and visuospatial short-term and working memory in children: are they separable? Child Development, 77 (6), 1698– 1716. doi:10.1111/j.1467-8624.2006.00968.x Astle, D.E., Nobre, A.C., & Scerif, G. (2010). Attentional con- trol constrains visual short-term memory: insights from developmental and individual differences. Quarterly Journal of Experimental Psychology, 65, 277–294. doi:10.1080/17470218. 2010.492622 Astle, D.E., & Scerif, G. (2011). Interactions between attention and visual short-term memory (VSTM): what can be learnt from individual and developmental differences? Neuropsych- ologia, 49 (6), 1435–1445. doi:10.1016/j.neuropsycholo- gia.2010.12.001 Baddeley, A. (2003). Working memory: looking back and looking forward. Nature Reviews Neuroscience, 4 (10), 829– 839. doi:10.1038/nrn1201 Second, we observed robust and substantial develop- ment between age 7 and 13 years in the ability to pre- cisely recall each item from a set of three, as well as development in the ability to precisely recall an item presented individually. This demonstrates that informa- Bays, P.M., Catalao, R.F.G., & Husain, M. (2009). The pre- cision of visual working memory is set by allocation of a shared resource. Journal of Vision, 9 (10), 7.1–11. doi:10. 1167/9.10.7 538 Stephanie Burnett Heyes et al. 538 Gathercole, S.E., Pickering, S.J., Ambridge, B., & Wearing, H. (2004). The structure of working memory from 4 to 15 years of age. Developmental Psychology, 40 (2), 177–190. doi:10.1037/0012-1649.40.2.177 Bays, P.M., Gorgoraptis, N., Wee, N., Marshall, L., & Husain, M. (2011a). Temporal dynamics of encoding, storage, and reallocation of visual working memory. Journal of Vision, 11 (10). doi:10.1167/11.10.6 Bays, P.M., & Husain, M. (2008). Dynamic shifts of limited working memory resources in human vision. Science, 321 (5890), 851–854. doi:10.1126/science.1158023 Gonzalez-Burgos, G., Kroener, S., Zaitsev, A.V., Povysheva, N.V., Krimer, L.S., Barrionuevo, G., & Lewis, D.A. (2008). Functional maturation of excitatory synapses in layer 3 pyramidal neurons during postnatal development of the primate prefrontal cortex. Cerebral Cortex, 18 (3), 626–637. doi:10.1093/cercor/bhm095 Bays, P.M., & Husain, M. (2009). Response to Comment on ‘Dynamic shifts of limited working memory resources in human vision’. Science, 323 (5916), 877. doi:10.1126/sci- ence.1166794 Gorgoraptis, N., Catalao, R.F.G., Bays, P.M., & Husain, M. (2011). Dynamic updating of working memory resources for visual objects. Journal of Neuroscience, 31 (23), 8502–8511. doi:10.1523/JNEUROSCI.0208-11.2011 Bays, P.M., Wu, E.Y., & Husain, M. (2011b). Storage and binding of object features in visual working memory. Neu- ropsychologia, 49 (6), 1622–1631. doi:10.1016/j.neuropsych- ologia.2010.12.023 Hitch, G.J., Halliday, S., Schaafstal, A.M., & Schraagen, J.M.C. (1988). 2012 Blackwell Publishing Ltd. References The develop- ment of visual short-term memory for multifeature items during middle childhood. Journal of Experimental Child Psychology, 108 (4), 802–809. doi:10.1016/j.jecp.2010. 11.006 Cowan, N., & Rouder, J.N. (2009). Comment on ‘Dynamic shifts of limited working memory resources in human vision’. Science, 323 (5916), 877. doi:10.1126/science.1166478 Enns, J.T., & Girgus, J.S. (1985). Developmental changes in selective and integrative visual attention. Journal of Experi- mental Child Psychology, 40 (2), 319–337. doi:10.1016/0022- 0965(85)90093-1 Rolls, E.T., & Deco, G. (2011). A computational neuroscience approach to schizophrenia and its onset. Neuroscience and Biobehavioral Reviews, 35 (8), 1644–1653. doi:10.1016/j.neu- biorev.2010.09.001 Fisher, N.I. (1996). Statistical analysis of circular data. Cam- bridge: Cambridge University Press. Schutte, A.R., Spencer, J.P., & Schçner, G. (2003). Testing the dynamic field theory: working memory for locations becomes more spatially precise over development. Child Development, 74 (5), 1393–1417. Fougnie, D., Asplund, C.L., & Marois, R. (2010). What are the units of storage in visual working memory? Journal of Vision, 10 (12), 27. doi:10.1167/10.12.27 Seung, H.S., & Sompolinsky, H. (1993). Simple models for reading neuronal population codes. Proceedings of the Na- tional Academy of Sciences of the United States of America, 90 (22), 10749–10753. Fry, A.F., & Hale, S. (2000). Relationships among processing speed, working memory, and fluid intelligence in children. Biological Psychology, 54 (1–3), 1–34. Gathercole, S.E., & Baddeley, A.D. (1993). Working memory and language. Essays in cognitive psychology. Hillsdale, NJ: Lawrence Erlbaum Associates. Stevens, S.S. (1958). Problems and methods of psychophysics. Psychological Bulletin, 55 (4), 177–196. 2012 Blackwell Publishing Ltd. Development of visual working memory precision 539 Wang, M., Gamo, N.J., Yang, Y., Jin, L.E., Wang, X.-J., Laubach, M., Mazer, J.A., Lee, D., & Arnsten, A.F.T. (2011). Neuronal basis of age-related working memory decline. Nature, 476 (7359), 210–213. doi:10.1038/ nature10243 Wright, I., Strand, S., & Wonders, S. (2005). Estimation of premorbid intellectual abilities in children. Educational and Child Psychology, 22 (2), 100–107. Zhang, W., & Luck, S.J. (2008). Discrete fixed-resolution rep- resentations in visual working memory. Nature, 453 (7192), 233–235. doi:10.1038/nature06860 Wilken, P., & Ma, W.J. (2004). A detection theory account of change detection. Journal of Vision, 4 (12), 1120–1135. doi:10:1167/4.12.11 Zhang, W., & Luck, S.J. (2009). Sudden death and gradual decay in visual working memory. Psychological Science, 20 (4), 423–428. doi:10.1111/j.1467-9280.2009.02322.x Wright, A.A., Santiago, H.C., Sands, S.F., Kendrick, D.F., & Cook, R.G. (1985). Memory processing of serial lists by pigeons, monkeys, and people. Science, 229 (4710), 287– 289. References Received: 13 October 2011 Accepted: 2 February 2012 Received: 13 October 2011 Accepted: 2 February 2012 2012 Blackwell Publishing Ltd. 2012 Blackwell Publishing Ltd.
https://openalex.org/W1987643439	https://www.scielo.br/j/abb/a/t4mfbWpSBjhM79Bb5kBM85G/?lang=pt&format=pdf	Portuguese	null	Taxonomia e distribuição de Cheilolejeunea aneogyna (Spruce) A. Evans (Lejeuneaceae, Marchantiophyta)	Acta Botanica Brasílica/Acta Botânica Brasílica	2,012	cc-by	3,524	Acta Botanica Brasilica 26(3): 709-713. 2012. ABSTRACT (Taxonomy and distribution of Cheilolejeunea aneogyna (Spruce) A. Evans (Lejeuneaceae, Marchantiophyta)). Chei- lolejeunea aneogyna (Spruce) A. Evans is a South American species found in the Amazon and Atlantic Forest. It is characterized mainly by the presence of the first and second teeth of the lobule that are adjacent to each other and the lack of innovations. Among the neotropical species, C. aneogyna is the only one that has ocelli; however, their presence is not constant. A description, illustration, and comments on the morphology and geographic distribution are provided. This species is reported for the first time for Bolivia. Key words: Bryophytes, Hepaticae, Lejeuneoideae, Neotropic, Bahia Introdução ram a sustentação desse gênero, tendo transferido algumas espécies de Trachylejeunea para Cheilolejeunea, e reviven- do o antigo status de Cheilolejeunea aneogyna (Spruce) A. Evans, como considerado por Grolle (1979). Cheilolejeunea aneogyna (Spruce) A. Evans é uma es- pécie neotropical, ocorrendo principalmente nas Florestas Amazônica e Atlântica. Foi circunscrita por Evans (1906) a partir de Lejeunea (Cheilo-Lejeunea) aneogyna, descrita por Spruce (1885) para o Estado do Amazonas, Brasil. No entanto, o conceito de Evans para esse táxon é controverso. O objetivo do trabalho é caracterizar morfologicamente a espécie, conceituando-a corretamente, conforme o con- ceito de Grolle (1979) para Trachylejeunea aneogyna, bem como discutir a sua ocorrência no Neotrópico. Cheilolejeunea aneogyna foi tratada por Grolle (1979) como Trachylejeunea aneogyna (Spruce) Grolle, o qual referiu que a espécie considerada por Krachoo & Schuster (1966) como Cheilolejeunea aneogyna (Spruce) A. Evans era, na verdade, Cheilolejeunea decidua (= C. adnata), e que a verdadeira Cheilolejeunea aneogyna apresenta o segundo e primeiro dentes contíguos, com a papila hialina situada entre os dois e, assim, deveria pertencer à Trachylejeunea (Spruce) Schiffn., transferindo-a, portanto, para esse gênero. Recentemente, Gradstein & Ilkiu-Borges (2009) questiona- RESUMO (Taxonomia e distribuição de Cheilolejeunea aneogyna (Spruce) A. Evans (Lejeuneaceae, Marchantiophyta)). Chei- lolejeunea aneogyna (Spruce) A. Evans é uma espécie sul americana, ocorrendo nas Florestas Amazônica e Atlântica. Caracteriza-se, principalmente, pela presença do segundo e primeiro dentes do lóbulo contíguos um ao outro e ausência de inovações. Entre as espécies neotropicais, C. aneogyna é a única que pode apresentar ocelos, porém, a presença destes não é constante. Descrição, ilustração, comentários sobre a morfologia e distribuição geográfica são fornecidos e discutidos. Está sendo referida pela primeira vez para a Bolívia. Palavras-chave: Briófitas, Hepáticas, Lejeuneoideae, Neotrópico, Bahia Recebido em 12/10/2011. Aceito em 18/06/2012 Recebido em 12/10/2011. Aceito em 18/06/2012 Taxonomia e distribuição de Cheilolejeunea aneogyna (Spruce) A. Evans (Lejeuneaceae, Marchantiophyta) Cid José Passos Bastos1 Cid José Passos Bastos1 1 Universidade Federal da Bahia, Instituto de Biologia, Laboratório de Taxonomia de Briófitas, Campus Universitário de Ondina, Salvador, BA, Brasil Autor para correspondência: cid-bastos@uol.com.br rsidade Federal da Bahia, Instituto de Biologia, Laboratório de Taxonomia de Briófitas, Campus Universitário de Ondina, Sal para correspondência: cid-bastos@uol.com.br Taxonomia Vilas Bôas-Bastos 2034 (ALCB). Espírito Santo: Domingos Martins, Parque Estadual Pedra Azul, 20º23’S, 40º59’W, 1200-1910m alt., 7/III/2009, Penha, L.T. Cheilolejeunea aneogyna (Spruce) A. Evans, Bull. Torrey Bot. Club 33(1): 2. 1906. Lejeunea aneogyna Spruce, Trans. & Proc. Bot. Soc. Edin- burgh 15: 254. 1884. Tipo: Brasil, Amazonas, San Carlos, in ramulis e mortuis, R. Spruce L103 (lectótipo MAN- CH!, designado por Grolle 1979; sintipo MANCH!, BM, E, JE, NY, W, M). Trachylejeunea aneogyna (Spruce) Grolle, J. Hattori Bot. Lab. 46: 338. 1979. =Cheilolejeunea surinamensis (Steph.) X.-L. He, Ann. Bot. Fennici 34: 65. 1997. =Rectolejeunea heteroclada (Spruce) Steph., Sp. Hepat. 5: 688. 1914. Brasil, Ad fl . Negro juxta S. Gabriel, S. Carlos, etc., etiam ad fl . Caipurú fl uvio Trombetus affl u- entem, in truncis praecipue inundatis, R. Spruce s.n. (US 70820!) (fi de Gradstein & Costa 2003). =Rectolejeunea roseo-alba (Spruce) Steph., Sp. Hepat. 5: 695. 1914 (fi de Gradstein & Costa 2003). Fig. 1A-J. Lejeunea aneogyna Spruce, Trans. & Proc. Bot. Soc. Edin- burgh 15: 254. 1884. Tipo: Brasil, Amazonas, San Carlos, in ramulis e mortuis, R. Spruce L103 (lectótipo MAN- CH!, designado por Grolle 1979; sintipo MANCH!, BM, E, JE, NY, W, M). Trachylejeunea aneogyna (Spruce) Grolle, J. Hattori Bot. Lab. 46: 338. 1979. =Cheilolejeunea surinamensis (Steph.) X.-L. He, Ann. Bot. Fennici 34: 65. 1997. =Rectolejeunea heteroclada (Spruce) Steph., Sp. Hepat. 5: 688. 1914. Brasil, Ad fl . Negro juxta S. Gabriel, S. Carlos, etc., etiam ad fl . Caipurú fl uvio Trombetus affl u- entem, in truncis praecipue inundatis, R. Spruce s.n. (US 70820!) (fi de Gradstein & Costa 2003). =Rectolejeunea roseo-alba (Spruce) Steph., Sp. Hepat. 5: 695. 1914 (fi de Gradstein & Costa 2003). Fig. 1A-J. Gametófitos pequenos, 0,8 − 1,1 mm de largura, verdes, prostrados, ramificações do tipo-Lejeunea. Caulídio em secção transversal com 7 células corticais e 9 células medu- lares. Merófito ventral com duas células de largura. Taxonomia Cheilolejeunea aneogyna é uma espécie caracterizada, principalmente, pela presença, no lóbulo, de dois dentes (primeiro e segundo dentes) contíguos e pela ausência de inovação. Apenas Grolle (1979, como Trachylejeunea aneogyna) e He (1997, como Cheilolejeunea surinamensis) apresentaram descrição e ilustração para essa espécie, porém, nenhum material procedente do Brasil foi ainda ilustrado ou descrito. Material examinado: BRASIL. Amazonas: São Gabriel, corticícola, R. Spruce L419 (MANCH). Manaus, Manaus- Caracaraí Road, km 45, Caatinga on white sand; on tree trunk, 5/IV/1971, G.T. Prance et al. 11374 p.p. (INPA); Mauá Road. Flooded river margin. Growing on tree trunk, 23/III/1971, G.T. Prance et al. 11550 (INPA); idem, Igapó, on tree trunk, 5/IV/1971, G.T. Prance et al. 11734 (INPA). Ponta Negra, on tree trunk, 31/III/1971, G.T. Prance et al. 11709, 11712 (INPA); idem, on spines on palm leaf bases, 31/ III/1971, G.T. Prance et al. 11707 (INPA). Tarumã Grande, along Rio Tarumã, a tributary of the Rio Negro, ca. 15 km E of Manaus; white sand igapó, 19/XI/1977, W.R. Buck et al. 1790 (INPA). Between Manaus and São Gabriel, along the Marié, at Manauná; primary Forest with large rokcs over wihte sand, 00º40’S, 66º45’W, 5/VII/1979, W.R. Buck 2402 (INPA); between Manaus and São Gabriel, along Rio Negro just E of Santa Isabel (Tapuruquara). Igapó and small, sand terra firme islands, 00º30’S, 65º00’W, 1/VII/1979, W.R. Buck 2270 (INPA). Ilha Acarabu, in the Rio Negro at mouth of the Rio Marié; flood plain forest, 00º25’S, 66º25’W, 4/ VII/1979, W.R. Buck 2326, 2328 (INPA). Rio Negro, Ilha Acaburu, 0º40’S, 66º40’W, sobre casca de árvore viva, 4/ VIII/1979, O. Yano 1667 (INPA; SP). Highway between Humaitá and Porto Velho, 60 km S Humaitá, at highway crossing of São João stream; ca. 8ºS, 63ºW; primary forest, white sand w/ root mat, closed canopy; level terra firme; on fallen log with intact bark, mesic, partial shade, 30/IV/1982, Alan J. Fife et al. 3982 (INPA). Highway between Humaitá and Porto Velho, 60 km S Humaitá, at highway crossing of São João stream; ca. 8ºS, 63ºW; 1º Igapó forest on bank of clear-water stream, immediately upstream of highway, canopy open; on bark of Sapotaceae, partial shade, 1-2 m, vertical surface, 1/V/1982, Alan J. Fife et al. 3992 (INPA). Bahia: Eunápolis, Estação Veracruz, nascente do rio dos Mangues, 10/III/2001, S.B. Vilas Bôas-Bastos & C. Bastos 1632 (ALCB). Igrapiúna, Reserva Ecológica da Michelin, 13º48’S, 39º10’W, 15/II/2006, C. Bastos 4239 (ALCB); idem, 16/II/2006, S.B. Material e métodos Foram estudados o material tipo de Cheilolejeunea ane- ogyna (MANCH) e espécimes de coleções dos Herbários ALCB, SP, INPA, NX, de acordo com as técnicas usuais para estudos morfológicos aplicados à taxonomia (fide Bastos & Yano 2006). As descrições e comentários morfológicos foram baseados no material estudado, e a distribuição geográfica e da Bahia, Instituto de Biologia, Laboratório de Taxonomia de Briófitas, Campus Universitário de Ondina, Salvador, BA, Brasil ncia: cid-bastos@uol.com.br Cid José Passos Bastos os dados sobre substrato colonizado foram extraídos da eti- queta das exsicatas. Os dados complementares de distribuição geográfica no Brasil estão de acordo com Yano (2008), sendo os Estados listados em ordem alfabética. A classificação da vegetação segue o conceito de “domínios fitogeográficos”, de acordo com Fiaschi & Pirani (2009). As ilustrações foram feitas em câmara-clara acoplada ao microscópio. segundo dentes proeminentes, contíguos, obtusos, primeiro dente às vezes não aparente, papila hialina entre o primeiro e segundo dentes, margem apical reta a curva, quilha reta; lóbulos ocasionalmente reduzidos. Anfigastros ovalados a oblongo-ovalados, 120 – 232 μm de largura, distanciados, sinus agudo, lobos com ápice agudo a obtuso, base cuneada, linha de inserção arqueada. Autóicos. Androécios laterais ou intercalares, 3−4 pares, lóbulos inflados, gibosos, hipos- táticos, bractéola 1−2 na base do ramo. Ginoécio lateral, inovação ausente, brácteas com lobo obovado, margem inteira, ápice arredondado, lóbulo oblongo, ápice obtuso a agudo, bractéola oblonga, bífida, sinus agudo; perianto obovado, 4 quilhas, rostro curto. Multiplicação vegetativa por filídios caducos. Taxonomia L103”). Relatou, ainda, que outra planta, etiquetada como “B”, apre- senta o dente proximal longo e hialino, e que esta planta parece ser idêntica a encontrada por Williams na Rodésia e que seria a planta que Evans aceitou como Cheilolejeunea aneogyna. Concluiu a sua nota referindo que Grolle (1979) identificou a planta “B” como Cheilolejeunea adnata, mas que a planta etiquetada como “B” é monóica, e que sua iden- tidade como Cheilolejeunea adnata deve ser questionada. Na verdade, essa planta foi recentemente definida como uma variedade de C. adnata por Gradstein & Ilkiu-Borges (2009), com base em outro material-tipo. Figura 1. Cheilolejeunea aneogyna (Spruce) A. Evans. A – Gametófito, vista ventral. B e C – Lóbulos. D – Anfigastros. E – Parte do filídio caduco com plântula. F – Margem do lobo do filídio evidenciando um rizoide emergente. G – Ginoécio. H – Células da lâmina do lobo do filídio próximo à margem. I – Seção transversal do caulídio. J –Ramo androecial e gonoecial, vista ventral. L – Ramo androecial isolado, vista ventral. M – Ginoécio com perianto, vista ventral. 390p.p., 425p.p. (ALCB). Pará: Santarém, s.d., R. Spruce s.n. (US 70819). Pernambuco: Felchter Sekundärwald an der Kürstenstraβe PE 40 nördlich Barreiras beim Reserva Bio- lógica do Saltinho, 70m, 10/VII/1990, Schäfer-Verwimp & Verwimp (Herb. Schäfer-Verwimp 12954, ALCB, SP). Mato Grosso: Nova Xavantina, Fazenda Aruama, 14º33’28.1”S, 51º54’36.4”W, 247m de alt., Rio das Mortes, 19/V/2009, F.P. Athayde Filho & L.R. Fernandes 2078 (NX); idem, Ilha da Viração, 14º33’30.2”S, 51º54’40”W, 233m de alt., Rio das Mortes, 19/V/2009, F.P. Athayde Filho & L.R. Fernandes 2131 (NX). BOLÍVIA. Beni: Vicinity of Guayaramerín city dump, just NW of town; border of flooded arroyo; low forest, 19/II/1978, W.D. Reese 13125 (INPA). Assim, quando se considera atualmente Cheilolejeu- nea aneogyna (Spruce) A. Evans, aceita-se, desse modo, o conceito de Evans (1906) para essa espécie, o que significa que estamos aceitando Cheilolejeunea adnata var. autoica para esse nome, consoante a descrição e comentários de Evans (1906) para a espécie. Considerando esse aspecto, os comentários feitos por Evans (1906) em relação ao dente apical de C. aneogyna foram equivocados e referem-se à “planta B”, ou seja, C. adnata var. autoica, e não devem ser considerados para a correta circunscrição da espécie; Cheilolejeunea aneogyna apresenta o primeiro e segundo dentes contíguos e curtos. Taxonomia Filídios imbricados, patentes; lobo oblongo-ovalado, 392 – 584 μm de comprimento × 304 − 416 μm de largura, margem dorsal arqueada, inteira, margem ventral reta, inteira, levemente ascendente junto à quilha, ápice arredondado; células oblon- gas a arredondadas, 18 – 36 de comprimento × 10 – 16 μm de largura, retangulares a quadráticas na margem, paredes espessas, trigônios radiados a confluentes, espessamentos intermediários nodulosos; oleocorpos não observados; oce- los em geral ausentes, raramente presentes, quando presen- tes 2-4 na base do lobo; lóbulo triangular-ovalado, 100 – 140 μm de comprimento × 80 – 92 μm de largura, margem livre fracamente involuta, levemente arqueada a reta, primeiro e Acta bot. bras. 26(3): 709-713. 2012. 710 Taxonomia e distribuição de Cheilolejeunea aneogyna (Spruce) A. Evans (Lejeuneaceae, Marchantiophyta) Figura 1. Cheilolejeunea aneogyna (Spruce) A. Evans. A – Gametófito, vista ventral. B e C – Lóbulos. D – Anfigastros. E – Parte do filídio caduco com plântula. F – Margem do lobo do filídio evidenciando um rizoide emergente. G – Ginoécio. H – Células da lâmina do lobo do filídio próximo à margem. I – Seção transversal do caulídio. J –Ramo androecial e gonoecial, vista ventral. L – Ramo androecial isolado, vista ventral. M – Ginoécio com perianto, vista ventral. referindo que “In C. aneogyna and its immediate allies the apical tooth is long and sharp (figure 4)”. Contudo, isto foi provocado em razão de existirem duas plantas diferentes em uma das amostras de Spruce. Examinando os tipos (sintipo) de Lejeunea (Cheilo-Lejeunea) aneogyna e o lec- tótipo designado por Grolle (1979), foi observado que, na exsicata Spruce L419 existem duas plantas, etiquetadas como planta “A” e planta “B” por E.W. Jones, sendo que a maior parte do material corresponde ao conceito de Cheilolejeu- nea aneogyna senso Grolle (planta “A”), e algumas plantas correspondem à Cheilolejeunea adnata var. autoica (planta “B”). Infelizmente, Spruce (1884) não descreve detalhes do dente apical de Lejeunea (Cheilo-Lejeunea) aneogyna. Entretanto, em uma extensa anotação feita por E.W. Jones, ele relata que a planta que ele designou como “A” tem dois dentes convergentes, de igual dimensão, que ele aceitou como Cheilolejeunea aneogyna; relatou, também, que essa mesma planta foi encontrada em outras exsicatas (nº 1682 e 1683) e que ele presume que esta seja a planta que Spruce designou como Lejeunea (Cheilo-Lejeunea) aneogyna (isto pode estar correto, porque uma anotação, provavelmente feita por Spruce, na exsicata L419 indica “sp. nov. aff. Taxonomia Um aspecto a ser considerado na morfologia de Cheilole- jeunea aneogyna é a presença ocasional de ocelos, reportado também por Moura (2010). Existem poucas espécies, que são paleotropicais (Zhu & So 2000), que apresentam ocelos: Cheilolejeunea falsinervis (Sande Lac.) Kachroo & R.M. Schust. e Cheilolejeunea inignis Ast & Tixier. Esses ocelos ge- ralmente ocorrem em uma fileira longitudinal (moniliados). Das espécies neotropicais, apenas Cheilolejeunea aneogyna pode apresentar ocelos. A presença de ocelos basais não é constante, mas em alguns espécimes examinados, estes es- Evans (1906) em seus comentários sobre o gênero Cheilolejeunea, referiu que Lejeunea (Cheilo-Lejeunea) aneogyna Spruce deveria ser o tipo do gênero por ter sido esse táxon o primeiro descrito por Spruce para o subgênero Cheilo-Lejeunea. Evans (1906) fez a nova combinação para esse táxon, transferindo-o de Lejeunea para Cheilolejeunea: Cheilolejeunea aneogyna (Spruce) A. Evans. Evans (op. cit.) também destacou caracteres do lóbulo para essa espécie, Acta bot. bras. 26(3): 709-713. 2012. 711 Cid José Passos Bastos tavam presentes, bem característicos, grandes, 2-3 na base do lobo. Outro aspecto é a presença do primeiro e segundo dentes no lóbulo, caráter esse utilizado na circunscrição de Trachylejeunea, recentemente sinonimizado a Cheilo- lejeunea (fide Gradstein & Ilkiu-Borges 2009). Embora a presença de dois dentes contíguos no lóbulo seja uma característica desta espécie, alguns exemplares portavam apenas o segundo dente, inclusive nos espécimes coletados por Spruce (sintipo MANCH). Esse caráter também pode ser observado em Cheilolejeunea neblinense Ilkiu-Borges & Gradst. (Ilkiu-Borges & Gradstein 2008), que se diferencia de C. aneogyna pela boca do lóbulo bem estreita e pelas grandes células da quilha. tela, estudos filogeográficos e de diversidade genética seriam necessários para se certificar qual a natureza do evento que deu origem ao atual padrão de distribuição de C. aneogyna. Cheilolejeunea surinamensis (Steph.) X.-L. He, descrita para o Suriname, foi sinonimizada a Trachylejeunea aneogy- na por Gradstein & Costa (2003). Material proveniente da coleção particular de A. Schäfer-Verwimp, com duplicata nos Herbários ALCB e SP, identificado como C. surinamen- sis, é perfeitamente idêntico a C. aneogyna. Habita tipicamente ambiente de floresta ombrófila (Flo- restas Amazônica e Atlântica), contudo foi encontrada no Estado de Mato Grosso, município de Nova Xavantina, em mata de galeria, possivelmente no Domínio Cerrado. Na Bahia, tem sido apenas encontrada em Floresta Atlântica, no sul do Estado. Coloniza, principalmente, tronco de árvore viva, que parece ser o substrato preferencial, mas alguns espécimes foram encontrados crescendo em tronco morto. Referências bibliográficas Bastos, C.J.P. 2008. Padrões de reprodução vegetativa em espécies de Lejeuneaceae (Marchantiophyta) e seu significado ecológico. Revista Brasileira de Botânica 31(2): 309-315. Bastos, C.J.P. 2008. Padrões de reprodução vegetativa em espécies de Lejeuneaceae (Marchantiophyta) e seu significado ecológico. Revista Brasileira de Botânica 31(2): 309-315. Bastos, C.J.P. & Yano, O. 2006. Lejeuneaceae holostipas (Marchantiophyta) no Estado da Bahia, Brasil. Acta Botanica Brasilica 20(3): 687-700. Dauphin, G. & Ilkiu-Borges, A.L. 2002. Hepaticae of Cerro Venamo, Venezuela, collected by J. Steyermark. Tropical Bryology 22: 115-123. Evans, A.W. 1906. Hepaticae of Puerto Rico VI. Cheilolejeunea, Rectole- jeunea, Ceratolejeunea, and Pycnolejeunea. Bulletin of the Torrey Botanical Club 33(1): 1-25. Fiaschi, P. & Pirani, J.R. 2009. Review of plant biogeographic studies in Brazil. Journal of Systematic and Evolution 47(5): 477-496. Gradstein, S.R. & Costa, D.P. 2003. The Hepaticae and Anthocerotae of Brazil. Memoirs of The New York Botanical Garden 87: 1-318. Gradstein, S.R. & Costa, D.P. 2003. The Hepaticae and Anthocerotae of Brazil. Memoirs of The New York Botanical Garden 87: 1-318. Gradstein, S.R. & Ilkiu-Borges, A.L. 2009. Guide to the Plants of Central French Guiana. Memoirs of the New York Botanical Garden 76(4): 1-140. C. aneogyna parece ser uma espécie tipicamente amazô- nica, e sua ocorrência na Floresta Atlântica do sul da Bahia, bem como em mata de galeria no Estado de Mato Grosso, reforça a ideia de uma antiga conexão entre a floresta amazô- nica e a floresta atlântica por uma rota através das matas de galeria do Domínio Cerrado (Oliveira-Filho & Ratter 1995, Fiaschi & Pirani 2009). No entanto, eventos recentes de dispersão à longa distância não podem ser desconsiderados. De acordo com Heinrichs et al. (2009), os atuais padrões de distribuição podem ser mais bem explicados por eventos de dispersão (a curta e a longa distância), extinção local e recolonização, do que por eventos vicariantes. No caso em Grolle, R. 1979. Miscellanea hepaticologica 191-200. Journal of the Hattori Botanical Laboratory 46: 337-355. Grolle, R. 1979. Miscellanea Hepaticologica 181-190. Journal of the Hat- tori Botanical Laboratory 45: 173-183. Heinrichs, J., Hentschel, J., Feldberg, K., Bombosch, A. & Schneider, H. 2009. Phylogenetic biogeography and taxonomy of disjunctly distribut- ed bryophytes. Journal of Systematics and Evolution 47(5): 497-508. 2009. Phylogenetic biogeography and taxonomy of disjunctly distribut- ed bryophytes. Journal of Systematics and Evolution 47(5): 497-508. Ilkiu-Borges, A.L. & Gradstein, S.R. 2008. A new species of Cheilolejeunea (Spruce) Schiffn. (Lejeuneaceae) from Cerro de la Neblina, Venezuela. Agradecimentos Na literatura só há registro da ocorrência de C. aneogy- na em outros países do Neotrópico para o Suriname (He 1997, como Cheilolejeunea surinamensis; Gradstein & Ilkiu- -Borges 2009) e Venezuela (Dauphin & ILkiu-Borges 2002, como Trachylejeunea aneogyna); contudo, foi examinado material procedente da Bolívia (INPA), sendo essa, portanto, a primeira referência da espécie para este país. É possível que ocorra em outros países da América do Sul. O autor é especialmente grato aos Curadores dos Herbários MANCH, SP, INPA e NX pelo empréstimo do material estudado, ao CNPq pela concessão da Bolsa de Produtividade em Pesquisa 2 e a Silvana B. Vilas Bôas- -Bastos pela confecção da ilustração, e aos revisores pelas valiosas sugestões. No Brasil C. aneogyna foi reportada na literatura ape- nas para os Estados do Amazonas, Bahia, Pará, Roraima e São Paulo (Grolle 1979, Yano 2008, como Trachylejeunea aneogyna), mas o estudo de material de Herbário revelou uma distribuição um pouco mais ampla, com ocorrência também nos Estados do Espírito Santo, Pernambuco e Mato Grosso. Contudo, é no Amazonas que a espécie apre- senta maior ocorrência, com 17 dos 25 registros. Embora a espécie tenha sido sempre registrada para florestas de terras baixas, no Espírito Santo foi encontrada em altitu- des entre 1200 – 1910 m. Esses resultados indicam que C. aneogyna tem no Estado de São Paulo o seu limite mais meridional de distribuição (20°S a 25°S). Até o momento, não foi reportada para a América Central e nem para a América do Norte. Taxonomia Em alguns espécimes examinados, foi observada mul- tiplicação vegetativa por filídios caducos, os quais apre- sentavam rizóides na margem. Esse tipo de reprodução foi relatado por Gradstein & Costa (2003, como Trachylejeunea aneogyna) e Bastos (2008). Moura, O.S. 2010. Diversidade e aspectos ecológicos da brioflora (Bryo- phyta e Marchantiophyta) da Ilha do Combu, Belém, Pará, Brasil. Dissertação de Mestrado, Museu Paraense Emílio Goeldi, Belém. Oliveira-Filho, A.T. & Ratter, J.A. 1995. A study of the origin of central Brazilian forests by the analysis of plant species distribution patterns. Edinburgh Journal of Botany 52: 141-194. Spruce, R. 1884. Hepaticae of the Amazon and Andes of Peru and Ecuador. Referências bibliográficas Nova Hedwigia 87(3-4): 521-528. Kachroo, P. & Schuster, R.M. 1961. The genus Pycnolejeunea and its affini- ties to Cheilolejeunea, Euosmolejeunea, Nipponolejeunea, Tuyamaella, Siphonolejeunea and Strepsilejeunea. Journal of Linnean Society, Botany 56(368): 475-511. Acta bot. bras. 26(3): 709-713. 2012. 712 Taxonomia e distribuição de Cheilolejeunea aneogyna (Spruce) A. Evans (Lejeuneaceae, Marchantiophyta) Taxonomia e distribuição de Cheilolejeunea aneogyna (Spruce) A. Evans (Lejeuneaceae, Marchantiophyta) Transactions and Proceedings of the Botanical Society 15: 1-588. Transactions and Proceedings of the Botanical Society 15: 1-588. Yano, O. 2008. Catálogo de Antóceros e Hepáticas Brasileiros: literatura original, basiônimo, localidade-tipo e distribuição geográfica. Boletim do Instituto de Botânica 19: 1-110. Zhu, R.-L. & So, M.L. 2000. Additions and corrections of Chinese Lejeuneaceae (Hepaticae). Botanical Bulletin of Academia Sinica 41: 243-250. Acta bot. bras. 26(3): 709-713. 2012. 713 Versão eletrônica do artigo em www.scielo.br/abb e http://www.botanica.org.br/acta/ojs Versão eletrônica do artigo em www.scielo.br/abb e http://www.botanica.org.br/acta/ojs Acta bot. bras. 26(3): 709-713. 2012. 713
https://openalex.org/W4286499631	https://link.springer.com/content/pdf/10.1007/s12519-022-00590-w.pdf	English	null	Human adenovirus (HAdV) infection in children with acute respiratory tract infections in Guangzhou, China, 2010–2021: a molecular epidemiology study	World Journal of Pediatrics	2,022	cc-by	6,480	Abstract Background Human adenovirus (HAdV) infection can cause a variety of diseases. It is a major pathogen of pediatric acute respiratory tract infections (ARIs) and can be life-threatening in younger children. We described the epidemiology and subtypes shifting of HAdV among children with ARI in Guangzhou, China. Methods We conducted a retrospective study of 161,079 children diagnosed with acute respiratory illness at the Guangzhou Women and Children’s Medical Center between 2010 and 2021. HAdV specimens were detected by real-time PCR and the hexon gene was used for phylogenetic analysis. Results Before the COVID-19 outbreak in Guangzhou, the annual frequency of adenovirus infection detected during this period ranged from 3.92% to 13.58%, with an epidemic peak every four to five years. HAdV demonstrated a clear seasonal distribution, with the lowest positivity in March and peaking during summer (July or August) every year. A significant increase in HAdV cases was recorded for 2018 and 2019, which coincided with a shift in the dominant HAdV subtype from HAdV-3 to HAdV-7. The latter was associated with a more severe disease compared to HAdV-3. The average mortal- ity proportion for children infected with HAdV from 2016 to 2019 was 0.38% but increased to 20% in severe cases. After COVID-19 emerged, HAdV cases dropped to 2.68%, suggesting that non-pharmaceutical interventions probably reduced the transmission of HAdV in the community. Conclusion Our study provides the foundation for the understanding of the epidemiology of HAdV and its associated risks in children in Southern China. Keywords Acute respiratory tract infection · Children · Human adenovirus · Severe acute hepatitis · * Rong Zhou zhourong@gird.cn * Bing Zhu zhubing0327@hotmail.com 1 Center Laboratory, Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, Guangzhou 510120, China 2 State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Health, Guangzhou Medical University, Guangzhou, China 3 Intensive Care Unit, Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, Guangzhou 510120, China 4 Guangzhou Medical University, Guangzhou 510120, China Human adenovirus (HAdV) infection in children with acute respiratory tract infections in Guangzhou, China, 2010–2021: a molecular epidemiology study Yi Chen1 · Tao Lin1 · Chang‑Bing Wang1 · Wan‑Li Liang1 · Guang‑Wan Lian1 · Mark Zanin2 · Sook‑San Wong2 · Xin‑Gui Tian2 · Jia‑Yu Zhong1 · Ying‑Ying Zhang1 · Jia‑Hui Xie1 · Ling‑Ling Zheng1 · Fei‑Yan Chen3 · Run Dang3 · Ming‑Qi Zhao1 · Yi‑Yu Yang3 · Rong Zhou4 · Bing Zhu1 Yi Chen1 · Tao Lin1 · Chang‑Bing Wang1 · Wan‑Li Liang1 · Guang‑Wan Lian1 · Mark Zanin2 · Sook‑San Wong2 · Xin‑Gui Tian2 · Jia‑Yu Zhong1 · Ying‑Ying Zhang1 · Jia‑Hui Xie1 · Ling‑Ling Zheng1 · Fei‑Yan Chen3 · Run Dang3 · Ming‑Qi Zhao1 · Yi‑Yu Yang3 · Rong Zhou4 · Bing Zhu1 Received: 16 March 2022 / Accepted: 29 June 2022 / Published online: 21 July 2022 © The Author(s) 2022 World Journal of Pediatrics (2022) 18:545–552 https://doi.org/10.1007/s12519-022-00590-w World Journal of Pediatrics (2022) 18:545–552 https://doi.org/10.1007/s12519-022-00590-w ORIGINAL ARTICLE 4 Guangzhou Medical University, Guangzhou 510120, China Clinical specimens Throat swabs were collected in 2.50 mL of viral transport medium whereas sputum was collected by aspiration. All specimens were delivered to the central diagnostic laboratory of GWCMC and were processed within 24 hours of collection. Detection of adenovirus and molecular typing with PCR HAdV molecular typing was performed on 557 HAdV-positive children randomly selected in respiratory wards from 2012 to 2019 and on 121 children diagnosed with severe adenovirus pneumonia in the ICU from 2014 to 2019. Total nucleic acid was extracted by the automated nucleic acid purification sys- tem Expure-20 (Shenzhen Huiyan Kechuang Biotechnology Co., China) using a total nucleic acid isolation kit (Shenzhen Huiyan Kechuang Biotechnology Co., China) according to the manufacturer's instructions. HAdV nucleic acid was detected by real-time quantitative polymerase chain reaction (RT-qPCR). Molecular typing of HAdV-3, -4, -7, -11, -14 and -55 was per- formed using the Taqman real-time PCR kit according to the manufacturer’s protocol (Guangzhou HuYanSuo Medical Tech- nology Co., China). Furthermore, the hypervariable regions (HVRs) of the HAdV hexon gene were amplified by PCR using the HVR forward (HVRF) and HVR reverse (HVRR) primers to amplify the 1.6 kbp fragment of the seven HVRs, as described previously [14]. The amplicon was submitted for sequencing (Invitrogen, Guangzhou). The positive samples were classified as HAdV-3, HAdV-7, or other HAdV subtypes. Introduction Human adenoviruses (HAdVs) cause a wide range of ill- nesses in individuals of all ages, including acute respira- tory infections (ARIs), gastroenteritis, conjunctivitis, cys- titis, and meningoencephalitis. Although HAdV infections often range from mild to moderate in severity, cases of severe pneumonia and death in otherwise healthy adults also have been reported [1, 2]. Life-threatening respira- tory disease related to HAdV infections mostly occurs in younger children, the elderly, and individuals with severely compromised immune systems [3–5]. For example, HAdV- associated ARI is one of the most common causes of mor- bidity and mortality in children. HAdV-3, -4, -14, -55 and * Bing Zhu zhubing0327@hotmail.com 1 Center Laboratory, Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, Guangzhou 510120, China 2 State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Health, Guangzhou Medical University, Guangzhou, China 3 Intensive Care Unit, Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, Guangzhou 510120, China 4 Guangzhou Medical University, Guangzhou 510120, China (0121 3456789) 3 546 World Journal of Pediatrics (2022) 18:545–552 Retrospective analysis of demographic data of HAdV infection was from 2014 to 2019, and the monthly incidence of HAdV infections was between 2012 and 2021. -7 are the major HAdV subtypes associated with ARI in children and in adults globally. The major HAdV subtypes detected in various countries and regions differ and change with time [6–9]. In China, HAdV-3 and -7 are the major subtypes, and they alternate in predominance from year to year. The subtype distributions in Southern and Northern China were occasionally not synchronous [10–13]. Ethical approval This project was approved by the Ethics Committee of the GWCMC and was carried out in accordance with the princi- ples of the Declaration of Helsinki. Clinical data and clinical specimens were de-identified and anonymous. In the second half of 2018, we found that the number of pediatric patients with ARI coming to pediatric hospitals was increasing, as well as the number of severe cases. This situation became more prominent in 2019 when the number of infections and severe cases had reached a record high. The purpose of this retrospective study was to determine the prevalence, epidemiology and subtypes of HAdV circulating among children with ARI in Guang- zhou, Southern China during the period 2010–2021. All the specimens were collected in Guangzhou Women and Children’s Medical Center (GWCMC), a large pediatric and women’s hospital that has a regional children’s medi- cal service network in Southern China. The hospital has about 2000 beds distributed in seven branches, receives over 4,700,000 pediatric outpatient person-times and admits 140,000 inpatients each year from Guangzhou as well as from other cities in Southern China. Therefore, our data provide a reasonable approximation of the HAdV activity in Southern China. Patient cohort Data were collected retrospectively from 161,079 chil- dren ≤ 14 years old who were diagnosed with ARI in the outpatient department, general ward and intensive care units (ICU) of the GWCMC between 1 January 2010 and 31 December 2021. Cases with repeated detections were not counted. Throat swabs or sputum collected from children were tested for HAdV, and samples from patients in ICU were detected for multiple respiratory viruses. We excluded the ICU cases who tested positive for influenza virus, respiratory syncytial virus, enterovi- rus, and known or suspected to have active tuberculosis, severe concomitant disease (chronic pulmonary disease except for asthma, severe cardiovascular disease, neo- plasia, and kidney or liver disease), primary immuno- deficiency, acquired immunodeficiency syndrome and patients taking immunosuppressive medications. Severe HAdV pneumonia was diagnosed when one of the fol- lowing conditions was present: hypoxemia, requiring either invasive mechanical ventilation or noninvasive positive pressure ventilation and fluid refractory shock. HAdV genome sequencing The hexon of HAdV was amplified and then the obtained sequences were used for the Basic Local Alignment Search Tool (BLAST) search (NCBI). The whole genome of HAdV was sequenced using Sanger sequencing. Assembly of the com- plete sequences of the HAdV genomes was accomplished using SeqMan software from the Lasergene package, as described previously [3]. Multiple sequence alignments and phylogenetic 1 3 547 World Journal of Pediatrics (2022) 18:545–552 laboratory-confirmed HAdV cases increased from 4543 in 2010 to 39,938 in 2019. Before the COVID-19 pandemic, the lowest annual frequency was in 2012 (3.92%) and the highest annual frequency in 2019 (13.58%) (Fig. 1). Among patients who were tested positive for HAdV between 2010 and 2018, the hospitalization frequency was 71.48% to 92.35%. Prior to 2019, when the frequency of positivity was highest, the proportion of HAdV-positive inpatients fell to 62.14% and the proportion of outpatients rose to 37.86%. This was attributed to the increased number of infections in the general population and to the hospital reaching full in-patient capacity during this time. tree construction were performed using Molecular Evolutionary Genetics Analysis (MEGA) version 11. The multiple sequence alignments were then revised using Clustal W. Phylogenetic trees were constructed by the Unweighted Pair Group Method with Arithmetic (UPGMA) method with 1000 bootstrap rep- licates, and default settings were used for all other parameters. Detection of other common respiratory pathogens with RT‑qPCR among ICU specimens The children admitted to ICU were tested for HAdV and 10 other respiratory pathogens: influenza A virus (infA), influ- enza B virus (infB), parainfluenza virus (PIV), respiratory syncytial virus (RSV), enterovirus (EV), human metapneu- movirus (hMPV), bocavirus (BOV), rhinovirus (RHV), Myco- plasma pneumoniae (MP), and Chlamydia pneumonia (CP) using a commercially available Taqman RT-qPCR kit (Guang- zhou HuYanSuo Medical Technology Co. LTD; China). Within the 2010–2021 time period, demographic data were available only for 102,469 patients from 2014 to Within the 2010–2021 time period, demographic data were available only for 102,469 patients from 2014 to Table 1 Positivity rate of human adenovirus (HAdV) infections in children seeking treatment at GWCMC for acute respiratory tract infection (ARI) between 2014 and 2019 GWCMC Guangzhou women and children’s medical center. Chi- square analysis, followed by Bonferroni corrections was used to cal- culate the P values. The letter †, ‡, §, ∥ indicated that there was a statis- tical difference, on the positivity rate amongst the ages Variables Samples tested (%) HAdV positive (%) P value Total 102,469 (100.00) 9965 (9.72) Age (y) < 0.001 0–1 25,822 (25.20) 2321 (8.99†) 1–3 36,171 (35.30) 4046 (11.18‡) 3–6 29,818 (29.10) 2252 (7.55§) 6–14 10,656 (10.40) 1335 (12.52\|\|) Gender 0.788 Male 66,604 (65.00) 6465 (9.71) Female 35,865 (35.00) 3500 (9.76) Table 1 Positivity rate of human adenovirus (HAdV) infections in children seeking treatment at GWCMC for acute respiratory tract infection (ARI) between 2014 and 2019 Statistical analysis Chi-square tests followed by post-hoc comparisons using Bonferroni’s adjustment were used to assess statistical sig- nificance. All tests were two-tailed with P < 0.05 considered to be statistically significant. Shift in the dominant HAdV subtype from HAdV‑3 to HAdV‑7 Fig. 2 Subtypes of the human adenovirus (HAdV) detected in the res- piratory samples in a respiratory ward and b in those admitted to the intensive care unit (ICU) of GWCMC in the specified years. GWCMC Guangzhou Women and Children’s Medical Center Molecular typing showed that among 557 patients in res- piratory wards, the proportion was (1) HAdV-3: 42.01% (234/557), (2) HAdV-7: 51.17% (285/557), (3) other HAdV subtypes: 4.85% (27/557), and HAdV-3 and HAdV-7 co- infection 1.97% (11/557). Among 121 ICU patients, the proportion was (1) HAdV-3: 16.53% (20/121), (2) HAdV- 7: 74.38% (90/121), (3) other HAdV subtypes: 8.26% (10/121), and there was one case of HAdV-3 and HAdV-7 co-infection. Overall, the majority of HAdV cases at GWCMC were caused by HAdV-3 and HAdV-7 (Fig. 2a), with relatively rare cases of HAdV-3 and HAdV-7 co-infec- tion being detected in 2014, 2016 and 2018, accounting for 3.66%, 1.03% and 4.93%, respectively. Cases of ARI caused by other HAdV subtypes were also relatively rare (< 10% each year). respectively, but in 2018 and 2019, HAdV-7 became the predominant subtype, causing an annual average of 82.45% ± 0.01% of cases (Fig. 2b). Our data from the ICU were collected between 2014 and 2019, and despite a lower overall rate of detection, the HAdV-7 positivity frequency amongst ICU admissions was significantly greater than that of HAdV-3 (60.5% ± 0.02% and 34.6% ± 0.04%, respectively, Fig. 2b). After 2017, HAdV-7 continued to cause a greater proportion of ICU cases compared to other subtypes. There- fore, regardless of the current epidemic strain, HAdV-7 was always dominant in the ICU. Of note, we also detected two cases of HAdV-55 during our study period and a single case of HAdV-14 in 2019 in the ICU. From 2012 to 2016, HAdV-3 was the most commonly detected subtype and was responsible for an annual aver- age of 74.30% ± 0.06% of cases (Fig. 2a). HAdV-3 fre- quency ranged from 58.54% (2014) to 94.29% (2015), while HAdV-7 made up the majority of the remaining cases. However, from 2017 onwards, we observed a change in the dominant subtype detected. The proportion of HAdV-7 and HAdV-3 cases was similar in 2017, at 50.00% and 45.45%, Monthly incidence of HAdV infections between 2012 and 2021 Based on data collected from January 2012 to December 2021, we studied the monthly incidence of HAdV and found that HAdV was detectable throughout the year (Fig. 1). Before the COVID -19 pandemic, the highest HAdV posi- tivity tended to occur during the summer months between June and September, whereas the lowest positivity occurred between February and March. The lowest HAdV positiv- ity was detected in February 2012 (0.92%) and April 2015 (1.10%), while high positivity was found in July (23.48%) and August (23.62%) of 2019, respectively. In most years HAdV activity peaked between July and August, except in 2018 when it peaked in June. After the COVID-19 pan- demic, HAdV activity showed less obvious seasonality, and the positivity frequency was below 5.00% each month between 2020 and 2021. Number of ARI cases associated with HAdV infection in children increased in 2018 and 2019 Number of ARI cases associated with HAdV infection in children increased in 2018 and 2019 GWCMC Guangzhou women and children’s medical center. Chi- square analysis, followed by Bonferroni corrections was used to cal- culate the P values. The letter †, ‡, §, ∥ indicated that there was a statis- tical difference, on the positivity rate amongst the ages GWCMC Guangzhou women and children’s medical center. Chi- square analysis, followed by Bonferroni corrections was used to cal- culate the P values. The letter †, ‡, §, ∥ indicated that there was a statis- tical difference, on the positivity rate amongst the ages Based on our retrospective data collected from pediatric patients diagnosed with ARI at GWCMC, the number of Fig 1 Monthly incidence of human adenovirus (HAdV) positive cases among children with acute respiratory tract infection (ARI) at GWCMC Fig. 1 Monthly incidence of human adenovirus (HAdV)-positive cases among children with acute respiratory tract infection (ARI) at GWCMC in Guangzhou between January 2012 and December 2021. GWCMC Guangzhou women and children’s medical center Fig. 1 Monthly incidence of human adenovirus (HAdV)-positive cases among children with acute respiratory tract infection (ARI) at GWCMC in Guangzhou between January 2012 and December 2021. GWCMC Guangzhou women and children’s medical center Fig. 1 Monthly incidence of human adenovirus (HAdV)-positive cases among children with acute respiratory tract infection (ARI) at GWCMC in Guangzhou between January 2012 and December 2021. GWCMC Guangzhou women and children’s medical center 1 548 World Journal of Pediatrics (2022) 18:545–552 Fig. 2 Subtypes of the human adenovirus (HAdV) detected in the res- piratory samples in a respiratory ward and b in those admitted to the intensive care unit (ICU) of GWCMC in the specified years. GWCMC Guangzhou Women and Children’s Medical Center 2019 (Table 1). The total HAdV positivity was 9.70%, with no significant differences detected by gender. The frequency of HAdV-positivity in those aged 0–1, 1–3, 3–6 and 6–14 years were 8.99%, 11.18%, 7.55% and 12.52%, respectively, with the positivity in 1–3- and 6–14-year-olds being significantly greater compared to the other age groups (P < 0.001). Mortality proportion in pediatric cases of ARI was greater in 2018 compared to previous years The proportion of ARI cases that tested positive for HAdV was also greater in 2019, at 13.58%, compared to previous years, at 6.37% ± 0.64%. The num- bers of HAdV cases in the ICU of GWCMC also increased during 2016, 2017, 2018 and 2019, with 15, 20, 34 and 35 cases, respectively, as did the number of ICU cases that were fatal, with 3, 3, 8 and 15 cases, respectively (Table 2). Of the 29 deaths in the ICU, 27 were HAdV-7 positive and the remaining two were HAdV-3 and HAdV-55. All of these deaths had typical symptoms of adenovirus pneumonia, such as bronchiolitis obliterans (BO) and bronchiectasis. The main cause of death was a respiratory failure (n = 24), while the other five cases died due to multiple organ dys- function syndromes (MODS) and shock. No co-infection with other common respiratory pathogens was detected (detailed in the patient cohort). HAdV cases were reduced significantly between 2020 and 2021 In February 2020, when the most stringent control meas- ures against COVID-19 were implemented in Guangzhou, HAdV positivity was only 2.68% (out of 15,804 total sam- ples tested) and was the lowest since 2010. The morbidity dropped to a very low level and without fatal cases (Table 2). Discussion The present study reported the recent twelve-year epide- miological profile of circulating HAdV strains in children with ARI at a regional children’s medical service network hospital in Guangzhou, China. The infection rate varies in different years. A previous study of 1778 hospitalized chil- dren with pneumonia in Guangzhou showed an infection rate of 2.50% from 2013 to 2017 and 6.00% from 2018 to 2019 [16]. Our study showed that the annual HAdV infection fre- quency in the Guangzhou area ranged from 3.92% (2012) to 13.58% (2019) between 2010 and 2019 before the COVID- 19 pandemic. Our data were collected during a twelve-year research period, where at least 4500 samples were tested Mortality proportion in pediatric cases of ARI was greater in 2018 compared to previous years With the increasing clinical need for pathogens detec- tion and with awareness of the population seeking more 1 3 1 3 World Journal of Pediatrics (2022) 18:545–552 549 Table 2 Numbers of pediatric cases of acute respiratory tract infection (ARI) and numbers positive for HAdV that were outpatients, hospitalized and admitted to the intensive care unit (ICU) of GWCMC between 2010 and 2021 HAdV Human adenovirus, GWCMC Guangzhou Women and Children’s Medical Center, NA not available. Total number of cases from 2010 to 2021 was 161,079 Year ARI cases HAdV positive (%) Outpatient (%) Hospitalized (%) ICU cases Fatal ICU cases (%) 2010 4543 266 (5.86) 28.52 71.48 NA NA 2011 7312 439 (6.00) 10.31 89.69 NA NA 2012 5682 223 (3.92) 8.86 91.14 NA NA 2013 6912 518 (7.49) 7.65 92.35 NA NA 2014 9668 918 (9.50) 12.81 87.19 NA NA 2015 10,149 434 (4.28) 13.99 86.01 NA NA 2016 10,180 773 (7.59) 17.59 82.41 15 3 (0.39) 2017 13,196 837 (6.34) 16.11 83.89 18 3 (0.36) 2018 19,338 1718 (8.88) 20.93 79.07 34 8 (0.47) 2019 39,938 5424 (13.58) 37.86 62.14 35 15 (0.28) 2020 15,804 423 (2.68) 17.86 82.14 4 0 2021 18,357 565 (3.08) 9.94 90.06 10 0 Table 2 Numbers of pediatric cases of acute respiratory tract infection (ARI) and numbers positive for HAdV tha and admitted to the intensive care unit (ICU) of GWCMC between 2010 and 2021 HAdV Human adenovirus, GWCMC Guangzhou Women and Children’s Medical Center, NA not available. *Total number of cases from 2010 to 2021 was 161,079 strains were deposited in the GenBank database under acces- sion numbers MK123978.1, MN164629.1 and MN135993.1. In our study, the two HAdV-7d isolates MN164629.1 and MN135993.1 had identical genomes (99.9%) with an earlier strain from a fatal acute respiratory distress case during an outbreak in China in 2009 (HAdV-7 0901 HZ) [15]. These data suggest relatively low mutation rates in the HAdV-7 genome. healthcare, especially the 2018/2019 adenovirus epidemic in children in Guangzhou, there was an interannual increase in the number of ARI cases at GWCMC from 4543 in 2010 to 19,338 in 2018 and 39,938 in 2019 (Table 2). The num- ber of ARI cases that tested HAdV positive was also greater in 2018 and 2019 compared to previous years, at 1718 and 5424, respectively, compared to an average of 551 ± 92.70 for previous years. Genomic characterization of HAdV from fatal cases The highest HAdV positive proportions were observed among children over six years of age, which was similar to some other studies done in Guangzhou [16, 18]. A study on seroprevalence and titer levels of neutralizing antibodies (NAb) against HAdV in healthy populations in 2017 from Guangzhou showed that the HAdV antibodies in children over five years were higher than those in other age groups, which was consistent with the results of the present study [19]. Genome variation analysis displayed the stable genome of HAdV-3 and HAdV-7 between 2018 and 2019 [20]. Sequence variation analysis indicated that three genes (pen- ton base, hexon, and fiber) of HAdV-7 were relatively stable across time and geographic space, particularly for viruses within sub-types, which shared almost the same variation sites [27]. Molecular typing of human adenoviruses among hospitalized patients with RTI (respiratory tract infection) in Guangzhou between 2017 and 2019 indicates stable con- servation of HAdV-3 and -7 [18]. Other research showed the nucleotide sequence homologies of the hexon, fiber and penton gene for HAdV-7 and HAdV-3 in Guangzhou between 2013 and 2019 were high (99.5–100%) [16], sug- gesting low strain variation among the circulating viruses. HAdV-7d should be of particular concern within the HAdV subtype. HAdV-7d was believed to be closely related to this HAdV epidemic in Southern China. In Hubei, a prov- ince in Southern China, restriction endonuclease analysis (REA) revealed that HAdV-7 belong to genome 7d [20]. We also detected HAdV-7d in the fatal cases, which had been reported to cause severe illness [5]. Between 2016 and 2019, we also detected HAdV-55 which caused a few severe cases. This study suggests that HAdV-55 has become a common pathogen causing life-threatening pneumonia in Guangzhou after the first outbreak in China in 2006 [28]. We have reported previously that HAdV-3 was respon- sible for most of the HAdV infection among hospitalized children in Guangzhou between 2012 and 2013 [12]. As an extension of that study, we found that HAdV-3 was still the main subtype until early 2017. The proportion of HAdV-7 infections began to rise in the second half of 2017, and by 2018 and 2019 HAdV-7 infections among hospitalized chil- dren had risen to more than 80%. The large epidemic in 2019 led to a sharp increase in pediatric patients in the region. Genomic characterization of HAdV from fatal cases During the course of our study, we noted that there were three particularly severe cases of ARI associated with HAdV infection in the ICU in 2018. To gain a greater insight into the causative viruses, we performed whole viral genome sequencing on samples obtained from these patients. One case was identified as HAdV-55 and the other two were iden- tified as HAdV-7d. The complete genome sequences of these 1 550 World Journal of Pediatrics (2022) 18:545–552 different subtypes may help these viruses to escape the pre- existing immune response. annually with more than 200 samples tested almost every month. This sampling period and study duration can accu- rately reflect the incidence of adenovirus infections in chil- dren. From 2010 to 2019, two years (2014 and 2019) were big epidemic years, suggesting that HAdV epidemics might peak every four or five years. These results provide a refer- ence for predicting the time of the next epidemic peak in the Guangzhou area. At the same time, our data also showed that the HAdV epidemic in the Guangzhou area has a distinct seasonal distribution, with a prominent activity peak in July and August every year and the lowest activity around March.f HAdV-7 was detected in a higher proportion of HAdV infections in the intensive care units, even when HAdV-7 was not the prevalent strain in circulation that year. This is also consistent with reports that HAdV-7 infection is usu- ally more severe than that of HAdV-3 [11, 24, 25]. Our data indicate the same, as 93% of the 29 fatal cases who were adenovirus positive in the ICU were HAdV-7. If infected with adenoviruses, the mortality proportion in children ranges from 0.28% to 0.47% but rises to about 20% in severe cases [26]. During the adenovirus epidemic from 2018 to 2019, there was a sharp increase in the number of children with severe cases in 2018, and the mortality proportion was relatively high (0.47%). However, from the experiences gained in treating severely infected children in 2018, our hospital began to use extracorporeal membrane oxygena- tion (ECMO) to rescue severe patients, which resulted in a slightly improved mortality proportion of severe adenovirus patients (0.28%) in 2019. We did not observe any gender difference in HAdV infec- tion, which is consistent with previous studies conducted in other provinces and countries [16, 17]. Declarations Ethical approval The project was approved by the Ethical Committee of Guangzhou Women and Children’s Medical Center, and the com- mittee’s reference number is 2020-43001. Conflict of interest No financial or non-financial benefits have been re- ceived or will be received from any party related directly or indirectly to the subject of this article. 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/. f In conclusion, our study highlights the transmission pat- tern and epidemiology of HAdV-infections in children in the Guangzhou area within the last 12 years. These data indicate that there was a shift in the dominant HAdV strain from HAdV-3 to HAdV-7 in 2018 and 2019 in our hospital, which may have been reflected in Guangzhou as a whole. HAdV-7 is associated with more severe disease and, as our data indi- cated that HAdV-3 was the dominant subtype, there may have also been limited immunity against HAdV-7, which could have been a contributing factor to the outbreak. Our findings suggest that the transmission pattern of HAdV-types may be associated with population immunity and should be investigated in future studies. Continued surveillance will be essential to predict the epidemic sizes, plan for adequate management of health care resources and to develop vac- cines to combat future outbreaks. Genomic characterization of HAdV from fatal cases LT: investigation, resources, supervi- sion, validation. WCB: investigation, methodology. LWL, ZYY, XJH, CFY, DR: investigation, resources. LGW: formal analysis, investi- gation. MZ: formal analysis, software, writing-original draft. WSS: formal analysis, writing-review&editing. TXG: methodology, fund- ing acquisition. ZJY: investigation, validation. ZLL: formal analysis, software. ZMQ: data curation. YYY: funding acquisition, methodology. The present study has some limitations. First, due to the nature of the retrospective analysis, some data were incom- plete or missing. For example, the demographic data of HAdV infection before 2014 and the number of ICU cases before 2015 were not available because they were not col- lected. Second, HAdV sequencing was not carried out in the early samples. With the increasing understanding of adeno- virus infection in children, we detected HAdV subtypes in the respiratory ward in 2012, and in the ICU ward in 2014. Third, considering the major HAdV subtypes in China and the relatively limited resources, we only detected the main common HAdV subtypes and classified the remaining unknown samples as other HAdV subtypes. Therefore, there is no in-depth discussion on other HAdV subtypes in this study. Finally, it is noteworthy that HAdV was effectively reduced after the emergence of SARS-CoV-2 infection in 2020. China implemented stringent non-pharmaceutical interventions (NPIs) in spring 2020 as part of the response to the emergence of SARS-CoV-2. In our study HAdV activities were reduced significantly during this period. This suggested that NPIs adopted to manage COVID-19 were also effective in preventing transmission of HAdV. Funding This work was supported by the National Natural Sci- ence Foundation of China: [grant number 82072264]; Natural Sci- ence Foundation of Guangdong Province, China: [grant number 2021A1515011071]; Guangzhou School (Institute) Joint Funding Project (No. 202102010202); Municipal Science and Technology Bureau Foundation of Guangzhou (201803040004); Guangzhou Basic Research Program Co-funded by Zhongnanshan Medical Foundation of Guangdong Province: [grant number 202102010364, ZNSA-2020003]. Data availability Condensed anonymized data are available from the corresponding author on reasonable request. Genomic characterization of HAdV from fatal cases In that year nearly 40,000 children were tested for HAdV in our laboratory, with a positive rate of 13.58%, which was the highest in the past decade. This epidemic caused a shortage of hospital beds, and many children could not be hospitalized. Thus, 37.86% of adenovirus-positive cases detected in 2019 came from outpatients. From 2018 to 2019, not only the Guangzhou area but also Hubei, Wenzhou and other places in Southern and Eastern China also witnessed a HAdV epidemic among children [20, 21] while Northern China did not. In North- ern China HAdV-3 was the most common subtype followed by HAdV-7 from 2018 to 2019 [22, 23]. According to our research, from 2012 to 2016, HAdV-3 was the dominant sub- type in Guangzhou, and HAdV-7 accounted for a relatively small proportion. Consequently, HAdV-7 immunity in chil- dren was likely low. A study in Guangzhou showed that the seroprevalence of neutralizing antibodies against HAdV-7 (10.90%) was much lower than HAdV-3 (29.40%) under the age of 18 in 2017 [19]. Some research revealed that HAdV subtype antisera showed no neutralizing activity to other subtype [4]. Immunity against HAdV-3 probably does not protect against HAdV-7. It is possible that the circulation of i Human adenoviruses are classified into seven species (A–G) and at least 110 genotypes, as defined by the Human Adenovirus Working Group (http://hadvwg.gmu.edu/). HAdV direct or indirect transmission can occur through the throat, feces, eyes or urine, depending on the virus subtype. Certain HAdV subtypes are predominantly associated with specific pathologies, such as acute respiratory outbreaks (HAdV-B/C/E) [8, 21], epidemic keratoconjunctivitis 1 3 3 551 World Journal of Pediatrics (2022) 18:545–552 (HAdV-D) [29, 30], gastroenteritis, and/or acute hemor- rhagic cystitis (HAdV-F/G) [31, 32]. On 31 March 2022 Public Health Scotland reported a cluster of cases of severe hepatitis of unknown origin in Scotland. Five children were adenovirus PCR-positive, which suggested that adenovirus also may be a possible pathogen causing unexplained severe acute liver injury in children [33]. formal analysis, investigation, project administration, writing-original draft, writing-review & editing. LT: investigation, resources, supervi- sion, validation. WCB: investigation, methodology. LWL, ZYY, XJH, CFY, DR: investigation, resources. LGW: formal analysis, investi- gation. MZ: formal analysis, software, writing-original draft. WSS: formal analysis, writing-review&editing. TXG: methodology, fund- ing acquisition. ZJY: investigation, validation. ZLL: formal analysis, software. ZMQ: data curation. YYY: funding acquisition, methodology. formal analysis, investigation, project administration, writing-original draft, writing-review & editing. References Yao LH, Wang C, Wei TL, Wang H, Ma FL, Zheng LS. Human adenovirus among hospitalized children with respiratory tract infections in Beijing, China, 2017–2018. Virol J. 2019;16:78. 8. Yamadera S, Yamashita K, Akatsuka M, Kato N, Inouye S. Trend of adenovirus type 7 infection, an emerging disease in Japan A report of the National epidemiological surveillance of infectious agents in Japan. Jpn J Med Sci Biol. 1998;51:43–51. 23. Huang Y, Wang C, Ma F, Guo Q, Yao L, Chen A, et al. Human adenoviruses in paediatric patients with respiratory tract infec- tions in Beijing. China Virol J. 2021;18:191. 9. Lynch JP 3rd, Kajon AE. Adenovirus: epidemiology, global spread of novel serotypes, and advances in treatment and preven- tion. Semin Respir Crit Care Med. 2016;37:586–602. 24. Fu Y, Tang Z, Ye Z, Mo S, Tian X, Ni K, et al. Human adenovirus type 7 infection causes a more severe disease than type 3. BMC Infect Dis. 2019;19:36. 25. Chen Q, Liu J, Liang W, Chen Y, Dou M, Liu Z, et al. Clini- cal features, replication competence, and innate immune responses of human adenovirus type 7 infection. J Infect Dis. 2021;223:1390–9. 10. Li Y, Zhou W, Zhao Y, Wang Y, Xie Z, Lou Y, et al. Molecular typing and epidemiology profiles of human adenovirus infection among paediatric patients with severe acute respiratory infection in China. PLoS ONE. 2015;10:e0123234. 26. Peng HY, Chen FY, Dang R, Zuo YL, Hu PD, Yang YY, et al. Effect of high-titer plasma in pediatric patients with severe adeno- virus pneumonia. Zhonghua Er Ke Za Zhi. 2020;58:392–7 (in Chinese). 11. Wo Y, Lu QB, Huang DD, Li XK, Guo CT, Wang HY, et al. Epi- demical features of HAdV-3 and HAdV-7 in pediatric pneumonia in Chongqing. China Arch Virol. 2015;160:633–8. 12. Chen Y, Liu F, Wang C, Zhao M, Deng L, Zhong J, et al. Molecular identification and epidemiological features of human adenoviruses associated with acute respiratory infections in hospitalized children in Southern China, 2012–2013. PLoS ONE. 2016;11:e0155412.i 27. Cai R, Mao N, Dai J, Xiang X, Xu J, Ma Y, et al. Genetic vari- ability of human adenovirus type 7 circulating in mainland China. PLoS ONE. 2020;15:e0232092. 28. Yang Z, Zhu Z, Tang L, Wang L, Tan X, Yu P, et al. Genomic analyses of recombinant adenovirus type 11a in China. J Clin Microbiol. 2009;47:3082–90. 13. References 1. Kajon AE, Ison MG. Severe infections with human adenovirus 7d in 2 adults in family, Illinois, USA, 2014. Emerg Infect Dis. 2016;22:730–3. 2. Scott MK, Chommanard C, Lu X, Appelgate D, Grenz L, Sch- neider E, et al. Human adenovirus associated with severe res- piratory infection, Oregon, USA, 2013–2014. Emerg Infect Dis. 2016;22:1044–51. Acknowledgements We thank Professor Lin Zhengshi for her role in editing our paper. We also thank every member of the central labora- tory of Guangzhou Women and Children’s Medical Center who helped with this Long-term work. 3. Chen SY, Liu W, Xu Y, Qiu S, Chen Y, Tian X, et al. Epidemiol- ogy and genetic variabilities of human adenovirus type 55 reveal relative genome stability across time and geographic space in China. Front Microbiol. 2020;11:606195. 4. Kajon AE, Lamson DM, Bair CR, Lu X, Landry ML, Menegus M, et al. Adenovirus type 4 respiratory infections among civilian adults, Northeastern United States, 2011–2015(1). Emerg Infect Dis. 2018;24:201–9. Author contributions CY and LT contributed equally to this paper. ZB: conceptualization, data curation, funding acquisition. RZ: conceptualization, funding acquisition, writing review & editing. CY: 1 3 3 552 World Journal of Pediatrics (2022) 18:545–552 indicates the low level of herd immunity in young children from Guangzhou. China Virol Sin. 2021;36:373–81. 5. Yu Z, Zeng Z, Zhang J, Pan Y, Chen M, Guo Y, et al. Fatal com- munity-acquired pneumonia in children caused by re-emergent human adenovirus 7d associated with higher severity of illness and fatality rate. Sci Rep. 2016;6:37216. g 20. Li Y, Wang D, Zhang J, Huang P, Du H, Xu J, et al. Human adenovirus type 7 infections in Hubei, China during 2018–2019: epidemic features and genetic characterization of the detected viruses. Front Cell Infect Microbiol. 2021;11:684606. 6. Cheng JL, Peng CC, Chiu NC, Weng LC, Chiu YY, Chang L, et al. Risk factor analysis and molecular epidemiology of respira- tory adenovirus infections among children in northern Taiwan, 2009–2013. J Microbiol Immunol Infect. 2017;50:418–26. 21. Wen S, Lin Z, Zhang Y, Lv F, Li H, Zhang X, et al. The epidemi- ology, molecular, and clinical of human adenoviruses in children hospitalized with acute respiratory infections. Front Microbiol. 2021;12:629971. 2009–2013. J Microbiol Immunol Infect. 2017;50:418–26. 7. Rojas LJ, Jaramillo CA, Mojica MF, Escalante MP, Delgado P. Molecular typing of adenovirus circulating in a Colombian paedi- atric population with acute respiratory infection. Epidemiol Infect. 2012;140:818–22. 22. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. References Deng J, Qian Y, Zhao LQ, Zhu RN, Sun Y, Tian R. Identifica- tion and typing of adenovirus from acute respiratory infections in pediatric patients in Beijing from 2003 to 2012. Bing Du Xue Bao. 2013;29:615–20 (in Chinese).i 29. Akello JO, Kamgang R, Barbani MT, Suter-Riniker F, Leib SL, Ramette A. Epidemiology of human adenoviruses: a 20-Year ret- rospective observational study in hospitalized patients in Bern. Switzerland Clin Epidemiol. 2020;12:353–66. 14. Han G, Niu H, Zhao S, Zhu B, Wang C, Liu Y, et al. Identification and typing of respiratory adenoviruses in Guangzhou, Southern China using a rapid and simple method. Virol Sin. 2013;28:103–8. 15. Tang L, An J, Yu P, Xu W. Complete genome sequence of human adenovirus type 7 associated with fatal infant pneumonia. Genome Announc. 2013;1:e00182–212. 30. Jonas RA, Ung L, Rajaiya J, Chodosh J. Mystery eye: human adenovirus and the enigma of epidemic keratoconjunctivitis. Prog Retin Eye Res. 2020;76:100826. 16. Zou L, Yi L, Yu J, Song Y, Liang L, Guo Q, et al. Adenovirus infection in children hospitalized with pneumonia in Guangzhou. China Influenza Other Respir Viruses. 2021;15:27–33. 31. Umekawa T, Kurita T. Acute hemorrhagic cystitis by adenovirus type 11 with and without type 37 after kidney transplantation. Urol Int. 1996;56:114–6. 32. Lee B, Damon CF, Platts-Mills JA. Pediatric acute gastro- enteritis associated with adenovirus 40/41 in low-income and middle-income countries. Curr Opin Infect Dis. 2020;33:398–403. l 17. Pan Y, Zhang Y, Shi W, Peng X, Cui S, Zhang D, et al. Human parainfluenza virus infection in severe acute respiratory infection cases in Beijing, 2014–2016: a molecular epidemiological study. Influenza Other Respir Viruses. 2017;11:564–8. 33. Marsh K, Tayler R, Pollock L, Roy K, Lakha F, Ho A, et al. Inves- tigation into cases of hepatitis of unknown aetiology among young children, Scotland, 1 January 2022 to 12 April 2022. Euro Sur- veill. 2022;27:2200318. l 18. Wang X, Wang D, Umar S, Qin S, Ling Q, Gray GC, et al. Molecular typing of human adenoviruses among hospitalized patients with respiratory tract infections in a tertiary Hospital in Guangzhou, China between 2017 and 2019. BMC Infect Dis. 2021;21:748. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 19. Tian X, Fan Y, Wang C, Liu Z, Liu W, Xu Y, et al. Seroprevalence of neutralizing antibodies against six human adenovirus types 1 3
https://openalex.org/W3000400837	https://journals.uic.edu/ojs/index.php/dad/article/download/10740/9508	English	null	Does pre-planning explain why predictability affects reference production?	Dialogue and discourse	2,019	cc-by	11,907	Abstract How does thematic role predictability affect reference production? This study tests a planning facilitation hypothesis – that the predictability effect on reference form can be explained in terms of the time course of utterance planning. In a discourse production task, participants viewed two sequential event pictures, listened to a description of the first picture (depicting a transfer event between two characters), and then provided a description of the second picture (continuing with one thematic role character, either goal or source). We replicated previous findings that goal continuations lead to more reduced forms of reference and shorter latency to begin speaking than source continuations. Additionally, we tracked speakers’ eye movements in two periods of utterance planning, early vs. late. We found that 1) early planning supports the use of reduced forms but is not affected by thematic role; 2) thematic role only affects late planning; and 3) in contrast with our hypothesis, planning does not account for predictability effects on reduced forms. We then speculate that discourse connectedness drives the thematic role predictability effect on reference form choice. Keywords: utterance planning, thematic role predictability, reference production, discourse Editor: Patrick Healey Submitted 08/2017; Accepted 06/2019; Published online 11/2019 Dialogue & Discourse 10(2) 34-55 Dialogue & Discourse 10(2) 34-55 doi: 10.5087/dad.2019.202 Sandra A. Zerkle SZERKLE@UNC.EDU Department of Psychology & Neuroscience, University of North Carolina – Chapel Hill Jennifer E. Arnold JARNOLD@EMAIL.UNC.EDU Department of Psychology & Neuroscience, University of North Carolina – Chapel Hill Editor: Patrick Healey 1 Introduction Variation in reference form exists in natural language production: an entity could be referred to with its proper name (Lady Mannerly), a noun phrase description (the duchess), a reduced form such as a pronoun (she), or even dropping the referent entirely (…and Ø left). What guides speakers as they make this choice? Previous work has supported the hypothesis that certain discourse conditions promote the use of particular referential forms (e.g., Ariel, 1990, 2001; Chafe, 1976, 1994; Gundel, Hedberg, & Zacharaski, 1993), where reduced forms tend to be used for salient or accessible referents. For example, speakers strongly prefer pronouns to refer to entities that appeared recently in the discourse, and especially in the previous subject position (Arnold, 1998). In addition, in some cases speakers are also more likely to use pronouns for thematic roles that are predictable (Arnold, 2001; Rosa & Arnold, 2017; but see Fukumura & van Gompel, 2010; Rohde & Kehler, 2014). However, there are no explicit models of the production mechanisms underlying reference production. y g p In particular, an unsolved puzzle concerns the role of predictability in pronoun production, and how and why predictability affects referential choices. The kind of predictability that matters here is referential predictability, i.e. the likelihood that the speaker will mention that entity at that point in the discourse. This is related to the calculation of referential probability, which includes probability estimates made both before and after the referring expression. There is good evidence that predictability is important for comprehension, for example listeners are faster to understand predictable information, and sometimes even anticipate it (e.g., Altmann & Kamide, 1999). However, it’s not obvious what predictability would mean to the speaker. People usually know what they are going to say ahead of time, so they aren’t predicting their message; they are planning it. That is, while predictability can be thought of as a property DOES PLANNING EXPLAIN WHY PREDICTABILITY AFFECTS REFERENCE PRODUCTION? of the referent within context, it is not known whether this leads to actual prediction in all cases. This raises questions about how factors related to predictability in comprehension might affect production, and why. 1.1 Thematic role predictability affects reference form p y Previous work has revealed that reduced forms of reference are more likely in certain linguistic contexts: if an entity is given (Fowler & Hossum, 1987), in the subject position (Arnold, 1998; Chafe, 1976), contextually salient, topical, in focus, accessible (Ariel, 1990; Arnold, 2010; Chafe, 1994; Frank & Goodman, 2012; Givón, 1983; Grosz et al., 1995), or predictable in context (Jurafsky, Bell, Gregory, & Raymond, 2001; Levy & Jaeger, 2007; Mahowald et al., 2013). These findings span multiple types of reduction, including durational shortening, the use of reduced syntactic structures, as well as the use of pronouns instead of longer referential expressions. The current study is focused on the contrast between explicit descriptions (the duchess) and pronouns or dropped (zero) references. pp ( ) We focus our attention on how thematic roles influence referential predictability, and whether this affects reference form. Thematic roles are assigned to a verb’s arguments (Dowty, 1991; Jackendoff, 1972), and represent a semantic characterization of the entities involved in an event. The current study examines the thematic roles associated with transfer verbs, where the verb of a sentence depicts the transfer of an object (the theme) from one character (the source) to another character (the goal), as in “the duchess handed a painting to the duke”. In a discourse context, these thematic role positions affect people’s expectations about who will be mentioned next, where the goal referent is more expected than the source (Rosa & Arnold, 2017; Stevenson et al., 1994). Rosa and Arnold measured this expectation by simply asking participants who they thought would be mentioned next, but it also correlates with a tendency for speakers to mention the goal more in either natural discourse (Arnold, 2001) or story-continuation tasks (Stevenson et al., 1994). There are a few possibilities for why goals are frequently mentioned, and perceived as predictable referents. One idea is that people expect goals because they frequently hear other people mention goals, so this high frequency affects future expectations of similar events (Arnold, 1998, 2001; Rosa & Arnold, 2017). Another possibility is that people have an inherent interest in goals, such that we expect the next event to involve the recipient of a transferred object. j When we consider transfer verbs, there is also excellent evidence that thematic roles affect reference form. 1 Introduction Despite the oddness of prediction during production, it is important to investigate the relationship between predictability and reference production, because referential probability underlies numerous models of reference production (Frank & Goodman, 2012; Tily & Piantadosi, 2009), other aspects of language production (Hale, 2001; Levy & Jaeger, 2007), and reference comprehension (Frank and Goodman, 2012; Kehler & Rohde, 2013). However, empirical findings show that referential probability only sometimes affects pronoun use (see e.g. Rosa & Arnold, 2017 on the one hand, and e.g. Rohde & Kehler, 2014 on the other), while it has a stronger effect on prosodic variation (e.g., Arnold & Watson, 2015). This raises questions about how predictability really affects reference form. Here we address this issue by examining the relation between reference form, referential predictability, and utterance planning. 1.1 Thematic role predictability affects reference form Speakers are more likely to use pronouns and zeros when referring to goals than sources (Arnold, 2001; Rosa & Arnold, 2017; Zerkle, Rosa, & Arnold, 2015). Some of this evidence comes from a paradigm (also used in the current study) in which participants saw pairs of pictures depicting a transfer in the first picture and a subsequent action in the second picture (see Figure 1). For half of the items, the goal character continued in the subsequent event, and for 35 ZERKLE & ARNOLD ZERKLE & ARNOLD ZERKLE & ARNOLD the other half the source character continued. Participants listened to a description of the first picture, and were instructed to provide a description of the second picture. In three experiments (Rosa & Arnold, 2017, exp. 1; Zerkle et al. 2015, exps. 1 and 2), speakers used reduced forms more for goal than source continuations. This finding occurred on top of the well-known tendency for subject continuations to use a pronoun. See Figure 2 for reference form choice results from these three studies. the other half the source character continued. Participants listened to a description of the first picture, and were instructed to provide a description of the second picture. In three experiments (Rosa & Arnold, 2017, exp. 1; Zerkle et al. 2015, exps. 1 and 2), speakers used reduced forms more for goal than source continuations. This finding occurred on top of the well-known tendency for subject continuations to use a pronoun. See Figure 2 for reference form choice results from these three studies. Figure 1. Example trial: A speaker listens to a description of the first panel (“The duchess handed a painting to the duke”) and then must provide a description of the second panel (e.g., “and then he threw it in the closet”). Figure 1. Example trial: A speaker listens to a description of the first panel (“The duchess handed a painting to the duke”) and then must provide a description of the second panel (e.g., “and then he threw it in the closet”). Figure 2. Speakers tend to use reduced forms more often for reference to goal continuations than source continuations, as well as for reference to subject continuations than non-subject continuations. (Note that Rosa & Arnold (2017) findings reflect pronoun use only, whereas the reduced forms in Zerkle, Rosa, & Arnold (2015) include both pronouns and zeros). Error bars are standard error of the subject means by condition. 0% 20% 40% 60% 80% 100% Subject Non-Subject Subject Non-Subject Subject Non-Subject Rosa & Arnold (2017) Zerkle, Rosa, & Arnold (2015, Exp1) Zerkle, Rosa, & Arnold (2015, Exp2) % pronoun/zero Goal Source Figure 2. Speakers tend to use reduced forms more often for reference to goal continuations than source continuations, as well as for reference to subject continuations than non-subject continuations. ZERKLE & ARNOLD (Note that Rosa & Arnold (2017) findings reflect pronoun use only, whereas the reduced forms in Zerkle, Rosa, & Arnold (2015) include both pronouns and zeros). Error bars are standard error of the subject means by condition. 0% 20% 40% 60% 80% 100% Subject Non-Subject Subject Non-Subject Subject Non-Subject Rosa & Arnold (2017) Zerkle, Rosa, & Arnold (2015, Exp1) Zerkle, Rosa, & Arnold (2015, Exp2) % pronoun/zero Goal Source Figure 2. Speakers tend to use reduced forms more often for reference to goal continuations than source continuations, as well as for reference to subject continuations than non-subject continuations. (Note that Rosa & Arnold (2017) findings reflect pronoun use only, whereas the reduced forms in Zerkle, Rosa, & Arnold (2015) include both pronouns and zeros). Error bars are standard error of the subject means by condition. One interpretation of these findings is that they reflect a preference to use reduced expressions for predictable information (Arnold, 2001; Rosa & Arnold, 2017). This is consistent with other evidence that speakers tend to use reduced forms for redundant information (Levy & Jaeger, 2007; Mahowald et al., 2013), and in particular that speakers use pronouns more for 36 DOES PLANNING EXPLAIN WHY PREDICTABILITY AFFECTS REFERENCE PRODUCTION? predictable referents (Arnold, 1998, 2010). However, this conclusion is complicated by the fact that not all types of predictability affect reference form. Several studies have examined implicit causality contexts, such as Val admired Elyce because.... or Val impressed Elyce because…. Listeners have a strong expectation that the speaker will mention the person who is the more likely cause of the event (i.e., Elyce in the first sentence; Val in the second). However, several studies have found that this expectation has no effect on the speakers’ use of pronouns (Fukumura & van Gompel, 2010; Kehler et al., 2008; Kehler & Rohde, 2013; Kravtchenko et al. 2017, but see Weatherford & Arnold, under review). Instead, speakers use pronouns more for the subject (Val) than the object (Elyce). ( ) j ( y ) While this difference between transfer verb experiments and implicit causality experiments may be attributable to the different verbtypes, it raises questions about why the thematic role effect occurs. In addition, it’s not clear how thematic role predictability affects language production mechanisms. ZERKLE & ARNOLD We assume that in most cases, speakers do not need to predict their own speech, because typically conceptual planning precedes formulation (for an alternate view see Pickering and Garrod, 2013). Instead, we expect that the types of conditions that make referents predictable to the listener may be associated with the production process. Here we test the hypothesis that the tendency to use reduced expressions for goals is not a direct result of prediction per se, but rather is a consequence of the mechanisms involved in utterance planning. Perhaps goal continuations are easier to plan (conceptually and/or lexically) than source continuations, and it is this facilitation that drives the choice to use a reduced form. The referential predictability previously found for goal continuations could affect both the time course of planning and reference form choice, raising the question of whether or not planning has a direct effect on reference form. 1.2 The role of planning facilitation Hypothetically, there are several reasons why ease of planning might affect reference form. One possibility is that early planning might increase the salience of information in the speaker’s discourse model. Classic theories about reference production suggest that pronouns are selected when the referent is salient, accessible, or in focus (for a review see Arnold, 2016; Arnold & Zerkle, 2019), where referential salience is often modeled in terms of the activation of the referent’s conceptual representation (Arnold & Griffin, 2007; van Rij et al., 2010). When planning is easy, it may leave greater mental resources for representing information in the discourse model, increasing the activation on those referents. Thus, if predictability makes planning easy, this might explain the effect of predictability on reference production. Another possibility, suggested by rational models of reference form, is that the production of reduced forms incurs a lower cognitive cost than the production of explicit forms (e.g., Frank & Goodman, 2012; Tily & Piantadosi, 2009). y ) One of the few studies to address the relation between utterance planning and reference form is Arnold & Nozari (2017). In their study, speakers described shapes moving on screen, e.g. The yellow pentagon flashes. Then it jumps over the yellow square. They found that for the sham subjects, who were not experiencing anodal transcranial direct current stimulation, pronouns and zeros were used more often when the timing of the stimuli and response enabled the speaker to do more pre-planning, i.e., when there was no gap between utterances, and when the speaker was planning one utterance while describing the previous event. In this task, these conditions also promoted other measures of discourse connectivity, such as the use of connectors and or then. However, this study used discourse contexts with relatively little semantic information, and did not address questions of how planning relates to predictability. 37 ZERKLE & ARNOLD The current study uses an eyetracking method to test whether planning facilitation explains the role of predictability on reference production. On the planning facilitation hypothesis, thematic roles promote the status of predictability, which in turn triggers earlier conceptual activation of the referent, making it more accessible for faster planning, which leads to increased pronoun choice. 1.2 The role of planning facilitation p In sum, the main question we will be asking in the current study is whether or not planning mechanisms can explain the predictability effect on reference form choice, and we will be using fine-grained measures of utterance planning with eye movements to test this. We will examine anticipatory looks to the target panel in two broad regions, representing relatively early and late planning windows. 1 Note that on this terminology, “in focus” refers to information that is salient or accessible. This contrasts with the linguistic term focus, which contrasts with topic and is associated with new information. 1.3 Traditional models of utterance planning Traditional models of utterance planning generally agree that the generation of an utterance is incremental; beginning with the transformation of a communicative intention into a preverbal conceptual message. The speaker then encodes the grammatical formulation of this message (which includes lexical/lemma selection and functional assignment), phonological encoding and syllabification occurs, and then the phonemes are encoded and articulated (Bock, Irwin, Davidson, & Levelt, 2002; Ferreira & Swets, 2002; Griffin & Bock, 2000; Levelt, 1989; Levelt, Roelofs, & Meyer, 1999; Wheeldon, 2013; see Figure 3). Visual input can affect the early conceptual message level, such as in picture naming tasks (Indefrey & Levelt, 2004; Roelofs, 1992). Figure 3. Model of utterance planning recreated from Levelt, Roelofs, & Meyer (1999). Figure 3. Model of utterance planning recreated from Levelt, Roelofs, & Meyer (1999). Following the general model in Figure 3, Schmitt, Meyer, & Levelt (1999) built a model of planning that is specific to reference production. They proposed that speakers must fit each message fragment into the current discourse, which involves marking certain concepts as old 38 DOES PLANNING EXPLAIN WHY PREDICTABILITY AFFECTS REFERENCE PRODUCTION? information (in focus), and others as new (not in focus; Chafe, 1976).1 These types of information can be referred to linguistically in different ways, from explicit noun phrases to reduced pronouns. Such marking helps speakers and listeners maintain a coherent discourse record (Levelt, 1989). In order to access pronouns, lexical concepts of the entities are marked as being in focus or not in focus depending on the discourse context. A pronoun is selected if and only if the corresponding lexical concept is accessible and marked as in focus within the discourse; otherwise, a noun is selected. The authors propose that a referent’s discourse accessibility status affects the message at the conceptual level; this accessibility node then affects the anaphoric link to lexical selection, in this case whether a reduced form vs. an explicit form is chosen (see Figure 4). However, this model did not address questions about the timing of utterance planning, and whether planning is faster for accessible referents. Neither does it include consideration of predictability as a component of discourse status. This raises questions about whether planning facilitation has any effect at all on reference production. Figure 4. An English-language adaptation of the model of reference production from Schmitt, Meyer, & Levelt (1999). Figure 4. 2 In Rosa & Arnold (2017), this effect of predictability on latency occurred even after controlling for the length of the context sentence (Arnold, 2017), which may have affected participants’ ability to pre-plan their responses. In the experiments from Zerkle, Rosa & Arnold (2015), the stimulus picture did not appear until after the context sentence, so the duration of the context sentence is not as relevant. See Zerkle, Rosa, & Arnold (2017) for a discussion of differences in planning across these two studies. ZERKLE & ARNOLD ZERKLE & ARNOLD In all three experiments, they found that speakers initiated goal continuations faster than source continuations, supporting the hypothesis that predictability affects the time course of utterance planning (see Figure 5). Response latency was measured from the offset of the initially heard description to the onset of the speaker’s fluent speech. 2 Figure 5. Main effect (indicated by asterisks) of goal continuation on response latency in Rosa & Arnold (2017) and Zerkle, Rosa, & Arnold (2015, Exp. 1); numerical trend in Zerkle, Rosa, & Arnold (2015, Exp. 2). Error bars are standard error of the subject means by condition. 0 400 800 1200 1600 2000 Subject Non-Subject Subject Non-Subject Subject Non-Subject Rosa & Arnold (2017) Zerkle, Rosa, & Arnold (2015, Exp1) Zerkle, Rosa, & Arnold (2015, Exp2) response latency (ms) Goal Source * * * * Figure 5. Main effect (indicated by asterisks) of goal continuation on response latency in Rosa & Arnold (2017) and Zerkle, Rosa, & Arnold (2015, Exp. 1); numerical trend in Zerkle, Rosa, & Arnold (2015, Exp. 2). Error bars are standard error of the subject means by condition. In sum, goal continuations both yield faster response latencies and are more likely to involve pronouns. The next question is whether response latency itself affects reference form choice. Yet all three of these experiments found that latency does not predict reference form – as latency to begin speaking increased, there was no significant change in the rate of pronoun/zero choice in any of these three experiments (analyzed separately). These findings provide strong evidence against one sort of hypothesis about planning time. However, planning in a discourse context can often occur earlier, while hearing/producing earlier utterances. For example, in Rosa & Arnold (2017, Exp. 1), participants could see both panels throughout the trial, allowing earlier planning. In both of the Zerkle et al. (2015) experiments, participants did not see the second panel until after the first sentence, but they had previewed the stimulus stories before the production task, which means they may have recalled the target sentence from memory before it appeared. Both of these tasks mirror the conditions of real life production, where the speaker typically knows what they are going to say before they say it. The current study tests whether earlier measures of planning pattern with reference form choices, and whether this explains the effects of predictability. 1.3 Traditional models of utterance planning An English-language adaptation of the model of reference production from Schmitt, Meyer, & Levelt (1999). 1.4 Does planning explain thematic role effects on reference form? The question behind our study is whether planning explains the relation between thematic roles and reference production choices. We have preliminary evidence about this question from previous studies (Rosa & Arnold, 2017; Zerkle, Rosa & Arnold, 2015), which used response latency as a proxy measure of planning time. In the storytelling task described above, participants hear the first sentence, and then provide a second sentence by describing the second panel of the cartoon picture. The time to respond provides a rough measure of the time needed to plan the response. Some planning could take place earlier, as participants listened to the first sentence. However, there was variable time to respond across trials, suggesting that considerable planning occurred immediately before the utterance. 39 39 1.5 Current study goals In all critical items, this event shows the continuing character (the target referent), as well as an action that they are performing. This motivates our use of the entire picture (as opposed to just the character) as our region of interest here. Additionally, there is enough variability in our visual stimuli that this broad region of interest is the most appropriate. p g p , g character (the target referent), as well as an action that they are performing. This motivates our use of the entire picture (as opposed to just the character) as our region of interest here. Additionally, there is enough variability in our visual stimuli that this broad region of interest is the most appropriate. p ( pp j ) g Additionally, there is enough variability in our visual stimuli that this broad region of interest is the most appropriate. We will also examine fixations to panel 2 in two different windows of time, which represent early vs. late planning. We speculate that when a speaker is still listening and comprehending the first sentence, they may likely be conceptually planning parts of their own upcoming utterance at this point. When this first sentence is finished there is a period of time that is silent right before the speaker begins speaking, and this may be where more lexical formulation planning occurs. These two periods are based on traditional models of utterance planning (e.g., Griffin & Bock, 2000; Meyer, Sleiderink, & Levelt, 1998; Wheeldon, 2013); however, we cannot link them directly to phases of production (message conceptualization vs. lexical formulation). Importantly, this is the first investigation into the time course of planning and how it relates to both thematic role predictability and reference form choice. To test the planning facilitation hypothesis, we will compare effects in both early and late stages of planning to determine if and where planning facilitation occurs, and how it relates to reference form. One challenge with studying language planning is that we don’t know precisely when planning begins, and thus, the direction of causation is not clear. If speakers plan a pronoun early, this choice might guide their looks during the planning periods. Alternatively, if speakers happen to look more at panel 2, this attention itself might drive the choice of pronoun. Nevertheless, we think it is plausible that events occurring earlier in time are likely to influence events occurring later in time. 1.5 Current study goals The current study tests the relation between utterance planning, predictability, and reference form. Even though latency did not predict reference form choice, eyetracking may provide an earlier and more sensitive measure for identifying planning effects on reference production. The 40 DOES PLANNING EXPLAIN WHY PREDICTABILITY AFFECTS REFERENCE PRODUCTION? conceptual planning of a message could begin earlier than the latency period that was used in previous studies. Indeed, speakers often pre-plan their utterances (e.g, Ferreira & Swets, 2002), at least at the message level (Brown-Schmidt & Konopka, 2015). If speaking about predictable entities leads to greater pre-planning than speaking about unpredictable entities, it might explain the link between predictability and reference production. To address this, in the current study we used eye tracking to each visual scene as a proxy for planning. Previous research has suggested that fixations to visual stimuli reflect the conceptual and linguistic planning that occurs before utterance production. In a simple picture-description task, trial-initial looks signify rapid planning: speakers may direct their attention to the part of the displayed picture that represents the first referent in their utterance (Bock et al., 2003). Speakers also tend to look at objects immediately before naming them (Griffin and Bock, 2000). In contrast, van der Meulen et al. (2001) found that speakers do not always look at the to-be-named objects: speakers allocated less visual attention to given objects than to new ones, and to objects they would later refer to with a pronoun than with a full noun phrase. This suggests that discourse focus (given vs. new) and referential form (pronoun vs. name) modulate the amount of visual attention allocated to a referent during utterance planning. Here we test whether planning, as measured by anticipatory eye movements, could be related to both reference form and predictability. This is the first study to examine these issues within a discourse context, so we do not have a priori expectations about how these are related. Current theories might suggest that accessible referents would lead to faster planning (e.g., Bell et al., 2009), but this question has not been addressed explicitly. We will be using looks to panel 2 (the right-most picture, Figure 1) as the metric of early planning, because looks ahead indicate that the speaker is attending to this event and preparing their own upcoming description turn. 1.5 Current study goals We therefore take this as our starting point. Our analyses look first at the effect of the discourse context, which occurs earlier than the participants’ response. How does the context affect both planning measures and utterance form? Then we look at the effect of planning, which 41 ZERKLE & ARNOLD is logically prior to the reference, and ask whether either early or late planning measures predict variation in reference form. is logically prior to the reference, and ask whether either early or late planning measures predict variation in reference form. A summary of our predictors and measures is shown in Figure 6. Using a variation on Rosa & Arnold (2017)’s paradigm, we expect to replicate the effect of thematic roles on reference form. We additionally test three measures of planning: early looks, late looks, and latency. These measures allow us to test three questions: q • Question 1. Does thematic role predict production planning measures (latency, early looks, and late looks)? • Question 1. Does thematic role predict production planning measures (latency, early looks, and late looks)? ) Question 2. Does each of the production planning measures predict reference form choice • Question 2. Does each of the production planning measures predict reference form choice? • Question 3: Can the predictability effect on reference form be explained in terms of utterance planning? • Question 3: Can the predictability effect on reference form be explained in terms of utterance planning? Figure 6. Visual summary of the measures used. If thematic role affects planning, we expect the goal continuation contexts to elicit shorter latencies (as in previous studies), and perhaps more looks to the target panel during either early or late planning regions. If planning facilitation drives reference form choice, we expect reference form to vary as a function of either latency or anticipatory fixations. Finally, if planning explains predictability effects on reference form, then we would expect planning to have a stronger effect on reference form than thematic role. Planning effects may occur for early measures of planning, late measures of planning, or both. Figure 6. Visual summary of the measures used. Figure 6. Visual summary of the measures used. If thematic role affects planning, we expect the goal continuation contexts to elicit shorter latencies (as in previous studies), and perhaps more looks to the target panel during either early or late planning regions. 1.5 Current study goals If planning facilitation drives reference form choice, we expect reference form to vary as a function of either latency or anticipatory fixations. Finally, if planning explains predictability effects on reference form, then we would expect planning to have a stronger effect on reference form than thematic role. Planning effects may occur for early measures of planning, late measures of planning, or both. 2.1 Participants 54 native English speakers at the University of North Carolina at Chapel Hill participated for course credit. 13 of these participants used only NP descriptions to refer to the target on critical trials, so these participants were excluded from analyses, leaving 41 participants with reference form variation. 6 of these participants were excluded due to mis-calibration of the eyetracker, and 1 participant was excluded for audio technical failure. This left 34 participants in the final analyses (19 females, and 15 males). 2.2 Materials and Design A narrative production paradigm designed by Rosa & Arnold (2017) was modified for use with eyetracking for the current study (for stimuli see http://jaapstimuli.web.unc.edu/transfer-verb- stimuli-and-paradigm/). In the primary task, participants viewed pairs of pictures (like Figure 1) and listened to a sentence describing the first (left) picture. Their task was to describe the second 42 DOES PLANNING EXPLAIN WHY PREDICTABILITY AFFECTS REFERENCE PRODUCTION? picture, and fixations to the first panel represented attention to the discourse context, while fixations to the second panel represented early planning of the target utterance. As in Rosa and Arnold’s task, participants first watched a background video that described the story context. They were given the role of tabloid photographer, and informed that they witnessed a murder in a mansion and they happened to capture pictures of the events from that day. They learned about six characters: three males (the duke, the butler, the driver) and three females (the duchess, the maid, the chef). Their task was to describe these pictures to a “detective” in order to help solve the crime. One critical change in this study was that the detective’s sentences were recorded, instead of spoken live (as in Rosa & Arnold, 2017). The recorded voice described the first of each picture pair, and participants were encouraged to “continue the story” when describing the second of each picture pair. Another critical change was that the instructions emphasized storytelling. In a similar task, Zerkle & Arnold (2016) found that some participants ignored the context and produced only descriptive noun phrases. Here we emphasized storytelling in order to encourage more connected discourse, and thus elicit greater variation in referential forms. The experimenter instructing each participant also gave two example trials (from filler trials of the main experiment), in which they used both connector words and pronouns in each continuation. The story in the main experiment consisted of 53 “evidence photos”: 29 fillers and 24 critical items. In the critical items, the first picture and sentence described a transfer event between two characters (from a source to a goal). The second panel only pictured the target character, which was either the goal or the source of the previous sentence (manipulated between items). 2.2 Materials and Design The grammatical placement of the target character in the detective’s sentence was manipulated between-subjects, such that half of the participants heard, “The Duchess handed a painting to the Duke” and half heard, “The Duke received a painting from the Duchess.” Thus there were four possible conditions of character continuation: goal/subject, source/subject, goal/non-subject, source/non-subject. However, the trials from both non-subject conditions were not included in analyses here (see section 3.2 for explanation). Filler trials pictured between one to three characters. All trials were presented in the same order for all participants to create a coherent story sequence. Character gender was controlled, such that half of the critical trials had two characters of the same gender, and half had two characters of different gender. 3 Results and Discussion In this section, we first describe the general procedure we used to analyze the data from this study. We then report the linguistic, planning, and reference form results. 2.3 Procedure Research assistants fitted each participant with an Eyelink II head-mounted eyetracker (SR Research) and a headset headphones/microphone. First participants viewed a slideshow in PowerPoint, which played the background video describing the story and characters, and then they viewed all 53 picture pairs silently in order (5s per pair). The purpose of this initial preview was to familiarize the participant with the series of the events and the outcome of the story. This imitates the characteristics of natural language production, in which speakers usually convey information they already know. After this, they heard two sample trials from the experimenter, and were instructed to continue the story as much as possible when they described each picture. Calibration of the eyetracker occurred right before the main experiment began. Before each trial, participants viewed a drift correct screen and pressed the space bar to continue. Then the pair of pictures appeared on the screen next to each other and remained there for the duration of the trial. After a preview period (average of 665ms), the context detective sentence played over the headphones. Participants spoke their description of the second picture into the microphone, and then used the mouse to click on a green circle in the bottom right corner to move on to the next 43 ZERKLE & ARNOLD trial, which began with another drift correct screen. See Figure 7 for a schematic diagram of the preview and trial procedures. Figure 7. Schematic diagrams of the preview procedure and an example trial procedure. Figure 7. Schematic diagrams of the preview procedure and an example trial procedure. 3.1.1 Response coding Trials were excluded if the participant did not refer to the target character in the subject position of their utterance. We excluded trials in which the speaker referred to the wrong character in the response (n=9), leaving 373 trials to be audio coded. Research assistants transcribed utterances and coded responses for type of reference (pronoun, zero, NP description) and use of connector before the reference (e.g., and, then, and then, now). For the analyses, we only analyzed the subject continuation trials (n=382), excluding the non-subject continuation trials (n=379) (see section 3.2). 3.1.2 Audio data coding Four research assistants analyzed the audio files in Praat (Boersma & Weenink, 2015). They segmented each trial in order to measure latency to begin speaking, which was calculated as the time in milliseconds between the end of the context sentence and the beginning of fluent speech. Any disfluencies prior to the response were considered as part of planning time, and thus were included in this latency measure (N=10). Outliers were excluded, where outliers were trials where the latency was less than or greater than 3 standard deviations from the grand mean (N=3). We also calculated track loss on a trial level, where trials with greater than 33% of looks in either time window were classified as No Data (blinks, looks not to the computer screen, etc.). There 44 DOES PLANNING EXPLAIN WHY PREDICTABILITY AFFECTS REFERENCE PRODUCTION? were 4 trials with < 33% track loss in the DS window, and 5 trials in the Latency window. Thus, 361 trials were included in statistical analyses. were 4 trials with < 33% track loss in the DS window, and 5 trials in the Latency window. Thus, 361 trials were included in statistical analyses. 3.1.3 Dependent measures Our analysis of reference form examined the binary contrast between reduced (pronoun and zero) and unreduced (description) forms. Pronouns and zeros were combined because they play a similar pragmatic role, and are both more common when the referent is accessible or predictable. Zeros in our task represent cases of syntactic coordination, e.g. The duke received the basket from the duchess…. and Ø threw it down the hallway. Some studies instead exclude zero continuations from analysis (e.g., Rosa & Arnold, 2017), especially when they are rare in the dataset. Here, however, doing so would exclude around 25% of the data, and misrepresent the rate of using reduced expressions. Moreover, an examination of pronoun vs. zero trials suggests that this decision did not change our findings, in that numerically similar patterns were found for pronouns and zeros. If zeros are excluded from the analyses, similar results obtain. y Our analysis of latency used the log of the latency as a dependent measure. For analyses of gaze, eye position was sampled every 4 ms, but the data were converted to samples at every 20 ms prior to analyses for faster processing (McMurray, 2002). Eye movement data is presented in terms of looks, where a look is defined as a fixation grouped together with the prior saccade. This practice is often used in language processing studies, where listeners are directing their attention to one referent and not another (e.g., Arnold, Hudson-Kam, & Tanenhaus, 2007; Arnold & Lao, 2015; Huettig, Rommers, & Meyer, 2011; McMurray, Tanenhaus, & Aslin, 2009). We used two areas of interest: Panel 1 and Panel 2 (see Figure 1). Ports used for analyses were rectangles exactly around each picture. Any looks to areas besides these were considered “other.” Average looks are presented graphically here for ease of interpretation, but empirical logits of looks were used in all statistical models. Empirical logits were calculated for both panels using the following formula based on Barr’s (2008) methods: 𝐿𝑂𝐺$ 𝑆𝑢𝑚 𝑜𝑓 𝑙𝑜𝑜𝑘𝑠 𝑡𝑜 𝑃𝑎𝑛𝑒𝑙 2 + 0.5 𝑇𝑜𝑡𝑎𝑙 𝑠𝑢𝑚 𝑜𝑓 𝑙𝑜𝑜𝑘𝑠−𝑆𝑢𝑚 𝑜𝑓 𝑙𝑜𝑜𝑘𝑠 𝑡𝑜 𝑃𝑎𝑛𝑒𝑙 2 + 0.5: 3.1.4 Model-building procedure g p Generalized linear mixed effects models were used to account for dependencies in repeated measures using SAS 9.4. Proc GLIMMIX was used for analyses of dichotomous dependent variables with a logit link, i.e. for all analyses of reference form. Proc MIXED was used for analyses of continuous dependent variables, i.e. for all analyses of eye gaze and response latency. We constructed models of each dependent variable with random intercepts of participant and item to account for nesting. Binary predictors were effects coded, such that each variable as reported in the models below are comparison group vs. reference group. All predictor variables were grand- mean centered. Random slopes of all included variables by participant and by item were included when appropriate, but if a slope was estimated to be zero it was excluded (Searle et al., 1992). The final fixed and random effects for each model are reported below each table. ZERKLE & ARNOLD In the first model, the main effect of thematic role is seen in the left bars (only using subject continuation trials). ZERKLE & ARNOLD ZERKLE & ARNOLD Within the subject continuation trials, the descriptive statistics by thematic role condition are reported in Table 1. Within the subject continuation trials, the descriptive statistics by thematic role condition are reported in Table 1. Variable Averages: Goal condition Source condition Overall Detective sentence length (ms) 2453.73 2242.10 2351.14 Latency to begin speaking (ms) 1454.71 1670.13 1559.14 Pronoun/zero use 64.52% 45.14% 55.12% Pronoun use 37.10% 22.29% 29.92% Zero use 27.42% 22.86% 25.21% Looks to P2 in DS window 47.65% 43.22% 45.50% Looks to P2 in Latency window 75.88% 68.14% 72.13% Table 1. Descriptive statistics by goal/source condition, subject continuation trials only. Percentages represent the total number of trials in which an event occurred in each thematic role condition (and overall). y Table 1. Descriptive statistics by goal/source condition, subject continuation trials only. Percentages represent the total number of trials in which an event occurred in each thematic role condition (and overall). Table 1. Descriptive statistics by goal/source condition, subject continuation trials only. Percentages represent the total number of trials in which an event occurred in each thematic role condition (and overall). First, we built a model of pronoun/zero use that showed that speakers used significantly more pronouns/zeros for goal continuations, (see Table 2, Figure 8). This finding replicated the effects found in both Rosa & Arnold (2017) and Zerkle, Rosa, & Arnold (2015). Effect Estimate SE DF t-value p-value Goal vs. Source continuation 1.1739 0.4486 19.88 2.62 0.0166 Table 2. Critical predictor of pronoun/zero use. Random intercepts of participant and of item. Effect Estimate SE DF t-value p-value Goal vs. Source continuation 1.1739 0.4486 19.88 2.62 0.0166 Table 2. Critical predictor of pronoun/zero use. Random intercepts of participant and of item. Figure 8. Reference form choice rates by grammatical role and thematic role. This includes 373 trials in the subject conditions, and 364 in the non-subject conditions. Error bars are standard error of the subject means by condition. In the first model, the main effect of thematic role is seen in the left bars (only using subject continuation trials). 65% 9% 45% 11% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Subject Non-Subject % pronoun/zero Goal Source Goal Source g choice rates by grammatical role and thematic role. This includes 373 trials in the subject conditions, and 364 in the non-subject conditions. Error bars are standard error of the subject means by condition. 3.2 Linguistic measures g Speakers rarely used reduced expressions for non-subject continuation trials (10%), so only subject continuation trials were included in all further analyses. Therefore, all subsequent analyses examined only the subject trials. See Figure 8 for a breakdown of pronoun/zero trials by non-subject and subject conditions. 45 3.3 Planning measures Time is on the x-axis, starting at the beginning of the trial for the left graph, and centered around the onset of participants’ response in the right graph. The gray box in the latency window represents the overall average response latency length. Figure 9. This figure shows the four time windows of a trial, averaged over all subject continuation items. Time is on the x-axis, starting at the beginning of the trial for the left graph, and centered around the onset of participants’ response in the right graph. The gray box in the latency window represents the overall average response latency length. Since we are interested in planning effects, analyses focused on the regions before the response. We have divided these measures into early planning: looks to panel 2 during the detective sentence window; and late planning: looks to panel 2 during the latency window, and the length of response latency (ms). We test these each in separate mixed effects regression analyses. 3.3 Planning measures g Our first question was whether thematic roles would predict variability in each of our three measures of planning: response latency, the speaker’s gaze to panel 2 during the context sentence, 46 DOES PLANNING EXPLAIN WHY PREDICTABILITY AFFECTS REFERENCE PRODUCTION? and speaker’s gaze to panel 2 during the latency period. Looks to panel 2 (which guided the response) were considered evidence of response planning (either message planning or formulation). Following Zerkle & Arnold (2016), we divided each trial into four different time windows (see Figure 9), but our analyses for this study focused primarily on the detective sentence window and latency window, as defined below. Time windows were calculated starting at the estimated average stimulus image onset. Due to a programming error, there was some variability in the image onset, which was estimated to occur an average of 110 ms after participants responded to the drift correct screen, with a standard deviation of ± 31 ms. The onset of the detective sentence was much less variable, occurring at an average of 665 ms after the estimated picture onset with a standard deviation of ± 3 ms , average of 665 ms after the estimated picture onset, with a standard deviation of ± 3 • The Detective Sentence (DS) window: the time between the onset (665ms from stimulus onset) and offset of the context detective sentence. • The Detective Sentence (DS) window: the time between the onset (665ms from stimulus onset) and offset of the context detective sentence. • The Latency window: the time between the offset of the detective sentence and the onset of the participant’s fluent speech. • The Latency window: the time between the offset of the detective sentence and the onset of the participant’s fluent speech. Figure 9. This figure shows the four time windows of a trial, averaged over all subject continuation items. Time is on the x-axis, starting at the beginning of the trial for the left graph, and centered around the onset of participants’ response in the right graph. The gray box in the latency window represents the overall average response latency length. i l d ll bj i i Figure 9. This figure shows the four time windows of a trial, averaged over all subject continuation items. 3.3.1 Early planning 3.3.1 Early planning Speakers were not significantly more likely to look at panel 2 during the detective sentence window for goal continuations (see Table 3, Figure 10). This shows that in answer to Question 1, we did not see strong effects of thematic role predictability on early planning. Effect Estimate SE DF t-value p-value Goal vs. Source continuation 0.1618 0.107 22 1.51 0.1449 Table 3. Critical predictors of looks to panel 2 in the detective sentence window. Random intercepts of participant and of item, random slope of goal continuation by subject. y p g Speakers were not significantly more likely to look at panel 2 during the detective sentence window for goal continuations (see Table 3, Figure 10). This shows that in answer to Question 1, we did not see strong effects of thematic role predictability on early planning. Effect Estimate SE DF t-value p-value Goal vs. Source continuation 0.1618 0.107 22 1.51 0.1449 Table 3. Critical predictors of looks to panel 2 in the detective sentence window. Random intercepts of participant and of item, random slope of goal continuation by subject. Table 3. Critical predictors of looks to panel 2 in the detective sentence window. Random intercepts of participant and of item, random slope of goal continuation by subject. 47 ZERKLE & ARNOLD Figure 10. Average looks to panel 2 during the detective sentence region by thematic role condition. Error bars are standard error of the subject means by condition. 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 goal source Average looks to P2 in DS window Figure 10. Average looks to panel 2 during the detective sentence region by thematic role condition. Error bars are standard error of the subject means by condition. 3 Separate models were also run with each individual predictor, and the results from each showed a similar pattern as the full model (only early looks significantly predict reference form). 3.3.2 Late planning p g Response latency was significantly predicted by thematic role, replicating findings from previous experiments using this task (see Table 4, Figure 11; Rosa & Arnold, 2017; Zerkle, Rosa, & Arnold, 2015). Effect Estimate SE DF t-value p-value Goal vs. Source continuation -0.06905 0.02803 359 -2.46 0.0142 Table 4. Critical predictors of log latency to begin speaking. Random intercepts of participant and of item, random slope of goal continuation by subject. Figure 11. Thematic role effect on response latency. Error bars are standard error of the subject means by condition. 0 200 400 600 800 1000 1200 1400 1600 1800 2000 Goal Source Latency (ms) Effect Estimate SE DF t-value p-value Goal vs. Source continuation -0.06905 0.02803 359 -2.46 0.0142 Table 4. Critical predictors of log latency to begin speaking. Random intercepts of participant and of item, random slope of goal continuation by subject. Figure 11. Thematic role effect on response latency. Error bars are standard error of the subject means by condition. 48 DOES PLANNING EXPLAIN WHY PREDICTABILITY AFFECTS REFERENCE PRODUCTION? Our next question was whether panel 2 looks during the latency period, which represent a later planning measure, would reflect thematic role condition. We found that indeed, they were significantly influenced by the goal/source manipulation (See Table 5, purple lines in right panel of Figure 9). Speakers looked more at panel 2 in this window for goal continuations than for source continuations. Effect Estimate SE DF t-value p-value Goal vs. Source continuation 0.2756 0.09201 21.4 3 0.0068 Table 5. Critical predictors of looks to panel 2 in latency window. Random intercepts of participant and of item. In sum, analyses of our three planning measures show that thematic role influences the duration of the latency period and looks to panel 2 during the latency period, but not looks to panel 2 during the context sentence. Thus, in answer to Question 1, we found that thematic role affected only late measures of planning, and not our early measure of planning. This suggests that predictability has an effect on utterance planning, but only immediately before utterance articulation. 3.4 What explains referential form choice? Our second question was whether our planning measures would help explain speakers’ choices of referential form. To assess this, we examined the three dependent measures from the previous section (early looks, late looks, and latency duration). We first tested the correlations between these three variables (using the transformed values) to assess multicollinearity. Early looks and late looks were marginally positively correlated, (r=0.10, p=0.052); early looks and latency duration were significantly negatively correlated (r=-0.23, p<.0001); and late looks and latency duration were significantly negatively correlated (r=-0.17, p=0.001). All three of these correlations reflect only small effect sizes (Cohen, 1988), given our large sample sizes. This suggests that multicollinearity is not an issue in a model with all three variables.3 gg y As shown in Table 6, we found that only early looks significantly predicted reference form choice, such that more looks to panel 2 in the DS window increased the likelihood of using a pronoun/zero (see Table 6). Effect Estimate SE DF t-value p-value Looks to P2 in DS window 0.5813 0.2666 357 2.18 0.0299 Looks to P2 in Latency window -0.04723 0.187 41.46 -0.25 0.8018 Log Latency (ms) -0.8283 0.7756 357 -1.07 0.2863 Table 6. Utterance planning predictors of pronoun/zero use. Random intercepts of participant and of item, random slopes of latency by subject and by item. Looks to P2 in Latency window -0.04723 0.187 41.46 -0.25 0.8018 Log Latency (ms) -0.8283 0.7756 357 -1.07 0.2863 Table 6. Utterance planning predictors of pronoun/zero use. Random intercepts of participant and of item, random slopes of latency by subject and by item. Table 6. Utterance planning predictors of pronoun/zero use. Random intercepts of participant and of item, random slopes of latency by subject and by item. Table 6. Utterance planning predictors of pronoun/zero use . Random intercepts of participant and of item, random slopes of latency by subject and by item. ZERKLE & ARNOLD ZERKLE & ARNOLD ZERKLE & ARNOLD dependent measure, and with three predictors: thematic role predictability, early looks, and the interaction between the two. Thematic role and early looks were significantly positively correlated, however this significance is contingent upon the large sample size and the effect size is rather small (r=0.124, p=0.019) (Cohen, 1988). dependent measure, and with three predictors: thematic role predictability, early looks, and the interaction between the two. Thematic role and early looks were significantly positively correlated, however this significance is contingent upon the large sample size and the effect size is rather small (r=0.124, p=0.019) (Cohen, 1988). As shown in Table 7, we again found a significant effect of goal continuation (more reduced forms for goals); and also a significant effect of looks to panel 2 in the DS window (more reduced forms for trials with more anticipatory looks). The interaction between thematic role and looks was not significant. g However, it is not the case that the thematic role effect can be entirely explained in terms of early planning. If it were, we would see the thematic role effect disappear in the presence of the DS-window looks predictor. Thus, in answer to our last question, planning does not account for the bias to use more reduced expressions for goals than sources. Instead, we see two independent effects of planning and thematic role. Effect Estimate SE DF t-value p-value Goal vs. Source continuation 1.173 0.4238 20.09 2.77 0.0118 Looks to P2 in DS window 0.7468 0.2912 357 2.56 0.0107 Goal continuationLooks to P2 DS 0.7385 0.5503 357 1.34 0.1804 Table 7. Critical predictors of pronoun/zero use, including eye movement predictors. Random intercepts of participant and of item. Table 7. Critical predictors of pronoun/zero use, including eye movement predictors. Random intercepts of participant and of item. 3.6. Empirical summary p y In sum, we found that goal thematic roles lead to both more reduced forms, and evidence of greater planning during the latency period, as indicated by both shorter latencies and greater anticipatory looks to the target panel. In addition, we found that one of our planning measures (early anticipatory looks) predicted the use of reduced forms. However, these findings are critically independent. The effect of thematic roles on planning occurred only for the late planning measures, whereas the effect of planning on reference form only occurred for the early planning measure. In addition, including both early looks and thematic role as predictors of reference form revealed that they are independent predictors. 5. Do planning measures account for the effect of thematic role on reference choices? 3.5. Do planning measures account for the effect of thematic role on reference choices? Our final question was how these planning measures, together with our thematic role manipulation, account for choices in reference form. Specifically, we wanted to know whether the planning measures would account for the effect of thematic role. We focused on early looks as a planning measure, since this was the only one to predict reference form choice in the previous model. We therefore built a model with reference form (pronoun/zero vs. description) as the p g ur final question was how these planning measures, together with our thematic role 49 4 General Discussion In sum, this study revealed three main findings: a) thematic role predictability affects late measures of planning, b) early planning supports the use of reduced forms; and c) planning does not account for predictability effects on reduced forms. We will discuss each in turn. 4.3 Early planning does not account for predictability effects 4.3 Early planning does not account for predictability effects In answer to Question 3, we found that early planning does not account for the effects of thematic roles on reference form. This is not consistent with the idea that early planning is the sole driving force. The relationship between predictability, planning, and reference form is complex, but this is the first step towards parsing the functions of early vs. late planning mechanisms for the production of reference form. In sum, planning facilitation is not the reason why predictability affects reference form, so there must be another characteristic of predictability that is responsible for the pattern of behaviors observed here. 4.1 Thematic role predictability affects late measures of planning 4.1 Thematic role predictability affects late measures of planning In answer to Question 1, this study found that thematic role predictability only affected planning during the latency window and the latency measure itself. This is consistent with earlier reports that response latencies are shorter for goal vs. source continuations (Rosa & Arnold, 2017; Zerkle, Rosa, & Arnold, 2015). We found that eye movements during the latency window reflected thematic role predictability, such that speakers looked more at the target stimulus (panel 2) in the goal condition than in the source condition. In other words, only at this late period of planning does the predictability of goal continuations increase looks to the target picture. It may be that speakers are more likely to look back to panel 1 for source continuations in this window because these items require additional comprehension of the context (beyond the length of the detective sentence) before planning. 4.2 Early planning supports the use of reduced forms 4.2 50 DOES PLANNING EXPLAIN WHY PREDICTABILITY AFFECTS REFERENCE PRODUCTION? In answer to Question 2, we found that only early planning supports the use of reduced referential forms. Importantly, this study is the first to examine reference production within a discourse context. Van der Meulen et al. (2001) also examined eye movements during the production of pronouns vs. noun phrases, but their study didn’t test natural language production within a discourse context. We found that early planning affects reference form (both pronouns and zeros) within a natural discourse context. This finding is consistent with Arnold & Nozari (2017) who also found that planning supported pronoun/zero use, in a different paradigm. This finding is consistent with classical models about reference form, which suggest that the discourse context influences decisions about when to use a pronoun. But critically, this is one of the first studies to link these decisions with inter-trial variation in production planning processes. When speakers are listening to the detective sentence, sometimes they begin planning earlier, as indicated by looks to panel 2. Those are the trials where they tend to use pronouns and zeros. It may be that advance planning is especially helpful when it co-occurs with processing the discourse context (Arnold & Nozari, 2017). Doing so helps establish links between the events, and increases the speaker’s desire to communicate these links with their linguistic choices, like the use of pronouns. 4.4 Conclusions The three main findings from this study rule out a production processing-based account, because planning facilitation does not directly account for pronoun choice. What, then is the likely explanation for predictability effects on reference form? Our speculations on this question build on the frequent observation that referential forms are selected on the basis of the referent’s cognitive status, i.e. its discourse salience or accessibility (Ariel, 1990; Chafe, 1994; Givón, 1983; Gundel, Hedberg, & Zacharaski, 1993). This classic view suggests that reduced forms are triggered by the representation of the discourse entity itself (Arnold, 2016). This view is consistent with our findings that early planning supports the use of pronouns and zeros, because we assume that participants are building a discourse representation of each story during the first part of the trial. When that representation is strong, speakers may have greater mental resources to begin pre-planning, and therefore look to panel 2 earlier. Thus, pre-planning does affect reference form, but only pre-planning that occurs during the first part of the trial. By contrast, the effect of thematic role predictability emerges later. We only see the effect of thematic roles on later measures of planning, and these planning measures are unrelated to reference form. Instead, thematic role must affect reference form through some other mechanism. The thematic role effect points to a property of references that derives from their participation in events, as opposed to questions about how activated or accessible each referent is alone. This raises the possibility that pronouns and zeros are used in cases where there are strong relationships between utterances. That is, goal references are predictable, and as a result are more tightly connected at a conceptual level. In support of this, Rosa & Arnold (2017) asked a group of subjects to rate the pairs of events in their stimuli (also used in the current study). They 51 ZERKLE & ARNOLD ZERKLE & ARNOLD found that the two events were perceived as more related in goal continuation than source continuation items. This suggests that speakers may perceive the goal continuations as more connected, which may have downstream effects on reduced form use. Further research is needed to better understand the relationship between thematic role predictability and reference form choice. Nevertheless, the current findings support the importance of early planning on discourse production. When speakers pre-plan, they are likely to connect events conceptually. 4.4 Conclusions In an unrelated finding, thematic roles may also lead to greater conceptual connection, but this effect is not driven by early planning. Both situations lead to the selection of linguistic forms that signal coherence, such as pronouns and zeros. Acknowledgements This work was funded by the National Science Foundation under Grant 1348549 to Jennifer E. Arnold. All procedures were performed in compliance with relevant laws and institutional guidelines, and the University of North Carolina at Chapel Hill Institutional Review Board has approved them. We would also like to thank Ana Medina Fetterman, Liz Reeder, Samuel Adam Smith, Jacob Pascual, Jenna Roller, Brianna Torres, and Kristen Bubak for their help preparing the stimuli and collecting and coding the data. References Ariel, M. (1990). Accessing noun-phrase antecedents. London: Routledge. Ariel, M. (2001). Accessibility theory: An overview. In Sanders, T., Schliperoord, J. and Spooren, W. (Eds.), Text representation. Amsterdam: John Benjamins (Human cognitive processing series), pp. 29-87. Arnold, J. E. (1998). Reference Form and Discourse Patterns. Doctoral dissertation, Stanford University, Stanford, California. Arnold, J. E. (2001). The effect of thematic role on pronoun use and frequency of reference continuation. Discourse Processes, 31, 137–162. https://doi.org/10.1207/ S15326950DP3102_02 Arnold, J. E. (2010). How speakers refer: The role of accessibility. Language and Linguistic Compass, 4, 187–203. DOI: https://doi.org/10.1111/j.1749-818X.2010.00193.x Arnold, J. E. (2016). Explicit and emergent mechanisms of information sta Cognitive Science, 8, 737–760. http://doi.org/10.1111/tops.12220 Arnold, J. E. (2017). Latency analysis for Rosa & Arnold (2017). Technical Report #1. UNC Language Processing Lab, Department of Psychology & Neuroscience, University of North Carolina – Chapel Hill, Chapel Hill, North Carolina. http://arnoldlab.web.unc.edu/files/2017/08/Arnold_TechReport1_2017.pdf Arnold, J. E., & Griffin, Z. M. (2007). The effect of additional characters on choice of referring expression: Everyone counts. Journal of Memory and Language, 56(4), 521-536. https://doi.org/10.1016/j.jml.2006.09.007 Arnold, J. E., Kam, C. L. H., & Tanenhaus, M. K. (2007). If you say thee uh you are describing something hard: the on-line attribution of disfluency during reference comprehension. Journal of Experimental Psychology. Learning, Memory, and Cognition, 33(5), 914–930. http://doi.org/10.1037/0278-7393.33.5.914 Arnold, J. E., & Lao, S.-Y. C. (2015). Effects of psychological attention on pronoun comprehension. Language, Cognition and Neuroscience, 30(7), 832–852. http://doi.org/10.1080/23273798.2015.1017511 52 DOES PLANNING EXPLAIN WHY PREDICTABILITY AFFECTS REFERENCE PRODUCTION? Arnold, J. E. & Nozari, N. (2017). The effects of utterance planning and stimulation of left prefrontal cortex on the production of referential expressions. Cognition, 160, 127–144. https://doi.org/10.1016/j.cognition.2016.12.008 Arnold, J. E. & Watson, D. G. (2015). Synthesizing meaning and processing approaches to prosody: performance matters. Language, Cognition, and Neuroscience, 30, 88-102. https://doi.org/10.1080/01690965.2013.840733 Arnold, J. E., & Zerkle, S. A. (2019). Why do people produce pronouns? Pragmatic selection vs Rational models. Language, Cognition and Neuroscience. https://doi.org/10.1080/23273798.2019.1636103 p g Barr D. J. (2008). Analyzing ‘visual world’ eyetracking data using multilevel logistic regression. Journal of Memory and Language, 59(4). 457–474. Bell, A., Brenier, J. M., Gregory, M., Girand, C. & Jurafsky, D. (2009). Predictability effects on durations of content and function words in conversational English. Journal of Memory and Language, 60(1), 92–111. https://doi.org/10.1016/j.jml.2008.06.003 Boersma, P. & Weenink, D. (2015). Praat: Doing phonetics by computer [Computer program]. Version 5.4.09, http://www.praat/org/. Bock, K., Irwin, D. E., Davidson, D. J., & Levelt, W.J. DOES PLANNING EXPLAIN WHY PREDICTABILITY AFFECTS REFERENCE PRODUCTION? ZERKLE & ARNOLD Hale, J. (2001). A probabilistic Earley parser as a psycholinguistic model. NAACL ’01: Second Meeting of the North American Chapter of the Association for Computational Linguistics on Language Technologies 2001, 1–8.: https://doi.org/10.3115/1073336.1073357 Huettig, F., Rommers, J., & Meyer, A. S. (2011). Using the visual world paradigm to study language processing: A review and critical evaluation. Acta Psychologica, 137(2), 151–171. http://doi.org/10.1016/j.actpsy.2010.11.003 Indefrey, P., & Levelt, W. J. (2004). The spatial and temporal signatures of word production components. Cognition, 92(1), 101-144. Jurafsky, D., Bell, A., Gregory, M., & Raymond, W. (2001). Probabilistic relations between words: Evidence from reduction in lexical production. In J. Bybee & P. Hopper (Eds.), Frequency and the emergence of linguistic structure (pp. 229- 254). Amsterdam, Netherlands: John Benjamins Publishing Company. j g y Kehler, A. & Rohde, H. (2013). A probabilistic reconciliation of coherence-driven and centering- driven theories of pronoun interpretation. Theoretical Linguistics, 39(1–2), 1–37. https://doi.org/10.1515/tl-2013-0001 p g Kehler, A., Kertz, L., Rohde, H. & Elman, J. L. (2008). Coherence and coreference revisited. Journal of Semantics, 25(1), 1–44. https://doi.org/10.1093/jos/ ffm018 Kravtchenko, E.; Modi, A.; Demberg V.; Titov, I., & Pinkal. M. (2017). Does referent predictability affect rate of pronominalization? Paper presented at the CUNY conference on human sentence processing, March 2017. Levelt, W. J. M. (1989). Speaking: From intention to articulation. Cambridge, MA: MIT Press. Levelt W J Roelofs A & Meyer A S (1999) A theory of lexical access in speech Levelt, W. J. M. (1989). Speaking: From intention to articulation. Cambridge, MA: MIT Press. Levelt, W. J., Roelofs, A., & Meyer, A. S. (1999). A theory of lexical access in speech production Behavioral and brain sciences 22(1) 1 38 Levelt, W. J. M. (1989). Speaking: From intention to articulation. Cambridge, MA: M , ( ) p g g , Levelt, W. J., Roelofs, A., & Meyer, A. S. (1999). A theory of lexical access in speech production. Behavioral and brain sciences, 22(1), 1-38. p oduct o . ehavio al and b ain sciences, ( ), 38. Levy, R., & Jaeger, T. F. (2007). Speakers optimize information density through syntactic reduction. Advances in neural information processing systems, 19, 849. Mahowald, K., Fedorenko, E., Piantadosi, S. T., & Gibson, E. (2013). Info/information theory: Speakers choose shorter words in predictive contexts Cognition 126(2) 313-318 Levy, R., & Jaeger, T. F. (2007). Speakers optimize information density through syntactic reduction. Advances in neural information processing systems, 19, 849. References M. (2003). Minding the clock. Journal of Memory and Language, 48(4), 653–685. https://doi.org/10.1016/S0749-596X(03)00007-X Brown-Schmidt, S., & Konopka, A. E. (2015). Processes of incremental message planning during conversation. Psychonomic Bulletin & Review, 22(3), 833–843. http://doi.org/10.3758/s13423-014-0714-2 p g Chafe, W. (1976). Givenness, contrastiveness, definiteness, subjects, topics, and point of view. In Charles N. Li (Ed.), Subject and topic, (pp. 25-56). New York: Academic Press Inc. ( ), j p , (pp ) Chafe, W. (1994). Discourse, consciousness, and time: The flow and displacement of conscious experience in speaking and writing. Chicago, IL: Chicago University Press. Cohen, J. (1988). Statistical power analysis for the behavioral sciences (2nd ed.). Hillsdale, NJ: Chafe, W. (1994). Discourse, consciousness, and time: The flow and displacement of conscious experience in speaking and writing. Chicago, IL: Chicago University Press. Cohen, J. (1988). Statistical power analysis for the behavioral sciences (2nd ed.). Hillsdale, NJ: Erlbaum. y, D. (1991). Thematic proto-roles and argument selection. Language, 67(3), 547-619. Ferreira, F. & Swets, B. (2002). How incremental is language production? Evidence from the production of utterances requiring the computation of arithmetic sums. Journal of Memory and Language, 46(1), 57–84. https://doi.org/10.1006/ jmla.2001.2797 Fowler, C. A. & Housum, J. (1987). Talkers’ signaling of “new” and “old” words in speech and listeners’ perception and use of the distinction. Journal of Memory and Language, 26, 489–504. https://doi.org/10.1016/0749-596X(87)90136-7 p g ( ) Frank, M. C., & Goodman, N. D. (2012). Predicting pragmatic reasoning in language games. Science, 336(6084), 998-998. Fukumura, K. & van Gompel, R. P. G. (2010). Choosing anaphoric expression: Do people take into account likelihood of reference? Journal of Memory and Language, 62, 52–66. https://doi.org/10.1016/j.jml.2009.09.001 Givón, T. (1983). Topic continuity in discourse: An introduction. In: Givón, T. (Ed.), Topic continuity in discourse: A quantitative cross-language study, 1–41. Amsterdam: John Benjamins. https://doi.org/10.1075/tsl.3 j g Griffin Z. M. & Bock K. (2000). What the eyes say about speaking. Psychological Science, 11(4). 274–279. Grosz, B. J., Weinstein, S., & Joshi, A. K. (1995). Centering: A framework for modeling the local coherence of discourse. Computational linguistics, 21(2), 203-225. p g ( ) Gundel, J. K., Hedberg, N., & Zacharski, R. (1993). Cognitive status and the form of referring expressions. Language, 69, 274-307. 53 ZERKLE & ARNOLD ZERKLE & ARNOLD f p g y Mahowald, K., Fedorenko, E., Piantadosi, S. T., & Gibson, E. (2013). Info/information theory: Speakers choose shorter words in predictive contexts. Cognition, 126(2), 313-318. McMurray, B. (2002). Analysis scripts written in Microsoft Access: EyelinkAnal [computer program]. Version 1.5. McMurray, B., Tanenhaus, M. K., & Aslin, R. N. (2009). Within-category VOT affects recovery from “lexical” garden-paths: Evidence against phoneme-level inhibition. Journal of Memory and Language, 60(1), 65–91. http://doi.org/10.1016/j.jml.2008.07.002 Meyer, A. S., Sleiderink, A. M., & Levelt, W. J. M. (1998). Viewing and naming objects: eye movements during noun phrase production. Cognition, 66(2), B25–B33. http://doi.org/10.1016/S0010-0277(98)00009-2 Nappa, R., & Arnold, J. E. (2014). The road to understanding is paved with the speaker’s intentions: Cues to the speaker’s attention and intentions affect pronoun comprehension. Cognitive Psychology, 70, 58–81. http://doi.org/10.1016/j.cogpsych.2013.12.003 Pickering, M. J., & Garrod, S. (2013). An integrated theory of language production on and comprehension. Behavioral and Brain Sciences, 36(4), 329–347. https://doi.org /10.1017/S0140525X12001495 Roelofs, A. (1992). A spreading-activation theory of lemma retrieval in speaking. Cognition, 42(1-3), 107-142. Rohde, H. & Kehler, A. (2014). Grammatical and information-structural influences on pronoun production. Language, Cognition, and Neuroscience, 29(8), 912–927. https://doi.org/10.1080/01690965.2013.854918 Rohde, H. & Kehler, A. (2014). Grammatical and information-structural influences on pronoun production. Language, Cognition, and Neuroscience, 29(8), 912–927. https://doi.org/10.1080/01690965.2013.854918 p g Rosa, E. & Arnold, J. E. (2017). Predictability affects production: Thematic roles can affect reference form selection. Journal of Memory and Language, 94, 43–60. https://doi. org/10.1016/j.jml.2016.07.007 Rosa, E. & Arnold, J. E. (2017). Predictability affects production: Thematic roles can affect reference form selection. Journal of Memory and Language, 94, 43–60. https://doi. org/10.1016/j.jml.2016.07.007 g j j Searle, S., Cassella, G. & McCullouch, C. (1992). Variance Components. New York: Jonh Wiley- Sons. https://doi.org/10.1002/9780470316856 g j j Searle, S., Cassella, G. & McCullouch, C. (1992). Variance Components. New York: Jonh Wiley- Sons. https://doi.org/10.1002/9780470316856 54 DOES PLANNING EXPLAIN WHY PREDICTABILITY AFFECTS REFERENCE PRODUCTION? Tily, H. J., & Piantadosi, S. T. (2009). Refer efficiently: Use less informative expressions for more predictable meanings. Proceedings of the Workshop on the Production of Referring Expressions: Bridging the Gap between Computational and Empirical Approaches to Reference, 1–8. van der Meulen, F. F., Meyer, A. S., & Levelt, W. J. (2001). Eye movements during the production of nouns and pronouns. Memory & Cognition, 29(3), 512–521. http://doi.org/10.3758/BF03196402 van Rij, J., van Rijn, H., & Hendriks, P. (2010). Cognitive architectures and language acquisition: a case study in pronoun comprehension. ZERKLE & ARNOLD Journal of Child Language, 37(3), 731–766. http://doi.org/10.1017/S0305000909990560 Weatherford, K., & Arnold, J. E. (under review). Semantic predictability of implicit causa affect referential form choice. Ms., University of North Carolina. Wheeldon, L. R. (2013). Producing spoken sentences: The scope of incremental planning. In S. Fuchs, M. Weirich, D. Pape, & P. Perrier (Eds.), Speech production and perception: Vol. 1. Speech planning and dynamics. Frankfurt, Germany: Peter Lang. Zerkle, S. A. & Arnold, J. E. (2016). Discourse attention during utterance planning affects referential form choice. Linguistics Vanguard, 2(s1). DOI: https://doi.org/10.1515/lingvan-2016-0067 Zerkle, S. A., Rosa, E. C., & Arnold, J. E. (2015). Do addressee gestures influence the effects of predictability on spoken reference form? Presented as a poster at the CUNY 2015 Conference on Sentence Processing, USC, Los Angeles, CA. March 19-21, 2015. g g Zerkle, S. A., Rosa, E. C., & Arnold, J. E. (2017). Thematic role predictability and planning affect word duration. Laboratory Phonology: Journal of the Association for Laboratory Phonology, 8(1): 17, 1-28. DOI: http://doi.org/10.5334/labphon.98 55 55
https://openalex.org/W2901326754	https://research-information.bris.ac.uk/files/182045801/polymers_10_01250.pdf	English	null	Exploring Structure–Property Relationships in Aromatic Polybenzoxazines Through Molecular Simulation	Polymers	2,018	cc-by	10,969	Thompson, S., Stone, C. A., Howlin, B. J., & Hamerton, I. (2018). Exploring Structure–Property Relationships in Aromatic Polybenzoxazines Through Molecular Simulation. Polymers, 10(11), Article 1250. https://doi.org/10.3390/polym10111250 Publisher's PDF, also known as Version of record License (if available): CC BY Link to published version (if available): 10.3390/polym10111250 Link to publication record on the Bristol Research Portal PDF-document This is the final published version of the article (version of record). It first appeared online via MDPI at https://www.mdpi.com/2073-4360/10/11/1250 . Please refer to any applicable terms of use of the publisher. Thompson, S., Stone, C. A., Howlin, B. J., & Hamerton, I. (2018). Exploring Structure–Property Relationships in Aromatic Polybenzoxazines Through Molecular Simulation. Polymers, 10(11), Article 1250. https://doi.org/10.3390/polym10111250 Thompson, S., Stone, C. A., Howlin, B. J., & Hamerton, I. (2018). Exploring Structure–Property Relationships in Aromatic Polybenzoxazines Through Molecular Simulation. Polymers, 10(11), Article 1250. https://doi.org/10.3390/polym10111250 Article Received: 26 September 2018; Accepted: 7 November 2018; Published: 12 November 2018 Abstract: A series of commercial difunctional benzoxazine monomers are characterized using thermal and thermo-mechanical techniques before constructing representative polymer networks using molecular simulation techniques. Good agreement is obtained between replicate analyses and for the kinetic parameters obtained from differential scanning calorimetry data (and determined using the methods of Kissinger and Ozawa). Activation energies range from 85 to 108 kJ/mol (Kissinger) and 89 to 110 kJ/mol (Ozawa) for the uncatalyzed thermal polymerization reactions, which achieve conversions of between 85% and 97%. Glass transition temperatures determined from differential scanning calorimetry and dynamic mechanical thermal analysis are comparable, ranging from BA-a (151 ◦C, crosslink density 3.6 × 10−3 mol cm−3) containing the bisphenol A moiety to BP-a, based on a phenolphthalein bridge (239 to 256 ◦C, crosslink density 5.5 to 18.4 × 10−3 mol cm−3, depending on formulation). Molecular dynamics simulations of the polybenzoxazines generally agree well with empirical data, indicating that representative networks have been modelled. Keywords: polybenzoxazines; polymerization kinetics; thermal stability; molecular dynamics simulation University of Bristol – Bristol Research Portal General rights This document is made available in accordance with publisher policies. Please cite only the published version using the reference above. Full terms of use are available: http://www.bristol.ac.uk/red/research-policy/pure/user-guides/brp-terms/ Polymers 2018, 10, 1250; doi:10.3390/polym10111250 polymers September 2018; Accepted: 7 November 2018; Published: 12 November 2018 A series of commercial difunctional benzoxazine monomers are characterized using thermal o-mechanical techniques before constructing representative polymer networks using simulation techniques. Good agreement is obtained between replicate analyses and for parameters obtained from differential scanning calorimetry data (and determined using ds of Kissinger and Ozawa). Activation energies range from 85 to 108 kJ/mol (Kissinger) 10 kJ/mol (Ozawa) for the uncatalyzed thermal polymerization reactions, which achieve s of between 85% and 97%. Glass transition temperatures determined from differential alorimetry and dynamic mechanical thermal analysis are comparable, ranging from BA-a sslink density 3.6 × 10−3 mol cm−3) containing the bisphenol A moiety to BP-a, based on thalein bridge (239 to 256 ◦C, crosslink density 5.5 to 18.4 × 10−3 mol cm−3, depending on n). Molecular dynamics simulations of the polybenzoxazines generally agree well with data, indicating that representative networks have been modelled. polybenzoxazines; polymerization kinetics; thermal stability; molecular dynamics simulation ion nzoxazines (PBZs) are modern thermosetting resins that are being explored [1] as possible s for common resins systems such as phenolic [2] and epoxy resins [3] in aerospace . When fully cured, PBZs offer a combination of favourable thermal and mechanical e (e.g., dry Tg values of 246 ◦C, hot/wet Tg = 207 ◦C are possible [4], coupled with re uptake), that gives an attractive property proﬁle. Although requiring toughening ngineering applications, PBZs can be combined with epoxy resins [5] or inherently thermoplastics (e.g., oligomeric polyethersulphone, KIC = 0.99 MPa m1/2) [6] to yield enhancements. PBZs are formed through step-growth ring-opening polyaddition from ine monomers (Scheme 1), which are in turn the products of the Mannich reaction between , formaldehyde and a primary amine [7]. Unlike many other commercial thermosetting h evolve condensation products such as water or ammonia, benzoxazine monomers react anly to form a polymer with few reaction by-products [8], although the exact mechanism merization reaction to form a network has not been fully elucidated. Recent work in as examined the inﬂuence of additives on the nature of the polymerization mechanism, n of the polymer network structure and the resulting ﬁnal properties [9]. Relationships in Aromatic olecular Simulation owlin 1 and Ian Hamerton 3,* Physical Sciences, University of Surrey, el.com (S.T.); b.howlin@surrey.ac.uk (B.J.H.) l.dstl.gov erospace, and Mechanical Engineering, k, Bristol BS8 1TR, UK xford, Cambridge CB22 4QD, UK. polymers Published: 12 November 2018 zine monomers are characterized using thermal ting representative polymer networks using s obtained between replicate analyses and for nning calorimetry data (and determined using ergies range from 85 to 108 kJ/mol (Kissinger) ermal polymerization reactions, which achieve on temperatures determined from differential al analysis are comparable, ranging from BA-a ning the bisphenol A moiety to BP-a, based on sity 5.5 to 18.4 × 10−3 mol cm−3, depending on e polybenzoxazines generally agree well with ks have been modelled. thermal stability; molecular dynamics simulation ng resins that are being explored [1] as possible henolic [2] and epoxy resins [3] in aerospace nation of favourable thermal and mechanical t Tg = 207 ◦C are possible [4], coupled with erty proﬁle. Although requiring toughening mbined with epoxy resins [5] or inherently rsulphone, KIC = 0.99 MPa m1/2) [6] to yield step-growth ring-opening polyaddition from n the products of the Mannich reaction between Unlike many other commercial thermosetting ater or ammonia, benzoxazine monomers react by-products [8], although the exact mechanism s not been fully elucidated. Recent work in the nature of the polymerization mechanism, e resulting ﬁnal properties [9]. polymers polymers e–Property Relationships in Aromatic Through Molecular Simulation tone 2, Brendan J. Howlin 1 and Ian Hamerton 3,* y of Engineering and Physical Sciences, University of Surrey, ; scott.thompson@hexcel.com (S.T.); b.howlin@surrey.ac.uk (B.J.H.) 4 0JQ, UK; cstone@mail.dstl.gov CIS), School of Civil, Aerospace, and Mechanical Engineering, ilding, University Walk, Bristol BS8 1TR, UK bristol.ac.uk ites, Ickleton Road, Duxford, Cambridge CB22 4QD, UK. ted: 7 November 2018; Published: 12 November 2018 difunctional benzoxazine monomers are characterized using thermal ues before constructing representative polymer networks using s. Good agreement is obtained between replicate analyses and for rom differential scanning calorimetry data (and determined using awa). Activation energies range from 85 to 108 kJ/mol (Kissinger) r the uncatalyzed thermal polymerization reactions, which achieve 97%. Glass transition temperatures determined from differential ic mechanical thermal analysis are comparable, ranging from BA-a 0−3 mol cm−3) containing the bisphenol A moiety to BP-a, based on 256 ◦C, crosslink density 5.5 to 18.4 × 10−3 mol cm−3, depending on cs simulations of the polybenzoxazines generally agree well with presentative networks have been modelled. ymerization kinetics; thermal stability; molecular dynamics simulation modern thermosetting resins that are being explored [1] as possible systems such as phenolic [2] and epoxy resins [3] in aerospace PBZs offer a combination of favourable thermal and mechanical of 246 ◦C, hot/wet Tg = 207 ◦C are possible [4], coupled with an attractive property proﬁle. Although requiring toughening ns, PBZs can be combined with epoxy resins [5] or inherently oligomeric polyethersulphone, KIC = 0.99 MPa m1/2) [6] to yield are formed through step-growth ring-opening polyaddition from e 1), which are in turn the products of the Mannich reaction between primary amine [7]. Unlike many other commercial thermosetting products such as water or ammonia, benzoxazine monomers react er with few reaction by-products [8], although the exact mechanism form a network has not been fully elucidated. Recent work in ing Structure–Property Relationships in Aromatic nzoxazines Through Molecular Simulation pson 1,†, Corinne A. Stone 2, Brendan J. Howlin 1 and Ian Hamerton 3,* ment of Chemistry, Faculty of Engineering and Physical Sciences, University of Surrey, d, Surrey GU2 7XH, UK; scott.thompson@hexcel.com (S.T.); b.howlin@surrey.ac.uk (B.J.H.) ton Down, Salisbury SP4 0JQ, UK; cstone@mail.dstl.gov omposites Institute (ACCIS), School of Civil, Aerospace, and Mechanical Engineering, ty of Bristol, Queen’s Building, University Walk, Bristol BS8 1TR, UK ondence: ian.hamerton@bristol.ac.uk address: Hexcel Composites, Ickleton Road, Duxford, Cambridge CB22 4QD, UK. 2.1. Materials 2.1. Materials All monomers were supplied by Huntsman (Basel, Switzerland) and used as received. The relevant product names are Araldite MT35600 (based on the benzoxazine of bisphenol A and aniline, BA-a), MT35700 (based on the benzoxazine of a mixture of three isomers of bisphenol F and aniline, BF-a), MT35800 (based on the benzoxazine of phenolphthalein and aniline, BP-a, blended with BF-a), MT35900 (based on the benzoxazine of thiodiphenol and aniline, BT-a), LME10140 (based on the benzoxazine of tricyclo[5.2.1.02,6]decane-4,8-diol and aniline, BD-a), and LMB6490 (batch 2 representing BP-a, blended with butan-1-ol). All monomers (see Figure 1 for structures), except BP-a, were placed in a domestic baking silicone mould, degassed at approximately 90 °C for 1 hour using a vacuum oven to reduce void formation during the subsequent curing process. BP-a was found to be very difficult to control under degassing conditions (excessive void formation) and so was simply melted and held at 120 °C over the same time period that the other samples were degassed. Following degassing, all samples were placed in an air circulating oven at 120 °C and the following cure schedule was applied: heating 2 K min−1 to 180 °C (isothermal 2 hours) followed by heating at 2 All monomers were supplied by Huntsman (Basel, Switzerland) and used as received. The relevant product names are Araldite MT35600 (based on the benzoxazine of bisphenol A and aniline, BA-a), MT35700 (based on the benzoxazine of a mixture of three isomers of bisphenol F and aniline, BF-a), MT35800 (based on the benzoxazine of phenolphthalein and aniline, BP-a, blended with BF-a), MT35900 (based on the benzoxazine of thiodiphenol and aniline, BT-a), LME10140 (based on the benzoxazine of tricyclo[5.2.1.02,6]decane-4,8-diol and aniline, BD-a), and LMB6490 (batch 2 representing BP-a, blended with butan-1-ol). All monomers (see Figure 1 for structures), except BP-a, were placed in a domestic baking silicone mould, degassed at approximately 90 ◦C for 1 hour using a vacuum oven to reduce void formation during the subsequent curing process. BP-a was found to be very difficult to control under degassing conditions (excessive void formation) and so was simply melted and held at 120 ◦C over the same time period that the other samples were degassed. 2.1. Materials 2.1. Materials Following degassing, all samples were placed in an air circulating oven at 120 ◦C and the following cure schedule was applied: heating 2 K min−1 to 180 ◦C (isothermal 2 hours) followed by heating at 2 K min−1 to 200 ◦C (isothermal 2 h). 1. Introduction Polybenzoxazines (PBZs) are modern thermosetting resins that are being explored [1] as possible replacements for common resins systems such as phenolic [2] and epoxy resins [3] in aerospace applications. When fully cured, PBZs offer a combination of favourable thermal and mechanical performance (e.g., dry Tg values of 246 ◦C, hot/wet Tg = 207 ◦C are possible [4], coupled with low moisture uptake), that gives an attractive property proﬁle. Although requiring toughening for some engineering applications, PBZs can be combined with epoxy resins [5] or inherently engineering thermoplastics (e.g., oligomeric polyethersulphone, KIC = 0.99 MPa m1/2) [6] to yield impressive enhancements. PBZs are formed through step-growth ring-opening polyaddition from bis-benzoxazine monomers (Scheme 1), which are in turn the products of the Mannich reaction between a bis-phenol, formaldehyde and a primary amine [7]. Unlike many other commercial thermosetting resins, which evolve condensation products such as water or ammonia, benzoxazine monomers react relatively cleanly to form a polymer with few reaction by-products [8], although the exact mechanism of the polymerization reaction to form a network has not been fully elucidated. Recent work in our group has examined the inﬂuence of additives on the nature of the polymerization mechanism, the formation of the polymer network structure and the resulting ﬁnal properties [9]. Polymers 2018, 10, 1250; doi:10.3390/polym10111250 www.mdpi.com/journal/polymers www.mdpi.com/journal/polymers 2 of 15 ng final Polymers 2018, 10, 1250 polymerization me ti [9] Scheme 1. Bisbenzoxazines homopolymerization reaction showing ring opening and crosslinking. Scheme 1. Bisbenzoxazines homopolymerization reaction showing ring opening and crosslinking. Scheme 1. Bisbenzoxazines homopolymerization reaction showing ring opening and crosslinking. Scheme 1. Bisbenzoxazines homopolymerization reaction showing ring opening and crosslinking. The aim of the present paper is to use thermal, mechanical and computational techniques to explore structure-property relationships in a series of well-characterized commercial benzoxazine monomers and to use these data to inform the construction of representative networks using molecular simulation. The aim of the present paper is to use thermal, mechanical and computational techniques to explore structure-property relationships in a series of well-characterized commercial benzoxazine monomers and to use these data to inform the construction of representative networks using molecular simulation. of 1 Hz. Cured polymer samples (2 × 10 × 17 mm3) were sca K/min 2.4. Construction of the Benzoxazines Using Molecular Mechanics K/min. 2.3. Molecular Simulation Molecular modelling was carried out using Materials Studio [11] (version 6.0, Accelrys, Cambridge, UK) on a PC. The potential energy for all models was calculated using the Condensed-phase Optimized Molecular Potential for Atomistic Simulation Studies (COMPASS) [12], a forcefield specifically designed for polymer calculations. Molecular models were all created using a combination of the Materials Studio modules and in-house programs to simulate a cure. 2.4. Construction of the Benzoxazines Using Molecular Mechanics Initially, the structures of the benzoxazine monomers were simulated at zero Kelvin using molecular mechanics. Molecular mechanics (MM) [13] treats the molecule as an array of atoms governed by a set of classical-mechanical potential functions while the force ﬁeld deﬁnes the mechanical model to represent the molecule. In order to optimize the structure of the molecule, the steepest-descents, conjugate gradients, and Newtonian [14] methods were performed to minimize the energy of the molecule. This yielded bond lengths that agreed well with published crystallographic data (Table 1) for similar monomers or compounds [15]. The Materials Studio Visualizer was used to create 3D models of the monomers, we then used 40 monomer models which were then packed into a periodic cell and repeated in a 3D space with voidless packing; the Ewald summation [16] was performed to neutralize the charges across the periodic boundaries. Initially, the structures of the benzoxazine monomers were simulated at zero Kelvin us lecular mechanics. Molecular mechanics (MM) [13] treats the molecule as an array of ato verned by a set of classical-mechanical potential functions while the force field defines chanical model to represent the molecule. In order to optimize the structure of the molecule, epest-descents, conjugate gradients, and Newtonian [14] methods were performed to minim energy of the molecule. This yielded bond lengths that agreed well with publish stallographic data (Table 1) for similar monomers or compounds [15]. The Materials Stu ualizer was used to create 3D models of the monomers, we then used 40 monomer models wh re then packed into a periodic cell and repeated in a 3D space with voidless packing; the Ew mmation [16] was performed to neutralize the charges across the periodic boundaries. Table 1. DSC First Heat Data of Monomers (20 to 300 ◦C at 10 K/min). K min−1 to 200 °C (iso 2.2. Instrumentation Differential scanning calorimetry (DSC) was performed on a TA Q1000 (TA Instruments, Crawley, West Sussex) with TA Q Series Advantage software on samples (4.0 ± 0.5 mg) in hermetically sealed aluminium pans. Samples were heated at 5, 8, 10, 12, and 15 K/min. from 25 to 300 ◦C (heat/cool/heat) in a nitrogen atmosphere (50 cm3/min.). Dynamic mechanical thermal analysis (DMTA) was performed on a TA Q800 in static air in single cantilever mode at a frequency of 1 Hz. Cured polymer samples (2 × 10 × 17 mm3) were scanned between −50 and 260 ◦C at 10 K/min. 3 of 15 3 of 15 Polymers 2018, 10, 1250 ymers 2018, 10, x FOR PEER REVIEW 3 of Figure 1. Structures of the monomers. N.B., BF-a is a mixture of isomers: typically 4,4′-, 2,4′-, and 2,2′-BF-a in the approximate ratio 5:4:1 [10]). Monomer spectral data are deposited as supplementary data. Figure 1. Structures of the monomers. N.B., BF-a is a mixture of isomers: typically 4,4′-, 2,4′-, and 2,2′-BF-a in the approximate ratio 5:4:1 [10]). Monomer spectral data are deposited as supplementary data. Figure 1. Structures of the monomers. N.B., BF-a is a mixture of isomers: typically 4,4′-, 2,4′-, and 2,2′-BF-a in the approximate ratio 5:4:1 [10]). Monomer spectral data are deposited as supplementary data. Figure 1. Structures of the monomers. N.B., BF-a is a mixture of isomers: typically 4,4′-, 2,4′-, and 2,2′-BF-a in the approximate ratio 5:4:1 [10]). Monomer spectral data are deposited as supplementary data. 2 2 Instrumentation 2.3. Molecular Simulation Differential scanning calorimetry (DSC) was performed on a TA Q1000 (TA Instruments, Crawley, West Sussex) with TA Q Series Advantage software on samples (4.0 ± 0.5 mg) in hermetically sealed aluminium pans. Samples were heated at 5, 8, 10, 12, and 15 K/min. from 25 to 300 °C (heat/cool/heat) in a nitrogen atmosphere (50 cm3/min.). Dynamic mechanical thermal analysis (DMTA) was performed on a TA Q800 in static air in single cantilever mode at a frequency Molecular modelling was carried out using Materials Studio [11] (version 6.0, Accelrys, Cambridge, UK) on a PC. The potential energy for all models was calculated using the Condensed-phase Optimized Molecular Potential for Atomistic Simulation Studies (COMPASS) [12], a forceﬁeld speciﬁcally designed for polymer calculations. Molecular models were all created using a combination of the Materials Studio modules and in-house programs to simulate a cure. p g p Key: Tm = Peak Melt Temperature, ∆Hm = Enthalpy of Melt, Tmax = Exothermic Peak Temperature, ∆Hp = Enthalpy of Polymerization. 2.6. Simulation of the Cured Polybenzoxazine Properties Using Molecular Dynamics Temperature ramped MD simulations were carried out in the Temperature Cycle option in the Amorphous Cell Protocols. A constant-temperature, constant-pressure (NPT) ensemble (0.0001 GPa) was used at each experimental temperature, with a time step of 1 fs, employing the Anderson thermostat and Barendsen barostat [20]. COMPASS was used with the atomic van der Waals summation, a cut-off at 9.50 Å, a spline width of 1.00 Å and a buffer width of 0.50 Å. The Ewald summation [16] was performed to neutralize the charges across the periodic boundaries. MD simulations were carried out at different temperatures, with decrements of 10 K from the starting temperature (set at between 573 and 773 K), a total of 31 to 51 MD simulations were carried out, between 773 and 273 K. At each temperature stage the structure was minimized before the MD simulation to allow equilibration; the energies have practically reduced to a minimum after 25 ps and the simulation time was 200 ps. of 1 Hz. Cured polymer samples (2 × 10 × 17 mm3) were sca K/min 2.4. Construction of the Benzoxazines Using Molecular Mechanics Monomer Tm (◦C) ∆Hm (J/g) Tmax (◦C) ∆Hp Tg (◦C) (J/g) kJ/mol Bz BA-a 36 3 241 309 70 151 BF-a - - 240 298 65 174 BP-a 70 12 228 266 74 239 BP-a batch 2 80 7 234 239 66 256 BT-a - - 220 326 74 172 BD-a 57 14 234 184 51 187 Key: Tm = Peak Melt Temperature, ∆Hm = Enthalpy of Melt, Tmax = Exothermic Peak Temperature, ∆Hp = Enthalpy of Polymerization. structures of the benzoxazine monomers were simulated at z nics. Molecular mechanics (MM) [13] treats the molecule as an Table 1. DSC First Heat Data of Monomers (20 to 300 ◦C at 10 K/min). p g p Key: Tm = Peak Melt Temperature, ∆Hm = Enthalpy of Melt, Tmax = Exothermic Peak Temperature, ∆Hp = Enthalpy of Polymerization. Polymers 2018, 10, 1250 4 of 15 2.5. Construction of the Cured Polybenzoxazines Using the Automated Cure Program 2.5. Construction of the Cured Polybenzoxazines Using the Automated Cure Program With the virtual space ﬁlled with the monomer mixture, control of the model was passed over to an in-house program to proceed with cure. A full discussion of the development of the automatic model building software falls beyond the scope of this paper, but it is outlined in more detail elsewhere [17]. The program selects and controls the reactions between benzoxazine groups to produce a realistic cured thermoset model. To model e.g., the polybenzoxazine based on BA-a the target density for the cell was set (within the automated program) at 1.15 g cm−3, consistent with the density found for the polymer (1.195 g cm−3) in the literature [18] and the monomers were packed to this value by the automated routine. To form the cross-linked polymer network to different selected degrees of conversion, the automated curing program was employed with the polymer-consistent force ﬁeld (PCFF) [19]. For the construction of the network, the automated program requires that a series of parameters be entered to determine the nature of the network formed. Thus, the cut-off distance (determining whether atoms were sufﬁciently close to undergo reaction) was set at between 5.5 and 7.5 Å depending on the experiment being formed; the dynamics duration was set at between 1000 and 10,000 fs and the simulated cure temperature was 453 K, reﬂecting the cure reaction. 3.1. Thermal Analysis of the Homopolymerization Reactions The monomers were each analyzed using DSC to determine (a) the degree of cure and (b) the glass transition temperature (Tg) for later use in the simulation study. In each case, the temperature program applied involved ‘heat-cool-heat’ with the sample being heated from room temperature to 300 ◦C at 10 K/min during each heat cycle. The DSC data produced from the ﬁrst step of the heat, cool, heat cycle are presented in Table 1 (average of three measurements) with examples shown in Figure 2. During the ﬁrst heating cycle it was apparent that two of the ﬁve monomers (BP-a) and (BD-a) underwent very visible endothermic melting transitions. All of the monomers used in this work are solid at room temperature and one might expect to see these endotherms in all of the samples. What these data demonstrate is that it takes more energy for the BP-a and BD-a samples to melt, which may be due to a higher degree of crystallinity in these monomers. These benzoxazines, containing phenolphthalein and dicyclopentadiene moieties, respectively, have much bulkier bridging groups than those of the other three monomers, this may be the cause for this greater requirement of energy. The exotherms, showing the polymerization reaction for all six monomers, display peak maxima within a narrow range of 218–242 ◦C. The monomers that display melting also show broad exotherms; the remaining monomers display much more pronounced peaks whilst curing. If a single exothermic peak is observed, then it is assumed that the curing results represent a single chemical process as a ﬁrst approximation, although two or more simultaneous or very close chemical reactions cannot be ruled 5 of 15 Polymers 2018, 10, 1250 out [21] and previously we have used mathematical modelling to deconvolute the thermal data [9] to reveal contributions from several processes to the reaction exotherm. In addition, it has to be assumed that all of the heat generated is a result of the curing reaction, which is irreversible ring-opening and formation of the methylene bridge in the case of benzoxazines. The most symmetrical, Gaussian curve appears to be produced by the BF-a monomer with BA-a and BT-a appearing quite symmetrical, although there does appear to be slight trailing on the curve, perhaps suggesting that there is a small change in viscosity occurring in the system which has resulted in the reaction becoming more diffusion controlled. 3.1. Thermal Analysis of the Homopolymerization Reactions Both BP-a batches and BD-a form especially broad peaks representing a slower reaction and perhaps a combination of more than one reaction process occurring. Polymers 2018, 10, x FOR PEER REVIEW 5 of 15 Figure 2. DSC of monomers, first heat of a heat cool heat cycle at 10 K/min. Figure 2. DSC of monomers, ﬁrst heat of a heat cool heat cycle at 10 K/min. Figure 2. DSC of monomers, first heat of a heat cool heat cycle at 10 K/min. Figure 2. DSC of monomers, ﬁrst heat of a heat cool heat cycle at 10 K/min. During the first heating cycle it was apparent that two of the five monomers (BP-a) and (BD-a) underwent very visible endothermic melting transitions. All of the monomers used in this work are solid at room temperature and one might expect to see these endotherms in all of the samples. What these data demonstrate is that it takes more energy for the BP-a and BD-a samples to melt, which may be due to a higher degree of crystallinity in these monomers. These benzoxazines, containing phenolphthalein and dicyclopentadiene moieties, respectively, have much bulkier bridging groups than those of the other three monomers, this may be the cause for this greater requirement of energy. The exotherms, showing the polymerization reaction for all six monomers, display peak The lowest energy transition occurs for the BD-a sample at 183.5 J/g (50.9 kJ/mol Bz ring) and the highest, almost double, occurs at 326.0 J/g (73.8 kJ/mol Bz ring) for BT-a, based on the sulphur containing benzoxazine. The exotherm for BA-a (Tmax ca. 240 ◦C and ∆Hp = 309.3 J/g) agrees reasonably closely with a literature value of Tmax = 243 ◦C, although the enthalpy is signiﬁcantly higher, ∆Hp = 240 J/g [22]. Pure benzoxazines typically show symmetrical exotherms between 200 and 250 ◦C, with ∆Hp = 150–600 J/g, although the exotherm may be skewed and reduced to lower temperatures in the presence of phenolic and amino impurities, arising from the synthesis of the monomer. In an extreme case, there will be multiple peaks, although none of the exotherms recorded here are truly multimodal. maxima within a narrow range of 218–242 C. The monome exotherms; the remaining monomers display much more 3.2. Determination of Glass Transition Temperature Using DSC single exothermic peak is observed, then it is assumed that the curing results represent a single chemical process as a first approximation, although two or more simultaneous or very close chemical reactions cannot be ruled out [21] and previously we have used mathematical modelling to deconvolute the thermal data [9] to reveal contributions from several processes to the reaction h I ddi i i h b d h ll f h h d i l f h i The second heating cycles of the DSC experiments were interpreted to yield the Tg of the monomers in their now cured resin form (Table 1). All the cured resins demonstrated a single Tg, which strongly implies that the samples are homogeneous. The Tg values given here experimentally have been interpreted using the inﬂexion as exempliﬁed by Figure 3. 6 of 15 mentally Polymers 2018, 10, 1250 which strongly impli have been interpreted Figure 3. DSC, second heat of monomer BF-a demonstrating the Tg transition and analysis. Figure 3. DSC, second heat of monomer BF-a demonstrating the Tg transition and analysis. Figure 3. DSC, second heat of monomer BF-a demonstrating the Tg transition and analysis. Figure 3. DSC, second heat of monomer BF-a demonstrating the Tg transition and analysis. The trend of the average glass transition temperature is: PBA-a < PBT-a < PBF-a < PBD-a BP-a, with PBP-a Batch 2 having the highest average Tg of all. This fits well with the knowledg hat PBP-a Batch 2 and PBP-a have the same polybenzoxazine backbone. It is interesting to note tha ncreasing the butanol content and decreasing the BF-a content of the BP-a formulation to creat P-a Batch 2 has resulted in a higher glass transition temperature by 17 K, presumably reflecting he increased reactivity (and higher crosslink density) of the system through a hydroxyl-initiated ng opening. The greatest Tg values found in this set of materials belong to the structures tha ontain the largest bisphenol linkages. The bulk of the dicyclopentadiene and phenolphthalein moieties no doubt restricts the molecular mobility of the chains with the ring structures limitin otation. This will particularly be the case for the phenolphthalein group of PBP-a, which contain oth a rigid aromatic ring and an oxygen atom, which may result in extra interactions with eighbouring chains (e.g., hydrogen bonding), which would also hinder chain motion and increas g. maxima within a narrow range of 218–242 C. The monome exotherms; the remaining monomers display much more 3.2. Determination of Glass Transition Temperature Using DSC When comparing the measured values to those available in the literature for the same monome h i d t f PBA (151 °C d 150 °C lit t [23]) d PBD (187 °C The trend of the average glass transition temperature is: PBA-a < PBT-a < PBF-a < PBD-a < PBP-a, with PBP-a Batch 2 having the highest average Tg of all. This ﬁts well with the knowledge that PBP-a Batch 2 and PBP-a have the same polybenzoxazine backbone. It is interesting to note that increasing the butanol content and decreasing the BF-a content of the BP-a formulation to create BP-a Batch 2 has resulted in a higher glass transition temperature by 17 K, presumably reﬂecting the increased reactivity (and higher crosslink density) of the system through a hydroxyl-initiated ring opening. The greatest Tg values found in this set of materials belong to the structures that contain the largest bisphenol linkages. The bulk of the dicyclopentadiene and phenolphthalein moieties no doubt restricts the molecular mobility of the chains with the ring structures limiting rotation. This will particularly be the case for the phenolphthalein group of PBP-a, which contains both a rigid aromatic ring and an oxygen atom, which may result in extra interactions with neighbouring chains (e.g., hydrogen bonding), which would also hinder chain motion and increase Tg. When comparing the measured values to those available in the literature for the same monomer there is good agreement for PBA-a (151 ◦C measured, 150 ◦C literature [23]) and PBD-a (187 ◦C measured, 183 ◦C literature [24]). 3.4. Determination of Cure Kinetics Using DSC 3.4. Determination of Cure Kinetics Using DSC 3.4. Determination of Cure Kinetics Using DSC 3.4. Determination of Cure Kinetics Using DSC Two different methods of kinetic analysis, proposed independently by Kissinger and Ozawa, were used in this work, which use variations of the Arrhenius equation [25]. The Kissinger method is depicted in Equation (1): ln β T2max = ln AR Ea − Ea RT2max (1) (1) where β = heating rate, Tmax = temperature of DSC peak maxium, Ea = activation energy, A = pre-exponential (collision) factor, R = gas constant. and employs thermal data determined using DSC. When lnβ is plotted against reciprocal temperature (Figure 4) the determination of activation energy is derived from the gradient and the pre-exponential factor from the intercept (Table 3). The Ozawa method is similar to the Kissinger method, where the inverse relationship between the logarithm of heating rate to exothermic peak temperature in Equation (2) (also Figure 4) allows graphical determination of activation from the slope of the plot of lnβ/T2max versus reciprocal temperature. The results are also presented in Table 3. Polymers 2018, 10, x FOR PEER REVIEW 8 of 15 change in the manner of cure. An obvious pattern to note is that BA-a, BF-a and BT-a all have small bisphenol linkages and have low activation energies, whereas the monomers with larger bisphenol linkages have the greater activation energies with the bulkiest linkage of BP-a having the greatest where β = heating rate, Tmax = temperature of DSC peak maxium, Ea = activation energy, A = pre-exponential (collision) factor, R = gas constant. and employs thermal data determined using DSC. When lnβ is plotted against reciprocal temperature (Figure 4) the determination of activation energy is derived from the gradient and the pre-exponential factor from the intercept (Table 3). The Ozawa method is similar to the Kissinger method, where the inverse relationship between the logarithm of heating rate to exothermic peak temperature in Equation (2) (also Figure 4) allows graphical determination of activation from the slope of the plot of lnβ/T2max versus reciprocal temperature. The results are also presented in Table 3. Polymers 2018, 10, x FOR PEER REVIEW 8 of 15 change in the manner of cure. here is good agreement for PBA a measured, 183 °C literature [24]). 3.3. Determination of Degree of Cure 3. Determination of Degree of Cure DSC was also used to ascertain the degree of cure of the monomers by comparing the curing xotherm (first heat) with the corresponding polybenzoxazine sample that had been produced sing a specific cure program. So, for instance: BA-a gave a cure exotherm of 309 J/g when cured to ompletion via DSC and was compared to the residual cure exotherm (31 J/g) of a sample cure in he laboratory using the cure program specified in the Experimental section which was used DSC was also used to ascertain the degree of cure of the monomers by comparing the curing exotherm (ﬁrst heat) with the corresponding polybenzoxazine sample that had been produced using a speciﬁc cure program. So, for instance: BA-a gave a cure exotherm of 309 J/g when cured to completion via DSC and was compared to the residual cure exotherm (31 J/g) of a sample cure in the laboratory using the cure program speciﬁed in the Experimental section, which was used throughout this work. Table 2 presents the resulting data, which demonstrate that in general the polybenzoxazines cure to a degree of approximately 90%. 7 of 15 Polymers 2018, 10, 1250 Table 2. Degree of cure of polybenzoxazines. Table 2. Degree of cure of polybenzoxazines. PBZ Cure exotherm (J/g) Degree of Cure (%) Monomer Cured Sample (rescan) PBA-a 309.30 31.11 89.9 PBF-a 298.30 23.24 92.2 PBP-a 265.37 24.08 90.9 PBP-a (Batch 2) 238.83 36.63 84.7 PBT-a 326.97 20.23 93.8 PBD-a 183.50 4.84 97.4 ination of Cure Kinetics Using DSC 3.4. Determination of Cure Kinetics Using DSC An obvious pattern to note is that BA-a, BF-a and BT-a all have small bisphenol linkages and have low activation energies, whereas the monomers with larger bisphenol linkages have the greater activation energies with the bulkiest linkage of BP-a having the greatest ln β = ln AEa R −lnF(α) −5.331 −1.052 Ea RTmax (2) herefore easy to assume that the size of the bisphenol linkage has a significant ion with larger groups hindering cure. This seems counterintuitive when one kinetic information to degree of cure where PBD-a gives by far the greatest (2) one where β = heating rate, Tmax = temperature of DSC peak maxium, Ea = activation energy, A = pre-exponential (collision) factor, R = gas constant, and F(α) is a constant function. conversion value. It has therefore been found that polymerization with a large activation energy does not necessarily lead to a lower degree of cure when cured under standard conditions. Figure 4. Kissinger and Ozawa plots for the polymerization of BA-a. Figure 4. Kissinger and Ozawa plots for the polymerization of BA-a. Figure 4. Kissinger and Ozawa plots for the polymerization of BA-a. Figure 4. Kissinger and Ozawa plots for the polymerization of BA-a. 8 of 15 Polymers 2018, 10, 1250 Table 3. Kinetic parameters for the polymerization of bis-benzoxazine monomers. Table 3. Kinetic parameters for the polymerization of bis-benzoxazine monomers. Monomer Kissinger Method Ozawa Method Ea (kJ mol−1) k × 10−2 (s−1) A × 108 (s−1) R2 Value Ea (kJ mol−1) R2 Value BA-a 85 21 1.53 0.9998 89 0.9999 BF-a 86 22 1.99 0.9995 90 0.9995 BP-a 135 50 559,425.15 0.9920 136 0.9929 BT-a 86 55 5.76 0.9998 90 0.9999 BD-a 108 34 602.97 0.9997 110 0.9997 Key: k = rate constant (s−1), A = pre-exponential factor (s−1). All analyses using both of the aforementioned methods showed a good linear relationship between heating rate and exothermic peak temperature with R2 values no lower than 0.99 throughout. All analyses using both of the aforementioned methods showed a good linear relationship between heating rate and exothermic peak temperature with R2 values no lower than 0.99 throughout. The activation energies calculated by the two methods are very similar for each sample giving a degree of conﬁdence in the results and the results for BA-a also match very closely to literature values (81 and 85 kJ mol−1 by Kissinger and Ozawa respectively. 3.4. Determination of Cure Kinetics Using DSC The similarity in activation energy of BA-a, BF-a and BT-a where only 1 kJ mol−1 separates both Kissinger and Ozawa values suggests that very similar cure processes occur in these materials. The values for BD-a are much greater at ~110 kJ mol−1 and greater still for BP-a at ~135 kJ mol−1, such a jump in activation energy suggests a change in the manner of cure. An obvious pattern to note is that BA-a, BF-a and BT-a all have small bisphenol linkages and have low activation energies, whereas the monomers with larger bisphenol linkages have the greater activation energies with the bulkiest linkage of BP-a having the greatest energy barrier. It is therefore easy to assume that the size of the bisphenol linkage has a signiﬁcant effect on polymerization with larger groups hindering cure. This seems counterintuitive when one attempts to relate the kinetic information to degree of cure where PBD-a gives by far the greatest conversion value. It has therefore been found that polymerization with a large activation energy does not necessarily lead to a lower degree of cure when cured under standard conditions. 3.5. Determination of the Glass Transition Temperature Using Dynamic Mechanical Thermal Analysis DMTA is the primary method for determining the glass transition temperature (Tg) of many polymers and has been identiﬁed to be several times more sensitive than DSC [26]. For this work a temperature range of −50 to 260 ◦C was used to allow identiﬁcation of Tg and where possible β-transitions whilst remaining within the calibration range of the instrument. Figure 5 shows the DMTA plot produced for PBA-a from which a clear a clear Tg can be ascertained as the storage and loss moduli and tan δ change dramatically in the region of 125 to 240 ◦C as the polybenzoxazine loses its stiff, glassy nature, ﬁrst becomes more plastic and ultimately more rubbery. Polymers 2018, 10, x FOR PEER REVIEW 9 of 15 Figure 5. DMA plot of PBA-a (2 K/min from −50 °C to 200 °C at 0.1% strain, 1Hz FF). Figure 5. DMA plot of PBA-a (2 K/min from −50 ◦C to 200 ◦C at 0.1% strain, 1Hz FF). Figure 5. DMA plot of PBA-a (2 K/min from −50 °C to 200 °C at 0.1% strain, 1Hz FF). Figure 5. DMA plot of PBA-a (2 K/min from −50 ◦C to 200 ◦C at 0.1% strain, 1Hz FF). 9 of 15 Polymers 2018, 10, 1250 Figure 5 Figure 6 shows the portion of the DMTA plots, which reveal the β-transitions, which occurs in the range −25 to 120 ◦C with the PBT-a β-transition occurring at the highest temperature and PBP-a (Batch 2) occurring at the lowest. The β-transition allows a much more limited degree of movement and is usually localised to side-chains or branches from the main polymer backbone. In Figure 6 an even more restricted transition (γ-transition) can be seen at lower temperatures in some of the materials e.g., in PBA-a centered around 0 ◦C, with the other materials all showing what might be the end of this peak at −50 ◦C. Figure 6 shows the portion of the DMTA plots, which reveal the β-transitions, which occurs in the range −25 to 120 °C with the PBT-a β-transition occurring at the highest temperature and PBP-a (Batch 2) occurring at the lowest. The β-transition allows a much more limited degree of movement and is usually localised to side-chains or branches from the main polymer backbone. 3.5. Determination of the Glass Transition Temperature Using Dynamic Mechanical Thermal Analysis In Figure 6 an even more restricted transition (γ-transition) can be seen at lower temperatures in some of the materials e.g., in PBA-a centered around 0 °C, with the other materials all showing what might be the end of this peak at −50 °C. Figure 6. Loss Modulus of PBZs via DMA (2 K/min from −50 °C to 200 °C at 0.1% strain, 1Hz FF). Figure 6. Loss Modulus of PBZs via DMA (2 K/min from −50 ◦C to 200 ◦C at 0.1% strain, 1Hz FF). Figure 6. Loss Modulus of PBZs via DMA (2 K/min from −50 °C to 200 °C at 0.1% strain, 1Hz FF). Figure 6. Loss Modulus of PBZs via DMA (2 K/min from −50 ◦C to 200 ◦C at 0.1% strain, 1Hz FF). Table 4 presents the DMTA data for all of the polybenzoxazine materials used in this work, from which it is clear that the sulfur- and phenolphthalein-containing polybenzoxazine backbones give rise to greater values of Tg than their counterparts. The phenolphthalein backbone in both of its formulations gives the highest Tg, with the ‘purified’ batch 2 version proving slightly superior (+3 K). This is to be expected as the Batch 1 sample contains a quantity of BF-a, which would depress Tg. The order of Tg by DMTA then is: PBP-a > PBT-a > PBA-a > PBF-a > PBD-a with the PBD-a value Table 4 presents the DMTA data for all of the polybenzoxazine materials used in this work, from which it is clear that the sulfur- and phenolphthalein-containing polybenzoxazine backbones give rise to greater values of Tg than their counterparts. The phenolphthalein backbone in both of its formulations gives the highest Tg, with the ‘puriﬁed’ batch 2 version proving slightly superior (+3 K). This is to be expected as the Batch 1 sample contains a quantity of BF-a, which would depress Tg. The order of Tg by DMTA then is: PBP-a > PBT-a > PBA-a > PBF-a > PBD-a with the PBD-a value being 20 K lower than the next lowest PBZ and PBP-a and PBT-a being 25 K higher than the next highest. It is difﬁcult to postulate why this trend is apparent. PBA-a and PBF-a which have very similar backbones give close matching Tg values (within 7 K) of each other, whilst PBP-a and PBT-a match even more closely (within 2 K) whilst having markedly different bridging groups. 3.5. Determination of the Glass Transition Temperature Using Dynamic Mechanical Thermal Analysis In theory, higher crosslink density of polymer networks can lead to increases in storage modulus and glass transition temperature (Tg)3. To gain a better understanding of these properties of the polybenzoxazines crosslink density has been calculated from the DMTA analyses using Equation (3). In theory, higher crosslink density of polymer networks can lead to increases in storage modulus and glass transition temperature (Tg)3. Key: Te is Tg + 50 C, Ge is storage modulus at equilibrium, v is the crosslink density. To gain a better understanding of these properties of the polybenzoxazines crosslink density has been calculated from the DMTA analyses using Equation (3). In theory, higher crosslink density f l k l d i i d l d l i i Ge = Ge = ΦvRTe (3) vRTe (3) s in storage modulus and glass transition temperature (3) (3) where Ge is the storage modulus at equilibrium, Φ is the front factor (unity for ideal rubbers), R is the gas constant, v is the crosslink density (number of moles of network chains per unit volume of cured polymer) and Te is Tg + 50 °C [27]. Th T d t (T bl 5) i ld th f l PBD b f th l t PBA d PBF where Ge is the storage modulus at equilibrium, Ge = ΦvRTe (3) where Ge is the storage modulus at equilibrium, Φ is the front factor (unity for ideal rubbers), R is the gas constant v is the crosslink density (number of moles of network chains per unit volume of cured is the front factor (unity for ideal rubbers), R is the gas constant, v is the crosslink density (number of moles of network chains per unit volume of cured polymer) and Te is Tg + 50 ◦C [27]. The Tg data (Table 5) yield three groups of values: PBD-a by far the lowest, PBA-a and PBF-a intermediate and PBP-a and PBT-a the greatest, the same grouping can be seen in the crosslink density. PBD-a gives a crosslink density of 1.4 × 10−3 mol cm−3, less than half the value of PBA-a and PBF-a (3.6 and 3.8 × 10−3 mol cm−3 respectively) which are ca. 1.7 × 10−3 mol cm−3 below that of PBP-a and PBT-a. This is consistent with the accepted view that crosslink density has an influence on Tg. 3.5. Determination of the Glass Transition Temperature Using Dynamic Mechanical Thermal Analysis As crosslink density increases chain movement/molecular mobility is further reduced resulting in more energy being required to overcome this obstacle. However, the results suggest that another factor gas constant, v is the crosslink density (number of moles of network chains per unit volume of cured polymer) and Te is Tg + 50 °C [27]. The Tg data (Table 5) yield three groups of values: PBD-a by far the lowest, PBA-a and PBF-a intermediate and PBP-a and PBT-a the greatest, the same grouping can be seen in the crosslink density. PBD-a gives a crosslink density of 1.4 × 10−3 mol cm−3, less than half the value of PBA-a and PBF-a (3.6 and 3.8 × 10−3 mol cm−3 respectively) which are ca. 1.7 × 10−3 mol cm−3 below that of PBP-a and PBT-a. This is consistent with the accepted view that crosslink density has an influence on Tg. As crosslink density increases chain movement/molecular mobility is further reduced resulting in more energy being required to overcome this obstacle. However, the results suggest that another factor The Tg data (Table 5) yield three groups of values: PBD-a by far the lowest, PBA-a and PBF-a intermediate and PBP-a and PBT-a the greatest, the same grouping can be seen in the crosslink density. PBD-a gives a crosslink density of 1.4 × 10−3 mol cm−3, less than half the value of PBA-a and PBF-a (3.6 and 3.8 × 10−3 mol cm−3 respectively) which are ca. 1.7 × 10−3 mol cm−3 below that of PBP-a and PBT-a. This is consistent with the accepted view that crosslink density has an inﬂuence on Tg. As crosslink density increases chain movement/molecular mobility is further reduced resulting in more energy being required to overcome this obstacle. However, the results suggest that another factor may be at work as PBT-a has a greater crosslink density than PBP-a by 20% yet their Tg values only differed by 2 K. It has been suggested that free volume, hydrogen bonding, chain interaction and intermolecular packing can also inﬂuence these properties [28]. It is interesting to note how in the PBP-a, the crosslink density changes with removal of the BF-a (20%) in its formulation. The crosslink density of the PBP-a Batch 1 is 5.5 × 10−3 mol cm−3 compared with the 18.4 × 10−3 mol cm−3 of PBP-a Batch 2 has an increase of more than 300%. 3.5. Determination of the Glass Transition Temperature Using Dynamic Mechanical Thermal Analysis When analyzed via differential scanning calorimetry (DSC) a similar trend in glass transition temperature was seen, however via DSC PBD-a gave the second highest Tg after PBP-a. It was suggested that this result showed a clear relationship between bisphenol linkage size and Tg, where the large linkages would restrict molecular motion and increase Tg. The change in PBD-a value when analyzed via DMA opposes this hypothesis as the materials with the two largest bisphenol linkages now give both the highest and lowest values for Tg. 10 of 15 Polymers 2018, 10, 1250 Table 4. Glass transition temperature data produced via DMTA. Sample Te (K) Ge (MPa) v (×10−3 mol cm−3) Table 5. Crosslink density calculated from DMTA data. Table 4. Glass transition temperature data produced via DMTA. Sample Te (K) Ge (MPa) v (×10−3 mol cm−3) Table 5. Crosslink density calculated from DMTA data Table 4. Glass transition temperature data produced via DMTA. PBZ Glass Transition Temperature (◦C) Storage Modulus Loss Modulus Tan δ PBA-a 173 174 191 PBA-a (Batch 2) 175 176 195 PBF-a 166 167 186 PBF-a (Batch 2) 161 161 179 PBP-a 196 201 225 PBP-a (Batch 2) 202 204 230 PBT-a 197 199 213 PBD-a 146 147 164 Sample Te (K) Ge (MPa) v (×10−3 mol cm−3) PBA-a 497 15.0 3.6 PBA-a (Batch 2) 498 15.2 3.7 PBF-a 490 15.4 3.8 PBF-a (Batch 2) 484 19.8 4.9 PBP-a 525 24.2 5.5 PBP-a (Batch 2) 527 80.8 18.4 PBT-a 522 28.7 6.6 PBD-a 470 5.4 1.4 Key: Te is Tg + 50 °C, Ge is storage modulus at equilibrium, v is the crosslink densit i b tt d t di f th ti f th l b i Table 5. Crosslink density calculated from DMTA data. Sample Te (K) Ge (MPa) v (×10−3 mol cm−3) PBA-a 497 15.0 3.6 PBA-a (Batch 2) 498 15.2 3.7 PBF-a 490 15.4 3.8 PBF-a (Batch 2) 484 19.8 4.9 PBP-a 525 24.2 5.5 PBP-a (Batch 2) 527 80.8 18.4 PBT-a 522 28.7 6.6 PBD-a 470 5.4 1.4 K T i T 50 °C G i t d l t ilib i i th li k d it Glass Transition Temperature (◦C) 97 15.0 3.6 98 15 2 3 7 K) Ge (MPa) v (×10−3 mol cm−3) To gain a better understanding of these properties of the polybenzoxazines crosslink density has been calculated from the DMTA analyses using Equation (3). 3.5. Determination of the Glass Transition Temperature Using Dynamic Mechanical Thermal Analysis Particularly intriguing is that the large increase in crosslink density only results in a 3 K (1.5%) increase in Tg, thus conﬁrming that although crosslink density does inﬂuence Tg it is not the only inﬂuence. Table 5. Crosslink density calculated from DMTA data. Sample Te (K) Ge (MPa) v (×10−3 mol cm−3) PBA-a 497 15.0 3.6 PBA-a (Batch 2) 498 15.2 3.7 PBF-a 490 15.4 3.8 PBF-a (Batch 2) 484 19.8 4.9 PBP-a 525 24.2 5.5 PBP-a (Batch 2) 527 80.8 18.4 PBT-a 522 28.7 6.6 PBD-a 470 5.4 1.4 Key: Te is Tg + 50 ◦C, Ge is storage modulus at equilibrium, v is the crosslink density. Table 5. Crosslink density calculated from DMTA data. Polymers 2018, 10, 1250 11 of 15 3.6. Determination of the Glass Transition Temperature Using Molecular Dynamics Simulation 3.6. Determination of the Glass Transition Temperature Using Molecular Dynamics Simulation Molecular dynamics (MD) has been used to estimate Tg by simulating the location and velocity vector for each atom within a molecular model over time at speciﬁed conditions of temperature and pressure. The region representing the Tg is typically determined by performing simulation experiments at different temperatures and calculating the density of the model at each simulation temperature; Tg is estimated as the point of intersection between the thermal expansion gradients for higher and lower temperature data. In this work, the method reported by Hall et al. [17] was used to ﬁnd the best point of gradient change (the ‘hinge point’) by ﬁnding when the ﬁt quality of a line is at its maximum, based on ﬁnding the best ﬁt for a gradient change as a function of temperature. An in-house program, written in Perl script, was used to analyze the raw data from the MD simulations, yielding a probability trace for Tg, mapped against temperature. A peak position may represent the Tg and the breadth of the peak also indicates the overall quality of the simulation data. The ‘quality of ﬁt’ is determined by centering an ellipse, of the same eccentricity as the standard deviation error bars and of sufﬁcient radius to make a tangent with the best ﬁt line. A Beckerman box reﬁnement method was employed to ﬁt the line and minimize the total of the semi minor axis radii, which quantify the ﬁt quality. Having calculated these parameters for a number of simulation temperatures, they were superimposed on the original density vs. 3.5. Determination of the Glass Transition Temperature Using Dynamic Mechanical Thermal Analysis temperature data. Examples of the calculations are shown on the simulated plots as the red line (the peak occurs where the gradient changes i.e., the transition midpoint certainty, TMC). Thus, the Tg is determined by the data rather than the experimentalist’s preconceived notion. A good example of this is shown for PBP-a (Figure 7) where two clear transitions are apparent, one over the range 190–240 ◦C and another above 290 ◦C. For comparison, the DMTA data for this material reveal an empirical Tg of 190–225 ◦C (Table 6). Polymers 2018, 10, x FOR PEER REVIEW 12 of 15 Figure 7. Plot of simulated density vs. temperature (black) for PBP-a with transition midpoint certainty aid (red) Figure 7. Plot of simulated density vs. temperature (black) for PBP-a with transition midpoint certainty aid (red). Figure 7. Plot of simulated density vs. temperature (black) for PBP-a with transition midpoint t i t id ( d) Figure 7. Plot of simulated density vs. temperature (black) for PBP-a with transition midpoint certainty aid (red). y aid (red). d l h l f b d h Table 6. Comparison of empirical Tg values with molecular dynamics simulations. second clear transition in the MD simulation for PBP-a is attributed to the onset ion. Whilst a discussion of this phenomenon falls outside the scope of the pres been shown to correlate well with thermal stability data determin avimetric analysis [27]. erally, the MD data show good agreement with the Tg data produced using D mulated transitions are more easily discerned than others (Figure 8). For insta BA-a, the density starts to fall between 160 and 190 °C, which matches the empi polymer measured by DMT; the TMC trace reveals a slightly lower value (150 Table 6. Comparison of empirical Tg values with molecular dynamics simulations. Sample Glass transition temperature (◦C) Storage Modulus Loss modulus Tan δ Simulation PBA-a 173 174 191 160–190 PBF-a 166 167 186 170–190 PBP-a 196 201 225 190–220 PBT-a 197 199 213 180–220 PBD-a 146 147 164 150–160 Polymers 2018, 10, 1250 12 of 15 The second clear transition in the MD simulation for PBP-a is attributed to the onset of thermal degradation. Whilst a discussion of this phenomenon falls outside the scope of the present paper, this has been shown to correlate well with thermal stability data determined using thermogravimetric analysis [27]. 3.5. Determination of the Glass Transition Temperature Using Dynamic Mechanical Thermal Analysis Generally, the MD data show good agreement with the Tg data produced using DMTA, but some simulated transitions are more easily discerned than others (Figure 8). For instance, in the case of PBA-a, the density starts to fall between 160 and 190 ◦C, which matches the empirical Tg for the same polymer measured by DMT; the TMC trace reveals a slightly lower value (150 ◦C). PBP-a shows a change in density between 190 and 220 ◦C (compared with a value of Tg of 200 ◦C determined using DMTA). The data for PBD-a are presented (Figure 8), but it is signiﬁcantly harder to discern changes in the density plot, which is more uniform in the observed changes. A small change can be identiﬁed between 150 and 160 ◦C, but the TMC trace shows a maximum at 170 ◦C. This may suggest that the model for PBD-a is not a good representation (in terms of crosslink density and bulk density) of the authentic network. It may be that our simpliﬁed model does not capture the complexity of the isomeric mix. The parent benzoxazine would originally have been prepared commercially from dicyclopentadiene and a phenolic derivative via reaction at the C=C double bonds on each ring (the resulting bisphenyl molecule would have been subsequently reacted with aniline and formaldeyde (or paraformaldehyde) to yield the benzoxazine monomer [29]. Consequently, not only may the structural motif, which makes up the bridge of the monomer exists in both exo and endo forms [30] (Figure 9), but the initial reaction of dicyclopentadiene with the phenol derivative at either ends of the double bonds might have led to different isomers (or more likely different isomeric mixtures). These structural differences would all potentially lead to polymers for which the free volume and Tg values would vary, but further work is required to conﬁrm this. Polymers 2018, 10, x FOR PEER REVIEW 13 of 15 Figure 8. Plot of density vs. temperature of PBZs with TMC. PBA-a (top left), PBF-a (top right), PBT-a (bottom left), PBD-a (bottom right). Figure 8. Plot of density vs. temperature of PBZs with TMC. PBA-a (top left), PBF-a (top right), PBT-a (bottom left), PBD-a (bottom right). Figure 8. Plot of density vs. temperature of PBZs with TMC. PBA-a (top left), PBF-a (top right), PBT-a (bottom left), PBD-a (bottom right). Figure 8. Plot of density vs. temperature of PBZs with TMC. 4. Conclusions 4. Conclusions The DSC analyses presented here have allowed determination of a number of properties of the bis-benzoxazine monomers and their respective polybenzoxazines. The first heat of DSC analyses has demonstrated that both BP-a batches, BD-a and BA-a undergo a visible melting stage when The DSC analyses presented here have allowed determination of a number of properties of the bis-benzoxazine monomers and their respective polybenzoxazines. The ﬁrst heat of DSC analyses has demonstrated that both BP-a batches, BD-a and BA-a undergo a visible melting stage when heated suggesting that these materials are more crystalline than the other materials under study. The exotherms representative of ring-opening for all the monomers present peak maxima within a narrow range of 218–242 ◦C and that the polymerization enthalpy of the monomers is in the order BT-a > BA-a > BF-a > BP-a > BD-a. The degree of cure of the materials studied here is around 90% however PBD-a gives a value of 97% which is surprising given that BD-a contains a large bisphenol linking group which one might imagine would lower cure degree, which is seen for PBP-a. The second heat of the heat-cool-heat cycle has shown that the glass transition temperatures of the cured materials range from 150 to 256 ◦C in the order PBA-a < PBT-a < PBF-a < PBD-a < PBP-a. The nature of bisphenol linkage in the polybenzoxazine structure has a dramatic effect on the structural properties of the material with larger bisphenol linkages imparting greater Tgs. Using the Kissinger and Ozawa methods activation energies of 80 kJ/mol−1 have been found for cure of BA-a, BF-a and BT-a with greater values of 110 and 135 kJ/mol−1 resulting for BD-a and BP-a respectively for the ring-opening polymerization process, with the bulkier bisphenol moieties yielding the greatest activation energies. Dynamic mechanical thermal analysis has revealed the glass transition temperature of the polybenzoxazines in this study to be in the order PBP-a > PBT-a > PBA-a > PBF-a > PBD-a in the range 147–204 ◦C. This suggests that the relationship between bisphenol linkage size and Tg is not as clear as had been seen via differential scanning calorimetry which gave rise to a much greater range in Tg between those of PBT-a and PBP-a. 3.5. Determination of the Glass Transition Temperature Using Dynamic Mechanical Thermal Analysis PBA-a (top left), PBF-a (top right), PBT-a (bottom left), PBD-a (bottom right). 13 of 15 ht), 13 of 15 ht), Polymers 2018, 10, 1250 Figure 8. Plot o b l PBT a (bottom left), PBD a (bottom right). Figure 9. Structure of BD-a monomer, ultimately derived from dicyclopentadiene (showing exo and endo isomers). Figure 9. Structure of BD-a monomer, ultimately derived from dicyclopentadiene (showing exo and endo isomers). Figure 9. Structure of BD-a monomer, ultimately derived from dicyclopentadiene (showing exo and endo isomers). Figure 9. Structure of BD-a monomer, ultimately derived from dicyclopentadiene (showing exo and endo isomers). References 1. Ishida, H.; Agag, T. (Eds.) Handbook of Polybenzoxazine Resins; Elsevier: New York, NY, USA, 2011. 2. Gardziella, A.; Pilato, L.A.; Knop, A. Phenolic Resins: Chemistry, Applications, Standardisation, Safety and Ecology; Springer: Berlin, Germany, 2000. 3. Hamerton, I. Recent Developments in Epoxy Resins; Rapra Review Reports Volume 8; Rapra: Shawbury, U 4. Henkel Benzoxazine Resins. Available online: http://www.henkelepsilonresin.com/pdf/Henkel%20BZ% 20Summary%20US.ppt.pdf (accessed on 20 February 2014). 5. Jubsilp, C.; Punson, K.; Takeichi, T.; Rimdusit, S. Curing kinetics of benzoxazine–epoxy copolymer investigated by non-isothermal differential scanning calorimetry. Polym. Degr. Stab. 2010, 95, 918–924. [CrossRef] 6. Hamerton, I.; McNamara, L.T.; Howlin, B.J.; Smith, P.A.; Cross, P.; Ward, S. Toughening mechanisms in aromatic polybenzoxazines using thermoplastic oligomers and telechelics. Macromolecules 2014, 47, 1946–1958. [CrossRef] 7. Ishida, H.; Ohba, O. Synthesis and characterization of maleimide and norbornene functionalized benzoxazines. Polymer 2005, 46, 5588–5595. [CrossRef] 8. Sudo, A.; Sudo, R.; Nakayama, H.; Arima, K.; Endo, T. Selective formation of poly(N,O-acetal) by polymerization of 1,3-benzoxazine and its main chain rearrangement. Macromolecules 2008, 41, 9030–9034. [CrossRef] 9. Hamerton, I.; McNamara, L.T.; Howlin, B.J.; Smith, P.A.; Cross, P.; Ward, S. Examining the initiation of the polymerization mechanism and network development in aromatic polybenzoxazines. Macromolecules 2013, 46, 5117–5132. [CrossRef] [PubMed] 10. Wan Hassan, W.A. Characterisation and Molecular Modelling of Selected Benzoxazines and Their Polymers. Ph.D. Thesis, University of Surrey, Surrey, UK, 2014. 11. Accelrys Inc. Accelrys Materials Studio Versions v 5.5.0.0 (2010) and v 6.0.0.0 (2012), Accelrys Inc.: San Diego, CA, USA, 2012. 12. Sun, H.; Mumby, S.J.; Maple, J.R.; Hagler, A.T. An ab initio CFF93 all-Atom force ﬁeld for polycarbonates. J. Amer. Chem. Soc. 1994, 116, 2978–2987. [CrossRef] 3. Burkert, U.; Allinger, N.L. Molecular Mechanics, ACS Monograph 177; American Chemical Society: Washing DC, USA, 1982. 14. Goodman, J.M. Chemical Applications of Molecular Modelling; Royal Society of Chemistry: Cambridge, UK, 1998; pp. 31–60. 15. Allen, F.H.; Watson, D.G.; Brammer, L.; Opren, A.G.; Taylor, R. Typical interatomic distances: Organic compounds. In International Tables for Crystallography; Springer: Dordrecht, The Netherlands, 2006; pp. 790–811. 16. Toukmaji, A.Y.; Board, J.A., Jr. Ewald summation techniques in perspective: a survey. Comput. Phys. Commun. 1996, 95, 73–92. [CrossRef] 17. Hall, S.A.; Howlin, B.J.; Hamerton, I.; Baidak, A.; Billaud, C.; Ward, S. Solving the Problem of Building Models of Crosslinked Polymers: An Example Focussing on Validation of the Properties of Crosslinked Epoxy Resins. PLoS ONE 2012, 7, e42928. [CrossRef] [PubMed] p y 18. Ishida, H.; Low, H.Y. 4. Conclusions 4. Conclusions The crosslink density of the materials was also determined from DMTA data, with PBP-a Batch 2 demonstrating a crosslink density of 18.4 × 10−3 mol cm−3, almost triple the value of the next greatest polymer, PBT-a at 6.6 × 10−3 mol cm−3. The trend in crosslink density data in general matches well with the Tg data and molecular simulation has been used with some success to simulate these thermal events. Polymers 2018, 10, 1250 14 of 15 14 of 15 Author Contributions: I.H., S.T., and B.J.H. conceived and designed the experiments; S.T. performed the experiments; S.T., I.H. and B.J.H. analyzed the data; I.H., B.J.H., S.T., C.A.S. wrote the paper. Funding: The authors thank the Defence Science Technology Laboratory (Dstl) for funding this work and supporting ST in the form of research contract Dstlx-10000065719. Acknowledgments: The authors thank Huntsman Advanced Materials (Basel, Switzerland) for supplying the monomers. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. References A study on the volumetric expansion of benzoxazine-based phenolic resin. Macromolecules 1997, 30, 1099–1106. [CrossRef] 19. Sun, H.J. Force ﬁeld for computation of conformational energies, structures, and vibrational frequencies of aromatic polyesters. Comput. Chem. 1994, 15, 752. [CrossRef] 15 of 15 Polymers 2018, 10, 1250 20. Berendsen, H.; Postma, J.; van Gunsteren, W.; Dinolaand, A.; Haak, J.J. Molecular dynamics with coupling to an external bath. Chem. Phys. 1984, 81, 3684–3690. [CrossRef] 21. Ishida, H.; Rodriguez, Y. Curing kinetics of a new benzoxazine-based phenolic resin by differential scanning calorimetry. Polymer 1995, 36, 3151. [CrossRef] 22. Takeichi, T.; Kano, T.; Agag, T. Synthesis and thermal cure of high molecular weight polybenzoxazine precursors and the properties of the thermosets. Polymer 2005, 46, 12172. [CrossRef] 23. Ning, X.; Ishida, H.J. Phenolic materials via ring-opening polymerization: Synthesis and characterization of bisphenol-A based benzoxazines and their polymers. Polym. Sci. Part A Polym. Chem. 1994, 32, 1121. [CrossRef] 24. Hwang, H.-J.; Lin, C.-Y.; Wang, C.-S. Flame retardancy and dielectric properties of dicyclopentadiene-based benzoxazine cured with a phosphorus-containing phenolic resin. J. Appl. Polym. Sci. 2008, 110, 2413. [CrossRef] 25. Jubsilp, C.; Damrongsakkul, S.; Takeichi, T.; Rimdusit, S. Curing kinetics of arylamine-based polyfunctional benzoxazine resins by dynamic differential scanning calorimetry. Thermochim. Acta 2006, 447, 131. [CrossRef] 26. Nair, C.P.R.; Kumar, K.S.S.; Ninan, K.N.; Kulkarni, A.D.; Wadgaonkar, P.P. Synthesis and properties of new 25. Jubsilp, C.; Damrongsakkul, S.; Takeichi, T.; Rimdusit, S. Curing kinetics of arylamine-based polyfunctional benzoxazine resins by dynamic differential scanning calorimetry. Thermochim. Acta 2006, 447, 131. [CrossRef] 26. Nair, C.P.R.; Kumar, K.S.S.; Ninan, K.N.; Kulkarni, A.D.; Wadgaonkar, P.P. Synthesis and properties of new polybenzoxazines containing (substituted) cyclohexyl moieties. Polym. Adv. Technol. 2009, 20, 1107. 27. Hamerton, I.; Thompson, S.; Howlin, B.J.; Stone, C.A. New method to predict the thermal degradation behavior of polybenzoxazines from empirical data using structure property relationships. Macromolecules 2013, 46, 7605–7615. [CrossRef] 28. Ishida, H.; Sanders, D.P.J. Physical and mechanical characterization of near-zero shrinkage polybenzoxazines. Polym. Sci. Part B Polym. Phys. 1996, 34, 1019. [CrossRef] 29. Ishida, H.; Liu, J.-P. Benzoxazine chemistry in solution and melt. In Phenolic Resins: Chemistry, Applications, Standardisation, Safety and Ecology; Chapter 2 in Reference 2; Springer: Berlin, Germany, 2000; pp. 85–102. 30. Li, Y.; Zhou, J.J.; Zhang, X.; Wang, L.; Mia, Z. Product distribution of tricyclopentadiene from cycloaddition of dicyclopentadiene and cyclopentadiene: A theoretical and experimental study. Fuel 2010, 89, 2522–2527. [CrossRef] © 2018 by the authors. © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). References Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W3112469500	http://www.scielo.br/pdf/pd/v38/0100-8358-PD-38-e020223617.pdf	English	null	Sourgrass phenological stage and efficacy of ACCase-inhibiting herbicides	Planta Daninha	2,020	cc-by	6,050	Corresponding author: Corresponding author: <sjpcarvalho@yahoo.com.br> Cite this article: Presoto JC, Andrade JF, Souza LA, Teixeira LS, Carvalho SJP. Sourgrass phenological stage and efficacy of ACCase-inhibiting herbicides. Planta Daninha. 2020:38:e020223617. https://doi.org/10.1590/S0100-83582020380100089 Cite this article: Presoto JC, Andrade JF, Souza LA, Teixeira LS, Carvalho SJP. Sourgrass phenological stage and efficacy of ACCase-inhibiting herbicides. Planta Daninha. 2020:38:e020223617. https://doi.org/10.1590/S0100-83582020380100089 Objective: This work was developed with the objective of evaluating the effect of the sourgrass phenological stage at the time of herbicide application on the efficacy of ACCase-inhibiting herbicides. Methods: Two independent experiments were conducted under greenhouse conditions to evaluate two ACCase-inhibiting herbicides: clethodim, and haloxyfop. The treatments were applied using a completely randomized block design, with an 8×4 factorial arrangement consisted of 8 herbicide rates (4, 2, 1, 1/2, 1/4, 1/8, and 1/16 times the recommended rate, and a control plot without herbicide application) and four phenological stages of sourgrass. Conflict of Interest: The authors declare that there is no conflict of interest regarding the publication of this manuscript. Conflict of Interest: The authors declare that there is no conflict of interest regarding the publication of this manuscript. Results: The development of sourgrass plants after tillering decreased the efficacy of the herbicide molecules; however, clethodim and haloxyfop were efficient to control sourgrass at earlier developmental stages. Morphological, anatomical, and physiological changes in older plants may explain the lower susceptibility to herbicides. Copyright: 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 that the original author and source are credited. Conclusions: Other control techniques should be considered for sourgrass plants at later developmental stages, such as sequential application or herbicide mixtures, to increase the efficacy of ACCase herbicides. 12 exotic species (Canto-Dorow and Longhi-Wagner, 2001). One of these species that have wide geographical distribution is the sourgrass (Digitaria insularis (L.) Fedde), which occurs in most <http://www.sbcpd.org> <http://www.sbcpd.org> Sourgrass phenological stage and efficacy of ACCase-inhibiting herbicides éssica C. Presotoa, Jeisiane F. Andradea, Laura A. Souzab, Laura S. Teixeirab, Saul J.P. Carvalh a Escola Superior de Agricultura “Luiz de Queiroz”, Universidade de São Paulo, Piracicaba-SP, Brasil; b Instituto Federal de Educação, Ciência e Tecnologia do Sul de Minas Gerais, Machado-MG, Brasil. ARTICLE INFORMATION  Herbicides are more efficient when applied before the sourgrass tillering or flowering. Received: May 6, 2019 Accepted: September 24, 2020  The greater the developmental stage of sourgrass plants, the more difficult the control. Keywords: Digitaria insularis clethodim haloxyfop management resistance  Herbicide mixtures or sequential applications may be needed for the control of sourgrass. Conflict of Interest: The authors declare that there is no conflict of interest regarding the publication of this manuscript. HIGHLIGHTS ARTICLE INFORMATION Received: May 6, 2019 Accepted: September 24, 2020 Planta Daninha ISSN 0100-8358 (print) 1806-9681 (online) ISSN 0100-8358 (print) 1806-9681 (online) Journal of The Brazilian Weed Science Society Copyright: 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 that the original author and source are credited. ABSTRACT Background: Sourgrass (Digitaria insularis) is a highly disseminated weed in Brazil. It is a perennial grass weed that has high infesting capacity in agricultural areas and presents glyphosate-resistant biotypes. An effective post-emergence control of sourgrass plants depends on their phenological stage at the time of herbicide application. *Corresponding author: 2 MATERIAL AND METHODS The experiment was conducted at a greenhouse of the Instituto Federal de Educação, Ciência e Tecnologia do Sul de Minas, in Machado, MG, Brazil (21º40'S, 45º55'W, and 850 m altitude). Sourgrass seeds were collected in an agricultural area of the Machado campus, in an area infested with plants susceptible to glyphosate herbicide (Gonçalves Netto et al., 2015). Glyphosate applications control susceptible young weeds grown from seeds; however, the control is difficult when the weeds are grown from rhizomes, (Andrade et al., 2019). Thus, the best time for controlling D. insularis plants is up to 45 days after emergence, when the rhizomes are not yet formed (Gazola et al., 2016). The importance of sourgrass infestations in agricultural areas can be great for soybean and maize crops and citrus orchards because of glyphosate-resistant biotypes (Carvalho et al., 2011; Heap, 2019). Thus, the species, which previously had low agronomic importance due to its easy control, became one of the most competitive and important weeds in the country (Andrade et al., 2019). Lacerda and Victoria Filho (2004) found that only 128.5 g ha-1 of glyphosate were enough to control 50% (DL50) of these plants. However, few years later, after a selection process of resistant biotypes, the required rates of glyphosate to obtain DL50 were higher than 2,880 g ha-1 (López-Ovejero et al., 2017; Costa et al., 2018). The sourgrass seeds were germinated in 2-liter plastic boxes filled with a commercial substrate (Pinus bark + turf + vermiculite) to select seedlings with adequate health; they were sown at different times to enable the joint application of herbicides to plants at different phenological stages. The seedlings were transplanted to 1-liter plastic pots when they had one fully expanded leaf; each pot with a mean of three plants represented one plot. The pots were filled with a mixture of commercial substrate, sieved clayey soil, vermiculite, and cattle manure at the proportion of 5:3:1:1, respectively. The soil of all plots was properly fertilized once and irrigated daily. Two similar and independent experiments were conducted from August 2017 to June 2018. The first experiment was conducted to evaluate the efficacy of herbicide clethodim, which is classified in the cyclohexanedione group; and the second experiment was conducted to evaluate the efficacy of haloxyfop, which is from the aryloxyphenoxypropionate group, both ACCase-inhibitor herbicides. 1 INTRODUCTION Brazil is the country that has the highest diversity of species of the Digitaria genus, with 26 native and Planta Daninha 2020;38:e020223617 - https://doi.org/10.1590/S0100-83582020380100089 1/7 SBCPD \| Planta Daninha Presoto JC, et al. Sourgrass phenology and herbicide efficacy environments favorable to agriculture (López-Ovejero et al., 2017). D. insularis plants have aggressive growth and may survive in environments with different types and intensities of limitation for plant growth and development (Licorini et al., 2015). They can form rhizomes that are short, but evident, forming pronounced clumps. These plants have a fast growth, production of large number of seeds, and high seed dissemination capacity during the whole summer (Gemelli et al., 2012; Ferreira et al., 2018). environments favorable to agriculture (López-Ovejero et al., 2017). D. insularis plants have aggressive growth and may survive in environments with different types and intensities of limitation for plant growth and development (Licorini et al., 2015). They can form rhizomes that are short, but evident, forming pronounced clumps. These plants have a fast growth, production of large number of seeds, and high seed dissemination capacity during the whole summer (Gemelli et al., 2012; Ferreira et al., 2018). herbicides regarding rates to be used, and about the effects of the plant phenology at the time of application. Thus, this work was developed with the objective of evaluating the effect of the sourgrass phenological stage at the time of herbicide application on the efficacy of ACCase-inhibiting herbicides. 2 MATERIAL AND METHODS                  c b x a Pmín y 1 where y = percent control or residual dry weight; x = herbicide rate; and Pmin, a, b, and c = parameters of the curve: Pmin is the lower limit of the curve (minimum point), a is the difference between the maximum and minimum points of the curve, b is the rate that provides 50% of the response of the variable, and c is the slope of the curve. where y = percent control or residual dry weight; x = herbicide rate; and Pmin, a, b, and c = parameters of the curve: Pmin is the lower limit of the curve (minimum point), a is the difference between the maximum and minimum points of the curve, b is the rate that provides 50% of the response of the variable, and c is the slope of the curve. Table 1 - Phenological stages of sourgrass plants at the time of application of the herbicides clethodim and haloxyfop. Machado, MG, Brazil, 2017-2018 Stage Description Days after emergence BBCH scale Clethodim 1 3 leaves 35 13 2 2 tillers 42 22 3 5 tillers + 4 visible internodes 55 34 4 5 internodes to pre-flowering 76 49 Haloxyfop 1 3 to 4 leaves 19 14 2 1 developed tiller 33 21 3 3 tillers + 2 visible internodes 47 32 4 full flowering 61 60 The BBCH scale was used as described by Hess et al. (1997). The analyses of each phenological stage were complemented with the mathematical calculation of the values GR50 and GR80 (GR = growth reduction), using the principle of inverse equation, as described by Carvalho et al. (2015). The successive values of GR50 and GR80 were then subjected to linear or polynomial regression models with high R2, correlating the variables of plant development with tolerance to herbicides. 2 MATERIAL AND METHODS Considering the resistance cases, the first changes in the management system should be the substitution of active ingredients or the combination of herbicides with different mechanisms of action. The best results of post-emergence control of glyphosate-resistant sourgrass in Brazil have been obtained with the use of ACCase-inhibitor herbicides, applied alone or in combination with glyphosate (Melo et al., 2012; Barroso et al., 2014; Zobiole et al., 2016; Carvalho et al., 2019). Each experiment was conducted in a completely randomized block design, with an 8×4 factorial arrangement and five replications (160 experimental plots in each experiment). The treatments of both experiments consisted of 8 herbicide rates (4, 2, 1, 1/2, 1/4, 1/8, and 1/16 times the recommended rate, and a control plot without herbicide application) and four phenological stages of sourgrass. The recommended rates used were 108 g ha-1 of the active ingredient for clethodim, and 60 g ha-1 of the acid equivalent for haloxyfop (Rodrigues and Almeida, 2018). Barroso et al. (2014) found that the herbicides quizalofop, haloxyfop, sethoxydim, and clethodim are good options for the control of sourgrass plants at initial developmental stages, showing efficacies higher than 90% at 28 days after application. An efficient control of weeds by using post-emergence herbicides depends mainly on the developmental stage of the target-plants at the time of application (Melo et al., 2012; Barroso et al., 2014; Andrade et al., 2019). However, little scientific information is available about the efficacy of ACCase-inhibitor Four plots of each phenological stage were sampled to measure the exact phenological stage, based on the scale of Hess et al. (1997) (BBCH), at Planta Daninha 2020;38:e020223617 - https://doi.org/10.1590/S0100-83582020380100089 2/7 SBCPD \| Planta Daninha SBCPD \| Planta Daninha Presoto JC, et al. Sourgrass phenology and herbicide efficacy stage were fitted to log-logistic non-linear regression models (Seefeldt et al., 1995): stage were fitted to log-logistic non-linear regression models (Seefeldt et al., 1995): the time of application of the herbicides clethodim (November 15, 2017) and haloxyfop (May 4, 2018) (Table 1). The herbicides were applied using a CO2- pressurized backpack sprayer equipped with a spray boom consisting of two nozzles (XR 110.02; Teejet®, Wheaton, USA) positioned at 0.50 m above the targets, using pressure of 2.5 bar and a solution volume of 200 L ha-1. Deionized water was used for the preparation of the solutions in all treatments to avoid contamination. 3 RESULTS AND DISCUSSION The susceptibility of sourgrass at different phenological stages to the herbicide clethodim is shown in Table 2, presenting the statistic parameters for the fit of weed control percentage and shoot dry weight (SDW) of sourgrass at 28 DAA. The maximum recommended rate of clethodim is 108 g ha-1 (Rodrigues and Almeida, 2018), which was enough to promote satisfactory control of sourgrass up to the two-tiller stage (BBCH 22). The later stages (BBCH 34 and 49) required rates higher than 150 g ha-1 for an 80% control, which is the minimum percentage required to base a recommendation for agricultural crops (Table 2). Weed control and residual plant shoot dry weight (SDW) were evaluated at 28 days after application (DAA), in all plots. The control was evaluated as percentages, with 0% representing absence of symptoms caused by the herbicides, and 100% representing the death of the plant. The SDW was obtained by taking the aerial part of remaining plants in the plots and drying it in an oven at 70 °C for 72 hours. The SDW was corrected to percentages by comparing the weight found in the treatments with the weight of the check plants (considered as 100%). The data of each experiment were subjected to analysis of variance by the F test. The data of weed control and SDW percentages in each phenological This denotes the effect of the sourgrass phenological stage on the efficiency of clethodim. The Table 2 - Statistic parameters of logistic models, coefficient of determination, GR50 and GR80 for efficacy of the herbicides clethodim applied to sourgrass plants at different phenological stages. Machado, MG, Brazil, 2017 Variable Stage Parameters R² (3)GR50 (3)GR80 Pmin a b c Control(1) 28 DAA(2) 13 X 99.94 13.28 -3.75 0.998 13.28 19.228 22 X 102.10 12.24 -1.65 0.993 11.94 26.736 34 X 102.16 38.35 -0.93 0.985 36.65 152.267 49 X 112.27 120.96 -0.57 0.983 82.52 588.189 Shoot dry weight percentage(1) 13 3.231 96.86 10.39 6.59 0.997 10.29 13.174 22 2.512 98.26 7.29 0.60 0.955 6.87 93.423 34 1.808 97.96 101.49 0.51 0.961 93.45 1894.910 49 -1.291 97.61 168.38 0.47 0.950 151.61 2575.836 (1) Model: y = a+(b/(1+(x/c)d). (2) DAA = days after application. (3) GR = growth reduction. Table 2 - Statistic parameters of logistic models, coefficient of determination, GR50 and GR80 for efficacy o clethodim applied to sourgrass plants at different phenological stages. Planta Daninha 2020;38:e020223617 - https://doi.org/10.1590/S0100-83582020380100089 3 RESULTS AND DISCUSSION Machado, MG, Brazil, 2017 c parameters of logistic models, coefficient of determination, GR50 and GR80 for efficacy of the herbicides d to sourgrass plants at different phenological stages. Machado, MG, Brazil, 2017 3/7 SBCPD \| Planta Daninha SBCPD \| Planta Daninha Presoto JC, et al. Sourgrass phenology and herbicide efficacy increasing evolution of GR50 and GR80, considering the control percentage and SDW at 28 DAA, respectively, is shown in Figures 1 and 2. The herbicide rates required to obtain these weed control levels increased significantly after the stage 20 of the BBCH scale (beginning of tillering) (Figure 1). of different herbicides on sourgrass plants with 3-5 tillers and found that clethodim applied alone (108 g ha-1) was not efficient, reaching a maximum control of 17.5%. Zobiole et al. (2016) found that single application of graminicide herbicides was not enough to control sourgrass plants at full flowering stage, and they indicated a sequential application system (two applications) for weed controls above 80%. In both cases, the combination of clethodim with other herbicides was needed to increase the weed control efficiency. Researches have frequently sought to control glyphosate-resistant sourgrass biotypes by using post-emergence application of ACCase-inhibitor herbicides (Barroso et al., 2014; Carvalho et al., 2019). However, the main limitation for this practice is the phenological stage of the plants at the time of application; the herbicide efficacy level decreases when applied on plants at later developmental stages, with fully developed clumps (Melo et al., 2012; Adegas and Gazziero, 2014). Gemelli et al. (2012) reported that ACCase-inhibitor herbicides affect sourgrass plants at later developmental stages, producing characteristic symptoms of necrosis in the plants' growth regions. However, despite they cause necrosis in the leaf ends, the other leaf parts remain only with chlorosis. Thus, the leaves remain erect, i.e., the herbicide application does not decrease significantly the plant leaf area, which is still able to intercept light and, potentially, affect the development of crops. Melo et al. (2012) evaluated the application The results found with application of the herbicide haloxyfop on sourgrass plants at different phenological stages were similar to those found with application of clethodim. The recommended rate of haloxyfop is 60 g ha-1; this rate was efficient to control plants up to the tillering stage (BBCH 22). 3 RESULTS AND DISCUSSION The fit of the SDW data for BBCH 60 was not possible because the maximum rate did not reach 50% decrease in SDW, which is required by the model (Table 3). The increasing evolution of GR50 and GR80, considering the control percentage and SDW at 28 DAA, is shown in Figures 3 and 4, respectively. The decreases in the efficacy of haloxyfop were even more significant than those found for clethodim, thus requiring applications to young plants. Figure 1 - Evolution of GR50 of sourgrass plants, estimated by the control percentage (A) and residual shoot dry weight (B) at 28 days after application, affected by the phenological stage at the time of applications of the herbicide clethodim. Machado, MG, Brazil, 2017. y = 0.0609x2 - 1.7982x + 25.068 R² = 0.9949 0 10 20 30 40 50 60 70 80 90 10 20 30 40 50 Clethodim (g ha-1) BBCH Phenological Scale y = 0.0464x2 + 1.4273x - 25.366 R² = 0.9422 0 20 40 60 80 100 120 140 160 180 10 20 30 40 50 Clethodim (g ha-1) BBCH Phenological Scale (A) (B) y = 0.0464x2 + 1.4273x - 25.366 R² = 0.9422 0 20 40 60 80 100 120 140 160 180 10 20 30 40 50 Clethodim (g ha-1) BBCH Phenological Scale (B) Figure 1 - Evolution of GR50 of sourgrass plants, estimated by the control percentage (A) and residual shoot dry weight (B) at 28 days after application, affected by the phenological stage at the time of applications of the herbicide clethodim. Machado, MG, Brazil, 2017. y = 0.0609x2 - 1.7982x + 25.068 R² = 0.9949 0 10 20 30 40 50 60 70 80 90 10 20 30 40 50 Clethodim (g ha-1) BBCH Phenological Scale y = 0.0464x2 + 1.4273x - 25.366 R² = 0.9422 0 20 40 60 80 100 120 140 160 180 10 20 30 40 50 Clethodim (g ha-1) BBCH Phenological Scale (A) (B) Figure 2 - Evolution of GR80 of sourgrass plants, estimated by the control percentage (A) and residual shoot dry weight (B) at 28 days after application, affected by the phenological stage at the time of applications of the herbicide clethodim. Machado, MG, Brazil, 2017. 3 RESULTS AND DISCUSSION y = 1.6413x - 21.291 R² = 0.9453 0 10 20 30 40 50 60 70 80 90 10 20 30 40 50 60 Haloxyfop (g ha-1) BBCH Phenological Scale y = 20.076x - 323.58 R² = 0.8871 0 50 100 150 200 250 300 350 400 10 15 20 25 30 35 Haloxyfop (g ha-1) BBCH Phenological Scale (A) (B) y = 1.6413x - 21.291 R² = 0.9453 0 10 20 30 40 50 60 70 80 90 10 20 30 40 50 60 Haloxyfop (g ha-1) BBCH Phenological Scale (A) y = 20.076x - 323.58 R² = 0.8871 0 50 100 150 200 250 300 350 400 10 15 20 25 30 35 Haloxyfop (g ha-1) BBCH Phenological Scale (B) (B) (A) Figure 3 - Evolution of GR50 of sourgrass plants, estimated by the control percentage (A) and residual shoot dry weight (B) at 28 days after application, affected by the phenological stage at the time of applications of the herbicide haloxyfop. Machado, MG, Brazil, 2018. Figure 4 - Evolution of GR80 of sourgrass plants, estimated by the control percentage (A) and residual shoot dry weight (B) at 28 days after application, affected by the phenological stage at the time of applications of the herbicide haloxyfop. Machado, MG, Brazil, 2017. y = -0.2526x2 + 31.817x - 442.14 R² = 0.9209 0 100 200 300 400 500 600 10 20 30 40 50 60 Haloxyfop (g ha-1) BBCH Phenological Scale y = 64.204x - 906.51 R² = 0.9987 0 200 400 600 800 1000 1200 1400 10 15 20 25 30 35 Haloxyfop (g ha-1) BBCH Phenological Scale (A) (B) y = 64.204x - 906.51 R² = 0.9987 0 200 400 600 800 1000 1200 1400 10 15 20 25 30 35 Haloxyfop (g ha-1) BBCH Phenological Scale (B) y = -0.2526x2 + 31.817x - 442.14 R² = 0.9209 0 100 200 300 400 500 600 10 20 30 40 50 60 Haloxyfop (g ha-1) BBCH Phenological Scale (A) (A) (B) Figure 4 - Evolution of GR80 of sourgrass plants, estimated by the control percentage (A) and residual shoot dry weight (B) at 28 days after application, affected by the phenological stage at the time of applications of the herbicide haloxyfop. Machado, MG, Brazil, 2017. These results agree with other studies that report that young plants developed from seeds are more easily controlled with the use of haloxyfop. 3 RESULTS AND DISCUSSION Machado, MG, Brazil, 2018 Variable Stage Parameters R² (3)GR50 (3)GR80 Pmin a b c Control(1) 28 DAA(2) 14 X 101.92 3.98 -1.68 0.985 3.89 8.610 21 X 101.77 3.79 -1.56 0.986 3.70 8.743 32 X 120.29 85.15 -0.46 0.974 40.80 374.614 60 X 126.48 179.87 -0.49 0.963 74.88 551.013 Shoot dry weight(1) percentage 14 3.139 96.34 3.08 1.87 0.996 2.95 7.052 21 -8.318 108.84 13.88 0.31 0.911 23.59 417.696 32 -17.485 113.14 191.20 0.39 0.935 347.78 1157.373 60 - - - - - > 240.00 - (1) Model: y = a+(b/(1+(x/c)d). (2) DAA = days after application. (3) GR = growth reduction. Figure 3 - Evolution of GR50 of sourgrass plants, estimated by the control percentage (A) and residual shoot dry weight (B) at 28 days after application, affected by the phenological stage at the time of applications of the herbicide haloxyfop. Machado, MG, Brazil, 2018. y = 1.6413x - 21.291 R² = 0.9453 0 10 20 30 40 50 60 70 80 90 10 20 30 40 50 60 Haloxyfop (g ha-1) BBCH Phenological Scale y = 20.076x - 323.58 R² = 0.8871 0 50 100 150 200 250 300 350 400 10 15 20 25 30 35 Haloxyfop (g ha-1) BBCH Phenological Scale Figure 4 - Evolution of GR80 of sourgrass plants, estimated by the control percentage (A) and residual shoot dry weight (B) at 28 days after application, affected by the phenological stage at the time of applications of the herbicide haloxyfop. Machado, MG, Brazil, 2017. y = -0.2526x2 + 31.817x - 442.14 R² = 0.9209 0 100 200 300 400 500 600 10 20 30 40 50 60 Haloxyfop (g ha-1) BBCH Phenological Scale y = 64.204x - 906.51 R² = 0.9987 0 200 400 600 800 1000 1200 1400 10 15 20 25 30 35 Haloxyfop (g ha-1) BBCH Phenological Scale (A) (B) (A) (B) Table 3 - Statistic parameters of logistic models, coefficient of determination, GR50 and GR80 for efficiency of the herbicide haloxyfop applied to sourgrass plants at different phenological stages. Machado, MG, Brazil, 2018 Table 3 - Statistic parameters of logistic models, coefficient of determination, GR50 and GR80 for efficiency of the herbicide haloxyfop applied to sourgrass plants at different phenological stages. 3 RESULTS AND DISCUSSION y = 0.6082x2 - 22.081x + 207.9 R² = 0.9991 0 100 200 300 400 500 600 700 10 20 30 40 50 Clethodim (g ha-1) BBCH Phenological Scale y = -0.1645x2 + 89.627x - 1326.6 R² = 0.9188 0 500 1000 1500 2000 2500 3000 10 20 30 40 50 Clethodim (g ha-1) BBCH Phenological Scale (A) (B) y = 0.0609x2 - 1.7982x + 25.068 R² = 0.9949 0 10 20 30 40 50 60 70 80 90 10 20 30 40 50 Clethodim (g ha-1) BBCH Phenological Scale (A) (A) (B) Figure 1 - Evolution of GR50 of sourgrass plants, estimated by the control percentage (A) and residual shoot dry weight (B) at 28 days after application, affected by the phenological stage at the time of applications of the herbicide clethodim. Machado, MG, Brazil, 2017. Figure 2 - Evolution of GR80 of sourgrass plants, estimated by the control percentage (A) and residual shoot dry weight (B) at 28 days after application, affected by the phenological stage at the time of applications of the herbicide clethodim. Machado, MG, Brazil, 2017. y = 0.6082x2 - 22.081x + 207.9 R² = 0.9991 0 100 200 300 400 500 600 700 10 20 30 40 50 Clethodim (g ha-1) BBCH Phenological Scale y = -0.1645x2 + 89.627x - 1326.6 R² = 0.9188 0 500 1000 1500 2000 2500 3000 10 20 30 40 50 Clethodim (g ha-1) BBCH Phenological Scale (A) (B) y = 0.6082x2 - 22.081x + 207.9 R² = 0.9991 0 100 200 300 400 500 600 700 10 20 30 40 50 Clethodim (g ha-1) BBCH Phenological Scale (A) y = -0.1645x2 + 89.627x - 1326.6 R² = 0.9188 0 500 1000 1500 2000 2500 3000 10 20 30 40 50 Clethodim (g ha-1) BBCH Phenological Scale (B) (B) (A) Figure 2 - Evolution of GR80 of sourgrass plants, estimated by the control percentage (A) and residual shoot dry weight (B) at 28 days after application, affected by the phenological stage at the time of applications of the herbicide clethodim. Machado, MG, Brazil, 2017. Planta Daninha 2020;38:e020223617 - https://doi.org/10.1590/S0100-83582020380100089 4/7 SBCPD \| Planta Daninha SBCPD \| Planta Daninha Presoto JC, et al. Sourgrass phenology and herbicide efficacy Table 3 - Statistic parameters of logistic models, coefficient of determination, GR50 and GR80 for efficiency of the herbicide haloxyfop applied to sourgrass plants at different phenological stages. 3 RESULTS AND DISCUSSION Machado, MG, Brazil, 2018 Table 3 - Statistic parameters of logistic models, coefficient of determination, GR50 and GR80 for efficiency of the herbicide haloxyfop applied to sourgrass plants at different phenological stages. Machado, MG, Brazil, 2018 Variable Stage Parameters R² (3)GR50 (3)GR80 Pmin a b c Control(1) 28 DAA(2) 14 X 101.92 3.98 -1.68 0.985 3.89 8.610 21 X 101.77 3.79 -1.56 0.986 3.70 8.743 32 X 120.29 85.15 -0.46 0.974 40.80 374.614 60 X 126.48 179.87 -0.49 0.963 74.88 551.013 Shoot dry weight(1) percentage 14 3.139 96.34 3.08 1.87 0.996 2.95 7.052 21 -8.318 108.84 13.88 0.31 0.911 23.59 417.696 32 -17.485 113.14 191.20 0.39 0.935 347.78 1157.373 60 - - - - - > 240.00 - (1) Model: y = a+(b/(1+(x/c)d). (2) DAA = days after application. (3) GR = growth reduction. Figure 3 - Evolution of GR50 of sourgrass plants, estimated by the control percentage (A) and residual shoot dry weight (B) at 28 days after application, affected by the phenological stage at the time of applications of the herbicide haloxyfop. Machado, MG, Brazil, 2018. y = 1.6413x - 21.291 R² = 0.9453 0 10 20 30 40 50 60 70 80 90 10 20 30 40 50 60 Haloxyfop (g ha-1) BBCH Phenological Scale y = 20.076x - 323.58 R² = 0.8871 0 50 100 150 200 250 300 350 400 10 15 20 25 30 35 Haloxyfop (g ha-1) BBCH Phenological Scale (A) (B) haloxyfop applied to sourgrass plants at different phenological stages. Machado, MG, Brazil, 2018 Variable Stage Parameters R² (3)GR50 (3)GR80 Pmin a b c Control(1) 28 DAA(2) 14 X 101.92 3.98 -1.68 0.985 3.89 8.610 21 X 101.77 3.79 -1.56 0.986 3.70 8.743 32 X 120.29 85.15 -0.46 0.974 40.80 374.614 60 X 126.48 179.87 -0.49 0.963 74.88 551.013 Shoot dry weight(1) percentage 14 3.139 96.34 3.08 1.87 0.996 2.95 7.052 21 -8.318 108.84 13.88 0.31 0.911 23.59 417.696 32 -17.485 113.14 191.20 0.39 0.935 347.78 1157.373 60 - - - - - > 240.00 - (1) Model: y = a+(b/(1+(x/c)d). (2) DAA = days after application. (3) GR = growth reduction. Figure 3 - Evolution of GR50 of sourgrass plants, estimated by the control percentage (A) and residual shoot dry weight (B) at 28 days after application, affected by the phenological stage at the time of applications of the herbicide haloxyfop. Machado, MG, Brazil, 2018. 3 RESULTS AND DISCUSSION Carvalho et al. (2019) found excellent control of sourgrass (higher than 90%) when applying 62.4 g ha-1 of haloxyfop to plants at tillering stage that grew from seeds. However, the control of adult plants with perennial clumps and presence of rhizomes is difficult. Thus, the best time for the control of D. insularis plants is up to 45 days after emergence, when the rhizomes are not fully formed (Gemelli et al., 2012; Andrade et al., 2019) and, in general, the plants are not yet at the flowering stage. Anatomical characteristics of sourgrass leaves can affect the absorption and translocation of herbicides. These characteristics, mainly presence of trichomes and layers of wax on leaves, are found in later developmental stages of the plants (Carvalho et al., 2012; Barroso et al., 2015). A negative correlation between presence of trichomes and absorption of herbicides is found in most studies (Hess and Falk, 1990). The cuticle, consisted of waxes, is an important barrier to the entry of microorganisms and agrochemicals; however, the herbicide absorption is Planta Daninha 2020;38:e020223617 - https://doi.org/10.1590/S0100-83582020380100089 5/7 SBCPD \| Planta Daninha SBCPD \| Planta Daninha Presoto JC, et al. Sourgrass phenology and herbicide efficacy not only related to the cuticle thickness, but to the cuticle lipidic constitution and level of prevention of entry of solutes (Carvalho et al., 2012; Barroso et al., 2015). Machado et al. (2008) reported that the difficulty to control sourgrass plants from rhizomes can be related to the epidermal thickness of the leaf adaxial and abaxial surfaces and to the leaf blade thickness, which are greater when compared to plants from seeds. In addition, they found large quantity of starch in rhizomes, which may hinder the translocation of herbicides, including glyphosate, and generate a fast shoot regrowth of weed plants. 4 CONCLUSIONS Carvalho SJP, Gonçalves Netto A, Nicollai M, Cavenaghi AL, López-Ovejero RF, Christoffoleti PJ. Detection of glyphosate-resistant Palmer amaranth (Amaranthus palmeri) in agricultural areas of Mato Grosso, Brazil. Planta Daninha. 2015;33:579-86. Carvalho SJP, Gonçalves Netto A, Nicollai M, Cavenaghi AL, López-Ovejero RF, Christoffoleti PJ. Detection of glyphosate-resistant Palmer amaranth (Amaranthus palmeri) in agricultural areas of Mato Grosso, Brazil. Planta Daninha. 2015;33:579-86. The herbicides clethodim and haloxyfop were efficiency in the controling of sourgrass plants at initial developmental stages. Satisfactory control of sourgrass was possible up to the two-tiller stage (stage 22 in the BBCH scale), when using the recommended rate of both herbicides tested; however, the efficacy of the herbicides decreases as the plant grows after the tillering stage, when the control becomes increasingly ineffective. More developed plants require the use of other control technics, such as sequential applications or combination of herbicides, to increase the efficacy of ACCase-inhibitor herbicides. Carvalho SJP, Andrade JF, Presoto JC. Efficacy and interaction of haloxyfop-clethodim tank mixtures to post emergence control of sourgrass in Brazil. Int J Agric Innov Res. 2019;8:115-21. Costa NV, Moratelli G, Ferreira SD, Salvalaggio AC, Rodrigues-Costa ACP. Resistance to glyphosate in populations of Digitaria insularis. Planta Daninha. 2018;36:e018175918. Ferreira SD, Exteckoetter V, Gibbert AM, Barbosa JA, Costa NV. Biological cycle of susceptible and glyphosate- resistant sourgrass biotypes in two growth periods. Planta Daninha. 2018;36:e018175923. Ferreira SD, Exteckoetter V, Gibbert AM, Barbosa JA, Costa NV. Biological cycle of susceptible and glyphosate- resistant sourgrass biotypes in two growth periods. Planta Daninha. 2018;36:e018175923. 7 REFERENCES Adegas FS, Gazziero DLP. Resistência de Digitaria insularis aos herbicidas inibidores da EPSPs. In: Agostinetto D, Vargas L, editores. Resistência de plantas daninhas a herbicidas no Brasil. Pelotas: UFPEL; 2014. p.304-13. Andrade JF, Presoto JC, Carvalho SJP. Interferência do estádio fenológico do capim-amargoso sobre a eficácia do herbicida glyphosate. Rev Bras Herb. 2019;18:1-7. Barroso AAM, Albrecht AJP, Reis FC, Victoria Filho R. Interação entre herbicidas inibidores de ACCase e diferentes formulações de glyphosate no controle de capim-amargoso. Planta Daninha. 2014;32:619-27. Barroso AAM, Galeano E, Albrecht AJP, Reis FC, Victoria Filho R. Does sourgrass leaf anatomy influence glyphosate resistance? Com Sci. 2015;6:445-53. Therefore, the use of measures for controlling sourgrass plants at initial developmental stages are needed in agricultural areas. Sourgrass plants developed from seeds can be controlled by using only one application of a grass herbicide, and they are more easily controlled than plants from clumps. After the tillering and flowering stages, the control of sourgrass is difficult, often demanding herbicide mixtures or sequential applications. This denotes the importance of the control of plants at initial growth stages, mainly before the tillering and flowering stages. Canto-Dorow TS, Longhi-Wagner HM. Novidades taxonômicas em Digitaria Haller (Poaceae) e novas citações para o gênero no Brasil. INSULA. 2001;30:21-34. Carvalho LB, Cruz-Hipolito H, Gonzalez-Torralva F, Alves PL, Costa A, Christoffoleti PJ, et al. Detection of sourgrass (Digitaria insularis) biotypes resistant to glyphosate in Brazil. Weed Sci. 2011;59:171-6. Carvalho LB, Cruz-Hipolito H, Gonzalez-Torralva F, Alves PL, Costa A, Christoffoleti PJ, et al. Pool of resistance mechanisms to glyphosate in Digitaria insularis. J Agric Food Chem. 2012;60:615-22. Carvalho LB, Cruz-Hipolito H, Gonzalez-Torralva F, Alves PL, Costa A, Christoffoleti PJ, et al. Pool of resistance mechanisms to glyphosate in Digitaria insularis. J Agric Food Chem. 2012;60:615-22. 5 CONTRIBUTIONS Gazola T, Belapart D, Castro EB, Cipola Filho ML, Dias MF. Características biológicas de Digitaria insularis que conferem sua resistência a herbicidas e opções de manejo. Científica. 2016;44:557-67. Gazola T, Belapart D, Castro EB, Cipola Filho ML, Dias MF. Características biológicas de Digitaria insularis que conferem sua resistência a herbicidas e opções de manejo. Científica. 2016;44:557-67. JCP: experiment conduction, data tabulation, and manuscript writing; JFA: experiment conduction and manuscript writing; Other authors: experiments conduction; SJPC: guidance, statistical analysis, paper writing and reviewing. Gemelli A, Oliveira Jr RS, Constantin J, Braz GBP, Jumes TMC, Oliveira Neto AM, et al. Aspectos da biologia de Digitaria insularis resistente ao glyphosate e implicações para o seu controle. Rev Bras Herb. 2012;11:231-40. Gemelli A, Oliveira Jr RS, Constantin J, Braz GBP, Jumes TMC, Oliveira Neto AM, et al. Aspectos da biologia de Digitaria insularis resistente ao glyphosate e implicações para o seu controle. Rev Bras Herb. 2012;11:231-40. Zobiole LHS, Krenchinski FH, Albrecht AJP, Pereira G, Lucio FR, Rossi C, et al. Controle de capim-amargoso perenizado em pleno florescimento. Rev Bras Herb. 2016;15:157-64. 6 ACKNOWLEDGEMENTS Gonçalves Netto A, Goveia YD, Carvalho SJP. Monitoring the occurrence of glyphosate-resistant sourgrass biotypes in the south region of Minas Gerais, Brazil. Rev Bras Herb. 2015;14:38-46. The authors thank the Brazilian National Council for Scientific and Technological Development (CNPq) for the financial support and granting of scholarship; and the Federal Institute of Education, Science and Technology of South of Minas Gerais (IFSULDEMINAS) for the financial support granted. Heap IM. International survey of herbicide-resistant weeds. [acesso em: 10 abr. 2019]. Disponível em: http://www.weedscience.org. Planta Daninha 2020;38:e020223617 - https://doi.org/10.1590/S0100-83582020380100089 6/7 SBCPD \| Planta Daninha SBCPD \| Planta Daninha Presoto JC, et al. Sourgrass phenology and herbicide efficacy Hess M, Barralis G, Bleiholder H, Buhr L, Eggers TH, Hack H, et al. Use of the extended BBCH scale - general for descriptions of the growth stages of mono-and dicotyledonous weed species. Weed Res. 1997;37:433- 41. Hess FD, Falk RH. Herbicide deposition on leaf surfaces. Weed Sci. 1990;38:280-8. Hess FD, Falk RH. Herbicide deposition on leaf surfaces. Weed Sci. 1990;38:280-8. Hess FD, Falk RH. Herbicide deposition on leaf surfaces. Weed Sci. 1990;38:280-8. Lacerda ALS, Victoria Filho R. Curvas dose-resposta em espécies de plantas daninhas com o uso do herbicida glyphosate. Bragantia. 2004;63:73-9. Lacerda ALS, Victoria Filho R. Curvas dose-resposta em espécies de plantas daninhas com o uso do herbicida glyphosate. Bragantia. 2004;63:73-9. Licorini LR, Gandolfo MA, Sorace MA, Cossa CA, Osipe JB. Identificação e controle de biótipos resistentes de Digitaria insularis (L.) Fedde ao glyphosate. Rev Bras Herb. 2015;14:141-7. López-Ovejero RF, Takano HK, Nicolai M, Ferreira A, Melo MS, Cavenaghi AL, et al. Frequency and dispersal of glyphosate-resistant sourgrass (Digitaria insularis) populations across Brazilian agricultural production areas. Weed Sci. 2017;65:285-94. Machado AFL, Meira RMS, Ferreira LR, Ferreira FA, Tuffi Santos LD, Fialho CMT, et al. Caracterização anatômica de folha, colmo e rizoma de Digitaria insularis. Planta Daninha. 2008;26:1-8. Melo MSC, Rosa LE, Brunharo CADCG, Nicolai M, Christoffoleti PJ. Alternativas para controle químico de capim-amargoso (Digitaria insularis) resistente ao glyphosate. Rev Bras Herb. 2012;11:195-203. Rodrigues BN, Almeida FS. Guia de herbicidas. 7. ed. Londrina: 2018. 764p. Seefeldt SS, Jensen SE, Fuerst EP. Log-logistic analysis of herbicide dose response relationship. Weed Technol. 1995;9:218-27. Zobiole LHS, Krenchinski FH, Albrecht AJP, Pereira G, Lucio FR, Rossi C, et al. Controle de capim-amargoso perenizado em pleno florescimento. Rev Bras Herb. 2016;15:157-64. Planta Daninha 2020;38:e020223617 - https://doi.org/10.1590/S0100-83582020380100089 Planta Daninha 2020;38:e020223617 - https://doi.org/10.1590/S0100-83582020380100089 7/7
https://openalex.org/W2101731663	https://europepmc.org/articles/pmc4474546?pdf=render	English	null	RDML-Ninja and RDMLdb for standardized exchange of qPCR data	BMC bioinformatics	2,015	cc-by	4,972	SOFTWARE SOFTWARE Open Access * Correspondence: andreas@untergasser.de 6Genomics Core Facility, European Molecular Biology Laboratory, Heidelberg 69117, Germany 8Center of Molecular Biology (ZMBH), Heidelberg University, Im Neuenheimer Feld 282, Heidelberg 69120, Germany Full list of author information is available at the end of the article Abstract Background: The universal qPCR data exchange file format RDML is today well accepted by the scientific community, part of the MIQE guidelines and implemented in many qPCR instruments. With the increased use of RDML new challenges emerge. The flexibility of the RDML format resulted in some implementations that did not meet the expectations of the consortium in the level of support or the use of elements. Results: In the current RDML version 1.2 the description of the elements was sharpened. The open source editor RDML-Ninja was released (http://sourceforge.net/projects/qpcr-ninja/). RDML-Ninja allows to visualize, edit and validate RDML files and thus clarifies the use of RDML elements. Furthermore RDML-Ninja serves as reference implementation for RDML and enables migration between RDML versions independent of the instrument software. The database RDMLdb will serve as an online repository for RDML files and facilitate the exchange of RDML data (http://www.rdmldb.org). Authors can upload their RDML files and reference them in publications by the unique identifier provided by RDMLdb. The MIQE guidelines propose a rich set of information required to document each qPCR run. RDML provides the vehicle to store and maintain this information and current development aims at further integration of MIQE requirements into the RDML format. Conclusions: The editor RDML-Ninja and the database RDMLdb enable scientists to evaluate and exchange qPCR data in the instrument-independent RDML format. We are confident that this infrastructure will build the foundation for standardized qPCR data exchange among scientists, research groups, and during publication. Keywords: qPCR, RDML, MIQE, RDML-Ninja, RDMLdb Keywords: qPCR, RDML, MIQE, RDML-Ninja, RDMLdb and exchange this collection of data was published in 2009 as the first version of Real-time PCR Data Markup Language (RDML) [1]. Since then, several journals have endorsed and advocated the use of RDML (e.g. Clinical Chemistry, Nucleic Acids Research, BioMed Central series of journals, etc.; see News section on RDML website). Of practical relevance, Bio-Rad (CFX96 and CFX384), Life Technologies (StepOne, ViiA7 and QuantStudio), Qiagen (Rotor-Gene Q) and Roche (LightCycler96) implemented RDML export capabilities into their instrument's software. Furthermore, third party software with RDML data file import and/or export functionality, such as LinRegPCR ([2]; http://LinRegPCR.nl), qbase + ([3]; http://www.qbase- plus.com) and an RDML R package, were developed to enable (part of the) data-analysis workflow, such as raw data quality control, amplification curve analysis, normalization and statistical analysis independent of the qPCR machine software (see [4] and [5] for an RDML-Ninja and RDMLdb for standardized exchange of qPCR data Jan M. Ruijter1, Steve Lefever2,3, Jasper Anckaert2,3, Jan Hellemans4, Michael W. Pfaffl5, Vladimir Benes6, Stephen A. Bustin7, Jo Vandesompele2,3,4, Andreas Untergasser6,8* and on behalf of the RDML consortium Ruijter et al. BMC Bioinformatics (2015) 16:197 DOI 10.1186/s12859-015-0637-6 Ruijter et al. BMC Bioinformatics (2015) 16:197 DOI 10.1186/s12859-015-0637-6 © 2015 Ruijter et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. ( p // g/ / y/ ), p , , p y , provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Background Real-time quantitative PCR (qPCR) is a powerful method for accurate measurement of nucleic acid concentra- tions. qPCR instruments collect a large set of data dur- ing each run, which provides the basis for quantification and PCR product validation. Further, the user has as- sembled data on biomaterials, targets and qPCR assays that are essential in determining data quality, down- stream statistical analysis and independent replication; target and sample names can be entered into the qPCR instrument software to be used in built-in data analysis routines. An instrument-independent format to store * Correspondence: andreas@untergasser.de 6Genomics Core Facility, European Molecular Biology Laboratory, Heidelberg 69117, Germany 8Center of Molecular Biology (ZMBH), Heidelberg University, Im Neuenheimer Feld 282, Heidelberg 69120, Germany Full list of author information is available at the end of the article Page 2 of 7 Ruijter et al. BMC Bioinformatics (2015) 16:197 overview of qPCR data analysis tools). As a design choice, many elements in the RDML data tree are op- tional and documentation fields are present in several places. This choice allows the flexible use of RDML even in non-qPCR applications. Primer3plus is an ex- ample of this intended use, where primer and amplicon sequences can be exported as RDML files containing only the “target” elements of the RDML format [6]. On the down side, this flexibility resulted in cases where the level of RDML support did not fully meet the ex- pectations and the original intentions of the RDML consortium. Some qPCR instrument software export only a small set of the collected data and ignore avail- able RDML elements, or the data are stored in differ- ent elements or in different formats than intended. However, due to the flexibility of RDML, the software would still export valid files in these cases. The RDML consortium aims to overcome these issues by clarifica- tion of the RDML elements that led to misinterpret- ation and by supporting the software developers with tools to create, analyze and validate RDML files. element contains run elements, each containing the set of reactions in the run with the fluorescence data, base- line values, quantification threshold and observed Cq value as sub-elements. A reaction element refers to a sam- ple element and a target element by their unique IDs. The release of a new RDML version is coordinated by the RDML consortium (www.rdml.org). Software organisation of RDML-Ninja Software organisation of RDML-Ninja RDML-Ninja is an editor that allows the researcher to view and to modify the contents of RDML data files. The RDML data is stored as a XML tree structure from where entries are read on demand and placed in the re- spective elements of the graphic interface ready for user interaction. For elements such as “sample” or “target”, RDML files allow one up to an unlimited number of en- tries. In such a case, the user first chooses one element from a list with all elements to access the sub-elements of this selected element (analogous to opening the branch of a folder tree in a computer operating system). The position of the chosen element in the XML tree is saved in memory upon selection. If the user decides to modify an entry via the graphic interface, the data can thus be written back to the corresponding position of the XML tree structure. Prior to modification of the XML tree, the validity of the user input is checked and the operation will proceed only when the input is valid. The majority of data collected by the instruments are stored within the “react” elements and their sub- elements. RDML-Ninja enables to view, but not modify, this part of the XML tree in a table view reconstructing the plate format. Furthermore, the collected fluorescence values can be plotted as amplification curves or melt curves and exported into the Scalable Vector Graphics (SVG) for- mat for further use in presentations or publications. One of the intentions for creating RDML was to facili- tate the exchange of raw fluorescence data free of smoothing or baseline subtraction. Raw data are the holy grail of qPCR analysis as they allow quality control, evaluation of the validity of conclusions and, if new methods or statistical analysis tools become available, re- evaluation of previously published results. Furthermore raw data open the door for meta-analysis of published qPCR experiments without any bias from the original analysis. Today the RDML format offers instrument in- dependency and free and straightforward data exchange, but publications with RDML files as supplemental data are still the exception. Furthermore, large experimental sets result in RDML files of significant size, and journals may prefer to not store these files on their website. A cen- tral repository dedicated to RDML files offers a better so- lution for easy exchange. Software organisation of RDML-Ninja Authors upload their RDML files into this database and provide the matching IDs in the art- icle, as is customarily done for microarray and RNA- sequencing data (through e.g. Gene Expression Omnibus, Sequence Read Archive, or European Nucleotide Archive). In this paper we describe the evolution of RDML up h d f l d RD N In this paper we describe the evolution of RDML up to version 1.2, present the data file editor RDML-Ninja and the database RDMLdb, a database dedicated to the storage and exchange of RDML files. The platform independence of RDML-Ninja as well as its native look was achieved by the use of the Qt cross-platform application framework (Qt; http://www.qt-project.org). Pre- compiled executable programs are available for Microsoft Windows and Apple OS X platform (http://sourceforge.net/ projects/qpcr-ninja/). Because of the design of the Qt framework, the support can be easily extended to other platforms if required. The application code was written in C++ making heavy use of Qt functionality, not only Background Any researcher, programmer or data-analyst in academia as well as in- dustry can join the consortium free of charge and can participate in the development of RDML. Suggestions are evaluated, discussed in the consortium and imple- mented by the RDML core group. Based on the commu- nity feedback, the new version is created and released. The consortium aims for an abstracted design that can be used with the majority of instruments and software packages available and thereby balances the interests of the different instrument providers and qPCR users. Overview of RDMLdb RDMLdb is an online database for exchanging RDML files (http://www.rdmldb.org). Users access the database via an interactive web interface. The database stores the original RDML files as well as key information extracted from these files required to index the database. Users can query the database based on the generated index to find a specific RDML file. The majority of the web interface scripts of RDMLdb are written in PHP 5.3.10 and use the JavaScript library jQuery 1.10.2 to enhance usability by providing auto- completion of search fields and form validation. At time of upload, the user only provides an RDML file. This file is parsed using a Perl 5.14.2 script to extract the meta information required to create a record such as version, target, sample and experiment description. The record is complemented with a unique ID, some additional fields such as the PubMed ID, the email address of the upload- ing user as well as the date of the upload. The meta in- formation record and the original file are stored in the database. The database functionality was provided by using MongoDB 2.6.7 (http://www.mongodb.org) be- cause its NoSQL structure allows varying number of fields in each record and it enables the storage of entire files within the database using a gridFS system. Users may query the database using the unique ID or search the above listed fields and then download the associated RDML files. RDML version development RDML files are compressed text files containing an XML-based hierarchical tree with elements for experi- menters, documentations, dyes, samples, targets, cycling conditions and experiments at the top level. Each elem- ent contains various sub-elements. The experiment Page 3 of 7 Ruijter et al. BMC Bioinformatics (2015) 16:197 element is no longer optional and all dyes must be regis- tered at top level. to display the graphical interface but also to store XML data or draw SVG graphics. The software is freely avail- able for commercial and academic use under GNU General Public licence (GPL). RDML version 1.2 addresses the need to classify sam- ples into groups, in order to facilitate downstream statis- tical analysis. Therefore, an annotation element was introduced containing a property and a value string as sub elements. Each defined sample may have several of these annotation elements. We envision the use, for ex- ample, in a mouse experiment, where a first annotation element could have the property “gender” with the values “male” or “female” and a second element could have the property “treatment” with the values “con- trol”,“condition1” or “condition2”. The free string format for properties and values allows flexible tagging of all samples and thus sub-groups of reactions. The annota- tion element also replaces the elements used to describe DNA or RNA quality. Furthermore the DNA and RNA quantity elements, which were already modified in RDML version 1.1, were united as one quantity element in the current version. Some elements in RDML version 1.2 were added to provide further MIQE compatibility [7]. An example is the element “amplificationEfficien- cySE” containing the uncertainty measure for the esti- mated PCR efficiency. For each target, this value results from the least-squares fit of the Cq versus log (input) observations of the standard curve or, alternatively, is calculated as the SEM of the observed efficiency values resulting from analysis of individual amplification curves [2, 8]. Finally, the documentation of several elements has been updated to clarify and avoid misinterpretation. This section highlights only the major changes; please check the online documentation as well as the supplemental data for a complete list of changes and the correspond- ing RDML standard definition (http://www.rdml.org). Despite the described changes, the majority of the ele- ments are unmodified since RDML version 1.0 and therefore upgrading to version 1.2 should be no big bur- den for software developers. Results RDML version development 1.1 and 1.2 RDML version development 1.1 and 1.2 Since the release of RDML version 1.0 in 2009 [1], the RDML standard was updated twice with the latest ver- sion 1.2 being released in Autumn 2014. The major change in version 1.1 was a complete redesign of the plate setup. In the initial RDML version 1.0, all possible plate setups were predefined and therefore each new in- strument making use of a new plate type required an up- date of the RDML standard. To overcome this instrument dependency, from version 1.1 onwards, the plate setup has been described by providing the number of available reac- tions in two dimensions. The identification of a single re- action was changed from the letter (row) + number (column) format to a number only format based on the re- action position in the two-dimensional matrix. Addition- ally, the handling of the dye element was redesigned. Being originally an optional element, the plate setup of multiplex reactions could not be reconstructed without dye information. To avoid ambiguous situations, the dye RDML-Ninja Th RDML The RDML format is flexible due to many optional ele- ments. On the one hand, this reduces the costs of imple- mentation because it allows focusing on the parts of RDML required for the qPCR instrument functionality. On the other hand, no software is available today that can display all possible entries in an RDML file. Further- more, the flexibility allowed some implementations to diverge from the intended use of RDML elements thus creating valid, but difficult to use, RDML files. RDML- Ninja was developed as reference implementation to fill this gap and serves to view and modify RDML files (http://sourceforge.net/projects/qpcr-ninja/). It provides access to all elements of an RDML file in an intuitive way (Fig. 1a-c). The majority of data can also be Page 4 of 7 Ruijter et al. BMC Bioinformatics (2015) 16:197 Fig. 1 Open source editor RDML-Ninja. a Target information is displayed after selection of an ID in the left section. b The annotation and results of a single plate are visualized in an interactive table view. c Amplification results and melt point measurements can be plotted in a graph and exported in SVG format. A high resolution image is available as supplemental data Fig. 1 Open source editor RDML-Ninja. a Target information is displayed after selection of an ID in the left section. b The annotation and results of a single plate are visualized in an interactive table view. c Amplification results and melt point measurements can be plotted in a graph and exported in SVG format. A high resolution image is available as supplemental data Page 5 of 7 Page 5 of 7 Ruijter et al. BMC Bioinformatics (2015) 16:197 supplemented and/or modified, which enables the user to complement and correct the collected information. All such modifications have to be confirmed by the user by clicking a “set Changes” button (Fig. 1a). The content of some elements in an RDML file is restricted to pre- defined input. These restrictions are checked once the “set Changes” button is clicked and before the file is modified. However, the RDML consortium cannot fore- see any reason to modify the raw fluorescence data col- lected by the instrument. Therefore, the entries of the “react” element cannot be modified by RDML-Ninja and are only displayed in a table view or in curve view (Fig. 1b, c). RDMLdb RDMLdb was designed for the exchange of RDML files (http://www.rdmldb.org). Users may upload their data and obtain a unique identifier (ID) referring to the uploaded file. This ID can be provided in publications and allows readers to extract the corresponding RDML file. Furthermore, users may add a PubMed ID to their files in order to link publications to the RDML files. The release of the file to the public can be delayed for up to one year to grant confidentiality during the reviewing and publication process. In addition to the ID, RDMLdb can be searched for PubMed ID, submitter name or RDML version (Fig. 2). RDML files can be downloaded from the website, either after searching for public files or using a direct link for private files in the review process. A statistics page keeps users informed on the number of records, linked publications, samples, targets and reactions stored in the database. RDML-Ninja Th RDML The table view can be exported as comma separated text files (CSV) that can be imported into other software packages such as Microsoft Office or other programs for analysis of amplification curves [8]. The curve view allows the export of graphics in the Scal- able Vector Graphics format (SVG) for easy inclusion in presentations and papers. The SVG format can be modi- fied by any vector graphics software without any loss of quality (e.g. Inkscape, http://www.inkscape.org). Discussion Over the past five years, the universal qPCR data ex- change file format RDML has been well accepted by the scientific user community and is implemented in many qPCR instruments available today. Further, being part of the MIQE guidelines, it is endorsed by scientific journals and publishers. Although this is a big achievement, the use of RDML should not stop at this point. Currently, we see the bottleneck at the level of handling RDML files and RDML file exchange. The editor, RDML-Ninja, has been designed with different user types in mind. In the laboratory, RDML-Ninja should allow researchers to enter information into RDML elements not supported by the software of their qPCR instrument. Furthermore, RDML-Ninja can form a bridge between software sup- porting different RDML versions by handling the In addition to the editor functionality, RDML-Ninja was enriched with RDML-specific functionality. An imported RDML file can be validated with the validator tool using the corresponding schema. The validator tool will state “validation successful” or provide information on the type of error that it has encountered. Currently, three versions of RDML exist and all were implemented into available qPCR systems and software. RDML-Ninja allows migrating files between all three versions at the cost of minor information loss if data were stored in ele- ments not supported by the target version. This feature allows users to migrate their RDML data to the most re- cent version for further analysis when their instrument software supports only an older version. Fig. 2 Search interface of RDMLdb. RDML files are found in the database based on query key elements Fig. 2 Search interface of RDMLdb. RDML files are found in the database based on query key elements Page 6 of 7 Ruijter et al. BMC Bioinformatics (2015) 16:197 overwhelming to researchers, RDML offers an easy help to handle this information. All the information is en- tered only once and stored in a basic RDML file. Re- searchers would not have to re-enter this information with every qPCR run, but can instead import from this RDML file only the parts they need for the current qPCR run. Furthermore, integration of MIQE would allow checking to what extent MIQE information is pro- vided by calculating the checklist completeness. Conclusions Our applications lower the barriers using RDML for data exchange. The open source editor RDML-Ninja allows visualisation of all RDML elements and migration be- tween RDML versions. The database RDMLdb will serve as public online repository for RDML files. RDMLdb will ease data exchange between research groups and facili- tate the use of RDML files in publications. Making qPCR data exchange more accessible will significantly enhance biology research, publication quality and qPCR data val- idation. With a tighter integration of MIQE a data for- mat is in reach allowing collecting and exchanging all data required by MIQE at one. RN seque c g. RDML has the potential to connect all qPCR-data- associated processes in a lab. We envision users start by designing their qPCR assays using primer3plus and obtaining an RDML file containing the primer se- quences. Then they extend the target information using RDML-Ninja with references to the gene ID and add- itional information. Many labs use a limited set of tar- gets and qPCR cycling programs that could be collected in one comprehensive RDML file and shared among re- searchers. Once researchers start a qPCR run, they im- port this information from the lab file, enter the sample names and annotations, choose targets and edit the plate layout, adding targets and samples to each reaction in the run. After the real-time PCR instrument completes the qPCR run, it combines the run informations and the reactions and saves it into RDML files. Finally, re- searchers can analyze the raw data using the instrument software or the third party software of their choice and perform final statistical analysis. An RDML based pipe- line is currently provided for RDML compatible qPCR instruments. The run, target and tissue information and the raw fluorescence data can be read into LinRegPCR [2]. This program performs qPCR quantification based on the analysis of the amplification curves and saves PCR efficiency values per target and Cq values per reac- tion back into the RDML file. When this RDML file is read into qbase + [3] the gene expression data can be normalized and further statistical analysis of the result- ing relative expression levels can be performed. Discussion This would serve researchers, enabling them to avoid the re- petitive burden of manual documentation, as well as re- viewers and readers, who require complete information to judge and replicate the published results. conversion. In the field of publishing, reviewers as well as readers can use RDML-Ninja to visualize and evaluate qPCR data in RDML files independent of instrument software. In the bioinformatics field, RDML-Ninja should assist software developers with the implementa- tion of the RDML standard. Programmers can use RDML-Ninja to create files to challenge their software or to evaluate the validity of the files created by their software. The online database RDMLdb will facilitate the storage and public exchange of RDML data. RDMLdb serves as a repository for RDML files where individual files are re- ferred to by a unique ID. RDMLdb can thus function for qPCR data like the repositories for other gene expression measurement technologies, such as microarrays and RNA-sequencing. Acknowledgement g We are grateful to Filip Pattyn for his help with the beta version of RDMLdb. We are grateful to Filip Pattyn for his help with the beta version of RDMLdb Author's contributions JMR, SL, JH, JV and AU drafted the new RDML versions. AU designed and programmed RDML-Ninja and JMR helped with beta testing. SL, JA, JH and JV designed, programmed and maintain RDMLdb. MWP, VB, SAB, JV and AU participated in qPCR standardisation efforts and in the definition of standardisation requirements in the perspective of RDML and MIQE. AU conceived of the study, and participated in its design and coordination. JMR, JV and AU helped to draft the manuscript. All authors read and approved the final manuscript. Any restrictions to use by non-academics: no Any restrictions to use by non-academics: no Conclusions When, at the point of publication, the RDML file has been uploaded to RDMLdb and its ID is referred to in the publication the reviewers and readers can download the RDML file from the database, visualize the raw data as well as the derived efficiency and Cq values and thus re- view the complete data analysis process. Availability and requirements Project name: RDML, RDML-Ninja, RDMLdb Project home page: http://www.rdml.org, http://source- forge.net/projects/qpcr-ninja/, http://www.rdmldb.org Operating system(s): Platform independent Programming language: Qt, C++, Perl, JavaScript, XML Other requirements: Microsoft Windows 7 or higher, Macintosh OS X Other requirements: Microsoft Windows 7 or higher, Macintosh OS X License: GNU GPL Ruijter et al. BMC Bioinformatics (2015) 16:197 Ruijter et al. BMC Bioinformatics (2015) 16:197 Ghent University, Ghent B-9000, Belgium. 4Biogazelle, Zwijnaarde 9052, Belgium. 5Physiology, Center of Life and Food Sciences, Technical University of Munich, Freising Weihenstephan, Munich 85354, Germany. 6Genomics Core Facility, European Molecular Biology Laboratory, Heidelberg 69117, Germany. 7Postgraduate Medical Institute, Anglia Ruskin University, Chelmsford CM1 1SQ, UK. 8Center of Molecular Biology (ZMBH), Heidelberg University, Im Neuenheimer Feld 282, Heidelberg 69120, Germany. Received: 1 April 2015 Accepted: 6 June 2015 Received: 1 April 2015 Accepted: 6 June 2015 8. Ruijter JM, Pfaffl MW, Zhao S, Spiess AN, Boggy G, Blom J, et al. Evaluation of qPCR curve analysis methods for reliable biomarker discovery: bias, resolution, precision, and implications. Methods. 2013;59:32–46. doi:10.1016/ j.ymeth.2012.08.011. Author details 1 Ultimately, RDML should be extended to store all in- formation required according to the MIQE guidelines. While the information required by MIQE may seem 1Department of Anatomy, Embryology & Physiology, Academic Medical Center, Amsterdam 1100AZ, Netherlands. 2Center for Medical Genetics, Ghent University, Gent B-9000, Belgium. 3Bioinformatics Institute Ghent, Page 7 of 7 Page 7 of 7 Ruijter et al. BMC Bioinformatics (2015) 16:197 Ruijter et al. BMC Bioinformatics (2015) 16:197 7. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009;55:611–22. doi:10.1373/clinchem.2008.112797. doi: 10.1093/nar/gkp045 3. Hellemans J, Mortier G, De Paepe A, Speleman F, Vandesompele J. qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol. 2007;8:R19. 4. Rödiger S, Burdukiewicz M, Blagodatskikh KA, Schierack P. R as an Environment for the Reproducible Analysis of DNA Amplification Experiments. The R Journal. 2015;7:1–24. ahead of print. 5. Pabinger S, Rödiger S, Kriegner A, Vierlinger K, Weinhäusel A. A survey of tools for the analysis of quantitative PCR (qPCR) data. Biomolecular Detection and Quantification. 2014;1:23–33. doi:10.1016/j.bdq.2014.08.002. 6. Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, et al. Primer3–new capabilities and interfaces. Nucleic Acids Res. 2012;40, e115. doi:10.1093/nar/gks596. 7. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009;55:611–22. doi:10.1373/clinchem.2008.112797. 8. Ruijter JM, Pfaffl MW, Zhao S, Spiess AN, Boggy G, Blom J, et al. Evaluation of qPCR curve analysis methods for reliable biomarker discovery: bias, resolution, precision, and implications. Methods. 2013;59:32–46. doi:10.1016/ j.ymeth.2012.08.011. References 1. Lefever S, Hellemans J, Pattyn F, Przybylski DR, Taylor C, Geurts R, et al. RDML: structured language and reporting guidelines for real-time quantita- tive PCR data. Nucleic Acids Res. 2009;37:2065–9. doi:10.1093/nar/gkp056. 1. Lefever S, Hellemans J, Pattyn F, Przybylski DR, Taylor C, Geurts R, et al. RDML: structured language and reporting guidelines for real-time quantita- tive PCR data. Nucleic Acids Res. 2009;37:2065–9. doi:10.1093/nar/gkp056. g p 2. Ruijter JM, Ramakers C, Hoogaars WM, Karlen Y, Bakker O, van den Hoff MJ and Moorman AF. Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data. Nucleic Acids Res. 2009; A37:e45. doi: 10.1093/nar/gkp045 3. Hellemans J, Mortier G, De Paepe A, Speleman F, Vandesompele J. qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol. 2007;8:R19. 4. Rödiger S, Burdukiewicz M, Blagodatskikh KA, Schierack P. R as an Environment for the Reproducible Analysis of DNA Amplification Experiments. The R Journal. 2015;7:1–24. ahead of print. 5. Pabinger S, Rödiger S, Kriegner A, Vierlinger K, Weinhäusel A. A survey of tools for the analysis of quantitative PCR (qPCR) data. Biomolecular Detection and Quantification. 2014;1:23–33. doi:10.1016/j.bdq.2014.08.002. 6. Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, et al. Primer3–new capabilities and interfaces. Nucleic Acids Res. 2012;40, e115. doi:10.1093/nar/gks596. 7. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009;55:611–22. doi:10.1373/clinchem.2008.112797. 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https://openalex.org/W4256456372	http://wrap.warwick.ac.uk/137865/1/WRAP-decomposing-educational-inequalities-factors-associated-severe-acute-malnutrition-Fagbamigbe-2020.pdf	English	null	Decomposing the educational inequalities in the factors associated with severe acute malnutrition among under-five children in Low- and Middle-Income Countries	Research Square (Research Square)	2,020	cc-by	9,217	Fagbamigbe et al. BMC Public Health (2020) 20:555 https://doi.org/10.1186/s12889-020-08635-3 Fagbamigbe et al. BMC Public Health (2020) 20:555 https://doi.org/10.1186/s12889-020-08635-3 Open Access Decomposing the educational inequalities in the factors associated with severe acute malnutrition among under-five children in low- and middle-income countries A. F. Fagbamigbe1,2* , N. B. Kandala3 and O. A. Uthman2 A. F. Fagbamigbe1,2* , N. B. Kandala3 and O. A. Uthman2 © 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. Abstract Background: Low- and Middle-Income Countries (LMIC) have remained plagued with the burden of severe acute malnutrition (SAM). The decomposition of the educational inequalities in SAM across individual, household and neighbourhood characteristics in LMIC has not been explored. This study aims to decompose educational-related inequalities in the development of SAM among under-five children in LMIC and identify the risk factors that contribute to the inequalities. Methods: We pooled successive secondary data from the Demographic and Health Survey conducted between 2010 and 2018 in 51 LMIC. We analysed data of 532,680 under-five children nested within 55,823 neighbourhoods. Severe acute malnutrition was the outcome variable while the literacy status of mothers was the main exposure variable. The explanatory variables cut across the individual-, household- and neighbourhood-level factors of the mother-child pair. Oaxaca-Blinder decomposition method was used at p = 0.05. (Continued on next page) (Continued on next page) * Correspondence: franstel74@yahoo.com 1Department of Epidemiology and Medical Statistics, Faculty of Public Health, College of Medicine, University of Ibadan, Ibadan, Nigeria 2Division of Health Sciences, Populations, Evidence and Technologies Group, Warwick Medical School, University of Warwick, Coventry, UK Full list of author information is available at the end of the article * Correspondence: franstel74@yahoo.com 1Department of Epidemiology and Medical Statistics, Faculty of Public Health, College of Medicine, University of Ibadan, Ibadan, Nigeria 2Division of Health Sciences, Populations, Evidence and Technologies Group, Warwick Medical School, University of Warwick, Coventry, UK Full list of author information is available at the end of the article Background and mothers’ employment status and income [1, 6–12]. In a recent hierarchical analysis of factors associated with SAM in 51 LMIC, Fagbamigbe et al. identified ma- ternal educational attainment, household wealth status and rural-urban differentials in the location of residence as the main determinants of whether a child has SAM or not [13]. These findings motivated the current study. Malnutrition among under-five children (U5C) remains both a social and public health burden [1, 2] especially in the Low- and Middle-Income Countries (LMIC). The World Health Organisation (WHO) maintains that mal- nutrition is responsible, directly or indirectly, for 35% of deaths among U5C [3], among which is Severe Acute Malnutrition (SAM). SAM is the most extreme and visible form of undernutrition among U5C. Under-five children with SAM usually “have very low weight for their height and severe muscle wasting” [4]. The likeli- hood that a child with SAM will eventually die is very high [4, 5]. Besides, “children with severe acute malnu- trition are nine times more likely to die than well- nourished children” [4]. The UNICEF (United Nations International Children’s Emergency Fund) reported that SAM affected more than 16 million children globally in 2016 [4]. Although this figure is staggering, it is likely to have been underestimated [5]. There is a paucity of data on SAM in LMIC, especially on its decomposition by maternal educational differ- ences, which has limited the understanding of the magnitude of the challenges of SAM for fact-based inter- ventions. This is despite UNICEF’s recommendation that complex social and political efforts are crucial to ending SAM [4]. The role(s) of educational inequalities in the distribution of SAM in the LMIC and factors associated with the inequalities have not received sufficient atten- tion. A recent Ghanaian study found a high level of influence of educational inequalities on all factors associ- ated with malnutrition in the study [14]. Amongst others, the study showed that the nutritional status of children from educated mothers are generally better than among those from uneducated mothers and some factors influence these differentials. To reduce the burden of SAM, there is a need to im- plement multi-sectoral evidence-based interventions which will enhance child and maternal health [3] in the long run. However, the development of the appropriate strategies, programmes and policies on the reduction of SAM, is hinged on the availability of information that can enhance child health interventions. (Continued from previous page) (Continued from previous page) Results: The proportion of children whose mothers were not educated ranged from 0.1% in Armenia and Kyrgyz Republic to as much as 86.1% in Niger. The overall prevalence of SAM in the group of children whose mothers had no education was 5.8% compared with 4.2% among those whose mothers were educated, this varied within each country. Fourteen countries (Cameroon(p < 0.001), Chad(p < 0.001), Comoro(p = 0.047), Burkina Faso(p < 0.001), Ethiopia(p < 0.001), India(p < 0.001), Kenya(p < 0.001), Mozambique(p = 0.012), Namibia(p = 0.001), Nigeria(p < 0.001), Pakistan(p < 0.001), Senegal(p = 0.003), Togo(p = 0.013), and Timor Leste(p < 0.001) had statistically significant pro- illiterate inequality while no country showed statistically significant pro-literate inequality. We found significant differences in SAM prevalence across child’s age (p < 0.001), child’s sex(p < 0.001), maternal age(p = 0.001), household wealth quintile(p 0 001) mother’s access to media(p 0 001) birth weight(p < 0 001) and alth quintile(p = 0.001), mother’s access to media(p = 0.001), birth weight(p < 0.001) and neighbourhood socioeconomic status disadvantage(p < 0.001). On the average, neighbourhood socioeconomic status disadvantage, location of residence were the most important factors in most countries. Other contributors to the explanation of educational inequalities are birth weight, maternal age and toilet type. Conclusions: SAM is prevalent in most LMIC with wide educational inequalities explained by individual, household and community-level factors. Promotion of women education should be strengthened as better education among women will close the gaps and reduce the burden of SAM generally. We recommend further studies of other determinate causes of inequalities in severe acute malnutrition in LMIC. Keywords: Severe acute malnutrition, LMIC, Educational inequalities, Blinder-Oaxaca decomposition © 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 14 Fagbamigbe et al. BMC Public Health (2020) 20:555 Background While the litera- ture is replete with the factors predisposing children to SAM and other poor nutrition outcomes, decomposition of these factors on key variables significant to poor nu- trition is scarce in the literature. The identified factors are largely individual and household factors such as food insecurity, inadequate care and feeding, unhealthy envir- onment, poor access to education, child’s age and sex, Inequalities in maternal education remain key barriers to the occurrence of SAM among U5C [9, 11, 12, 15– 18]. However, the underlying causes of educational in- equalities in the development of SAM among U5C re- main poorly operationalized, studied and understood. There is, therefore, a need to understand what influences the wide gap in the development of SAM among children from educated and uneducated mothers. In an attempt to understand the factors that explain the educational-related inequalities in the development of Fagbamigbe et al. BMC Public Health (2020) 20:555 Fagbamigbe et al. BMC Public Health (2020) 20:555 Fagbamigbe et al. BMC Public Health (2020) 20:555 Page 3 of 14 SAM among U5C in the LMIC and propose necessary strategies for interventions, we assessed the level of edu- cational inequalities in LMIC and examined the factors associated with these inequalities in the development of SAM among U5C in LMIC. We were motivated to iden- tify the causes and the extent of the variabilities of the educational-related inequalities in the development of SAM among U5C in the LMIC beyond compositional characteristics. A good understanding of the gaps in the development of SAM among U5C in the LMIC would guide interventions for improving child nutrition. Main determinant variable Maternal education was used as a proxy for literacy in this study. Literacy is a key skill and an important meas- ure of a population’s level of education. Literacy is the ability to both read and write a short, simple statement about one’s own life [25]. We, therefore, categorized education as having no formal education (Illiterate) and educated (can read and write: have a minimum of com- pleted primary education - Literate). Neighbourhood-level factors In this study, the term “neighbourhood” was used to de- pict people living in the same cluster within the same geographical setting. The neighbourhoods were mapped out to include households of the same clusters otherwise referred to as sharing the same PSU across each of the countries studied [19, 20]. Operationally, we defined “neighbourhood” as clusters and “neighbours” as a mem- ber of the same cluster. The PSUs were identified using the most recent census in each country where DHS was carried out. Among the community-level variables gen- erated is the neighbourhood socioeconomic disadvan- tage. It was generated using principal component analysis of the proportion of respondents living in rural areas, with no education, unemployed, and belonging to the lowest two wealth quintiles. Independent variables Individual-level factors These include sex of the children (male or female), children age (under 1 year and 12–59 months), mater- nal age (15–24, 25--34, 35–49 years), occupation (cur- rently working or not), access to media (at least one of radio, television, or newspaper), sources of drinking water (improved or unimproved), toilet type (im- proved or unimproved), weight at birth (average+, small, and very small), ability to pay for health care, health insurance coverage, birth interval (firstborn, < 36 months, and > 36 months) and birth order (1, 2, 3, and 4+). We used the DHS-generated wealth index as an alternative indicator for the households’ Socio-Economic Status (SES). The methods used in the computation of the DHS wealth index have been published previously [26]. Dependent variable The dependent variable in this study is severe acute mal- nutrition defined as “a very low weight for height score (WHZ) below -3 z-scores of the median WHO growth standards, by visible severe wasting, or by the presence of nutritional oedema” [3]. The z-scores are composite scores computed using the weight and height of the chil- dren. We generated z-scores using the WHO-approved methodologies [24] and categorized children with z- scores <−3 standard deviation as having SAM (Yes = 1) and as No = 0 if otherwise. Study design and data y The nationally representative cross-sectional data ob- tained from successive Demographic and Health Surveys (DHS) conducted in LMIC was used for this study. We extracted data from 51 most recent successive DHS con- ducted between 2010 and 2018 which were available as of March 2019 and these included under-five children (U5C) anthropometry data. Typically, the DHS uses stratified multi-stage sample drawing techniques with clusters (neighbourhoods) as the primary sampling unit (PSU) [19, 20]. Country-specific sampling method- ologies are also available at dhsprogram.com and in re- port forms [21–23]. Within each sampled household, individuals that meet the inclusion criteria were inter- viewed. Sampling weights were calculated to adjust for unequal probabilities of selection including non- responses. Application of sample weights and adjust- ment for non-responses make the findings from the sur- veys to fully represent the target populations. All the DHS questionnaires were standardized and implemented across the various countries using similar training of the interviewers, supervision of the interviewers as well as the implementation protocols. In this study, we used the DHS children recode data. The data covered the health experiences of under-five children born to sampled women within five years preceding the survey date. The anthropometry measurements were taken using standard procedures. Statistical analyses We found sig- nificant pro-illiterate inequalities in fourteen countries: Cameroon (p < 0.001), Chad (p < 0.001), Comoro (p = 0.047), Burkina Faso (p < 0.001), Ethiopia (p < 0.001), India (p < 0.001), Kenya (p < 0.001), Mozambique (p = 0.012), Namibia (p = 0.001), Nigeria (p < 0.001), Pakistan (p < 0.001), Senegal (p = 0.003), Togo (p = 0.013), and Timor Leste (p < 0.001) but no country has pro-educated inequalities as shown in Table 1. development of SAM between U5C whose mothers were literate and the others that were not literate. A risk dif- ference (RD) greater than (RD > 0) suggests that SAM is more prevalent among children born to mothers with no formal education (pro-illiterate inequality). Conversely, an RD < 0 indicates that SAM is more prevalent among children whose mothers were educated (pro-educated inequality). q y) Thirdly, in the multivariabe analysis, the logistic re- gression analysis using the pooled data from the 51 LMIC was used to carry out a Blinder-Oaxaca decom- position analysis (BODA) [27, 28]. The Blinder-Oaxaca decomposition assumes that children whose mothers were uneducated share the same characteristics with the children of educated mothers. Our choice of the Blinder-Oaxaca method is hinged on the fact that it al- lows for the decomposition of the differentials in the de- terminate variable between the two groups of the children into two components so that the gaps can be seen more clearly [29–31]. The first component of the decomposition is the “explained” portion (also known as the “compositional” or “endowments”) of the gap that shows the differentials in the distributions of the quanti- fiable characteristics of interest among these groups. The BODA method enabled the quantification of the magnitude of the gap between “the advantaged” and “the disadvantaged” groups is attributable to differentials in the specific quantifiable characteristics. The second component of the decomposition analysis is the “unex- plained” part (also referred to as the structural compo- nent) which shows the magnitude of the gap caused by the differentials in the regression coefficients and the unmeasured characteristics between these two groups of children being compared. The methods used in the current study have been used in previous and related studies [13, 29–31]. Statistical analyses In this study, we carried out analytical analyses compris- ing descriptive statistics and multivariate analysis. Uni- variable and bivariable analysis were used to describe the study population. Descriptive statistics was used to de- pict the distribution of respondents by country and the explanatory variables. Estimates of the frequencies were expressed as percentages and confidence intervals. Secondly, we computed the risk difference in the Fagbamigbe et al. BMC Public Health (2020) 20:555 Page 4 of 14 LMIC studied (Table 1 and Fig. 1). The overall SAM prevalence was 4.7% with a median prevalence of 1.8% ranging from 0.1% in Guatemala to 9.9% in Timor- Leste as shown in Table 1. The prevalence of SAM among children of uneducated mothers ranged from 0.0% in Lesotho, Zimbabwe, Kyrgyz Republic , Armenia and Guatemala to 12.7% in Timor-Leste, while the prevalence ranged from 0.1% in Peru and Guatemala to 9.4% in Timor-Leste among children of the educated mothers. We used the Mantel Haenszel test of homo- geneity of odds ratio to test the statistical significance of the association between the explanatory variables with literacy level as an effect modifier. We found sig- nificant pro-illiterate inequalities in fourteen countries: Cameroon (p < 0.001), Chad (p < 0.001), Comoro (p = 0.047), Burkina Faso (p < 0.001), Ethiopia (p < 0.001), India (p < 0.001), Kenya (p < 0.001), Mozambique (p = 0.012), Namibia (p = 0.001), Nigeria (p < 0.001), Pakistan (p < 0.001), Senegal (p = 0.003), Togo (p = 0.013), and Timor Leste (p < 0.001) but no country has pro-educated inequalities as shown in Table 1. LMIC studied (Table 1 and Fig. 1). The overall SAM prevalence was 4.7% with a median prevalence of 1.8% ranging from 0.1% in Guatemala to 9.9% in Timor- Leste as shown in Table 1. The prevalence of SAM among children of uneducated mothers ranged from 0.0% in Lesotho, Zimbabwe, Kyrgyz Republic , Armenia and Guatemala to 12.7% in Timor-Leste, while the prevalence ranged from 0.1% in Peru and Guatemala to 9.4% in Timor-Leste among children of the educated mothers. We used the Mantel Haenszel test of homo- geneity of odds ratio to test the statistical significance of the association between the explanatory variables with literacy level as an effect modifier. Prevalence of SAM by children characteristics and maternal education In Table 2, we present the descriptive statistics of the characteristics of children across the 51 LMIC. About 51% of the children were males while only 20% were in- fants. About 53% of the children are from mothers were aged 25–34 years old while about 31% had no formal education. Nearly one-third of the mothers were un- employed at the time of data collection. The overall prevalence of SAM in the group of children whose mothers had no education was 5.8% compared with 4.2% among those whose mothers were educated. The Mantel Haenszel test of homogeneity of odds ratio showed that all the characteristics considered were independently sig- nificant. For instance child’s age (p < 0.001), child’s sex (p < 0.001), maternal age (p = 0.001), household wealth quintile (p = 0.001), mother’s access to media (p = 0.001), birth weight (p < 0.001) and neighbourhood socioeco- nomic status disadvantage (p < 0.001) had significant dif- ferences in SAM prevalence viz-a-viz mothers’ literacy (Table 2). Infants, male children and mothers at extreme age intervals; 15 to 24 and 34 to 49 had overall higher SAM prevalence. For wealth index, births of women from lowest wealth quintile had the highest rate of SAM within the “uneducated” group compared with those from richest wealth quintile (6.8% vs 3.4%) but the mar- gins were closer within the “educated” group. Sample characteristics We analysed data of 532,680 under-five children nested within 55,823 neighbourhoods from 51 LMIC who par- ticipated in the DHS between 2010 and 2018. The re- gions of the world, countries, year of data collection, numbers of neighbourhoods, number of under-five children, percentage of the uneducated mothers and the weighted prevalence of SAM among children of unedu- cated and educated mothers are listed in Table 1. The proportions of children whose mothers had no formal education ranged from 0.1% in Armenia and Kyrgyz Re- public to 86.1% in Niger and a median of 20.1% in Haiti. Magnitude and variations in educational inequality in SAM Magnitude and variations in educational inequality in SAM Prevalence of SAM by countries and maternal education We found differences in the prevalence of SAM among children of educated and uneducated mothers in the 51 Figures 1 and 2 show the RD between the children of uneducated and educated mothers across the 51 LMIC. Fagbamigbe et al. Magnitude and variations in educational inequality in SAM BMC Public Health (2020) 20:555 Page 6 of 14 Table 1 Description of Demographic and Health Surveys data by countries and SAM prevalence among under-five children in LMIC, 2010–2018 (Continued) Country Year of Survey Number of Under-5 Children Weighted SAM prevalence (%) Weighted Uneducated (%) Weighted SAM (%) Uneducated Weighted SAM (%) Educated Tajikistan 2017 5867 1.8 2.7 1.0 1.8 South-Eastern Asia 4324 2.4 13.2 2.9 2.4 Cambodia 2014 4324 2.4 13.2 2.9 2.4 Southern Asia 240,849 7.1 29.4 7.8 6.8 Bangladesh 2014 6965 3.1 16.3 3.0 3.1 India 2016 225,002 7.4 29.7 8.1 7.1 Maldives 2016 2362 2.0 1.2 0.0 2.0 Nepal 2016 2369 1.9 34.5 1.7 2.0 Pakistan 2018 4151 2.3 48.6 2.6 2.1 Western Asia 1561 1.5 0.1 0.0 1.5 Armenia 2016 1561 1.5 0.1 0.0 1.5 Central America 21,717 0.2 12.6 0.1 0.2 Guatemala 2012 11,744 0.1 18.6 0.0 0.1 Honduras 2016 9973 0.3 4.9 0.4 0.3 South America 9213 0.1 3.1 0.3 0.1 Peru 2012 9213 0.1 3.1 0.3 0.1 South Europe 2462 0.5 1.1 2.7 0.5 Albania 2018 2462 0.5 1.1 2.7 0.5 Caribbean 18,700 3.9 17.7 6.7 3.3 Dominica 2013 3187 0.6 2.2 1.2 0.6 Haiti 2016 5598 0.9 20.1 1.2 0.8 Myanmar 2016 4197 1.4 16.6 1.4 1.4 Timor-Leste 2016 5718 9.9 24.4 13.4 8.8 Significant at 0.05 in Mantel Haenszel test of homogeneity of the odds ratio Table 1 Description of Demographic and Health Surveys data by countries and SAM prevalence among 2010–2018 (Continued) Table 1 Description of Demographic and Health Surveys data by countries and SAM prevalence among under-five children in LMIC, 2010–2018 (Continued) Among the 51 countries, 14 countries had statistically significant pro-illiterate inequality (that is, prevalence of SAM is higher among children from uneducated mothers). None of the countries had statistically signifi- cant pro-literacy while 37 countries had no statistically significant inequality. As shown in Fig. 1, the educational difference was largest for Ethiopia (20.55 per 1000 children) and lowest for Malawi (−0.50/1000) in the Eastern Africa. In Western Africa, the largest educa- tional difference was in Nigeria (48.22/1000) and lowest for Cote d’Ivoire (−6.41/1000). In the Caribbean, the dif- ference was largest for Timor-Leste (32.60/1000) and lowest for Myanmar (−0.96/1000). Burundi and Senegal with 2.5% weight each had the largest contribution to the pooled result. In the pooled analysis, Nigeria still had the highest pro-illiterate inequality (48.22/1000) and followed by Namibia (44.75/1000) as shown in Fig. 2. Magnitude and variations in educational inequality in SAM Overall, there was significant pro-illiterate in the total pooled sample of children in this study. The risk differ- ence was 7.18 (95% Confidence Interval (CI): 3–12) per 1000 children among children of uneducated mothers compared with those of educated mothers as shown in the random effects in Fig. 1. The random effect shows the overall risk difference among all children born to ed- ucated and uneducated mothers irrespective of their countries. In Fig. 2, we used the colours blue, yellow and red to indicate statistically significant pro-illiterate in- equality, no significant inequality and statistically signifi- cant pro-literate inequality respectively. Two of the nine countries in Eastern Africa inequality, 2 of the countries in Middle Africa, none in Northern Africa, and only Namibia in Southern Africa showed sta- tistically significant pro-illiteracy inequalities. Two of the 13 Western Africa countries and 2 of the five countries in Southern Asia showed statistically significant pro- illiterate inequality compared with only one country among the four countries studied in the Caribbean. Magnitude and variations in educational inequality in SAM BMC Public Health (2020) 20:555 Page 5 of 14 Table 1 Description of Demographic and Health Surveys data by countries and SAM prevalence among under-five children 2010–2018 Country Year of Survey Number of Under-5 Children Weighted SAM prevalence (%) Weighted Uneducated (%) Weighted SAM (%) Uneducated Weighted S Educated All 532,680 4.7 31.1 5.8 4.2 Eastern Africa 67,418 1.5 29.4 2.5 1.1 Burundi 2016 6052 0.9 47.5 0.9 0.9 Comoro 2012 2387 3.9 47.8 4.9 2.9 Ethiopia 2016 8919 3.0 65.8 3.5 2.0 Kenya 2014 18,656 1.0 11.9 2.3 0.8 Malawi 2016 5178 0.6 13.3 0.5 0.6 Mozambique 2011 9313 2.1 37.6 2.6 1.9 Rwanda 2015 3538 0.6 14.4 0.9 0.6 Tanzania 2016 8962 1.3 21.5 1.5 1.2 Uganda 2016 4413 1.4 11.2 2.0 1.3 Middle Africa 37,136 2.5 32.4 4.1 1.8 Angola 2016 6407 1.0 28.9 1.4 0.9 Cameroon 2010 5033 1.9 26.2 4.3 1.0 Chad 2015 9826 4.3 65.3 5.2 2.3 Congo 2012 4475 1.6 7.0 2.8 1.5 DRC 2014 8059 2.7 19.3 2.7 2.7 Gabon 2012 3336 1.2 6.9 1.6 1.1 Northern Africa 13,682 3.8 17.9 4.3 3.7 Egypt 2014 13,682 3.8 17.9 4.3 3.7 Southern Africa 20,273 1.7 7.2 2.3 1.6 Lesotho 2016 1312 0.7 0.9 0.0 0.7 Namibia 2013 1558 2.2 6.8 7.9 1.7 South Africa 2016 1082 0.5 2.1 3.1 0.5 Zambia 2014 11,407 2.1 11.2 2.0 2.1 Zimbabwe 2015 4914 1.1 1.2 0.0 1.1 Western Africa 85,462 4.7 60.8 5.4 3.7 Benin 2018 12,033 1.1 65.7 1.2 0.9 Burkina Faso 2010 6532 5.8 83.8 6.1 4.5 Cote d’Ivoire 2012 3200 1.8 64.8 1.7 2.0 Gambia 2013 3098 4.7 59.6 4.9 4.4 Ghana 2014 2720 0.7 28.8 0.9 0.7 Guinea 2012 3085 3.7 78.7 4.1 2.4 Liberia 2013 3171 2.2 42.5 2.1 2.3 4.5 Mali 2013 4306 5.1 82.9 5.2 4.5 Niger 2012 4771 6.2 86.1 6.2 6.2 Nigeria 2013 24,505 8.8 46.4 11.9 6.2 Senegal 2017 10,787 1.5 61.6 1.9 1.0 Sierra Leone 2013 4069 3.8 69.8 3.6 4.3 Togo 2014 3185 1.6 40.6 2.2 1.1 Central Asia 9883 1.5 1.7 1.0 1.6 Kyrgyz Republic 2012 4016 1.1 0.1 0.0 1.1 Table 1 Description of Demographic and Health Surveys data by countries and SAM prevalence among under-five children in LMIC, 2010–2018 Table 1 Description of Demographic and Health Surveys data by countries and SAM prevalence among under-five children in LMIC, 2010 2018 Fagbamigbe et al. Relationship between prevalence of SAM and magnitude of the educational inequality Figure 3 shows the level of relationship between the prevalence of SAM and the magnitude of the inequality Fagbamigbe et al. BMC Public Health (2020) 20:555 Page 7 of 14 Fig. 1 Risk difference between children from uneducated and educated mothers in the prevalence of SAM by countries ed and educated mothers in the prevalence of SAM by countries Fig. 1 Risk difference between children from uneducated and educated mothers in the prevalence of SAM by countries Fig. 1 Risk difference between children from uneducated and educated mothers in the prevalence of SAM by countries Fagbamigbe et al. Relationship between prevalence of SAM and magnitude of the educational inequality The 51 countries were categorized into 4: (1) High severe acute malnutri- tion and high pro-illiterate inequality countries such as Timor-Leste and Nigeria; (2) High severe acute malnu- trition and high pro-literate inequality was not found in any country; (3) Low severe acute malnutrition and high pro-illiterate inequality in countries such as Namibia and Kenya; and (4) Low severe acute malnutrition and high pro-literate inequality was not found in any coun- try. In Fig. 3, colours cyan, orange and red were used to depict statistically significant pro-illiterate inequality, no significant inequality and statistically significant pro- literate inequality respectively. Relationship between prevalence of SAM and magnitude of the educational inequality BMC Public Health (2020) 20:555 Page 9 of 14 Table 2 Summary of pooled sample characteristics of the studied children in 51 LMIC (Continued) Characteristics Weighted n Weighted % Weighted (%) Uneducated Weighted SAM (%) Uneducated Weighted SAM (%) Educated Birth Order 1 157,065 30.4 17.0 6.3 4.5 2 134,436 26.0 23.3 5.9 4.6 3 83,134 16.1 34.7 6.0 3.9 4 142,462 27.6 52.0 5.5 3.1 Have money for health care Not Problem 101,954 20.5 21.2 7.0 6.2 Problem 395,445 79.5 33.2 5.8 3.7 Has Health Insurance No 409,359 87.3 32.8 6.1 4.5 Yes 59,643 12.7 16.1 6.3 3.9 Community SES Quintiles 1 (Highest) 117,186 20.2 9.6 4.5 4.2 2 101,302 20.0 17.8 4.8 4.2 3 103,795 20.1 28.9 5.0 3.9 4 100,611 20.0 42.6 6.0 4.2 5 (Lowest) 94,203 19.7 62.4 6.7 4.2 Total 532,680 100.0 31.1 5.8 4.2 Significant at 0.05 in Mantel Haenszel test of homogeneity of the odds ratio Table 2 Summary of pooled sample characteristics of the studied children in 51 LMIC (Continued) Characteristics Weighted n Weighted % Weighted (%) Uneducated Weighted SAM (%) Uneducated Weighted SAM (%) Educated Table 2 Summary of pooled sample characteristics of the studied children in 51 LMIC (Continued) Characteristics Weighted n Weighted % Weighted (%) Uneducated Weighted SAM (%) Uneducated Weighted SAM (%) Educated significant factors in most of the countries studied. In Senegal, the highest contributions to the educational in- equality in the prevalence of SAM was by neighbour- hood socioeconomic disadvantage, followed by the location of residence, wealth index and access to media. Wealth index and media access narrowed the inequality in the development of SAM between children from edu- cated and uneducated mothers. In Togo, location of resi- dence was the highest contributor to the educational inequality followed by neighbourhood socioeconomic status disadvantage and then access to media. Marital status, child age and sex, birth weight and employment status of the mothers did not show any significant con- tribution to educational inequality in the development of SAM in any of the countries. across the 51 countries in this study. Relationship between prevalence of SAM and magnitude of the educational inequality BMC Public Health (2020) 20:555 Page 8 of 14 Table 2 Summary of pooled sample characteristics of the studied children in 51 LMIC Characteristics Weighted n Weighted % Weighted (%) Uneducated Weighted SAM (%) Uneducated Weighted SAM ( Individual Level Age < 12 Months 103,379 20.0 29.0 9.0 6.7 12–59 Months 413,718 80.0 31.7 5.1 3.5 Sex Female 252,541 48.8 31.5 5.4 3.8 Male 264,556 51.2 30.8 6.3 4.5 Maternal Age 15–24 160,133 31.0 22.4 6.7 4.8 25–34 273,802 52.9 31.8 5.8 4.1 35–49 83,162 16.1 45.7 5.1 2.7 Wealth Index Poorest 122,991 23.8 54.5 6.8 4.3 Poorer 112,755 21.8 37.0 5.7 4.4 Middle 104,194 20.1 26.4 5.3 4.2 Richer 96,896 18.7 18.3 4.4 4.2 Richest 80,261 15.5 8.8 3.4 3.8 Employment Yes 366,033 70.8 31.7 5.9 4.6 No 151,064 29.2 31.1 5.5 3.2 Access To Media No 188,357 36.5 55.8 6.1 4.3 Yes 328,311 63.5 17.0 5.3 4.1 Drinking-Water Sources Unimproved 95,544 19.2 43.9 5.4 3.1 Improved 402,688 80.8 28.7 5.9 4.3 Toilet Type Unimproved 248,331 49.9 45.3 6.0 4.4 Improved 249,753 50.1 18.1 5.2 3.9 Marital Status Never Married 12,199 2.4 10.0 3.5 1.7 Currently Married 484,949 93.8 32.0 5.9 4.3 Formerly Married 19,946 3.9 23.5 4.1 1.8 Weight At Birth Average+ 423,017 85.4 30.4 5.7 4.2 Small 52,939 10.7 33.5 6.0 4.4 Very Small 19,624 4.0 43.7 7.7 5.4 Birth Interval 1st 157,067 30.4 17.0 6.3 4.5 < 36 193,030 37.4 39.9 5.8 4.4 36+ 165,780 32.1 34.5 5.6 3.5 Table 2 Summary of pooled sample characteristics of the Characteristics Weighted n Weighted % Weighte Individual Level Age < 12 Months 103,379 20.0 29.0 12–59 Months 413,718 80.0 31.7 Sex Female 252,541 48.8 31.5 Male 264,556 51.2 30.8 Maternal Age 15–24 160,133 31.0 22.4 25–34 273,802 52.9 31.8 35–49 83,162 16.1 45.7 Wealth Index Poorest 122,991 23.8 54.5 Poorer 112,755 21.8 37.0 Middle 104,194 20.1 26.4 Richer 96,896 18.7 18.3 Richest 80,261 15.5 8.8 Employment Yes 366,033 70.8 31.7 No 151,064 29.2 31.1 Access To Media No 188,357 36.5 55.8 Yes 328,311 63.5 17.0 Drinking-Water Sources Unimproved 95,544 19.2 43.9 Improved 402,688 80.8 28.7 Toilet Type Unimproved 248,331 49.9 45.3 Improved 249,753 50.1 18.1 Marital Status Never Married 12,199 2.4 10.0 Currently Married 484,949 93.8 32.0 Formerly Married 19,946 3.9 23.5 Weight At Birth Average+ 423,017 85.4 30.4 Small 52,939 10.7 33.5 Very Small 19,624 4.0 43.7 Birth Interval 1st 157,067 30.4 17.0 < 36 193,030 37.4 39.9 36+ 165,780 32.1 34.5 Table 2 Summary of pooled sample characteristics of the studied children in 51 LMIC Characteristics Weighted n Weighted % Weighted (%) Uneducated Weighted SAM (%) Uneducated Weighted SAM (%) Educated Table 2 Summary of pooled sample characteristics of the studied children in 51 LMIC Characteristics Weighted n Weighted % Weighted (%) Uneducated Weighted SAM (%) Uneducated Weighted SAM (%) Educated Fagbamigbe et al. Discussion Figure 4 shows a detail decomposition of the inequality due to compositional effects of the factors associated with SAM among the under-five children. There were variations in the important factors associated with the educational inequal- ities across the 51 countries. The “explained” (compositional component) and the “unexplained” (structural component) portions of the educational inequalities are depicted by red and blue colours respectively; the lighter the red colour the lower the percentage contribution of the “explained” portion and the lighter the blue colour, the lower the percentage contribution of the “unexplained” portion. The goal of this study was to use the DHS data to de- compose educational inequalities in the development of SAM across the 51 low- and middle-income countries. This study was carried out to improve the knowledge of the compositional and structural factors that are associ- ated with educational inequalities in the development of SAM in the countries. The study is premised on the fact that SAM has continued to be a major public health challenge. The prevalence of SAM among children of illiterate and literate mothers varied significantly. We found significant educational-related differences that are better explained by structural and compositional factors On the average, neighbourhood socioeconomic status disadvantage and, location of residence were the most Fagbamigbe et al. BMC Public Health (2020) 20:555 Page 10 of 14 Fig. 2 Risk difference between children born to uneducated and educated mothers in the prevalence of SAM by countries with 7 more children of every 1000 children of uneducated mothers developing SAM compared with children born to educated mothers. Educational attainment of caregivers is an important factor in whether a child develops SAM or not. Our finding aligns with previous studies which re- ported that children of uneducated mothers were associ- ated with a poor range of nutritional outcomes such as stunting, wasting and malnutrition [7, 12, 17, 32–35]. This finding has several implications; first, there is a need for LMIC to develop child nutrition public health policies, in- terventions and programmes that particularly inform and train uneducated mothers on the need to provide their children with adequate nutrition. which were nested both at the neighbourhood-level and the country-level. We also found wide inter-country dif- ferences viz-a-viz literacy level in the prevalence of SAM. The inter-country variations could be ascribed to the prevalent differences in individual country’s socio- economic distribution, policies, strategies and existing level of intervention on child nutrition. Discussion Our findings are corroborated by some previous research which found similar differentials in the prevalence of SAM. In particular, the analysis in this study shows the un- equal distribution in the prevalence of SAM between the children of the educated and uneducated mothers. This suggests the presence of educational inequalities in the development of SAM among the children. In 13 of the 51 countries, pro-illiterate inequality SAM was signifi- cantly prevalent but pro-literate inequality, although higher in 16 countries, was insignificant in any of the countries. Among the countries which had significant pro- illiterate inequalities, risk difference used as the measure of inequality in our study showed that 8 to 48 per 1000 of children whose mothers were not educated will develop SAM compared with children from educated mothers. Also, there is a need to increase the knowledge of mothers and households in general so that they can have a higher capacity to afford good nutrition for their children. Besides, governments may wish to subsidize children foods as a means of relieving a huge household burden of getting food for their wards. Nonetheless, such public health interventions should be all- encompassing. It should include health education and promotion, adequate communication, seminars, political will and the community and religious leaders’ participa- tion. This is consistent with a UNICEF report that Overall, there was significant pro-illiterate inequality among the total pooled sample of children in this study Page 11 of 14 Fagbamigbe et al. BMC Public Health (2020) 20:555 Fig. 3 Scatter plot of rate of SAM and risk difference between children born to uneducated and educated mothers in LMIC Fig. 3 Scatter plot of rate of SAM and risk difference between children born to uneducated and educated mothers composition and context in which the children live. A wholesome approach should be used to address the chal- lenges of educational inequalities in child health in gen- eral and in SAM in particular. This finding from our study underscores the advantage of enhancing both the compositional and structural characteristics if educational-related inequalities in SAM are to be re- duced. Earlier reports on child malnutrition have clearly indicated the nuances of individual, community and country-level factors associated with child nutrition [2, 4, 8, 10]. Discussion prevention and long term solutions to the burden of SAM will involve “dismantling unequal power struc- tures, improving equitable access to health services and nutritious foods, promoting breastfeeding and optimal infant and young child feeding practices, improving water and sanitation, and planning for cyclic food short- ages and emergencies” [4]. It is very evident from our analysis that compositional effects of the additional explanatory variables explored contributed to the majority of the inequalities in SAM between the children of the educated and the unedu- cated mothers in Chad, Timor-Leste and Mozambique. While in Togo, and Kenya, structural effects of the iden- tified characteristics contributed mostly to the educational-inequalities in the development of SAM. y 4, 8, 10]. We find interesting results in our attempt to map the relationships between the prevalence of SAM and educa- tional inequality. Countries such as Namibia and Kenya had low prevalence of SAM and high pro-illiterate in- equalities while countries such as Timor-Leste and Nigeria had a high prevalence of SAM and high pro- illiterate inequality. These variations can be explained by access to media, household wealth status, country-level policies and programmes for child nutrition, famine, war, internal displacement, political and economic in- stability. It is quite understandable that we did not find significant pro-literate inequality in any of the countries The decomposition analysis has shown that compos- itional factors including the neighbourhood SES, loca- tion of residence, wealth index and access to media were the greatest contributors to educational-related inequal- ities across the countries. Obviously, to attain a mean- ingful reduction in educational inequalities in SAM, there is a need to look outside the box and properly understand the connection among the structure, Page 12 of 14 Fagbamigbe et al. BMC Public Health (2020) 20:555 Fig. 4 Contributions of differences in the distribution of the determinants of SAM to the total gap between children from uneducated and educated mothers by countries ons of differences in the distribution of the determinants of SAM to the total gap between children from uneducated and s by countries Fig. 4 Contributions of differences in the distribution of the determinants of SAM to the total gap between children from uneducated and educated mothers by countries studied. An educated mother should engage in good nu- tritional practices for her wards. measurement of household income is available. While multilevel analysis has proved to be an efficient method for assessing disparities and to monitor health care indi- cators, Blinder-Oaxaca decomposition analysis is not an alternative measure of causality. It, however, gives robust evidence of inequalities after controlling for the expos- ure variables. There may be a need for further studies to examine the influence and association of structural and compositional factors with educational-inequalities in the prevalence of SAM. Nonetheless, our study has major strengths. As shown in Fig. 4, we quantified the magnitude of the explained and unexplained factors as- sociated with our outcome measure. The study covered 51 LMIC using the DHS data is reputed for accuracy and comparability across countries. Conclusions We identified that SAM is prevalent in most LMIC with wide educational variations. The occurrence of SAM was explained by the individual, household and community- level factor. The overall significance of our exposure variable in explaining the difference in SAM prevalence is a pointer that education of the whole population, es- pecially the girl child who is a potential mother, is very y 4, 8, 10]. Our findings on the effect of neighbourhood SES on the likelihood that children of an educated mother have SAM are in consonance with existing findings [36, 37]. These studies showed that the odds of better health outcomes are higher among residents in high socioeco- nomic areas than persons who reside in socioeconomi- cally disadvantaged areas [36, 37]. It is therefore important that the countries with high SAM and high pro-illiterate inequalities in SAM rework their child nu- trition policies by taking a cue from countries with a low SAM and low pro-literate inequalities. For instance, re- searchers and health programmers in such countries may wish to explore the differentials in child health and nutrition in Nigeria and Kenya. Why is SAM higher in Nigeria than in Kenya even though both countries have pro-illiteracy inequalities? Funding 9. Binns P, Myatt M. Does treatment of short or stunted children aged 6–59 months for severe acute malnutrition using ready to use therapeutic food make them overweight? Data from Malawi. Arch. Public Health [Internet]. BioMed Central; 2018 [cited 2019 Jun 28];76:78. Available from: http://www. ncbi.nlm.nih.gov/pubmed/30559964. The authors receive no funding for this study. Consent for publication Not applicable. Consent for publication Not applicable. Authors’ contributions AFF and OU conceived and designed the study and analysed the data; AFF retrieved and merged the data, drew the Figures and wrote the results; AFF, OU, and NBK carried out the literature search, data interpretation, and writing of the manuscript. All authors read and consented to the final version of the manuscript. 6. Titaley CR, Ariawan I, Hapsari D, Muasyaroh A, Dibley MJ. Determinants of the Stunting of Children Under Two Years Old in Indonesia: A Multilevel Analysis of the 2013 Indonesia Basic Health Survey. Nutrients. 2019 [cited 2019 Jun 21];11:1106. Available from: http://www.ncbi.nlm.nih.gov/ pubmed/31109058. 6. Titaley CR, Ariawan I, Hapsari D, Muasyaroh A, Dibley MJ. Determinants of the Stunting of Children Under Two Years Old in Indonesia: A Multilevel Analysis of the 2013 Indonesia Basic Health Survey. Nutrients. 2019 [cited 2019 Jun 21];11:1106. Available from: http://www.ncbi.nlm.nih.gov/ pubmed/31109058. Abbreviations CI: Confidence Interval; DHS: Demographic and Health Survey; IRB: Institutional Review Board; LMIC: Low- And Middle-Income Countries; PSU: Primary Sample Unit; RD: Risk Difference; SAM: Severe Acute Malnutrition; SES: Socioeconomic Status; U5C: Under-Five Children; UNICEF: United Nations International Children’s Emergency Fund; WHO: World Health Organization; WHZ: Weight for Height Score 2. Akombi B, Agho K, Hall J, Wali N, Renzaho A, Merom D. Stunting, Wasting and Underweight in Sub-Saharan Africa: A Systematic Review. Int. J. Environ. Res. Public Health [Internet]. 2017 [cited 2019 Jun 21];14:863. Available from: http://www.ncbi.nlm.nih.gov/pubmed/28788108. Competing interests The authors declare that they have no competing interests. Acknowledgements 3. WHO. Severe acute malnutrition [Internet]. Geneva, Switzerland; 2019. Available from: https://www.who.int/nutrition/topics/severe_malnutrition/ en/. The authors are grateful to ICF Macro, USA, for permitting the authors to use the data. No further ethics approval was necessary to access the data. We appreciate the Consortium for Advanced Research and Training in Africa (CARTA) for providing logistic supports to AFF in the course of writing this paper. 4. UNICEF. Severe acute malnutrition [Internet]. Nutrition. 2015 [cited 2019 Jun 29]. p. 1. Available from: https://www.unicef.org/nutrition/index_sam.html. 4. UNICEF. Severe acute malnutrition [Internet]. Nutrition. 2015 [cited 2019 Jun 29]. p. 1. Available from: https://www.unicef.org/nutrition/index_sam.html. 5. Bulti A, Briend A, Dale NM, De Wagt A, Chiwile F, Chitekwe S, et al. Improving estimates of the burden of severe acute malnutrition and predictions of caseload for programs treating severe acute malnutrition: experiences from Nigeria. Arch. Public Heal. [Internet]. BioMed Central; 2017 [cited 2019 Jun 29];75:66. Available from: http://archpublichealth. biomedcentral.com/articles/10.1186/s13690-017-0234-4. References 1. Khan S, Zaheer S, Safdar NF. Determinants of stunting, underweight and wasting among children < 5 years of age: evidence from 2012–2013 Pakistan demographic and health survey. BMC Public Health [Internet]. 2019 [cited 2019 Jun 21];19:358. Available from: http://www.ncbi.nlm.nih.gov/ pubmed/30935382. 1. Khan S, Zaheer S, Safdar NF. Determinants of stunting, underweight and wasting among children < 5 years of age: evidence from 2012–2013 Pakistan demographic and health survey. BMC Public Health [Internet]. 2019 [cited 2019 Jun 21];19:358. Available from: http://www.ncbi.nlm.nih.gov/ pubmed/30935382. 1. Khan S, Zaheer S, Safdar NF. Determinants of stunting, underweight and wasting among children < 5 years of age: evidence from 2012–2013 Pakistan demographic and health survey. BMC Public Health [Internet]. 2019 [cited 2019 Jun 21];19:358. Available from: http://www.ncbi.nlm.nih.gov/ pubmed/30935382. Author details 1 Author details 1Department of Epidemiology and Medical Statistics, Faculty of Public Health, College of Medicine, University of Ibadan, Ibadan, Nigeria. 2Division of Health Sciences, Populations, Evidence and Technologies Group, Warwick Medical School, University of Warwick, Coventry, UK. 3Department of Mathematics, Physics & Electrical Engineering, Northumbria University, Newcastle upon Tyne, UK. Received: 10 October 2019 Accepted: 1 April 2020 Authors information AFF is a Senior Lecturer and Medical Statistician at the Department of Epidemiology and Medical Statistics, College of Medicine, Ibadan, University of Ibadan, Nigeria and a Visiting Academic under CARTA Fellowship to the Division of Health Sciences, Populations, Evidence and Technologies Group, Warwick Medical School, University of Warwick, Coventary, United Kingdom. NBK is a Professor of Biostatistics at the Department of Mathematics, Physics & Electrical Engineering (MPEE), Northumbria University, Newcastle Upon Tyne, United Kingdom. OAU is an Associate Professor of Public Health at the Division of Health Sciences, Populations, Evidence and Technologies Group, University of Warwick, Coventry, United Kingdom. 7. Fantay GK, Mekonnen HW, Haftom TA, Oumer SA, Afework MB. Determinants of stunting among under-five children in Ethiopia: a multilevel mixed-effects analysis of 2016 Ethiopian demographic and health survey data. BMC Pediatr. 2019 [cited 2019 Jun 21];19:176. Available from: http://www.ncbi.nlm.nih.gov/pubmed/31153381. 8. Motedayen M, Dousti M, Sayehmiri F, Pourmahmoudi AA. An Investigation of the Prevalence and Causes of Malnutrition in Iran: a Review Article and Meta-analysis. Clin. Nutr. Res. [Internet]. 2019 [cited 2019 Jun 21];8:101. Available from: http://www.ncbi.nlm.nih.gov/pubmed/31089464. Availability of data and materials The data supporting this article is available at http://dhsprogram.com. The data is publicly available but permission to use the data is required. The authors obtained permission from the data owners to use the data. 10. Harimbola DR, Mizumoto K. Individual and Household Risk Factors for Severe Acute Malnutrition among Under-Five Children in the Analamanga Region, Madagascar. Int. J. MCH AIDS [Internet]. Global Health and Education Projects, Inc.; 2018 [cited 2019 Jun 28];7:217–225. Available from: http://www.ncbi.nlm.nih.gov/pubmed/30631640. 10. Harimbola DR, Mizumoto K. Individual and Household Risk Factors for Severe Acute Malnutrition among Under-Five Children in the Analamanga Region, Madagascar. Int. J. MCH AIDS [Internet]. Global Health and Education Projects, Inc.; 2018 [cited 2019 Jun 28];7:217–225. Available from: http://www.ncbi.nlm.nih.gov/pubmed/30631640. Study limitations and strengths We have used household wealth status as a proxy for household income as the DHS survey did not collect any information on household income. Hence, our findings may not be generalizable in countries where direct Page 13 of 14 Fagbamigbe et al. BMC Public Health (2020) 20:555 Page 13 of 14 Fagbamigbe et al. BMC Public Health (2020) 20:555 important to child health. The advantages of education in human endeavour cannot be overemphasized. The low- and middle-income countries must improve their tactics in child nutrition with the goal of eradication of severe acute malnu- trition which would eventually reduce child morbidity, op- portunistic infections and mortality. To address the educational inequalities in SAM, an urgent child nutrition intervention is a must in the low- and middle- income coun- tries, especially in those identified as having pro-illiterate in- equalities as better education among all women will close the gaps and reduce the burden of SAM generally. We rec- ommend further studies of other determinate causes of in- equalities in severe acute malnutrition in low- and middle- income countries. Publisher’s Note 18. Nhampossa T, Sigaúque B, Machevo S, Macete E, Alonso P, Bassat Q, et al. Severe malnutrition among children under the age of 5 years admitted to a rural district hospital in southern Mozambique. Public Health Nutr. [Internet]. 2013 [cited 2019 Jun 28];16:1565–1574. Available from: https://www.cambridge.org/ core/product/identifier/S1368980013001080/type/journal_article. 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. g ore/product/identifier/S1368980013001080/type/journal_article. 19. Croft TN, Marshall AMJ, Allen CK. Guide to DHS statistics [Internet]. 2018. Available from: https://dhsprogram.com/pubs/pdf/DHSG1/Guide_to_DHS_ Statistics_DHS-7.pdf. 20. ICF International. Demographic and Health survey: sampling and household listing manual [Internet]. Calverton; 2012. Available from: https://www.dhsprogram. com/pubs/pdf/DHSM4/DHS6_Sampling_Manual_Sept2012_DHSM4.pdf. 21. ICF International Inc. Uganda Demographic and Health Survey 2011. Kampala, Uganda: UBOS and Calverton, Maryland:; 2012. 22. National Bureau of Statistics Tanzania and ICF - Macro. Tanzania Demographic and Health Survey 2010. Natl. Bur. Stat. Dar es Salaam, Tanzania ICF Macro Calverton, Maryland, USA. 2011. 23. National Population Commission (Nigeria) and ICF International. Nigeria Demographic and Health Survey 2013. Abuja, Nigeria; 2014. 23. National Population Commission (Nigeria) and ICF International. Nigeria Demographic and Health Survey 2013. Abuja, Nigeria; 2014. 24. WHO. WHO AnthroPlus for personal computers Manual: Software for assessing growth of the world’s children and adolescents [Internet]. Geneva, Switzerland; 2009. Available from: http://www.who.int/growthref/tools/en/. 25. Roser M, Ortiz-Ospina E. Global Rise of Education [Internet]. Ourworldindata. org. 2019 [cited 2019 Jun 29]. p. 1. Available from: https://ourworldindata. org/global-rise-of-education. 26. Vyass S, Kumaranayake L. Constructing socioeconomic status indexes: how to use principal component analysis. Health Policy Plan. 2006;21:459–68. 26. Vyass S, Kumaranayake L. Constructing socioeconomic status indexes: how to use principal component analysis. Health Policy Plan. 2006;21:459–68. 27. Oaxaca R. Male-female wage differentials in urban labor markets. Intl Econ Rev. 1973;14:693–709. 27. Oaxaca R. Male-female wage differentials in urban labor markets. Intl Econ Rev. 1973;14:693–709. 28. Blinder AS. Wage discrimination: reduced form and structural estimates. J Hum Resour. 1973;8:436–55. 28. Blinder AS. Wage discrimination: reduced form and structural estimates. J Hum Resour. 1973;8:436–55. 29. Sambala EZ, Uthman OA, Adamu AA, Ndwandwe D, Wiyeh AB, Olukade T, et al. Mind the gap: what explains the education-related inequality in missed opportunities for vaccination in sub-Saharan Africa? Compositional and structural characteristics. Hum. Vaccines Immunother. [Internet]. Taylor & Francis; 2018;14:2365–2372. Available from: https://doi.org/10.1080/ 21645515.2018.1460985. 30. Ethics approval and consent to participate This study was based on the analysis of existing survey data. The Institutional Review Board (IRB) of ICF Macro at Fairfax, Virginia in the USA reviewed and approved the MEASURE Demographic and Health Surveys Project Phase III. The 2010–2018 DHS’s are categorized under that approval. The IRB of ICF Macro complied with the United States Department of Health and Human Services requirements for the “Protection of Human Subjects” (45 CFR 46). Written informed consent was obtained from every study participant before participation and all information was collected without identifiers and kept confidentially. The full details of the ethical approvals can be found at http://dhsprogram.com. A copy of the IRB approval is available as a supplementary file. 11. Kshatriya GK, Acharya SK. Gender Disparities in the Prevalence of Undernutrition and the Higher Risk among the Young Women of Indian Tribes. PLoS One [Internet]. Public Library of Science; 2016 [cited 2019 Jun 28]; 11:e0158308. Available from: http://www.ncbi.nlm.nih.gov/pubmed/27379521. 11. Kshatriya GK, Acharya SK. Gender Disparities in the Prevalence of Undernutrition and the Higher Risk among the Young Women of Indian Tribes. PLoS One [Internet]. Public Library of Science; 2016 [cited 2019 Jun 28]; 11:e0158308. Available from: http://www.ncbi.nlm.nih.gov/pubmed/27379521. 12. Alemayehu M, Tinsae F, Haileslassie K, Seid O, Gebregziabher G, Yebyo H. Undernutrition status and associated factors in under-5 children, in Tigray, Northern Ethiopia. Nutrition [Internet]. Elsevier; 2015 [cited 2019 Jun 28];31: 964–970. Available from: https://www.sciencedirect.com/science/article/abs/ pii/S0899900715000817?via%3Dihub. 12. Alemayehu M, Tinsae F, Haileslassie K, Seid O, Gebregziabher G, Yebyo H. Undernutrition status and associated factors in under-5 children, in Tigray, Northern Ethiopia. Nutrition [Internet]. Elsevier; 2015 [cited 2019 Jun 28];31: 964–970. Available from: https://www.sciencedirect.com/science/article/abs/ pii/S0899900715000817?via%3Dihub. Page 14 of 14 Page 14 of 14 Fagbamigbe et al. BMC Public Health (2020) 20:555 Page 14 of 14 13. Fagbamigbe AF, Kandala NB, Uthman O. Severe acute malnutrition among under-five children in low- and middle-income countries: A hierarchical analysis of associated risk factors. Nutrition [Internet]. Elsevier; 2020 [cited 2020 Feb 15];110768. Available from: https://linkinghub.elsevier.com/ retrieve/pii/S0899900720300514. 34. Abdulahi A, Shab-Bidar S, Rezaei S, Djafarian K. Nutritional status of under five children in Ethiopia: a systematic review and meta-analysis. Ethiop. J. Health Sci. [Internet]. 2017 [cited 2019 Jun 21];27:175. Available from: https://www.ajol.info/index.php/ejhs/article/view/153152. 35. Meshram II, Arlappa N, Balakrishna N, Mallikharjuna Rao K, Laxmaiah A, Brahmam GNV. Trends in the prevalence of undernutrition, nutrient and food intake and predictors of undernutrition among under five year tribal children in India. Asia Pac. Ethics approval and consent to participate J. Clin. Nutr. [Internet]. 2012 [cited 2019 Jun 21]; 21:568–576. Available from: http://www.ncbi.nlm.nih.gov/pubmed/2301 7315. 14. Novignon J, Aboagye E, Agyemang OS, Aryeetey G. Socioeconomic-related inequalities in child malnutrition: evidence from the Ghana multiple indicator cluster survey. Health Econ. Rev. [Internet]. BioMed Central; 2015 [cited 2019 Jun 29];5:34. Available from: http://www.ncbi.nlm.nih.gov/ pubmed/26603158. 36. Uthman OA, Sambala EZ, Adamu AA, Ndwandwe D, Wiyeh AB, Olukade T, et al. Does it really matter where you live? A multilevel analysis of factors associated with missed opportunities for vaccination in sub-Saharan Africa. Hum. Vaccin. Immunother. [Internet]. Taylor & Francis; 2018 [cited 2019 Jun 22];14:2397–2404. Available from: https://www.tandfonline.com/doi/full/10.1 080/21645515.2018.1504524. 15. Khan T, Khan R, Raza MA. Gender analysis of malnutrition: a case study of school- going children in Bahawalpur. Asian Dev. Policy Rev. [Internet]. 2015 [cited 2019 Jun 28];3:29–48. Available from: http://www.aessweb.com/journals/5008. 16. Jawaregowda KS, Angadi M. Gender differences in nutritional status among under five children in rural areas of Bijapur district, Karnataka, India. Int. J. Community Med. Public Heal. [Internet]. 2015 [cited 2019 Jun 28];2:506–509. Available from: http://www.ijcmph.com. 37. Fagbamigbe AF, Bamgboye EA, Yusuf BO, Akinyemi JO, Issa BK, Ngige E, et al. The Nigeria wealth distribution and health seeking behaviour : evidence from the 2012 national HIV / AIDS and reproductive health survey. Health Econ Rev. 2015;5:e1–10. 17. Eshete H, Abebe Y, Loha E, Gebru T, Tesheme T. Nutritional status and effect of maternal employment among children aged 6–59 months in Wolayta Sodo Town, Southern Ethiopia: a cross-sectional study. Ethiop. J. Health Sci. [Internet]. 2017 [cited 2019 Jun 28];27:155. Available from: http://www.ncbi. nlm.nih.gov/pubmed/28579711. Publisher’s Note Ndwandwe D, Uthman OA, Adamu AA, Sambala EZ, Wiyeh AB, Olukade T, et al. Decomposing the gap in missed opportunities for vaccination between poor and non-poor in sub-Saharan Africa: A multicountry analyses. Hum. Vaccines Immunother. [Internet]. Taylor & Francis; 2018;14:2358–2364. Available from: https://doi.org/10.1080/21645515.2018.1467685. 31. Yaya S, Uthman OA, Okonofua F, Bishwajit G. Decomposing the rural-urban gap in the factors of under-five mortality in sub-Saharan Africa? Evidence from 35 countries. BMC Public Health BMC Public Health. 2019;19:1–10. 32. Mawa R, Lawoko S. Malnutrition among children under five years in Uganda. Am J Heal Res. 2018;6:56–66. 33. Man SL, Guo Y. Research on the social determinants of malnutrition among children under the age of 5 in China. Beijing Da Xue Xue Bao. [Internet]. 2016; 48:418–23 Available from: https://www.ncbi.nlm.nih.gov/pubmed/27318901. 33. Man SL, Guo Y. Research on the social determinants of malnutrition among children under the age of 5 in China. Beijing Da Xue Xue Bao. [Internet]. 2016; 48:418–23 Available from: https://www.ncbi.nlm.nih.gov/pubmed/27318901.
https://openalex.org/W3082217150	https://digitalcommons.usf.edu/context/mme_facpub/article/1881/viewcontent/biomolecules_10_01312_v2.pdf	English	null	Why COVID-19 Transmission Is More Efficient and Aggressive Than Viral Transmission in Previous Coronavirus Epidemics?	Biomolecules	2,020	cc-by	23,050	University of South Florida University of South Florida Digital Commons @ University of Digital Commons @ University of South Florida South Florida Molecular Medicine Faculty Publications Molecular Medicine 2020 Why COVID-19 Transmission Is More Efficient and Aggressive Why COVID-19 Transmission Is More Efficient and Aggressive Than Viral Transmission in Previous Coronavirus Epidemics? Than Viral Transmission in Previous Coronavirus Epidemics? Fatma Elrashdy Cairo University Elrashdy M. Redwan King Abdulaziz University Vladimir N. Uversky University of South Florida, vuversky@usf.edu Follow this and additional works at: https://digitalcommons.usf.edu/mme_facpub Part of the Medicine and Health Sciences Commons Scholar Commons Citation Scholar Commons Citation Elrashdy, Fatma; Redwan, Elrashdy M.; and Uversky, Vladimir N., "Why COVID-19 Transmission Is More Efficient and Aggressive Than Viral Transmission in Previous Coronavirus Epidemics?" (2020). Molecular Medicine Faculty Publications. 884. https://digitalcommons.usf.edu/mme_facpub/884 Fatma Elrashdy Cairo University Elrashdy M. Redwan King Abdulaziz University Vladimir N. Uversky University of South Florida, vuversky@usf.edu Follow this and additional works at: https://digitalcommons.usf.edu/mme_facpub Part of the Medicine and Health Sciences Commons Part of the Medicine and Health Sciences Commons Part of the Medicine and Health Sciences Commons Fatma Elrashdy 1 , Elrashdy M. Redwan 2,* and Vladimir N. Uversky 2,3,4,* Keywords: severe acute respiratory syndrome coronavirus 2; SARS-CoV-2; coronavirus disease 2019; COVID-19; viral infection; virus-host interaction Keywords: severe acute respiratory syndrome coronavirus 2; SARS-CoV-2; coronavirus disease 2019; COVID-19; viral infection; virus-host interaction Fatma Elrashdy 1 , Elrashdy M. Redwan 2,* and Vladimir N. Uversky 2,3,4,* 1 Department of Endemic Medicine and Hepatogastroenterology, Kasr Alainy School of Medicine, Cairo University, Cairo 11562, Egypt; fatmaelrashdy@kasralainy.edu.eg 1 Department of Endemic Medicine and Hepatogastroenterology, Kasr Alainy School of Medicine, Cairo University, Cairo 11562, Egypt; fatmaelrashdy@kasralainy.edu.eg Cairo University, Cairo 11562, Egypt; fatmaelrashdy@kasralainy.edu.eg 2 Biological Science Department, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia 3 Department of Molecular Medicine and USF Health Byrd Alzheimer’s Research Institute, Morsani College of Medicine, University of South Florida, Tampa, FL 33612, USA 3 Department of Molecular Medicine and USF Health Byrd Alzheimer’s Research Institute, Morsani College o Medicine, University of South Florida, Tampa, FL 33612, USA y p 4 Institute for Biological Instrumentation of the Russian Academy of Sciences, Federal Research Center “Pushchino Scientiﬁc Center for Biological Research of the Russian Academy of Sciences”, Pushchino, 142290 Moscow, Russia y p 4 Institute for Biological Instrumentation of the Russian Academy of Sciences, Federal Research Center “Pushchino Scientiﬁc Center for Biological Research of the Russian Academy of Sciences”, Pushchino, 142290 Moscow, Russia * Correspondence: lradwan@kau.edu.sa (E.M.R.); vuversky@usf.edu (V.N.U.) * Correspondence: lradwan@kau.edu.sa (E.M.R.); vuversky@usf.edu (V.N.U.) Received: 3 September 2020; Accepted: 8 September 2020; Published: 11 September 2020 Received: 3 September 2020; Accepted: 8 September 2020; Published: 11 September 2020 Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing a pandemic of coronavirus disease 2019 (COVID-19). The worldwide transmission of COVID-19 from human to human is spreading like wildﬁre, aﬀecting almost every country in the world. In the past 100 years, the globe did not face a microbial pandemic similar in scale to COVID-19. Taken together, both previous outbreaks of other members of the coronavirus family (severe acute respiratory syndrome (SARS-CoV) and middle east respiratory syndrome (MERS-CoV)) did not produce even 1% of the global harm already inﬂicted by COVID-19. There are also four other CoVs capable of infecting humans (HCoVs), which circulate continuously in the human population, but their phenotypes are generally mild, and these HCoVs received relatively little attention. These dramatic diﬀerences between infection with HCoVs, SARS-CoV, MERS-CoV, and SARS-CoV-2 raise many questions, such as: Why is COVID-19 transmitted so quickly? Is it due to some speciﬁc features of the viral structure? Are there some speciﬁc human (host) factors? Are there some environmental factors? The aim of this review is to collect and concisely summarize the possible and logical answers to these questions. Why COVID-19 Transmission Is More Eﬃcient and Aggressive Than Viral Transmission in Previous Coronavirus Epidemics? Fatma Elrashdy 1 , Elrashdy M. Redwan 2,* and Vladimir N. Uversky 2,3,4,* www.mdpi.com/journal/biomolecules Scholar Commons Citation Scholar Commons Citation Elrashdy, Fatma; Redwan, Elrashdy M.; and Uversky, Vladimir N., "Why COVID-19 Transmission Is More Efficient and Aggressive Than Viral Transmission in Previous Coronavirus Epidemics?" (2020). Molecular Medicine Faculty Publications. 884. https://digitalcommons.usf.edu/mme_facpub/884 This Article is brought to you for free and open access by the Molecular Medicine at Digital Commons @ University of South Florida. It has been accepted for inclusion in Molecular Medicine Faculty Publications by an authorized administrator of Digital Commons @ University of South Florida. For more information, please contact digitalcommons@usf.edu. biomolecules 1. Introduction In addition to the seasonal ﬂu that annually infects 9% of the world population and causes 291,000–600,000 deaths each year (death rate around 0.1%), the past 100 years witnessed several outbreaks of viral infections, such as the 1918 inﬂuenza pandemic (500 million infected; 50 million died; mortality rate 10%), 2002–2004 severe acute respiratory syndrome (SARS) outbreak (8098 cases; 774 deaths; mortality rate 9.5%), 2009–2010 H1N1 inﬂuenza pandemic (1.649 billion infected; i.e., 24% of the global population (~61 million cases in the USA); 284,000 died (~12,500 deaths in the USA); mortality rate 0.02%), 2012–2020 middle east respiratory syndrome (MERS) outbreak (2519 cases; 866 deaths; mortality rate 34.4%), 2014–2016 Ebola outbreak (~28,650 cases across 10 countries; 11,325 deaths; mortality rate 39.5%), and currently developing coronavirus disease 2019 (COVID-19) pandemic. It is diﬃcult to make a projection of the ﬁnal outcomes of the COVID-19 pandemic, which is still Biomolecules 2020, 10, 1312; doi:10.3390/biom10091312 www.mdpi.com/journal/biomolecules 2 of 32 Biomolecules 2020, 10, 1312 developing, but the currently available data are staggering (as of 4 September 2020): there are almost 26.8 million COVID-19 cases in 213 countries and territories around the world and two international conveyances, with more than 877,000 patients died. Although current statistics indicate that 3.3% of the SARS-CoV-2 infected have died worldwide, the COVID-19 mortality rates are not equal in all aﬀected territories and vary in a wide range in diﬀerent countries (from 0.56% in Iceland to >18% in France). Of these six global outbreaks of viral infections, three were caused by coronaviruses (SARS, MERS, and COVID-19), of which COVID-19 is characterized by the most eﬃcient and aggressive transmission. In fact, COVID-19, which is caused by the infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, also known as 2019 new CoV, 2019-nCoV), is spreading like wildﬁre worldwide, aﬀecting almost every country in the world. Taken together, both previous outbreaks of other members of the coronavirus family (SARS-CoV and MERS-CoV) did not produce even 1% of the global harm already inﬂicted by COVID-19. Furthermore, in addition to SARS-CoV, MERS-CoV, and SARS-CoV-2 (all are β-CoVs of the B and C lineage), there are four other coronaviruses (CoVs) capable of infecting humans (HCoVs), which circulate continuously in the human population. These are HCoV-OC43 [1,2] and HCoV-HKU1 [3] (β-CoVs of the A lineage or β1CoVs), and HCoV-229E [4,5] and HCoV-NL63 [6,7] (α-CoVs). 1. Introduction Being identiﬁed in the late 1960s (HCoV-229E and the HCoV-OC43) [8–12] and in 2004-2005 (HCoV-NL63 [6,7,13] and HCoV-HKU1 [3]), these HCoVs are known to be responsible for 3–10% cases of the common cold and short-term upper respiratory infections that occur mainly in winter, with a short incubation time [14,15], with about 2% of the human population being healthy carriers of an HCoV [16,17]. Although these HCoV strains can also cause more serious diseases of the lower respiratory tract, such as bronchitis, bronchiolitis, and pneumonia, especially in newborns or infants, elderly people, and immunocompromised patients [16,17], their phenotypes are generally mild, and as a result, these four HCoVs received relatively little attention. These dramatic diﬀerences between infection with HCoVs, SARS-CoV, MERS-CoV, and SARS-CoV-2 raise many questions, such as: Why is COVID-19 transmitted so quickly? Is it due to some speciﬁc features of the viral structure? Are there some speciﬁc human (host) factors? Are there some environmental factors? The aim of this study is to collect and concisely summarize the possible and logical answers to these questions. 2. Intrinsic Viral Factors Based on the evaluation of the levels of intrinsic disorder in the nucleocapsid (N) and membrane (M) proteins of SARS-CoV-2, it was proposed that this virus is characterized by high resilience to the conditions outside the body and in body ﬂuids, suggesting that SARS-CoV-2 belongs to viruses with intermediate levels of both respiratory and fecal-oral transmission potentials [34,35], which favor alternative ways for the COVID-19 transmission vertically and horizontally. y y y An important feature that diﬀerentiates β-CoVs of the B and C lineages (SARS-CoV, MERS-CoV, and SARS-CoV-2) from β-CoVs of the A lineage (β1CoVs) is the lack of hemagglutinin-esterase (HE) protein, which is present in toroviruses, inﬂuenza C and D viruses, and in β1CoVs [36–40]. HE is a receptor-binding/receptor-destroying viral protein interacting with the 9-O-acetylated sialic acids (9-O-Ac-Sias) [38], which are the glycan components commonly present in mammals and birds [41]. Therefore, in β1CoVs, both spike and HE proteins bind 9-O-Ac-Sias, whereas virus elution is promoted by receptor destruction via the action of the HE esterase domain. These opposing activities of receptor binding and receptor destruction deﬁne dynamic and reversible attachment of β1CoV to sialoglycans. The sialate-O-acetyl-esterase activity promotes escape from attachment to non-permissive host cells or decoy and facilitates the release of viral progeny from infected cells [42]. Curiously, it was shown that the HE lectin function is progressively lost during the in-host evolution of the human β1CoVs, HCoV-OC43, and HCoV-HKU1 [43]. Spike proteins of MERS-CoV interact with a speciﬁc receptor, dipeptidyl peptidase-4 (DPP4), which is a key factor in the signaling and activation of the acquired and innate immune responses in infected patients [44]. On the other hand, the host cell entry of SARS-CoV, HCoV-NL63, and SARS-CoV-2 is mediated by interaction with the angiotensin-converting enzyme-2 (ACE2) receptors, which are expressed in the brain, gut, heart, kidney, lung (particularly in type 2 pneumocytes and macrophages), vessels, and testis [45]. However, besides this protein membrane receptor, the host cell entry of HCoVs, including SARS-CoV-2, also depends on the sialic-acid-containing glycoproteins and gangliosides, which might act as primary attachment factors for viruses along the respiratory tract [38]. In fact, the N-terminal domain (NTD) of the spike (S) glycoprotein of SARS-CoV-2 was shown to contain a ganglioside-binding site that can be eﬃciently blocked by chloroquine (CLQ) and its more active derivative, hydroxychloroquine (CLQ-OH) [46]. 2. Intrinsic Viral Factors CoVs belong to the subfamily Coronavirinae of the Coronaviridae family (which also includes the Torovirinae subfamily) in the order Nidovirales. They are divided into four genera, namely α-, β-, γ-, and δ-CoVs, with β-CoVs being further separated into A, B, C, and D lineages or clades [18]. Of four CoV genera, α- and β-CoV are able to infect mammals (including humans and domestic animals), while γ- and δ-CoV tend to infect birds. The emergence of human-infecting CoVs is likely associated with cross-species transmission events [19]. For example, SARS-CoV-2 shows close genetic similarity to bat coronaviruses [20–23]. SARS-CoV and MERS-CoV are zoonotic viruses that crossed the species barrier using bats/palm civets [24] and dromedary camels [25], respectively. Similarly, HCoV-OC43 originated from a zoonotic transmission event of a bovine coronavirus (BCoV) [26,27], HCoV-HKU1 from a bat coronavirus [28], and HCoV-NL63 originated from ARCoV.2 (Appalachian Ridge CoV) detected in North American tricolored bat (Perimyotis subﬂavus) [29]. Finally, HCoV-229E originated in hipposiderid bats, with camelids serving as potential intermediate hosts [30]. The single-stranded RNA genome of SARS-CoV-2 includes 29,903 nucleotides and encodes three structural proteins, such as spike glycoprotein (S), an envelope protein (E), membrane protein (M), and nucleocapsid protein (N), six accessory proteins, encoded by ORF3a, ORF6, ORF7a, ORF7b, and ORF8 genes, and several non-structural proteins (NSPs) in the form of a polyprotein encoded by a large, 5′-located ORF1ab replicase gene that covers more than two-thirds of the viral genome [31–33]. This ORF1ab replicase gene encodes a set of NSPs that play a number of important roles in viral replication. This replicase gene encodes the overlapping polyproteins named pp1a and pp1ab, which are necessary for viral replication and transcription. The longer pp1ab is a 7073 amino acid-long polypeptide 3 of 32 Biomolecules 2020, 10, 1312 containing 15 non-structural proteins. NSP1, NSP2, and NSP3 are released from polyprotein via proteolytic processing using a viral papain-like proteinase (NSP3/PLPro), whereas the rest of NSPs are cleaved by another viral 3C-like proteinase—NSP5/3CLPro or main protease Mpro—that utilizes 11 or more conserved sites to digest the polyprotein. This digestion starts with an autocatalytic cleavage of this enzyme itself from pp1a and pp1ab. 2. Intrinsic Viral Factors Furthermore, the presence of the two capping loops in the RBD of the SARS-CoV-2 spike protein is likely to produce a higher stabilization eﬀect over the interaction with the cellular receptor. These two loops around the RBD of SARS-CoV-2 might promote interaction with the ACE2 receptor, improving the binding to the ACE2 by increasing the number of groups involved. Therefore, these amino acid substitutions and the longer capping loops could explain the increase in the binding aﬃnities in SARS-CoV-2 compared to SARS-CoV. These higher values of aﬃnity might be related to the higher dynamics of the infection and the rapid spread observed for this virus [53]. This is in line with the outputs of the computational analysis showing that when all the residues favoring interaction of the CoV S protein with human ACE2 would be combined into one RBD, this RBD would bind to ACE2 with super aﬃnity, and the corresponding spike protein would mediate viral entry into human cells with super eﬃciency [54]. p g p p y p y Furthermore, SARS-CoV-2 uses the transmembrane protease serine 2 (TMPRSS2, also known as serine protease 10) for the viral spike glycoprotein priming, a process crucial for the viral entry [55]. In fact, host TMPRSS2 priming of the S glycoprotein causes irreversible conformational changes and activation of the S2 subunit, thereby facilitating the fusion of the virus to the cell membrane. The virus with the processed S protein then enters the cell [56,57]. Importantly, S protein of SARS-CoV-2 contains a polybasic cleavage site (RRAR) at the junction of S1 and S2 [51,52,58,59], which deﬁnes the eﬀective cleavage by furin and other proteases and has a role in determining viral infectivity and host range [60]. The presence of this unique furin cleavage site within the SARS-CoV-2 spike protein, which is a novel feature setting this virus apart from SARS-CoV, and the almost ubiquitous expression of furin-like proteases could participate in expanding cell and tissue tropism of SARS-CoV-2 and increasing transmissibility and/or altering pathogenicity of this virus [51,52,58,59]. While S2 facilitated the fusion step after proteolysis by TMPRSS2 and furin proteases in a sequential pattern [51,52], there is also evidence suggesting that these enzymes are not the exclusive players in priming S protein for the eﬃcient COVID-19 entry. 2. Intrinsic Viral Factors Therefore, the SARS-CoV-2 S protein acts on both protein and 9-O-acetylated sialic acid-containing receptors, with the receptor-bind domain (RBD) being involved in ACE2 receptor recognition, and the NTD being responsible for ﬁnding a ganglioside-rich landing area (lipid raft) at the cell surface [46]. It was hypothesized that the interaction of S protein with the lipid rafts deﬁnes an adequate positioning of the viral S protein at the ﬁrst step of the infection process [46]. Importantly, the evolutionary analysis revealed that the ganglioside-binding subdomain (residues 111–162) of the NTD is completely conserved in 11 clinical isolates of SARS-CoV-2 of various geographic origins. Furthermore, this subdomain is also completely conserved in the bat coronavirus RaTG13, but noticeable variability is detected in other bat SARS-like and human SARS-CoVs, suggesting that higher levels of SARS-CoV-2 contagiousness in comparison with previously characterized HCoVs can be attributed to recent evolution [46]. Just a few weeks after the ﬁrst reports on COVID-19 infection, it was revealed that the virus enters the lung alveolar type II (AT2) via the ACE2, which is expressed on the surfaces of the heart, kidneys, intestine, and lung alveolar epithelial cells. Here, a speciﬁc role is played by the spike glycoprotein S. In fact, the S glycoproteins of coronaviruses have two subunits—S1 and S2. The S1 subunit binds to the ACE2 enzyme, via its receptor-binding domain (RBD), on the cell membrane [47,48], and S2 fuses with the cell membrane [49]. Although the genome of SARS-CoV-2 shares 79.6% sequence identity to SARS-CoV, and although SARS-CoV-2 is capable of using the same cell entry receptor (ACE2) as SARS-CoV to infect humans [21,50], the aﬃnity of SARS-CoV-2 spike protein to the human ACE2 is 4 of 32 Biomolecules 2020, 10, 1312 ~10–20 fold higher than that of the SARS-CoV spike protein [51,52]. This is because of the presence of the distinctive structural diﬀerences between the receptor-binding domains (RBDs) of the spike proteins from SARS-CoV and SARS-CoV-2, which represent energetically favorable changes in the amino acid sequence for the more eﬃcient interaction of the SARS-CoV-2 spike protein with the ACE2 receptor. In fact, the local environment within the ACE2 receptor allows SARS-CoV-2-speciﬁc residues in the RBD of the spike protein to make a signiﬁcant number of electrostatic stabilizing interactions. 2. Intrinsic Viral Factors For example, this protease is known to cleave the subunits of the epithelial sodium channel (ENaC, which is also known as the amiloride-sensitive sodium channel) [64]. ENaC is a heterotrimer of three homologous subunits α or δ, β, and γ, which can be found at the apical membranes of epithelial cells of many tight epithelia of the airway, kidney, and lung. Such plasmin-induced cleavage of the ENaC subunits promotes the ﬂow of Na+ ions into the epithelial cells, leading to the dehydration of the air-facing surface of the lungs and alveoli, which is normally lined by a thin ﬁlm of liquid, and hypertension [64]. Plasmin is a potent protease that cleaves the human γ ENaC subunit at 16 sites, including the cleavage sites of trypsin, chymotrypsin, prostasin, and elastases [66]. Signiﬁcant harm is induced by the uncontrolled proteolysis of these proteins, which are highly expressed on epithelial cells, considered as the major pathways for Na+ entry, and play important roles in maintaining the proper depth of airway and alveolar lining ﬂuids, the reabsorption of edema ﬂuid in injured lungs, and the regulation of salt retention in the collecting tubules [64,67–74]. Of note, the renin-angiotensin system (RAS) is mainly known to regulate blood pressure and Na+ reabsorption via its roles in maintaining blood pressure homeostasis [75] and salt and ﬂuid balance [76]. The role of plasmin in the pathogenesis of other viruses is rather well established. For example, it is known that plasmin cleaves the inﬂuenza virus hemagglutinin (HA) proteins to enable fusion with the target host endosome [77–82]. Besides, the plasminogen (ﬁbrinolytic zymogen, the precursor of plasmin) has been shown to cleave the inﬂuenza HA proteins [82–84]. The cleavage of HA from the A/WSN/1933 H1N1 inﬂuenza virus governs the virus spread in a plasmin-dependent manner [83]. In addition, the replication of both plasmin-sensitive and plasmin-insensitive inﬂuenza A virus strains was shown to be enhanced by the plasmin fragment (mini-plasmin), which is preferentially found in the bronchiole epithelial cells, providing further support to the idea that plasmin has several crucial roles in the spread and pathogenicity of the inﬂuenza virus [80]. Furthermore, there is a place for other non-furin proteases in viral pathogenesis too. For example, HA proteins from the H1, H2, and H3 subtypes of the inﬂuenza virus are sensitive to kallikreins cleavage and can be activated by this protease [85]. 2. Intrinsic Viral Factors It is known that airway and alveolar type I and II epithelial cells are expressing other proteases, such as trypsin, kallikrein, and plasminogen, which are also expressed in endothelial cells and which might contribute to the priming of S glycoprotein. The possibility for non-furin proteases to cleave viral envelope proteins is supported by the evidence that the plasmin cleaves the S proteins of SARS-CoV in vitro [61]. Furthermore, S protein of HCoV-HKU1 is cleaved by kallikrein within the S1/S2 region and mediates the entry of HCoV-HKU1 to non-permissive rhabdomyosarcoma cells [62]. Altogether, the S protein of coronaviruses may be cleaved by plasmin, trypsin, cathepsins, elastase, and TMPRSS family members, with such cleavage of S protein mediating the enhancement of the virus entry into the bronchial epithelial cells [61]. One should keep in mind, though, that since the currently available information on the roles of plasmin and other non-furin proteases in cleavage of SARS-CoV in vivo is rather limited, the clinical relevance of such non-furin cleavage is not strictly established. Furthermore, the capability of plasmin to cleave the SARS-CoV-2 envelope proteins remains to be demonstrated [63]. Meanwhile, there is evidence indicating the presence of at least some interplay between SARS-CoV-2 and plasmin. In fact, the enhanced plasmin(ogen) levels and resulting alterations in the ﬁbrin D-dimer levels are the common features observed in the COVID-19 patients [64]. Plasmin proteolytically breaks down excess ﬁbrin and elevates levels of D-dimer (which is a cross-linked dimer of the two smallest ﬁbrin degradation products, with increased D-dimer levels indicating increased ﬁbrinolysis or inability to clear the products from the circulation, and with D-dimer assays being commonly used in clinical practice [65]) and other ﬁbrin degradation products in both bronchoalveolar lavage ﬂuid and plasma, which decreases platelets and results in hemorrhage [64]. Clinical data showed that in the COVID-19 5 of 32 Biomolecules 2020, 10, 1312 patients, the lungs are the most injured organs, followed by the moderate injury in the heart, liver, kidney, and brain. Systemic microthrombi in the circulatory system and hemorrhage in the aﬀected organs result from the miscoordinated responses between the coagulation and ﬁbrinolysis systems [64]. Coagulation and hemorrhage rank among the top three leading causes of COVID-19-associated death [58]. In addition, elevated levels of plasmin can be related to some other pathological conditions. 2. Intrinsic Viral Factors In fact, an additional layer of complexity has been added to the interplay between the CoV S protein and host proteases by the observations that not only S of SARS-CoV-2 but also its receptor, ACE2, is proteolytically processed. ACE2 is known to be shed into the extracellular space upon cleavage by the sheddase ADAM17/TACE (disintegrin and metalloproteinase domain-containing protein 17 or tumor necrosis factor (TNF)-alpha-converting enzyme) [93,97,98]. ADAM17 is a 610-residue-long protein that was initially described in 1997 by Black et al. to speciﬁcally cleave the precursor of the tumor necrosis factor-α (pro-TNF-α) [99,100]. ACE2 shedding by ADAM17 was ﬁrst described by Lambert et al., when they studied human HEK293 cells (embryonic kidney cells) expressing human ACE2 (HEK-ACE2) in 2005 [98,99]. In 2008, Haga et al. demonstrated that binding of S protein from SARS-CoV also induced ACE2 shedding by ADAM17 and provided evidence that the ACE2 shedding is important for the uptake of SARS-CoV into the target cells [101]. The up-regulation of ACE2 shedding by ADM17 may inhibit the infectivity of the SARS-CoV [98,99]. Furthermore, it was demonstrated that an ADAM17 inhibitor displays modest antiviral activity in SARS-CoV-infected mice [102]. Furthermore, it was found that TMPRSS2 competes with the metalloprotease ADAM17 for ACE2 processing, but the only cleavage by TMPRSS2 resulted in the augmented SARS-S-driven entry [93]. Since the ACE2 expression levels within the main COVID-19 target, lungs, is relatively low, some researchers suggested that there could be some co-receptors needed for the SARS-CoV-2 entry [103]. Using single-cell RNA sequencing of 13 human tissues, it was established that ANPEP (alanyl aminopeptidase), ENPEP (glutamyl aminopeptidase), and DPP4 (dipeptidyl peptidase-4) are the top three genes correlated with ACE2 [103]. It is known that both ANPEP (which is a membrane-bound broad speciﬁcity aminopeptidase) and DPP4 (which is a cell surface glycoprotein receptor) can serve as receptors for HCoVs [104], whereas the involvement of the membrane-bound peptidase ENPEP in virus infection is unclear [103]. One should also keep in mind that human coronaviruses regularly use peptidases as their receptors [48]. ANPEP is the targeted receptor for many viruses belonging to the Coronaviridae family, such as porcine epidemic diarrhea virus, HCoV-229E, feline coronavirus, canine coronavirus, transmissible gastroenteritis virus, and infectious bronchitis virus. It is mainly expressing in the colon, ileum, rectum, kidney, liver, and skin [103], demonstrating that the receptor of coronavirus may have similar expression proﬁles in the human body. 2. Intrinsic Viral Factors Similar to CoVs and inﬂuenza viruses, plasmin, trypsin, thrombin, and furin were shown to enhance cytopathology induced by a respiratory syncytial virus (RSV) [86]. Curiously, the cleavage of a target protein by diﬀerent proteases may enhance or decrease its activities. For example, prostasin (which is a serine protease with trypsin-like substrate speciﬁcity that is found in the prostate gland, kidney, bronchi, colon, liver, lung, pancreas, and salivary glands) increases the activity (60–80%) of human ENaC, whereas TMPRSS2 markedly decreases ENaC function and protein levels [87]. Similarly, plasmin is capable of cleaving the subunit of human ENaC at the furin sites [64,88], which may increase the patient complications and subsequently promote viral vertical (and maybe horizontal) tissue tropism and transmissibility [64,89]. TMPRSS2, TMPRSS4, TMPRSS11A, and HAT (human airway tryptase) belong to the type II transmembrane serine proteases (TTSP) family, which includes 19 members, and most of them are expressed in the human respiratory tract [90]. These TTSP can cleave and activate inﬂuenza A virus hemagglutinin as well as S proteins of CoVs for host cell entry [91,92]. A comprehensive study detected extensive coexpression of ACE2, TMPRSS2, and HAT in the epithelia of the aerodigestive tract, although exceptions were noted, including the epithelia of the trachea, vocal folds, and epiglottis [92]. Therefore, TMPRSS2 and HAT are present in major viral target cells and could promote viral spread in infected humans [93]. Both enzymes were shown to cleave and activate the HCoV-229E S-protein for cathepsin L-independent virus-cell fusion [93]. Furthermore, TMPRSS2 and HAT were shown to 6 of 32 Biomolecules 2020, 10, 1312 activate all inﬂuenza virus subtypes previously pandemic in humans [94,95], and TMPRSS4 was found to activate the HA protein of the 1918 inﬂuenza virus [96]. These observations on the roles of various non-furin proteases in the pathogenesis of diﬀerent viruses raise important questions, such as: Can the plasmin increase the pathogenicity of COVID-19 by cleaving the SARS-CoV-2 S glycoprotein extracellularly, and thereby modulating the ability of this protein to interact with ACE2 receptors of host cells and probably facilitating virus entry and fusion? Can the elevated plasmin(ogen) levels in patients with some pre-existing conditions be considered as one of the avenues for the enhanced susceptibility to SARS-CoV-2 infection and fatality? There are also some other players from the host protease realm that can contribute to the COVID-19 pathogenesis. 2. Intrinsic Viral Factors Are these data consistent with the fact that CoVs infect similar types of cells and CoV-infected patients share similar clinical symptoms [103]? Some reports discussed the non-peptidase SARS-CoV receptors as potential avenues for the COVID-19 entry to the host cells. Among such SARS-CoV receptors are DC-SIGN1 (dendritic cell-speciﬁc intercellular adhesion molecule (ICAM)-3-grabbing non-integrin 1), CLEC4G (C-type lectin domain family 4 member G), and CLEC4M (C-type lectin domain family 4 member M) [103,105,106]. Furthermore, SARS-CoV-2 may also use integrins as cell receptors via binding to them through a conserved RGD motif (Arg-Gly-Asp, residues 403–405) that is exclusively present in SARS-CoV-2, being absent from other coronaviruses [107]. Curiously, the RGD motif is used by various human non-CoV viruses to interact with their receptors, proteins from the integrin family [108]. Among such human viruses utilizing RGD motifs in their binding to integrins are human adenovirus type 2/5 [109], coxsackievirus A9 [110], human metapneumovirus (HMPV) [111,112], Epstein–Barr virus (EBV, also known as human herpesvirus type 4 (HHV-4)) [113], human cytomegalovirus (HCMV, also known 7 of 32 Biomolecules 2020, 10, 1312 as human herpesvirus type 5 (HHV-5)) [114], Kaposi’s sarcoma-associated virus (HHV-8) [108], and rotavirus (RV) [115]. It is known that the RNA enveloped viruses are using extracellular vesicles (exosomes) to translocate into new host cells [116–118]. These vesicles enable the viruses to infect cells in both receptor-dependent and receptor-independent manner and promote viral persistence. They modulate the host immune response, transport populations of viral particles and genomes, increase multiplicities of the ways of viral infection, facilitate cooperative interactions, and enhance the viral replicative ﬁtness [116]. Is SARS-CoV-2 (which is an enveloped RNA virus) follow this pathway to cellular entry and to propagate very quickly? If so, is it dependent or independent on receptor entry? Are there any additional factors that would be increasing the virus entry into the cell? In line with these considerations, we proposed recently that a cellular transport pathway associated with the release of the SARS-CoV-2-loaded exosomes and other extracellular vesicles might represent potential mechanisms for the relapse of the COVID-19 infection [119]. Utilization of such a “Trojan horse” strategy provides SARS-CoV-2 with means to hide viral material within such exosomes or extracellular vesicles during the “silence” time, followed by the re-appearance of the viral RNA in the recovered and discharged COVID-19 patients [119]. 2. Intrinsic Viral Factors In the search for the additional receptor for SARS-CoV cellular entry, SARS pseudovirus or HCoV-NL63 [120,121] were used to explore the possibility of additional routes of the viral entry. It was found that the SARS virus used both S spike and membrane (M) proteins for interaction with common cellular receptors, heparan sulfate proteoglycans (HSPGs), which are present on most cells [122]. These results demonstrated that HSPGs could serve as adhesion receptors that provide the binding sites for SARS-CoV invasion at the early attachment phase. HSPG blockage results in the failure of SARS virus entry even in the presence of the internalization factor ACE2 [123]. From this perspective, it is important to note that lactoferrin/lactotransferrin (LTF) is known to co-localize with the widely distributed cell-surface HSPGs [124–126]. SARS-CoV infection activates a host immune response against the virus, where an essential role in the inhibition of the viral infection is played by the innate immune response. In fact, the infection causes up-regulation of several innate immune response-related genes, such as LFT, S100A9, and LCN2, with their corresponding proteins (lactoferrin, S100A9, and lipocalin 2) being involved in the SARS-CoV clearance. As an example, in comparison with the healthy controls, the SARS patients typically showed a 150-fold increase in the LTF expression [127]. This is an important observation since lactoferrin is known for its broad virucidal activity, being able to play a role in the suppression of a wide variety of RNA and DNA viruses, such as cytomegalovirus, echovirus, herpes simplex virus, hepatitis C virus, human immunodeﬁciency virus, human papillomavirus, human polyomavirus, rotavirus, Semliki forest virus, and Sindbis virus [124–126]. The entrance of these diﬀerent viruses into the host cells depends on interaction with common receptors located on the surface of the cells. Among these common receptors that provide the ﬁrst anchoring sites on the cell surface and thereby promote primary contacts of the virus with the host cells [122] are HSPGs that are broadly distributed on the host cells [124–126]. Since lactoferrin can bind to HSPGs, leading to the eﬃcient inhibition of the internalization of some viruses [128], it was hypothesized that such molecular mechanisms could be responsible for the anti-SARS-CoV eﬀects of this protein [120,121]. Is it possible that the SARS-CoV-2 can use a similar entry pathway and utilize HSPGs as its host cell receptors? 2. Intrinsic Viral Factors Finally, there is compelling evidence that CoVs can use multiple pathways to enter the host cell (see Figure 1). In one scenario, the entry of SARS-CoV into cells might occur by direct fusion of envelopes with the plasma membrane at the cell surface [129–131]. However, this virus can also take advantage of the endocytic machinery of the target cell. Here, SARS-CoV enters cells by endosomal pathways, where the S protein is activated for fusion by trypsin-like protease in an acidic endosomal environment [130]. The endocytic pathways used by viruses to get into the host cells include macropinocytosis, clathrin-dependent endocytosis, and caveolae-dependent endocytosis, as well as clathrin- and caveolae-independent endocytosis [132,133]. It was pointed out that in the most cases, only one of these pathways is used by a given virus to enter cells, and some viruses might 8 of 32 Biomolecules 2020, 10, 1312 use multiple endocytic pathways to gain entry into host cells [134–137], with one of these viruses being SARS-CoV [138]. Furthermore, there is also a possibility for the non-endosomal entry of a virus into the host cell. Here, proteases, such as trypsin and thermolysin, promote SARS-CoV cell entry directly from the site where this virus is adsorbed onto the cell surface [139]. Furthermore, protease-mediated SARS-CoV entry from the cell surface was shown to result in a 100- to 1000-fold more eﬃcient infection than entry through endosome [139]. Therefore, SARS-CoV can enter cells via clathrin- and caveolae-independent endocytic pathway or by the non-endosomal pathways that depend on the presence of the proteases [139]. It is known that SARS-CoV-2, which is an enveloped RNA virus, follows this non-endosomal pathway of cellular entry [140]. Figure 1. Suggested scenarios for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cellular entry pathways and their potential eﬀects on the viral load and transmission capability. Figure 1. Suggested scenarios for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cellular entry pathways and their potential eﬀects on the viral load and transmission capability. 3. Human (Host) Factors The outcome of SARS-CoV-2 infection is primarily deﬁned by the virus-host interaction, with transmissibility and pathogenicity of SARS-CoV-2 being related to its interplay with host antiviral defense [141]. The ﬁrst requirements for the successful COVID-19 transmission are the susceptible host with a permissive cell, which carries its receptor. If all these requirements are met, then other factors (such as the receptor orientation, distribution, and structure) will come to play, deﬁning the capabilities of viral particles to be distributed vertically (within the host tissues) and horizontally (within the host population). All this could underhandedly help the virus to be more aggressive (virulent). p p y p gg Often, the viruses emerging from more resistant hosts have lower overall virulence than viruses emerging from more susceptible hosts. There is correlative evidence supporting the link between the host resistance and virulence evolution [142–144]. For example, since virulent strains can be favored over avirulent pathogen strains as a result of the within-host competition, resistant hosts may limit competitive interactions between co-infecting pathogens, thereby hampering the evolution of virulence [145]. The largest adaptive responses in a viral pathogen are achieved via the serial passage of the virus through resistant vs. susceptible hosts, and such adaptive responses are often linked to the most dramatic increases in virulence [146]. It is also possible that the optimal environment for virus adaptation is provided by the hosts with intermediate levels of immunity. This is because such individuals represent an appropriate environment for the optimization of both the pathogen population size and the strength of the immune-mediated selection [147]. All the accumulated data indicate that 9 of 32 Biomolecules 2020, 10, 1312 SARS-CoV-2 may gain some adaptation and enhanced virulence, which globally contributes to its pathogenicity and transmission. p g y Adaptive T cell immune responses play important roles in the pathogenesis of infectious disease and long-term protective immunity as well as in the development of eﬀective vaccines and therapeutics. The importance of the adaptive T cell immune responses is in the capability of memory T cells induced by the previous pathogens to become activated in the course of new infection with an unrelated heterologous virus, and these memory T cells might be related to the protective immunity and immunopathology [148]. To control the virus, the priming and expansion of the adaptive T cell immune responses are required, and these processes typically take 7–10 days [149]. 3. Human (Host) Factors Viral clearance and capability to reduce the severity of symptoms represent the basis for the T cell-based partial protection against many of acute viral infections, including inﬂuenza [150–152]. In ten COVID-19 patients placed to an intensive care unit, the presence of SARS-CoV-2-speciﬁc cluster of diﬀerentiation 4 and 8 (CD4+ and CD8+) T cells was reported, with the spike surface glycoprotein generating the strongest T-cell responses, and with such SARS-CoV-2-speciﬁc T cells predominantly producing Th1 and eﬀector cytokines [153]. The SARS-CoV-2-speciﬁc T cells appeared relatively early, and their level increased over time [153]. Curiously, two out of 10 healthy control subjects with no previous exposure to SARS-CoV-2 were shown to also possess low levels of SARS-CoV-2-reactive T cells, suggesting the presence of some cross-reactivity with other human ‘common cold’ causing CoVs [153]. These ﬁndings were further validated in an independent study comparing 16 healthy control donors with 42 COVID-19 patients, including 28 mild and 14 severe cases [154]. This study found that in comparison with mild COVID-19 cases, patients with severe cases were characterized by the signiﬁcantly higher frequency, breadth, and magnitude of memory T cell responses, with the most notable responses being generated by the spike, membrane, and ORF3a proteins [154]. Based on the analysis of the T cell responses against the structural (nucleocapsid protein N) and non-structural (NSP7 and NSP13) proteins of SARS-CoV-2 in 36 individuals recovering from COVID-19, the capability of CD4 and CD8 T cells to recognize multiple regions of the N protein was pointed out [155]. Although there is no information on the duration of the adaptive T cell immune responses against SARS-CoV-2, the recent analysis of the patients recovered from SARS-CoV (i.e., 17 years after the outbreak of SARS in 2003) showed to possess long-lasting memory T cells reactive to the N protein of SARS-CoV [155]. Furthermore, these memory T cells showed vigorous cross-reactivity to the SARS-CoV-2 N protein [155]. Finally, SARS-CoV-2-speciﬁc T cells were found in 37 uninfected donors as well. In these individuals with no history of SARS, COVID-19, or known contacts with SARS and/or COVID-19 patients, the SARS-CoV-2-speciﬁc T cells possessed a diﬀerent pattern of immunodominance, being capable of recognition of NSP7 and NSP13 [155]. g ACE2 represents the conﬁrmed protein receptor for the SARS-CoV-2 entry into the host cells. 3. Human (Host) Factors p ) y p p [ ] To address the role of SARS-CoV-2 tropism in the eﬃciency of COVID-19 transmission, Sungnak et al. looked at the single-cell transcriptome expression data in scRNA-seq datasets from diﬀerent tissues, such as the respiratory tree, ileum, colon, liver, placenta/decidua, kidney, testis, pancreas, and prostate gland of healthy donors [166]. This analysis revealed that TMPRSS2, the primary protease important for SARS-CoV-2 entry, is highly expressed in diﬀerent tissues, whereas the SARS-CoV-2 entry receptor ACE2 is characterized by relatively low expression levels in all the tissues analyzed [166]. These ﬁndings indicated that at the initial stage of infection, ACE2, and not TMPRSS2, represents a limiting factor for viral entry [166]. The authors also showed that ACE2 is more highly expressed (and co-expressed with viral entry-associated protease TMPRSS2) in nasal epithelial cells, speciﬁcally in a goblet and ciliated cells. This important ﬁnding explains an apparent contradiction between the rapid spread of the SARS-CoV-2 and the dependency of this virus on alveolar epithelial cells as the primary point of entry and viral replication. The fact that the SARS-CoV-2 entry receptor ACE2 is more highly expressed and co-expressed with the viral entry-associated protease TMPRSS2 in nasal epithelial cells indicates that these cells can serve as loci of original SARS-CoV-2 infection and also act as possible reservoirs for virus dissemination within a given patient and from person to person [166]. It was also pointed that reported data describe the peculiarities of ACE2 expression in various tissues of healthy donors and that the gene expression landscape in the nose and other tissues can be drastically changed in the course of viral infection [166]. Furthermore, since in addition to lung and airways, ACE2 is expressed in the ileum, colon, and kidney [166], other modes of COVID-19 transmission, which involve intestine, kidney, testis, and other tissues, should be considered. Special attention should be paid to the intestines, which express the highest level of ACE2. Earlier studies demonstrated that diarrhea was present in up to 70% of patients infected with SARS-CoV [167]. Furthermore, a recent case report demonstrated the presence of SARS-CoV-2 in the feces of a COVID-19 patient with an initial diarrhea episode [168]. Similar ﬁndings have been reported in other studies, indicating that tests of feces and urine samples for the presence of SARS-CoV-2 are warranted [169]. 3. Human (Host) Factors The susceptibility of diﬀerent cohorts of patients to SARS-CoV-2 is correlated with the ACE2 level, and the distribution of target organs that are susceptible to the SARS-CoV-2 infection and the spread of COVID-19-related complications are similar to that of the ACE2 [156]. In fact, entry of the SARS-CoV-2 into the lung alveolar type 2 (AT2) cells is determined by the presence of this receptor. Although ACE2 is reported to be expressed in lung AT2 cells, liver cholangiocyte, colon colonocytes, esophagus keratinocytes, ileal epithelial cells (ECs), rectum ECs, stomach ECs, testis, gallbladder cells, and kidney proximal tubules, its expressing levels are rather low, especially in the lung AT2 cells, where the ACE2 expression levels are 4.7-fold lower than the average expression levels of all ACE2 expressing cell types [103,157]. AT2 cells are considered as alveolar stem cells [158]. They comprise only 5% of the alveoli, but produce the surfactant, a factor essential to maintain lung elasticity, and, most importantly, act as progenitors for AT1 cells, the latter covering 95% of the alveoli and responsible for gas exchange. Therefore, SARS-CoV-2 that targets AT2 cells attacks and kills the lung regenerative pool. Depletion in the AT2 cells and corresponding deﬁcit of surfactants have been previously shown to be associated with the incomplete repair of injured alveolar epithelium and ﬁbrotic obliteration [159]. Therefore, these mechanisms could also explain the development of lung injury in COVID-19 [160]. The low 10 of 32 Biomolecules 2020, 10, 1312 expression of ACE2 in the lung may also suggest the presence of selected cells with up-regulated ACE2 expression under certain conditions. In fact, obese young patients showing increased ACE2 expression in lung epithelial cells are typically characterized by the increased severity of COVID-19 [161,162]. On the other hand, relative to the upper airway epithelial cells, the human olfactory epithelium shows higher levels of the expressed ACE2 protein, suggesting that the initial site of SARS-CoV-2 infection is the upper, rather than the lower, airway [163]. These important ﬁndings provide an explanation for the COVID-19-associated olfactory dysfunction, such as common disturbances in the sense of smell, which were reported in 85% COVID-19 patients who participated in a large, multicenter European survey [164]. Furthermore, the lower prevalence of COVID-19 children can be explained (at least in part) by the lower levels of ACE2 expression in the nasal epithelium of children relative to adults [165]. 3. Human (Host) Factors Another important question is whether the ACE polymorphism can serve as one of the factors promoting the high eﬃciency of the COVID-19 spread? Besides serving as a CoV receptor, ACE2 plays an important role in the regulation of the renin-angiotensin-aldosterone system (RAAS), which includes a cascade of vasoactive peptides, which coordinates key processes in human physiology and maintains plasma sodium concentration, arterial blood pressure, and extracellular volume [170]. Angiotensin I is a physiologically inactive decapeptide derived from angiotensinogen by the action of renin. It serves as a precursor for an octapeptide angiotensin II, which is the main RAAS eﬀector that acts as an agonist for both angiotensin II receptors type 1 and type 2 (AT1R and AT2R, respectively). Angiotensin II is generated from angiotensin I by the action of ACE1. Angiotensin II is converted, by ACE2, to the heptapeptide angiotensin-(1–7), which is a vasodilator. ACE2 also converts angiotensin I to the nonapeptide angiotensin-(1–9), which is further processed by ACE1 to generate angiotensin-(1–7) that serves as an antagonist for the AT1R receptors and an agonist for the MAS1 receptor (also known as proto-oncogene Mas). Therefore, in RAAS, ACE2 acts as an inhibitor by cleaving a single residue from angiotensin I to generate angiotensin-(1–9) and via degrading angiotensin II to the 11 of 32 Biomolecules 2020, 10, 1312 angiotensin-(1–7) [171]. Therefore, down-regulation or depletion of ACE2 results in the distortions of the angiotensin II levels, which are linked to an overwhelming number of chronic and acute diseases [170]. SARS-CoV-2 infection down-regulates ACE2 expression, leading to the subsequent elevation of the plasma angiotensin II levels, which, in turn, correlate with the total viral load and deterioration of lung tissues [75,172]. In fact, plasma of the COVID-19 patients was shown to contain signiﬁcant levels of angiotensin II when compared with healthy individuals [173]. Importantly, in addition to ACE2, ACE1 may also be related to the eﬃcient spread of COVID-19. In fact, it is known that circulating and tissue concentrations of ACE1 can be altered by a genetic deletion/insertion (D/I) polymorphism in intron 16 of the ACE1 gene, with the D allele being associated with reduced expression of ACE2 [174]. Based on the analysis of the D-allele frequency of the ACE1 gene in samples from 25 diﬀerent European countries, Delanghe et al. 3. Human (Host) Factors concluded that 38% of the variability of the COVID-19 prevalence could be attributed to the relative frequency of the ACE1 D-allele and that there is a signiﬁcant correlation between COVID-19-associated mortality and the prevalence of the ACE1 D-allele [174]. These data suggest that ACE1 D/I polymorphism may be regarded as a confounder in the spread of COVID-19 [174]. These observations are in agreement with the known role of ACE1 in pulmonary infections caused by coronaviruses [175]. Therefore, the ACE1 D/I genotype may aﬀect the clinical course of the infection. In contrast to this conclusion, the analysis of the ACE2 genomic structure revealed that some allelic variants of this gene would potentially oﬀer resistance against SARS-CoV-2 [176]. To address an issue of the ACE2 multifunctionality that not only serves as a SARS-CoV-2 receptor but also acts as a key RAAS component participating in the generation of a multitude of vasoactive peptides coordinating several physiological processes, we recently conducted a comprehensive bioinformatics analysis of the predisposition of major players related to the SARS-CoV-2-AAS axis to intrinsic disorder and showed that all these proteins contain functional intrinsically disordered regions [177]. These observations represent a unique protein intrinsic disorder-based view of the RAAS-SARS-CoV-2 interplay and indicate the importance of the consideration of the intrinsic disorder phenomenon [177]. p An important feature of SARS-CoV-2 is the ability of this virus to be transmitted from human to household pets (speciﬁcally cats and dogs) [178–187], indicating that such susceptibility of domesticated animals to SARS-CoV-2 would increase the transmissibility of this virus and worsen the infection-related situation because these pets and other domestic animals are almost in constant contact with family members and especially with the children [188–190]. It is known that the ACE2 is expressed in most vertebrates, and not all ACE2 can be equally eﬃciently utilized by SARS-CoV-2 as the receptors. It was also pointed out that not all pets are equally susceptible to SARS-CoV-2, with chimpanzees and monkeys being the most sensitive to this infection, and with mice being shown to be the least susceptible to SARS-CoV-2 [178,191]. Although previous studies were focused on the structural part of the interactions between the SARS-CoV-2 spike protein and the ACE2 proteins from diﬀerent organisms, a diﬀerent approach was utilized in a recent study, where the intrinsic disorder predispositions ACE2 proteins from diﬀerent species were compared [190]. 3. Human (Host) Factors In CoVs, such a high frequency of homologous recombination, which can reach the level of 25% through the entire CoV genome [201], can be attributed to the commonly observed discontinuous RNA synthesis [202]. Epidemic outbreaks caused by the pathogenic HCoVs, such as HCoV-OC43 [44], HCoV-NL63 [27], SARS-CoV [27,203,204], and MERS-CoV [205], are reported to be characterized by frequent genomic rearrangements of HCoVs. It should be mentioned that the S protein of SARS-CoV is the most divergent viral protein in all strains infecting humans [206,207]. The variations arise quickly in both C- and N-terminal domains of S protein, providing important means for the immunological escape [208]. Furthermore, the N-terminal region of S protein hosts a recombination hot-spot, indicating the genomic instability of SARS-CoV-2 over the poly-A and poly-U regions [192]. Often, the progress of infection is associated with virus adaptation to host environments. Variants of the same virus can diﬀer in disease potential (virulence) [209,210]. The COVID-19 tropism based on gender is a controversy. In fact, one study linked COVID-19 infection and transmission power to gender [211], whereas other researchers did not ﬁnd any dependency of ACE2 expression on gender on a single cell level [140], suggesting that the inter- and intra-gender viral transmission is equally eﬃcient until this moment. However, the situation is completely diﬀerent when comparing the patient susceptibility and the eﬃciency of COVID-19 transmission based on age (see Figure 2). It has been suggested that diﬀerential levels of ACE2 in the cardiac and pulmonary tissues of younger versus older adults maybe, at least, partially responsible for the spectrum of disease virulence observed among patients with COVID-19 [212]. Persons older than 60 years with chronic diseases, such as hypertension, diabetes, chronic obstructive pulmonary disease (COPD), as well as cardiovascular, cerebrovascular, liver, kidney, and gastrointestinal diseases, are more susceptible to the infection by SARS-CoV-2 and experience higher mortality when they develop COVID-19 [64,213,214]. In addition, patients older than 65 years generally have higher viral load lasting up to 14 days [215] in comparison to the younger patients, who have a much lower viral load that is undetectable within 1 week after onset [216]. The association between the viral load and the severity of COVID-19 has been reported [217]. Collectively, it seems the older people are more susceptible than younger people to COVID-19 hijacking, which may make them better hosts for virus passage. 3. Human (Host) Factors Based on this comparative intrinsic disorder predisposition analysis of the ACE2 proteins from diﬀerent organisms, it was concluded that despite the overall rather high similarity between the resulting disorder proﬁles, there is a noticeable diﬀerence between these proteins in the disorder predispositions of their N-terminal regions (residues 19–83) involved in the interaction with the SARS-CoV-2 S protein [190]. These observations suggested that the aﬃnity of ACE2-protein S interaction could be, at least in part, determined by the local peculiarities of the intrinsic disorder distribution within the S protein-binding region of ACE2 [190]. These data also provide important indications that the analysis of the intrinsic disorder predisposition in ACE2 can help to predict which species could be infected with SARS-CoV-2 via the ACE2 binding rout and, therefore, could serve as an intermediate host in the transmission of this virus [190]. It was recently indicated that, at least in part, the COVID-19 success in transmission could be attributed to the intra-host genomic diversity and plasticity of SARS-CoV-2 and its ability to 12 of 32 Biomolecules 2020, 10, 1312 form low-frequency polymorphic quasispecies [192,193]. This may mean three things [194]: (i) The presence of such viral quasispecies characterized by some sequence diversity can be responsible for the diﬀerences in coping with innate host defenses, packaging, replication kinetics, translation eﬃciency, and response to the antiviral therapies. (ii) The genetic diversity of such viral quasispecies that entered the cytoplasm could be responsible for their genetic cooperation, resulting in an increase in viral replication eﬃciency. (iii) Under selection pressure, population ﬁtness can be enhanced via the group cooperation among the viral quasispecies, with such group cooperation being frequently seen when the number of infecting viral particles between passages is high [195]. The structure and dynamics of quasispecies of replicating RNA enable virus populations to persist in their hosts and cause disease. In fact, there is a critical interplay between the host and virus mutual inﬂuences (including, in some cases, the quasispecies organization), which represent the main driving force for the long-term survival of viruses in nature. The stability of virus particles may also play a relevant role in successful transmission [196]. The presence of quasispecies has previously been reported for SARS-CoV and MERS-CoV [193,197,198]. It is known that the substantial genetic diversity of RNA viruses is driven by recombination events [199,200]. 3. Human (Host) Factors Generally, older persons, and especially those with chronic illness, are more susceptible to COVID-19. In fact, while many younger people experience no or mild symptoms of infection, older adults are highly susceptible to life-threatening respiratory and systemic conditions [218]. It seems that there are many factors deﬁning why older people are more susceptible to COVID-19 and experience higher mortality when they develop COVID-19. 13 of 32 Biomolecules 2020, 10, 1312 Figure 2. Suggested scenarios for the coronavirus disease 2019 (COVID-19) pathogenicity in old and young patients. Figure 2. Suggested scenarios for the coronavirus disease 2019 (COVID-19) pathogenicity in old and young patients. In fact, although aging is associated with many changes, one of the most pronounced transformations is the decline of the immune system, aﬀecting both the innate and adaptive immune responses [219,220]. The process of chronological aging is known to aﬀect various components of the immune response, leading to impaired host defense, defective vaccine responses, and a signiﬁcantly higher risk of elderly persons developing life-threatening bacterial infections [219,221,222]. Aging aﬀects all immune cells, including hematopoietic stem cells (HSCs), which maintain the immune system by producing all blood cells throughout the lifetime of an organism [223]. There are also age-related changes in the T cell compartment that are characterized by three main hallmarks: (i) Decrease in the number of naïve T cells related to the thymic involution [224,225]. (ii) Shrinking of the T-cell receptor (TCR) repertoire that determines antigenic diversity broadness and thus preconditions the successful elimination of pathogens from the system [226]. (iii) Increased proportion of the terminally diﬀerentiated oligoclonal eﬀector memory T-cell population, especially those related to the control of persistent viral infections [227]. In old age, there is a decrease in the number and/or frequency as well as delay in the generation of the antigen-speciﬁc CD4 and CD8 T cell responses [228]. This generates a signiﬁcant disturbance of a link between the early innate immune response and the recruitment of the antigen-speciﬁc T cells to the site of infection. Furthermore, the overall population of memory CD8 T cells is known to signiﬁcantly change with age. Even though the total percentage of memory CD8 T cells is increased with age, the diversity of the repertoire of the naïve and memory CD8 T cell receptors is noticeably reduced in old age [229–231]. 3. Human (Host) Factors These changes in the immune system with age are allied with the poor immune responses of aged hosts to vaccines and viral infections [232–234]. Besides, in old humans, the number of peripheral B cells decreases, and the antigen-recognition repertoire of B cells and optimal pro-inﬂammatory cytokines production is altered [235]. As a consequence of the decreased generation of early progenitor B cells, the output of new naïve B cells is reduced [236,237], and, consequently, the longevity of the antigen-experienced memory B cells is increased [237]. Since class-switch recombination is impaired in memory B cells with aging [237,238], this may also contribute to the decline of the quality of humoral immune response [239]. The production of higher aﬃnity protective antibodies in elderly individuals is impaired [240] due to the age-associated down-regulation of the activation-induced cytidine deaminase (AID), which is the enzyme for class switching, and its transcription factor E47 [241,242]. All these alterations can be related to the increased susceptibility of elderly people to infection with various pathogens [243,244]. Furthermore, as individuals age, they experience an increase in basal inﬂammation [245], which is now recognized as a global phenomenon known as inﬂammaging [246]. Inﬂammatory 14 of 32 Biomolecules 2020, 10, 1312 cytokines, including TNF and interleukin 6 (IL-6), are associated with increased risk for many diseases, including sarcopenia, osteoarthritis, and many infectious diseases [247–249]. The elderly are more susceptible to many infections, from those that are commonly diagnosed (inﬂuenza and pneumococcal pneumonia) [250,251] to those considered more exotic (such as anthrax and SARS) [248,252], due to their poor response to and control of infectious agents [253]. There are also some other age-related changes that can contribute to the increased susceptibility to infection. The NLRP3 (NACHT, LRR, and PYD domains-containing protein 3, where NACHT reﬂects a set of proteins containing this domain, e.g., (NLP family apoptosis inhibitor protein), CIITA (that is, C2TA or MHC class II transcription activator), HET-E (incompatibility locus protein from Podospora anserina) and TEP1 (that is, TP1 or telomerase-associated protein), whereas LRR and PYD stay for leucine-rich repeat and pyrin domain, respectively) inﬂammasome is a multiprotein complex consisting of the nucleotide-binding domain leucine-rich repeat-containing (NLR) family member NLRP3, the adaptor protein ASC (an apoptosis-associated speck-like protein containing a caspase recruitment domain (CARD) domain, also known as PYD and CARD domain-containing protein), and the cysteine protease caspase 1 [254]. 3. Human (Host) Factors The NLRP3 inﬂammasome can activate caspase 1 in response to cellular danger, resulting in the processing and secretion of proinﬂammatory cytokines—IL1β and IL18 [255–257]. Many studies reported high IL18 and IL1β levels in SARS, MERS, and COVID-19 patients, not only in the blood but also in lungs and lymphoid tissues, indicating the increased inﬂammasome activation. Maturation of IL1β (interleukin-1β) is achieved through the proteolytic cleavage of pro-IL1β by caspase 1, activation of which requires the formation of the NLRP3 inﬂammasome. When danger signals are sensed in the cells, NLRP3 is activated to recruit ASC and facilitate its oligomerization. For the full activation of the inﬂammasome, two signals are needed. The ﬁrst of these signals stimulates the pro-IL1β transcription, whereas the second signal leads to the pro-IL1β cleavage [258]. p p g p g A diverse array of stimuli can activate the NLRP3 inﬂammasome, including both pathogen-associated molecular patterns (PAMPs) and endogenous host-derived molecules indicative of cellular damage [259,260]. NLRP3 inﬂammasome responses are tightly regulated [261]. Using aged murine models of infection (inﬂuenza A virus (A/PR/8/1934(H1N1)), it was demonstrated that aged mice within 48 h post-secondary Streptococcus pneumoniae infection possessed increased morbidity and mortality. Increased susceptibility of aged mice was associated with decreased Toll-like receptors 1, 6, and 9 (TLR1, TLR6, and TLR9, respectively) mRNA expression and diminished IL1β mRNA expression. Examination of NLRP3 inﬂammasome expression illustrated decreased NLRP3 mRNA expression and decreased IL1β production in the aged lung in response to secondary S. pneumoniae infection [261]. Hoegen et al. used a pneumococcal meningitis model to demonstrate that the NLRP3 inﬂammasome could contribute to the increased host pathology instead of pathogen protection and clearance [262]. NLRP3 inﬂammasome is believed to be one of the major pathophysiologic components in the clinical course of patients with COVID-19 [263,264]. It has been shown that the NLRP3 inﬂammasome serves an important instrument in the development of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) [265]. It was also demonstrated that SARS-CoV viroporins (i.e., viral proteins with ion channel activity) E protein, ORF3a, and ORF8A act as ion-conductive pores in planar lipid bilayers and are required for maximal SARS-CoV replication and virulence [266]. Furthermore, there are data showing that these three proteins provoke the activation of the NLRP3 inﬂammasome [263]. 3. Human (Host) Factors For example, it was recently shown that the SARS-CoV ORF3a protein activates the NLRP3 inﬂammasome in lipopolysaccharide-primed macrophages by aﬀecting K+ eﬄux and mitochondrial reactive oxygen species [267]. Another study showed that the SARS-CoV ORF3a accessory protein activates the NLRP3 inﬂammasome by promoting the TNF receptor associated factor 3 (TRAF3)-mediated ubiquitination of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) [268]. Although the ORF8 protein of SARS-CoV-2 does not contain known functional domain or motifs, an aggregation motif VLVVL (residues 75–79) has been found in SARS-CoV ORF8B, which was shown to trigger intracellular stress pathways and activate the NLRP3 inﬂammasomes. However, this motif is apparently absent in ORF8 of the SARS-CoV-2 [264,269]. 15 of 32 15 of 32 Biomolecules 2020, 10, 1312 Apart from the cytokine storm observed in patients infected by the highly pathogenic HCoVs, other cell death programs, such as apoptosis and necrosis, might also contribute to the pathogenesis. Cell death is a double-edged sword that can play both antiviral and proviral roles during viral infection [270]. For example, ORF8a from the SARS-CoV was shown to trigger cellular apoptosis [271]. It was shown that the largest of the SARS-CoV accessory proteins, ORF3a, shares membrane insertion characteristics and channel functionality with necrotic eﬀector molecules and interacts with receptor-interacting protein 3 (Rip3), which augments the oligomerization of ORF3a, causing causes necrotic cell death, lysosomal damage, and caspase-1 activation [272]. Apoptosis was detected in various HCoV-infected samples derived from not only the respiratory tract but also from the extrapulmonary sites [273]. Autopsy studies of SARS-CoV-infected tissues revealed the presence of apoptosis in the lung, spleen, and thyroid [274,275]. The apoptosis induced by SARS-CoV is caspase-dependent and could be inhibited by the Bcl2 overexpression or using the caspase inhibitors [276,277]. In 293 of ACE2 cells infected with SARS-CoV, several apoptosis-associated events were activated [278], namely cleavage of caspase-3, caspase-8, and poly(ADP-ribose) polymerase 1 (PARP), phosphorylation and inactivation of the eukaryotic translation initiation factor 2α (eIF2α), leading to the chromatin condensation, as well as activation of protein kinase R (PKR) and PKR-like endoplasmic reticulum kinase (PERK) [278]. Furthermore, HCoV-induced apoptosis was reported for several immune cells, such as macrophages, monocytes, T lymphocytes, and dendritic cells [279]. Infection of primary T lymphocytes by MERS-CoV induced DNA fragmentation and caspase 8 and 9 activation, indicating that, in this case, both extrinsic and intrinsic apoptotic pathways were activated [280]. 3. Human (Host) Factors Furthermore, MERS-CoV infection was shown to induce pyroptosis (which is a lytic and inﬂammatory mode of regulated cell death catalyzed by the caspase family) and over-activation of complement (which is an ancient molecular cascade that, being a part of the immune system, enhances the clearance potential of antibodies and phagocytic cells against microbes and damaged cells, as well as promotes inﬂammation and regulates attack at the membrane of pathogenic cells) in human macrophages [281]. The physical environment of the lung may also contribute to the eﬃciency of viral transmission. In fact, the elderly are more susceptible to many infections due to the aging-related changes in this environment [282,283], such as decreased strength of respiratory muscles, reduced lung elasticity, and lowered vital capacity [283]. As a result of all these changes, the expulsion of infectious agents through breathing, cough reﬂex, or sneezing is impaired. This is further complicated by the increased probability of the ﬂuid and/or solid aspiration into the lungs, as well as age-associated inﬂammatory diseases, such as pulmonary ﬁbrosis or chronic obstructive pulmonary disease (COPD) [284]. In fact, it was emphasized that both susceptibility to SARS-CoV-2 and severity of COVID-19 are systematically increased in the patients with COPD [285]. Alveolar epithelial cells (AETs) are responsible for the generation, secretion, and recycling of the lung mucosa or alveolar lining ﬂuid (ALF), which is crucial for the correct lung maintenance [286]. Senescence of the AETs in the aged individuals is associated with the decreased lung recycling [286] that might lead to the inﬂammatory response in the lung tissue [287], which represents a part of the chronic low-grade inﬂammation that develops with advanced age and is known as systemic inﬂammaging [288]. These considerations imply that in old age, ALF might be characterized by an elevated inﬂammatory proﬁle. In agreement with this hypothesis, signiﬁcantly increased levels of TNF, IL-6, IL-1β, and other inﬂammatory cytokines were found in pulmonary ﬂuids of aged humans [289]. Such increased inﬂammation within the lung mucosa is strongly connected to the speciﬁc changes in various innate molecular defense mechanisms. For example, ALF from elderly human subjects contained increased levels of the components of the complement system (e.g., complement C3β chain) and surfactant proteins A and D (SP-A, SP-D) [289]. Among diﬀerent factors potentially aﬀecting the susceptibility to SARS-CoV-2 and changing the outcomes and mortality amongst COVID-19 patients are smoking and vaping [285,290–292]. 3. Human (Host) Factors This is in line with the well-known general correlation between smoking and increased prevalence and mortality of infectious diseases [291], and with the fact that many COPD patients are smokers [285]. One should keep in mind, though, that existing data on the prevalence of smokers among COVID-19 patients and 16 of 32 16 of 32 Biomolecules 2020, 10, 1312 on the association between the COVID-19 outcomes and smoking are rather contradictory [290]. In fact, although some studies showed that smokers are more susceptible to COVID-19, and smoking is associated with more severe disease outcomes [293], several other studies pointed out the underrepresentation of active smokers among the COVID-19 patients [290] and indicated that active smoking is not associated with the COVID-19 severity [294]. Since these observations of smokers being protected from infection and severe complications of COVID-19 contradict the known association between morbidity and mortality of respiratory infections and cigarette smoking, the existence of a ‘smoker’s paradox’ in COVID-19 was proposed [290]. Among the possible molecular mechanisms of such protection are inhibition of SARS-CoV-2 entry into cells and replication caused by the smoking-induced increase in the nitric oxide levels in the respiratory tract, anti-inﬂammatory eﬀects of nicotine, and reduced risk of a cytokine storm in COVID-19 associated with the dampened immune response in smokers [290]. However, systematic analysis of the existing literature pointed out that many results used in support of the smoker’s paradox-related claims are questionable and limited, indicating that extreme caution should be used while considering the protective eﬀects of active smoking against COVID-19 [290]. As a continuation of the discussion of a link between smoking and COVID-19, it was shown that the lung and oral epithelial tissue samples of smokers are characterized by the up-regulation of ACE2 and TMPRSS2, which are the SARS-CoV-2 receptor and the transmembrane protease needed for the virus entry into host cells, respectively [295]. Importantly, this ACE2 and TMPRSS2 up-regulation was also associated with the up-regulation of the androgen pathway, suggesting that the smoking-mediated increased activity of the androgen signaling pathway itself and up-regulation of the central regulators of androgen pathways (e.g., HDAC6, CTNNB1, and SMARCA4) paired with the increased ACE2 and TMPRSS2 expression could represent a mechanism for the increased susceptibility of smokers to SARS-CoV-2 [295]. 3. Human (Host) Factors Importantly, the opportunity for SARS-CoV-2 infection of being androgen-mediated [296] via the androgen receptor-TMPRSS2 link, where the transcription of the TMPRSS2 is controlled by the androgen receptor activity [297], can represent a mechanistic explanation for the known sex-related diﬀerences in the COVID-19 vulnerability and lethality, with males typically being more susceptible to the infection [298–300]. This also suggests that androgen deprivation therapy, leading to the reduction of the TMPRSS2 expression, thereby limiting SARS-CoV-2 cellular entry, could potentially protect against severe complications from COVID-19 [301–303]. p y p g p We conclude this overview of the pathogenic pathways and transmission potentials of HCoVs by considering an interplay between epigenetics and the coronavirus infection. This short section complements the description of molecular mechanisms regulating the pathogenesis of the emerging coronaviruses, which are complex processes that include virus–host interactions associated with the entry, egress, innate immune regulation, and control of various types of programmed cell death. Epigenetics studies how the genetic and non-genetic factors can regulate phenotypic variation. Typically, epigenetic eﬀects are caused by external and environmental factors that alter host expression patterns and performance without any change in the underlying genotype. Therefore, epigenetic regulation links genotype and phenotype by promoting changes in the function of the gene locus without aﬀecting the sequence of the underlying DNA. Some of the most common epigenetic modiﬁcations include chromatin remodeling, DNA methylation, histone modiﬁcations, and non-coding RNAs. These factors act as important regulators of the remodeling of host chromatin and alter host expression patterns and networks in a highly ﬂexible manner. It was pointed out that viruses are able to regulate the host epigenome via a set of highly evolved, intricate, and well-coordinated processes, aiming at promotion of the robust virus replication and pathogenesis [304]. Some of these viral mechanisms to disturb and antagonize epigenetic regulatory programs of the host include interference with the histone modiﬁcation enzymes of the host [305], interference with the chromatin remodeling machinery [306], and the presence of viral proteins that directly bind to the modiﬁed histones of the host [307,308]. 3. Human (Host) Factors For example, it was shown that the highly pathogenic H3N2 inﬂuenza A virus interferes with the epigenetic control of the gene expression to inhibit the initiation of the host innate immune response using histone 17 of 32 17 of 32 Biomolecules 2020, 10, 1312 mimicry (the C-terminal region of viral NS1 protein mimics the H3 histone tail and interacts with the transcription complex) [309,310]. SARS-CoV and MERS-CoV were shown to delay and/or antagonize pathogen recognition by successfully delaying interferon (IFN)-stimulated gene response [311]. This was achieved by modulation of the histone modiﬁcations (such as enrichment in H3K27me3 and depletion in H3K4me3) for a subset of genes, favoring a closed chromatin conformation that inhibits interferon-stimulated gene (ISG) expression [304,311]. In patients with systemic lupus erythematosus, who already have elevated ACE2 levels due to the hypomethylation and overexpression of ACE2, oxidative stress induced by SARS-CoV-2 infection resulted in exacerbation of these lupus-induced DNA methylation defects, leading to further ACE2 hypomethylation accompanied by the overexpression of ACE2 and enhanced viremia [312]. 4. Concluding Remarks Data collected in this review clearly indicate that SARS-CoV-2 uses multiple ways for eﬃcient transmission. It has a virion structure optimized for various environmental conditions, allowing this virus to use both respiratory and fecal-oral transmission modes. Its S protein has an amended structure for eﬃcient interaction with the ACE2 receptor and is optimized for furin cleavage. Furthermore, S protein can be primed and activated by TMPRSS2, furin, and multiple non-furin proteases (e.g., plasmin). In addition to ACE2, SARS-CoV-2 can interact with other cellular peptidase receptors, such as ANPEP and DPP4, and also can utilize non-peptidase receptors, such as DC-SIGN1, CLEC4G, and CLEC4M. SARS-CoV-2 utilizes multiple ways for cellular entry (both non-endosomal and endosomal) and potentially uses various means of epigenetic control to inhibit the initiation of the host innate immune response. During the course of the pandemic, this CoV eﬃciently undergoes genomic rearrangements, thereby developing important means for the immunological escape. SARS-CoV-2 is engaged in intricate interplay with various host systems and pathways. It initiates cytokine storm and promotes various cell death programs, such as pyroptosis, apoptosis, and necrosis, which might contribute to the COVID-19 pathogenesis. This remarkably broad spectrum of means for the eﬃcient SARS-CoV-2 transmission indicates that it is very unlikely that COVID-19 can be cured by targeting just one segment of this complex mosaic. A better understanding of various molecular mechanisms associated with all stages of SARS-CoV-2 infection is needed for ﬁnding the most appropriate approaches for COVID-19 prevention and treatment. Author Contributions: Conceptualization, F.E., E.M.R., and V.N.U.; Literature collection and analysis, F.E., E.M.R., and V.N.U.; Writing—Original Draft Preparation, F.E., E.M.R., and V.N.U.; Writing—Review and Editing, F.E., E.M.R., and V.N.U.; Visualization, E.M.R. and V.N.U.; Supervision, E.M.R. and V.N.U. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Funding: This research received no external funding. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. Author Contributions: Conceptualization, F.E., E.M.R., and V.N.U.; Literature collection and analysis, F.E., E.M.R., and V.N.U.; Writing—Original Draft Preparation, F.E., E.M.R., and V.N.U.; Writing—Review and Editing, F.E., E.M.R., and V.N.U.; Visualization, E.M.R. and V.N.U.; Supervision, E.M.R. and V.N.U. All authors have read and agreed to the published version of the manuscript. 3. Woo, P.C.; Lau, S.K.; Chu, C.M.; Chan, K.H.; Tsoi, H.W.; Huang, Y.; Wong, B.H.; Poon, R.W.; Cai, J.J.; Luk, W.K.; et al. Characterization and complete genome sequence of a novel coronavirus, coronavirus HKU1, from patients with pneumonia. J. Virol. 2005, 79, 884–895. [CrossRef] [PubMed] References 1. Bruckova, M.; McIntosh, K.; Kapikian, A.Z.; Chanock, R.M. The adaptation of two human coronavirus strains (OC38 and OC43) to growth in cell monolayers. Proc. Soc. Exp. Biol. Med. 1970, 135, 431–435. [CrossRef] [PubMed] 2. Zhu, Y.; Li, C.; Chen, L.; Xu, B.; Zhou, Y.; Cao, L.; Shang, Y.; Fu, Z.; Chen, A.; Deng, L.; et al. A novel human coronavirus OC43 genotype detected in mainland China. Emerg. Microbes Infect. 2018, 7, 173. [CrossRef] [PubMed] 3. Woo, P.C.; Lau, S.K.; Chu, C.M.; Chan, K.H.; Tsoi, H.W.; Huang, Y.; Wong, B.H.; Poon, R.W.; Cai, J.J.; Luk, W.K.; et al. Characterization and complete genome sequence of a novel coronavirus, coronavirus HKU1, from patients with pneumonia. J. Virol. 2005, 79, 884–895. [CrossRef] [PubMed] 4. Hierholzer, J.C. Puriﬁcation and biophysical properties of human coronavirus 229E. Virology 1976, 75, 155–165. [CrossRef] 18 of 32 18 of 32 Biomolecules 2020, 10, 1312 5. Kaye, H.S.; Ong, S.B.; Dowdle, W.R. Detection of coronavirus 229E antibody by indirect hemagglutination. Appl. Microbiol. 1972, 24, 703–707. [CrossRef] 6. Fouchier, R.A.; Hartwig, N.G.; Bestebroer, T.M.; Niemeyer, B.; de Jong, J.C.; Simon, J.H.; Osterhaus, A.D. A previously undescribed coronavirus associated with respiratory disease in humans. Proc. Natl. Acad. Sci. USA 2004, 101, 6212–6216. [CrossRef] 7. Van der Hoek, L.; Pyrc, K.; Jebbink, M.F.; Vermeulen-Oost, W.; Berkhout, R.J.; Wolthers, K.C.; Wertheim-van Dillen, P.M.; Kaandorp, J.; Spaargaren, J.; Berkhout, B. Identiﬁcation of a new human coronavirus. Nat. Med. 2004, 10, 368–373. [CrossRef] 8. Larson, H.E.; Reed, S.E.; Tyrrell, D.A. Isolation of rhinoviruses and coronaviruses from 38 colds in adults. J. Med. Virol. 1980, 5, 221–229. [CrossRef] 9. Bradburne, A.F.; Bynoe, M.L.; Tyrrell, D.A. Eﬀects of a “new” human respiratory virus in volunteers. Br. Med. J. 1967, 3, 767–769. [CrossRef] 10. McIntosh, K.; Dees, J.H.; Becker, W.B.; Kapikian, A.Z.; Chanock, R.M. Recovery in tracheal organ cultures of novel viruses from patients with respiratory disease. Proc. Natl. Acad. Sci. USA 1967, 57, 933–940. [CrossRef] 11. Almeida, J.D.; Tyrrell, D.A. The morphology of three previously uncharacterized human respiratory viruses 10. McIntosh, K.; Dees, J.H.; Becker, W.B.; Kapikian, A.Z.; Chanock, R.M. Recovery in tracheal organ cultures of novel viruses from patients with respiratory disease. Proc. Natl. Acad. Sci. USA 1967, 57, 933–940. [CrossRef] 11. Almeida, J.D.; Tyrrell, D.A. The morphology of three previously uncharacterized human respiratory viruses that grow in organ culture. J. Gen. Virol. 1967, 1, 175–178. [CrossRef] [PubMed] 2. Hamre, D.; Procknow, J.J. References A new virus isolated from the human respiratory tract. Proc. Soc. Exp. Biol. M 1966, 121, 190–193. [CrossRef] [PubMed] 13. Esper, F.; Weibel, C.; Ferguson, D.; Landry, M.L.; Kahn, J.S. Evidence of a novel human coronavirus that is associated with respiratory tract disease in infants and young children. J. Infect. Dis. 2005, 191, 492–498. [CrossRef] [PubMed] 14. Gerna, G.; Percivalle, E.; Sarasini, A.; Campanini, G.; Piralla, A.; Rovida, F.; Genini, E.; Marchi, A.; Baldanti, F. Human respiratory coronavirus HKU1 versus other coronavirus infections in Italian hospitalised patients. J. Clin. Virol. 2007, 38, 244–250. [CrossRef] 15. Gerna, G.; Campanini, G.; Rovida, F.; Percivalle, E.; Sarasini, A.; Marchi, A.; Baldanti, F. Genetic variability of human coronavirus OC43-, 229E-, and NL63-like strains and their association with lower respiratory tract infections of hospitalized infants and immunocompromised patients. J. Med. Virol. 2006, 78, 938–949. [CrossRef] [PubMed] 16. Geller, C.; Varbanov, M.; Duval, R.E. Human coronaviruses: Insights into environmental resistance and its inﬂuence on the development of new antiseptic strategies. Viruses 2012, 4, 3044–3068. [CrossRef] 17. Zumla, A.; Chan, J.F.; Azhar, E.I.; Hui, D.S.; Yuen, K.Y. Coronaviruses—Drug discovery and therapeutic options. Nat. Rev. Drug Discov. 2016, 15, 327–347. [CrossRef] 18. De Groot, R.J.; Baker, S.C.; Baric, R.; Enjuanes, L.; Gorbalenya, A.E.; Holmes, K.V.; Perlman, S.; Poon, L.; Rottier, P.J.M.; Talbot, P.J.; et al. Family Coronaviridae. In Virus Taxonomy: Classiﬁcation and Nomenclature of Viruses: Ninth Report of the International Committee on Taxonomy of Viruses; King, A., Adams, M., Carstens, E.B., Lefkowitz, E.J., Eds.; Elsevier: Amsterdam, The Netherlands; Academic Press: Boston, MA, USA, 2012; pp. 806–820. 19. Drexler, J.F.; Corman, V.M.; Drosten, C. Ecology, evolution and classiﬁcation of bat coronaviruses in the aftermath of SARS. Antivir. Res. 2014, 101, 45–56. [CrossRef] 20. Andersen, K.G.; Rambaut, A.; Lipkin, W.I.; Holmes, E.C.; Garry, R.F. The proximal origin of SARS-CoV-2. Nat. Med. 2020, 26, 450–452. [CrossRef] 21. Zhou, P.; Yang, X.L.; Wang, X.G.; Hu, B.; Zhang, L.; Zhang, W.; Si, H.R.; Zhu, Y.; Li, B.; Huang, C.L.; et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 2020, 579, 270–273. [CrossRef] 22. Benvenuto, D.; Giovanetti, M.; Ciccozzi, A.; Spoto, S.; Angeletti, S.; Ciccozzi, M. The 2019-new coronavirus epidemic: Evidence for virus evolution. J. Med. Virol. 2020, 92, 455–459. [CrossRef] [PubMed] 23. Perlman, S. Another Decade, Another Coronavirus. N. Engl. J. Med. 2020, 382, 760–762. [CrossRef] [PubMed] 23. Perlman, S. Another Decade, Another Coronavirus. N. Engl. J. References The 29-nucleotide deletion present in human but not in animal severe acute respiratory syndrome coronaviruses disrupts the functional expression of open reading frame 8. J. Virol. 2007, 81, 13876–13888. [CrossRef] [PubMed] 33. Khailany, R.A.; Safdar, M.; Ozaslan, M. Genomic characterization of a novel SARS-CoV-2. Gene Rep. 2020, 100682. [CrossRef] [PubMed] 34. Goh, G.K.; Dunker, A.K.; Foster, J.A.; Uversky, V.N. Shell disorder analysis predicts greater resilience of the SARS-CoV-2 (COVID-19) outside the body and in body ﬂuids. Microb. Pathog. 2020, 144, 104177. [CrossRef] [PubMed] 35. Goh, G.K.; Dunker, A.K.; Foster, J.A.; Uversky, V.N. Rigidity of the outer shell predicted by a protein intrinsic disorder model sheds light on the COVID-19 (Wuhan-2019-nCoV) infectivity. Biomolecules 2020, 10. [CrossRef] 36. De Groot, R.J. Structure, function and evolution of the hemagglutinin-esterase proteins of corona- and toroviruses. Glycoconj. J. 2006, 23, 59–72. [CrossRef] 37. Hause, B.M.; Collin, E.A.; Liu, R.; Huang, B.; Sheng, Z.; Lu, W.; Wang, D.; Nelson, E.A.; Li, F. Characterization of a novel inﬂuenza virus in cattle and Swine: Proposal for a new genus in the Orthomyxoviridae family. mBio 2014, 5, e00014–e00031. [CrossRef] 38. Matrosovich, M.; Herrler, G.; Klenk, H.D. Sialic acid receptors of viruses. Top. Curr. Chem. 2015, 367, 1–28. [CrossRef] 39. Vlasak, R.; Luytjes, W.; Spaan, W.; Palese, P. Human and bovine coronaviruses recognize sialic acid-containing receptors similar to those of inﬂuenza C viruses. Proc. Natl. Acad. Sci. USA 1988, 85, 4526–4529. [CrossRef] receptors similar to those of inﬂuenza C viruses. Proc. Natl. Acad. Sci. USA 1988, 85, 4526–4529. [CrossRef] 40. Wan, H.; Perez, D.R. Quail carry sialic acid receptors compatible with binding of avian and human inﬂuenza viruses. Virology 2006, 346, 278–286. [CrossRef] 40. Wan, H.; Perez, D.R. Quail carry sialic acid receptors compatible with binding of avian and human inﬂuenza viruses. Virology 2006, 346, 278–286. [CrossRef] 41. Traving, C.; Schauer, R. Structure, function and metabolism of sialic acids. Cell Mol. Life Sci. 1998, 54, 1330–1349. [CrossRef] 42. Desforges, M.; Desjardins, J.; Zhang, C.; Talbot, P.J. The acetyl-esterase activity of the hemagglutinin-esterase protein of human coronavirus OC43 strongly enhances the production of infectious virus. J. Virol. 2013, 87, 3097–3107. [CrossRef] 43. Bakkers, M.J.; Lang, Y.; Feitsma, L.J.; Hulswit, R.J.; de Poot, S.A.; van Vliet, A.L.; Margine, I.; de Groot-Mijnes, J.D.; van Kuppeveld, F.J.; Langereis, M.A.; et al. Betacoronavirus adaptation to humans involved progressive loss of hemagglutinin-esterase lectin activity. Cell Host Microbe 2017, 21, 356–366. [CrossRef] [PubMed] 44. References Med. 2020, 382, 760–762. [CrossRef] [PubMed] 24 Ge X Y; Li J L ; Yang X L ; Chmura A A ; Zhu G ; Epstein J H ; Mazet J K ; Hu B ; Zhang W ; Peng C ; 23. Perlman, S. Another Decade, Another Coronavirus. N. Engl. J. Med. 2020, 382, 760 762. [CrossRef] [PubMed] 24. Ge, X.Y.; Li, J.L.; Yang, X.L.; Chmura, A.A.; Zhu, G.; Epstein, J.H.; Mazet, J.K.; Hu, B.; Zhang, W.; Peng, C.; et al. Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor. Nature 2013, 503, 535–538. [CrossRef] [PubMed] 25. Haagmans, B.L.; Al Dhahiry, S.H.; Reusken, C.B.; Raj, V.S.; Galiano, M.; Myers, R.; Godeke, G.J.; Jonges, M.; Farag, E.; Diab, A.; et al. Middle East respiratory syndrome coronavirus in dromedary camels: An outbreak investigation. Lancet. Infect. Dis. 2014, 14, 140–145. [CrossRef] 19 of 32 19 of 32 Biomolecules 2020, 10, 1312 26. Vijgen, L.; Keyaerts, E.; Moes, E.; Thoelen, I.; Wollants, E.; Lemey, P.; Vandamme, A.M.; Van Ranst, M. Complete genomic sequence of human coronavirus OC43: Molecular clock analysis suggests a relatively recent zoonotic coronavirus transmission event. J. Virol. 2005, 79, 1595–1604. [CrossRef] 27. Lau, S.K.; Lee, P.; Tsang, A.K.; Yip, C.C.; Tse, H.; Lee, R.A.; So, L.Y.; Lau, Y.L.; Chan, K.H.; Woo, P.C.; et al. Molecular epidemiology of human coronavirus OC43 reveals evolution of diﬀerent genotypes over time and recent emergence of a novel genotype due to natural recombination. J. Virol. 2011, 85, 11325–11337. [CrossRef] 28. Woo, P.C.; Lau, S.K.; Huang, Y.; Yuen, K.Y. Coronavirus diversity, phylogeny and interspecies jumping. Exp. Biol. Med. 2009, 234, 1117–1127. [CrossRef] 29. Huynh, J.; Li, S.; Yount, B.; Smith, A.; Sturges, L.; Olsen, J.C.; Nagel, J.; Johnson, J.B.; Agnihothram, S.; Gates, J.E.; et al. Evidence supporting a zoonotic origin of human coronavirus strain NL63. J. Virol. 2012, 86, 12816–12825. [CrossRef] 30. Corman, V.M.; Baldwin, H.J.; Tateno, A.F.; Zerbinati, R.M.; Annan, A.; Owusu, M.; Nkrumah, E.E.; Maganga, G.D.; Oppong, S.; Adu-Sarkodie, Y.; et al. Evidence for an ancestral association of human coronavirus 229E with bats. J. Virol. 2015, 89, 11858–11870. [CrossRef] 31. Li, F.; Li, W.; Farzan, M.; Harrison, S.C. Structure of SARS coronavirus spike receptor-binding domain complexed with receptor. Science 2005, 309, 1864–1868. [CrossRef] 32. Oostra, M.; de Haan, C.A.; Rottier, P.J. References 2020, 19, 410–417. [CrossRef] 54. Wan, Y.; Shang, J.; Graham, R.; Baric, R.S.; Li, F. Receptor recognition by the novel coronavirus from Wuhan: An analysis based on decade-long structural studies of SARS coronavirus. J. Virol. 2020, 94. [CrossRef] 54. Wan, Y.; Shang, J.; Graham, R.; Baric, R.S.; Li, F. Receptor recognition by the novel coronavirus from Wuhan: An analysis based on decade-long structural studies of SARS coronavirus. J. Virol. 2020, 94. [CrossRef] 55. Hoﬀmann, M.; Kleine-Weber, H.; Schroeder, S.; Kruger, N.; Herrler, T.; Erichsen, S.; Schiergens, T.S.; Herrler, G.; Wu, N.H.; Nitsche, A.; et al. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell 2020. [CrossRef] [PubMed] 56. Glowacka, I.; Bertram, S.; Muller, M.A.; Allen, P.; Soilleux, E.; Pfeﬀerle, S.; Steﬀen, I.; Tsegaye, T.S.; He, Y.; Gnirss, K.; et al. Evidence that TMPRSS2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response. J. Virol. 2011, 85, 4122–4134. [CrossRef] [PubMed] [ ] [ ] 57. Heurich, A.; Hofmann-Winkler, H.; Gierer, S.; Liepold, T.; Jahn, O.; Pohlmann, S. TMPRSS2 and ADAM17 cleave ACE2 diﬀerentially and only proteolysis by TMPRSS2 augments entry driven by the severe acute respiratory syndrome coronavirus spike protein. J. Virol. 2014, 88, 1293–1307. [CrossRef] [PubMed] 58. Zhang, B.; Zhou, X.; Qiu, Y.; Feng, F.; Feng, J.; Jia, Y.; Zhu, H.; Hu, K.; Liu, J.; Liu, Z.; et al. Clinical characteristics of 82 death cases with COVID 19 medRxiv 2020 [CrossRef] 57. Heurich, A.; Hofmann-Winkler, H.; Gierer, S.; Liepold, T.; Jahn, O.; Pohlmann, S. TMPRSS2 and ADAM17 cleave ACE2 diﬀerentially and only proteolysis by TMPRSS2 augments entry driven by the severe acute respiratory syndrome coronavirus spike protein. J. Virol. 2014, 88, 1293–1307. [CrossRef] [PubMed] 58. Zhang, B.; Zhou, X.; Qiu, Y.; Feng, F.; Feng, J.; Jia, Y.; Zhu, H.; Hu, K.; Liu, J.; Liu, Z.; et al. Clinical characteristics of 82 death cases with COVID-19. medRxiv 2020. [CrossRef] 59. Yan, R.; Zhang, Y.; Li, Y.; Xia, L.; Guo, Y.; Zhou, Q. Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2. Science 2020, 367, 1444–1448. [CrossRef] 60. Nao, N.; Yamagishi, J.; Miyamoto, H.; Igarashi, M.; Manzoor, R.; Ohnuma, A.; Tsuda, Y.; Furuyama, W.; Shigeno, A.; Kajihara, M.; et al. Genetic predisposition to acquire a polybasic cleavage site for highly pathogenic avian inﬂuenza virus hemagglutinin. mBio 2017, 8. References Mubarak, A.; Alturaiki, W.; Hemida, M.G. Middle east respiratory syndrome coronavirus (MERS-CoV): Infection, immunological response, and vaccine development. J. Immunol. Res. 2019, 2019, 6491738. [CrossRef] [PubMed] 45. Verdecchia, P.; Cavallini, C.; Spanevello, A.; Angeli, F. The pivotal link between ACE2 deﬁciency and SARS-CoV-2 infection. Eur. J. Intern. Med. 2020. [CrossRef] 20 of 32 20 of 32 Biomolecules 2020, 10, 1312 46. Fantini, J.; Di Scala, C.; Chahinian, H.; Yahi, N. Structural and molecular modelling studies reveal a new mechanism of action of chloroquine and hydroxychloroquine against SARS-CoV-2 infection. Int. J. Antimicrob. Agents 2020, 105960. [CrossRef] [PubMed] 7. Li, F. Receptor recognition and cross-species infections of SARS coronavirus. Antivir. Res. 2013, 100, 246– [CrossRef] [PubMed] 48. Li, F. Receptor recognition mechanisms of coronaviruses: A decade of structural studies. J. Virol. 2015, 89, 1954–1964. [CrossRef] 49. Kuba, K.; Imai, Y.; Rao, S.; Gao, H.; Guo, F.; Guan, B.; Huan, Y.; Yang, P.; Zhang, Y.; Deng, W.; et al. A crucial role of angiotensin converting enzyme 2 (ACE2) in SARS coronavirus-induced lung injury. Nat. Med. 2005, 11, 875–879. [CrossRef] 50. Hui, D.S.; Azhar, E.I.; Madani, T.A.; Ntoumi, F.; Kock, R.; Dar, O.; Ippolito, G.; McHugh, T.D.; Memish, Z.A.; Drosten, C.; et al. The continuing 2019-nCoV epidemic threat of novel coronaviruses to global health—The latest 2019 novel coronavirus outbreak in Wuhan, China. Int. J. Infect. Dis. 2020, 91, 264–266. [CrossRef] 51. Wrapp, D.; Wang, N.; Corbett, K.S.; Goldsmith, J.A.; Hsieh, C.L.; Abiona, O.; Graham, B.S.; McLellan, J.S. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science 2020, 367, 1260–1263. [CrossRef] 52. Walls, A.C.; Park, Y.J.; Tortorici, M.A.; Wall, A.; McGuire, A.T.; Veesler, D. Structure, function, and anti of the SARS-CoV-2 spike glycoprotein. Cell 2020. [CrossRef] 53. Ortega, J.T.; Serrano, M.L.; Pujol, F.H.; Rangel, H.R. Role of changes in SARS-CoV-2 spike protein in the interaction with the human ACE2 receptor: An in silico analysis. EXCLI J. 2020, 19, 410–417. [CrossRef] 53. Ortega, J.T.; Serrano, M.L.; Pujol, F.H.; Rangel, H.R. Role of changes in SARS-CoV-2 spike protein in the interaction with the human ACE2 receptor: An in silico analysis. EXCLI J. 2020, 19, 410–417. [CrossRef] 54. Wan, Y.; Shang, J.; Graham, R.; Baric, R.S.; Li, F. Receptor recognition by the novel coronavirus from Wuhan: An analysis based on decade long structural studies of SARS coronavirus J Virol 2020 94 [CrossRef] interaction with the human ACE2 receptor: An in silico analysis. EXCLI J. References [CrossRef] 61. Kam, Y.W.; Okumura, Y.; Kido, H.; Ng, L.F.; Bruzzone, R.; Altmeyer, R. Cleavage of the SARS coronavirus spike glycoprotein by airway proteases enhances virus entry into human bronchial epithelial cells in vitro. PLoS ONE 2009, 4, e7870. [CrossRef] 62. Milewska, A.; Falkowski, K.; Kalinska, M.; Bielecka, E.; Naskalska, A.; Mak, P.; Lesner, A.; Ochman, M.; Urlik, M.; Potempa, J.; et al. Kallikrein 13: A new player in coronaviral infections. bioRxiv 2020. [CrossRef] 62. Milewska, A.; Falkowski, K.; Kalinska, M.; Bielecka, E.; Naskalska, A.; Mak, P.; Lesner, A.; Ochman, M.; Urlik, M.; Potempa, J.; et al. Kallikrein 13: A new player in coronaviral infections. bioRxiv 2020. [CrossRef] 63. Millet, J.K.; Whittaker, G.R. Host cell proteases: Critical determinants of coronavirus tropism and pathogenesis. Virus Res. 2015, 202, 120–134. [CrossRef] 63. Millet, J.K.; Whittaker, G.R. Host cell proteases: Critical determinants of coronavirus tropism and pathogenesis. Virus Res. 2015, 202, 120–134. [CrossRef] 64. Ji, H.L.; Zhao, R.; Matalon, S.; Matthay, M.A. Elevated plasmin(ogen) as a common risk factor for COVID-19 susceptibility. Physiol. Rev. 2020, 100, 1065–1075. [CrossRef] [PubMed] 65. Linkins, L.A.; Takach Lapner, S. Review of D-dimer testing: Good, bad, and ugly. Int. J. Lab. Hematol. 2017, 39 (Suppl. S1), 98–103. [CrossRef] 66. Zhao, R.; Ali, G.; Nie, H.G.; Chang, Y.; Bhattarai, D.; Su, X.; Zhao, X.; Matthay, M.A.; Ji, H.L. Plasmin improves oedematous blood-gas barrier by cleaving epithelial sodium channels. Br. J. Pharmacol. 2020. [CrossRef] [PubMed] 21 of 32 Biomolecules 2020, 10, 1312 21 of 32 67. Pavlov, T.S.; Staruschenko, A. Involvement of ENaC in the development of salt-sensitive hypertension. Am. J. Physiol. Ren. Physiol. 2017, 313, F135–F140. [CrossRef] 68. Matalon, S.; Bartoszewski, R.; Collawn, J.F. Role of epithelial sodium channels in the regulation of lung ﬂuid homeostasis. Am. J. Physiol. Lung Cell. Mol. Physiol. 2015, 309, L1229–L1238. [CrossRef] 69. Kleyman, T.R.; Kashlan, O.B.; Hughey, R.P. Epithelial Na(+) channel regulation by extracellular and intracellular factors. Annu. Rev. Physiol. 2018, 80, 263–281. [CrossRef] 70. Kone, B.C. Epigenetics and the control of the collecting duct epithelial sodium channel. Semin. Nephrol. 2013, 33, 383–391. [CrossRef] 71. Hanukoglu, I.; Hanukoglu, A. Epithelial sodium channel (ENaC) family: Phylogeny, structure-function, tissue distribution, and associated inherited diseases. Gene 2016, 579, 95–132. [CrossRef] 72. Eaton, D.C.; Helms, M.N.; Koval, M.; Bao, H.F.; Jain, L. The contribution of epithelial sodium channels to alveolar function in health and disease. Annu. Rev. Physiol. 2009, 71, 403–423. [CrossRef] [PubMed] 73. References Ding, Y.; Zhao, R.; Zhao, X.; Matthay, M.A.; Nie, H.G.; Ji, H.L. ENaCs as both eﬀectors and regulators of MiRNAs in lung epithelial development and regeneration. Cell. Physiol. Biochem. 2017, 44, 1120–1132. [CrossRef] [PubMed] 74. Boscardin, E.; Alijevic, O.; Hummler, E.; Frateschi, S.; Kellenberger, S. The function and regulation of acid-sensing ion channels (ASICs) and the epithelial Na(+) channel (ENaC): IUPHAR review 19. Br. J. Pharmacol. 2016, 173, 2671–2701. [CrossRef] [PubMed] 75. Forrester, S.J.; Booz, G.W.; Sigmund, C.D.; Coﬀman, T.M.; Kawai, T.; Rizzo, V.; Scalia, R.; Eguchi, S. Angiotensin II signal transduction: An update on mechanisms of physiology and pathophysiology. Physiol. Rev. 2018, 98, 1627–1738. [CrossRef] [PubMed] 76. Lin, Y.C.; Lin, J.W.; Wu, M.S.; Chen, K.C.; Peng, C.C.; Kang, Y.N. Eﬀects of calcium channel blockers comparing to angiotensin-converting enzyme inhibitors and angiotensin receptor blockers in patients with hypertension and chronic kidney disease stage 3 to 5 and dialysis: A systematic review and meta-analysis. PLoS ONE 2017, 12, e0188975. [CrossRef] [PubMed] 77. Berri, F.; Rimmelzwaan, G.F.; Hanss, M.; Albina, E.; Foucault-Grunenwald, M.L.; Le, V.B.; Vogelzang-van Trierum, S.E.; Gil, P.; Camerer, E.; Martinez, D.; et al. Plasminogen controls inﬂammation and pathogenesis of inﬂuenza virus infections via ﬁbrinolysis. PLoS Pathog. 2013, 9, e1003229. [CrossRef] y g 78. Goto, H.; Wells, K.; Takada, A.; Kawaoka, Y. Plasminogen-binding activity of neuraminidase determines the pathogenicity of inﬂuenza A virus. J. Virol. 2001, 75, 9297–9301. [CrossRef] 79. LeBouder, F.; Lina, B.; Rimmelzwaan, G.F.; Riteau, B. Plasminogen promotes inﬂuenza A virus replication through an annexin 2-dependent pathway in the absence of neuraminidase. J. Gen. Virol. 2010, 91, 2753–2761. [CrossRef] 80. Murakami, M.; Towatari, T.; Ohuchi, M.; Shiota, M.; Akao, M.; Okumura, Y.; Parry, M.A.; Kido, H. Mini-plasmin found in the epithelial cells of bronchioles triggers infection by broad-spectrum inﬂuenza A viruses and Sendai virus. Eur. J. Biochem. 2001, 268, 2847–2855. [CrossRef] 81. Su, H.; Yang, X.; Wang, S.; Shi, H.; Liu, X. Eﬀect of annexin II-mediated conversion of plasmin from plasminogen on airborne transmission of H9N2 avian inﬂuenza virus. Vet. Microbiol. 2018, 223, 100–106. [CrossRef] 82. Tse, L.V.; Marcano, V.C.; Huang, W.; Pocwierz, M.S.; Whittaker, G.R. Plasmin-mediated activation of pandemic H1N1 inﬂuenza virus hemagglutinin is independent of the viral neuraminidase. J. Virol. 2013, 87, 5161–5169. [CrossRef] 83. Sun, X.; Tse, L.V.; Ferguson, A.D.; Whittaker, G.R. Modiﬁcations to the hemagglutinin cleavage site control the virulence of a neurotropic H1N1 inﬂuenza virus. J. Virol. 2010, 84, 8683–8690. [CrossRef] [PubMed] 83. References Sun, X.; Tse, L.V.; Ferguson, A.D.; Whittaker, G.R. Modiﬁcations to the hemagglutinin cleavage site control the virulence of a neurotropic H1N1 inﬂuenza virus. J. Virol. 2010, 84, 8683–8690. [CrossRef] [PubMed] 84. Nagai, Y.; Klenk, H.D.; Rott, R. Proteolytic cleavage of the viral glycoproteins and its signiﬁcance for the virulence of Newcastle disease virus. Virology 1976, 72, 494–508. [CrossRef] the virulence of a neurotropic H1N1 inﬂuenza virus. J. Virol. 2010, 84, 8683 8690. [CrossRef] [PubMed] 84. Nagai, Y.; Klenk, H.D.; Rott, R. Proteolytic cleavage of the viral glycoproteins and its signiﬁcance for the virulence of Newcastle disease virus. Virology 1976, 72, 494–508. [CrossRef] 85. Hamilton, B.S.; Whittaker, G.R. Cleavage activation of human-adapted inﬂuenza virus subtypes by kallikrein-related peptidases 5 and 12. J. Biol. Chem. 2013, 288, 17399–17407. [CrossRef] [PubMed] 86. Dubovi, E.J.; Geratz, J.D.; Tidwell, R.R. Enhancement of respiratory syncytial virus-induced cytopathology by trypsin, thrombin, and plasmin. Infect. Immun. 1983, 40, 351–358. [CrossRef] [PubMed] 87. Donaldson, S.H.; Hirsh, A.; Li, D.C.; Holloway, G.; Chao, J.; Boucher, R.C.; Gabriel, S.E. Regulation of the epithelial sodium channel by serine proteases in human airways. J. Biol. Chem. 2002, 277, 8338–8345. [CrossRef] p [CrossRef] 22 of 32 Biomolecules 2020, 10, 1312 88. Zhang, H.; Penninger, J.M.; Li, Y.; Zhong, N.; Slutsky, A.S. Angiotensin-converting enzyme 2 (ACE2) as a SARS-CoV-2 receptor: Molecular mechanisms and potential therapeutic target. Intensive Care Med. 2020, 46, 586–590. [CrossRef] 89. Wang, D.; Hu, B.; Hu, C.; Zhu, F.; Liu, X.; Zhang, J.; Wang, B.; Xiang, H.; Cheng, Z.; Xiong, Y.; et al. Clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in Wuhan, China. Jama 2020. [CrossRef] 90. Antalis, T.M.; Bugge, T.H.; Wu, Q. Membrane-anchored serine proteases in health and disease. Prog. Mol. Biol. Transl. Sci. 2011, 99, 1–50. [CrossRef] 91. Zmora, P.; Hoﬀmann, M.; Kollmus, H.; Moldenhauer, A.S.; Danov, O.; Braun, A.; Winkler, M.; Schughart, K.; Pohlmann, S. TMPRSS11A activates the inﬂuenza A virus hemagglutinin and the MERS coronavirus spike protein and is insensitive against blockade by HAI-1. J. Biol. Chem. 2018, 293, 13863–13873. [CrossRef] 92. Bertram, S.; Heurich, A.; Lavender, H.; Gierer, S.; Danisch, S.; Perin, P.; Lucas, J.M.; Nelson, P.S.; Pohlmann, S.; Soilleux, E.J. Inﬂuenza and SARS-coronavirus activating proteases TMPRSS2 and HAT are expressed at multiple sites in human respiratory and gastrointestinal tracts. PLoS ONE 2012, 7, e35876. [CrossRef] 93. Simmons, G.; Zmora, P.; Gierer, S.; Heurich, A.; Pohlmann, S. References Proteolytic activation of the SARS-coronavirus spike protein: Cutting enzymes at the cutting edge of antiviral research. Antivir. Res. 2013, 100, 605–614. [CrossRef] 94. Bottcher, E.; Matrosovich, T.; Beyerle, M.; Klenk, H.D.; Garten, W.; Matrosovich, M. Proteolytic activation of inﬂuenza viruses by serine proteases TMPRSS2 and HAT from human airway epithelium. J. Virol. 2006, 80, 9896–9898. [CrossRef] [PubMed] 95. Bottcher-Friebertshauser, E.; Freuer, C.; Sielaﬀ, F.; Schmidt, S.; Eickmann, M.; Uhlendorﬀ, J.; Steinmetzer, T.; Klenk, H.D.; Garten, W. Cleavage of inﬂuenza virus hemagglutinin by airway proteases TMPRSS2 and HAT diﬀers in subcellular localization and susceptibility to protease inhibitors. J. Virol. 2010, 84, 5605–5614. [CrossRef] 96. Chaipan, C.; Kobasa, D.; Bertram, S.; Glowacka, I.; Steﬀen, I.; Solomon Tsegaye, T.; Takeda, M.; Bugge, T.H.; Kim, S.; Park, Y.; et al. Proteolytic activation of the 1918 inﬂuenza virus hemagglutinin. J. Virol. 2009, 83, 3200. [CrossRef] [PubMed] 97. Jia, H.P.; Look, D.C.; Tan, P.; Shi, L.; Hickey, M.; Gakhar, L.; Chappell, M.C.; Wohlford-Lenane, C.; McCray, P.B., Jr. Ectodomain shedding of angiotensin converting enzyme 2 in human airway epithelia. Am. J. Physiol. Lung Cell. Mol. Physiol. 2009, 297, L84–L96. [CrossRef] [PubMed] 98. Lambert, D.W.; Yarski, M.; Warner, F.J.; Thornhill, P.; Parkin, E.T.; Smith, A.I.; Hooper, N.M.; Turner, A.J. Tumor necrosis factor-alpha convertase (ADAM17) mediates regulated ectodomain shedding of the severe-acute respiratory syndrome-coronavirus (SARS-CoV) receptor, angiotensin-converting enzyme-2 (ACE2). J. Biol. Chem. 2005, 280, 30113–30119. [CrossRef] 99. Palau, V.; Riera, M.; Soler, M.J. ADAM17 inhibition may exert a protective eﬀect on COVID-19. Nephrol. Dial. Transplant. 2020. [CrossRef] 100. Black, R.A.; Rauch, C.T.; Kozlosky, C.J.; Peschon, J.J.; Slack, J.L.; Wolfson, M.F.; Castner, B.J.; Stocking, K.L.; Reddy, P.; Srinivasan, S.; et al. A metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells. Nature 1997, 385, 729–733. [CrossRef] 101. Haga, S.; Yamamoto, N.; Nakai-Murakami, C.; Osawa, Y.; Tokunaga, K.; Sata, T.; Yamamoto, N.; Sasazuki, T.; Ishizaka, Y. Modulation of TNF-alpha-converting enzyme by the spike protein of SARS-CoV and ACE2 induces TNF-alpha production and facilitates viral entry. Proc. Natl. Acad. Sci. USA 2008, 105, 7809–7814. [CrossRef] 102. Haga, S.; Nagata, N.; Okamura, T.; Yamamoto, N.; Sata, T.; Yamamoto, N.; Sasazuki, T.; Ishizaka, Y. TACE antagonists blocking ACE2 shedding caused by the spike protein of SARS-CoV are candidate antiviral compounds. Antivir. Res. 2010, 85, 551–555. [CrossRef] [PubMed] 103. Qi, F.; Qian, S.; Zhang, S.; Zhang, Z. Single cell RNA sequencing of 13 human tissues identify cell types and receptors of human coronaviruses. Biochem. Biophys. Res. References Commun. 2020. [CrossRef] [PubMed] 104. Peck, K.M.; Scobey, T.; Swanstrom, J.; Jensen, K.L.; Burch, C.L.; Baric, R.S.; Heise, M.T. Permissivity of dipeptidyl peptidase 4 orthologs to Middle East respiratory syndrome coronavirus is governed by glycosylation and other complex determinants. J. Virol. 2017, 91. [CrossRef] [PubMed] 23 of 32 23 of 32 Biomolecules 2020, 10, 1312 105. Marzi, A.; Gramberg, T.; Simmons, G.; Moller, P.; Rennekamp, A.J.; Krumbiegel, M.; Geier, M.; Eisemann, J.; Turza, N.; Saunier, B.; et al. DC-SIGN and DC-SIGNR interact with the glycoprotein of Marburg virus and the S protein of severe acute respiratory syndrome coronavirus. J. Virol. 2004, 78, 12090–12095. [CrossRef] 106. Wang, S.; Guo, F.; Liu, K.; Wang, H.; Rao, S.; Yang, P.; Jiang, C. Endocytosis of the receptor-binding do SARS-CoV spike protein together with virus receptor ACE2. Virus Res. 2008, 136, 8–15. [CrossRef] 107. Sigrist, C.J.; Bridge, A.; Le Mercier, P. A potential role for integrins in host cell entry by SARS-CoV-2. Antivir. Res. 2020, 177, 104759. [CrossRef] 108. Hussein, H.A.; Walker, L.R.; Abdel-Raouf, U.M.; Desouky, S.A.; Montasser, A.K.; Akula, S.M. Beyond RGD: Virus interactions with integrins. Arch. Virol. 2015, 160, 2669–2681. [CrossRef] 109. Wickham, T.J.; Filardo, E.J.; Cheresh, D.A.; Nemerow, G.R. Integrin alpha v beta 5 selectively promotes adenovirus mediated cell membrane permeabilization. J. Cell Biol. 1994, 127, 257–264. [CrossRef] 110. Williams, C.H.; Kajander, T.; Hyypia, T.; Jackson, T.; Sheppard, D.; Stanway, G. Integrin alpha v beta 6 is an RGD-dependent receptor for coxsackievirus A9. J. Virol. 2004, 78, 6967–6973. [CrossRef] 111. Wei, Y.; Zhang, Y.; Cai, H.; Mirza, A.M.; Iorio, R.M.; Peeples, M.E.; Niewiesk, S.; Li, J. Roles of the putative integrin-binding motif of the human metapneumovirus fusion (f) protein in cell-cell fusion, viral infectivity, and pathogenesis. J. Virol. 2014, 88, 4338–4352. [CrossRef] 112. Chang, A.; Masante, C.; Buchholz, U.J.; Dutch, R.E. Human metapneumovirus (HMPV) binding and infection are mediated by interactions between the HMPV fusion protein and heparan sulfate. J. Virol. 2012, 86, 3230–3243. [CrossRef] 113. Xiao, J.; Palefsky, J.M.; Herrera, R.; Berline, J.; Tugizov, S.M. The Epstein-Barr virus BMRF-2 protein facilitates virus attachment to oral epithelial cells. Virology 2008, 370, 430–442. [CrossRef] [PubMed] 114. Feire, A.L.; Koss, H.; Compton, T. Cellular integrins function as entry receptors for human cytomegalovirus via a highly conserved disintegrin-like domain. Proc. Natl. Acad. Sci. USA 2004, 101, 15470–15475. [CrossRef] [PubMed] 115. Zarate, S.; Romero, P.; Espinosa, R.; Arias, C.F.; Lopez, S. References VP7 mediates the interaction of rotaviruses with integrin alphavbeta3 through a novel integrin-binding site. J. Virol. 2004, 78, 10839–10847. [CrossRef] 116. Altan-Bonnet, N. Extracellular vesicles are the Trojan horses of viral infection. Curr. Opin. Microbiol. 2016, 32, 77–81. [CrossRef] [PubMed] 117. Altan-Bonnet, N.; Perales, C.; Domingo, E. Extracellular vesicles: Vehicles of en bloc viral transmission. Virus Res. 2019, 265, 143–149. [CrossRef] 118. Gunasekaran, M.; Bansal, S.; Ravichandran, R.; Sharma, M.; Perincheri, S.; Rodriguez, F.; Hachem, R.; Fisher, C.E.; Limaye, A.P.; Omar, A.; et al. Respiratory viral infection in lung transplantation induces exosomes that trigger chronic rejection. J. Heart Lung Transplant. 2020, 39, 379–388. [CrossRef] 119. Elrashdy, F.; Aljaddawi, A.A.; Redwan, E.M.; Uversky, V.N. On the potential role of exosomes in the COVID-19 reinfection/reactivation opportunity. J. Biomol. Struct. Dyn. 2020, 1–12. [CrossRef] 120. Naskalska, A.; Dabrowska, A.; Szczepanski, A.; Milewska, A.; Jasik, K.P.; Pyrc, K. Membrane protein of human coronavirus NL63 is responsible for interaction with the adhesion receptor. J. Virol. 2019, 93, e00319–e00355. [CrossRef] 121. Milewska, A.; Zarebski, M.; Nowak, P.; Stozek, K.; Potempa, J.; Pyrc, K. Human coronavirus NL63 utilizes heparan sulfate proteoglycans for attachment to target cells. J. Virol. 2014, 88, 13221–13230. [CrossRef] 122. Belting, M. Heparan sulfate proteoglycan as a plasma membrane carrier. Trends Biochem. Sci. 2003, 28, 145 151 [CrossRef] 121. Milewska, A.; Zarebski, M.; Nowak, P.; Stozek, K.; Potempa, J.; Pyrc, K. Human coronavirus NL63 utilizes heparan sulfate proteoglycans for attachment to target cells. J. Virol. 2014, 88, 13221–13230. [CrossRef] 22. Belting, M. Heparan sulfate proteoglycan as a plasma membrane carrier. Trends Biochem. Sci. 2003 145–151. [CrossRef] [ ] 123. Lang, J.; Yang, N.; Deng, J.; Liu, K.; Yang, P.; Zhang, G.; Jiang, C. Inhibition of SARS pseudovirus cell entry by lactoferrin binding to heparan sulfate proteoglycans PLoS ONE 2011 6 e23710 [CrossRef] [PubMed] 23. Lang, J.; Yang, N.; Deng, J.; Liu, K.; Yang, P.; Zhang, G.; Jiang, C. Inhibition of SARS pseudovirus cell e by lactoferrin binding to heparan sulfate proteoglycans. PLoS ONE 2011, 6, e23710. [CrossRef] [PubMe 123. Lang, J.; Yang, N.; Deng, J.; Liu, K.; Yang, P.; Zhang, G.; Jiang, C. Inhibition of SARS pseudovirus cell entry by lactoferrin binding to heparan sulfate proteoglycans. PLoS ONE 2011, 6, e23710. [CrossRef] [PubMed] 124. Redwan, E.M.; Uversky, V.N.; El-Fakharany, E.M.; Al-Mehdar, H. Potential lactoferrin activity against pathogenic viruses. Comptes Rendus Biol. 2014, 337, 581–595. [CrossRef] 124. Redwan, E.M.; Uversky, V.N.; El-Fakharany, E.M.; Al-Mehdar, H. References Potential lactoferrin activity against pathogenic viruses. Comptes Rendus Biol. 2014, 337, 581–595. [CrossRef] 125. Albar, A.H.; Almehdar, H.A.; Uversky, V.N.; Redwan, E.M. Structural heterogeneity and multifunctionality of lactoferrin. Curr. Protein Pept. Sci. 2014, 15, 778–797. [CrossRef] [PubMed] 126. Liao, Y.; El-Fakkarany, E.; Lonnerdal, B.; Redwan, E.M. Inhibitory eﬀects of native and recombinant full-length camel lactoferrin and its N and C lobes on hepatitis C virus infection of Huh7.5 cells. J. Med. Microbiol. 2012, 61, 375–383. [CrossRef] 24 of 32 24 of 32 Biomolecules 2020, 10, 1312 127. Reghunathan, R.; Jayapal, M.; Hsu, L.Y.; Chng, H.H.; Tai, D.; Leung, B.P.; Melendez, A.J. Expression proﬁle of immune response genes in patients with severe acute respiratory syndrome. BMC Immunol. 2005, 6, 2. [CrossRef] 28. Jenssen, H.; Hancock, R.E. Antimicrobial properties of lactoferrin. Biochimie 2009, 91, 19–29. [CrossRef] 128. Jenssen, H.; Hancock, R.E. Antimicrobial properties of lactoferrin. Biochimie 2009, 91, 19–29. [CrossRef] 129. Qinfen, Z.; Jinming, C.; Xiaojun, H.; Huanying, Z.; Jicheng, H.; Ling, F.; Kunpeng, L.; Jingqiang, Z. The life cycle of SARS coronavirus in vero E6 cells. J. Med. Virol. 2004, 73, 332–337. [CrossRef] 129. Qinfen, Z.; Jinming, C.; Xiaojun, H.; Huanying, Z.; Jicheng, H.; Ling, F.; Kunpeng, L.; Jingqiang, Z. The life cycle of SARS coronavirus in vero E6 cells. J. Med. Virol. 2004, 73, 332–337. [CrossRef] 130. Simmons, G.; Reeves, J.D.; Rennekamp, A.J.; Amberg, S.M.; Piefer, A.J.; Bates, P. Characterization of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike glycoprotein-mediated viral entry. Proc. Natl. Acad. Sci. USA 2004, 101, 4240–4245. [CrossRef] 131. Ng, M.L.; Tan, S.H.; See, E.E.; Ooi, E.E.; Ling, A.E. Early events of SARS coronavirus infection in vero cells. J. Med. Virol. 2003, 71, 323–331. [CrossRef] 132. Pelkmans, L.; Helenius, A. Insider information: What viruses tell us about endocytosis. Curr. Opin. Cell Biol. 2003, 15, 414–422. [CrossRef] 133. Sieczkarski, S.B.; Whittaker, G.R. Dissecting virus entry via endocytosis. J. Gen. Virol. 2002, 83, 1535–1545. [CrossRef] 134. Nunes-Correia, I.; Eulalio, A.; Nir, S.; Pedroso de Lima, M.C. Caveolae as an additional route for inﬂuenza virus endocytosis in MDCK cells. Cell. Mol. Biol. Lett. 2004, 9, 47–60. [PubMed] 135. Sieczkarski, S.B.; Whittaker, G.R. Inﬂuenza virus can enter and infect cells in the absence of clathrin-mediated endocytosis. J. Virol. 2002, 76, 10455–10464. [CrossRef] [PubMed] y 136. Fackler, O.T.; Peterlin, B.M. Endocytic entry of HIV-1. Curr. Biol. 2000, 10, 1005–1008. [CrossRef] 137. Matlin, K.S.; Reggio, H.; Helenius, A.; Simons, K. References Infectious entry pathway of inﬂuenza virus in a canine kidney cell line. J. Cell Biol. 1981, 91, 601–613. [CrossRef] [PubMed] 138. Wang, H.; Yang, P.; Liu, K.; Guo, F.; Zhang, Y.; Zhang, G.; Jiang, C. SARS coronavirus entry into host cells through a novel clathrin- and caveolae-independent endocytic pathway. Cell Res. 2008, 18, 290–301. [CrossRef] 139. Matsuyama, S.; Ujike, M.; Morikawa, S.; Tashiro, M.; Taguchi, F. Protease-mediated enhancement of severe acute respiratory syndrome coronavirus infection. Proc. Natl. Acad. Sci. USA 2005, 102, 12543. [CrossRef] 140. Lukassen, S.; Chua, R.L.; Trefzer, T.; Kahn, N.C.; Schneider, M.A.; Muley, T.; Winter, H.; Meister, M.; Veith, C.; Boots, A.W.; et al. SARS-CoV-2 receptor ACE2 and TMPRSS2 are primarily expressed in bronchial transient secretory cells. EMBO J. 2020, e105114. [CrossRef] 141. Fung, S.Y.; Yuen, K.S.; Ye, Z.W.; Chan, C.P.; Jin, D.Y. A tug-of-war between severe acute respiratory syndrome coronavirus 2 and host antiviral defence: Lessons from other pathogenic viruses. Emerg. Microbes Infect. 2020, 9, 558–570. [CrossRef] 142. Gandon, S.; Michalakis, Y. Evolution of parasite virulence against qualitative or quantitative host resistance. Proc. Biol. Sci. R. Soc. 2000, 267, 985–990. [CrossRef] [PubMed] 143. Thrall, P.H.; Burdon, J.J. Evolution of virulence in a plant host-pathogen metapopulation. Science 2003, 299, 1735–1737. [CrossRef] [PubMed] 144. Thrall, P.H.; Burdon, J.J.; Bever, J.D. Local adaptation in the Linum marginale—Melampsora lini host-pathogen interaction. Evolution. 2002, 56, 1340–1351. [CrossRef] [PubMed] 145. Gandon, S.; van Baalen, M.; Jansen, V.A. The evolution of parasite virulence, superinfection, and host resistance. Am. Nat. 2002, 159, 658–669. [CrossRef] 146. Kubinak, J.L.; Potts, W.K. Host resistance inﬂuences patterns of experimental viral adaptation and virulence evolution. Virulence 2013, 4, 410–418. [CrossRef] 147. Grenfell, B.T.; Pybus, O.G.; Gog, J.R.; Wood, J.L.; Daly, J.M.; Mumford, J.A.; Holmes, E.C. Unifying the epidemiological and evolutionary dynamics of pathogens. Science 2004, 303, 327–332. [CrossRef] 148. Welsh, R.M.; Selin, L.K. No one is naive: The signiﬁcance of heterologous T-cell immunity. Nat. Rev. Immunol. 2002, 2, 417–426. [CrossRef] 149. St. John, A.L.; Rathore, A.P.S. Adaptive immune responses to primary and secondary dengue virus infections. Nat. Rev. Immunol. 2019, 19, 218–230. [CrossRef] 150. Kim, C.W.; Yoo, H.J.; Park, J.H.; Oh, J.E.; Lee, H.K. Exogenous interleukin-33 contributes to protective immunity via cytotoxic T-cell priming against mucosal inﬂuenza viral infection. Viruses 2019, 11. [CrossRef] 151. Mullbacher, A.; Lobigs, M.; Alshariﬁ, M.; Regner, M. Cytotoxic T-cell immunity as a target for inﬂuenza 150. References Kim, C.W.; Yoo, H.J.; Park, J.H.; Oh, J.E.; Lee, H.K. Exogenous interleukin-33 contributes to protective immunity via cytotoxic T-cell priming against mucosal inﬂuenza viral infection. Viruses 2019, 11. [CrossRef] 151. Mullbacher, A.; Lobigs, M.; Alshariﬁ, M.; Regner, M. Cytotoxic T-cell immunity as a target for inﬂuenza vaccines. Lancet. Infect. Dis. 2006, 6, 255–256. [CrossRef] 25 of 32 Biomolecules 2020, 10, 1312 152. McMichael, A.J.; Gotch, F.M.; Noble, G.R.; Beare, P.A. Cytotoxic T-cell immunity to inﬂuenza. N. Engl. J. Med. 1983, 309, 13–17. [CrossRef] [PubMed] 153. Weiskopf, D.; Schmitz, K.S.; Raadsen, M.P.; Grifoni, A.; Okba, N.M.A.; Endeman, H.; van den Akker, J.P.C.; Molenkamp, R.; Koopmans, M.P.G.; van Gorp, E.C.M.; et al. Phenotype and kinetics of SARS-CoV-2-speciﬁc T cells in COVID-19 patients with acute respiratory distress syndrome. Sci. Immunol. 2020, 5. [CrossRef] [PubMed] 154. Peng, Y.; Mentzer, A.J.; Liu, G.; Yao, X.; Yin, Z.; Dong, D.; Dejnirattisai, W.; Rostron, T.; Supasa, P.; Liu, C.; et al. Broad and strong memory CD4 (+) and CD8 (+) T cells induced by SARS-CoV-2 in UK convalescent COVID-19 patients. bioRxiv 2020. [CrossRef] 155. Le Bert, N.; Tan, A.T.; Kunasegaran, K.; Tham, C.Y.L.; Hafezi, M.; Chia, A.; Chng, M.H.Y.; Lin, M.; Tan, N.; Linster, M.; et al. SARS-CoV-2-speciﬁc T cell immunity in cases of COVID-19 and SARS, and uninfected controls. Nature 2020, 584, 457–462. [CrossRef] [PubMed] 156. Li, Y.; Zhou, W.; Yang, L.; You, R. Physiological and pathological regulation of ACE2, the SARS-CoV-2 receptor. Pharm. Res. 2020, 157, 104833. [CrossRef] [PubMed] 157. Zou, X.; Chen, K.; Zou, J.; Han, P.; Hao, J.; Han, Z. Single-cell RNA-seq data analysis on the receptor ACE2 expression reveals the potential risk of diﬀerent human organs vulnerable to 2019-nCoV infection. Front. Med. 2020. [CrossRef] 158. Barkauskas, C.; Cronce, M.; Rackley, C.; Bowie, E.; Keene, D.; Stripp, B.; Randell, S.; Noble, P.; Hogan, B. Type 2 alveolar cells are stem cells in adult lung. J. Clin. Investig. 2013, 123. [CrossRef] 159. Li, Y.; Wu, Q.; Sun, X.; Shen, J.; Chen, H. Organoids as a powerful model for respiratory diseases. Stem Cells Int. 2020, 2020, 5847876. [CrossRef] 160. Rivellese, F.; Prediletto, E. ACE2 at the centre of COVID-19 from paucisymptomatic infections to severe pneumonia. Autoimmun. Rev. 2020, 102536. [CrossRef] 161. Kass, D.A.; Duggal, P.; Cingolani, O. Obesity could shift severe COVID-19 disease to younger ages. Lancet 2020, 395, 1544–1545. [CrossRef] 162. References Heialy, S.A.; Hachim, M.; Senok, A.; Tayoun, A.A.; Hamoudi, R.; Alsheikh-Ali, A.; Hamid, Q. Regulation of angiotensin converting enzyme 2 (ACE2) in obesity: Implications for COVID-19. bioRxiv 2020. [CrossRef] 163. Chen, M.; Shen, W.; Rowan, N.R.; Kulaga, H.; Hillel, A.; Ramanathan, M., Jr.; Lane, A.P. Elevated ACE2 expression in the olfactory neuroepithelium: Implications for anosmia and upper respiratory SARS-CoV-2 entry and replication. Eur. Respir. J. 2020. [CrossRef] 164. Lechien, J.R.; Chiesa-Estomba, C.M.; De Siati, D.R.; Horoi, M.; Le Bon, S.D.; Rodriguez, A.; Dequanter, D.; Blecic, S.; El Aﬁa, F.; Distinguin, L.; et al. Olfactory and gustatory dysfunctions as a clinical presentation of mild-to-moderate forms of the coronavirus disease (COVID-19): A multicenter European study. Eur. Arch. Otorhinolaryngol. 2020, 277, 2251–2261. [CrossRef] [PubMed] 165. Bunyavanich, S.; Do, A.; Vicencio, A. Nasal gene expression of angiotensin-converting enzyme 2 in children and adults. Jama 2020. [CrossRef] [PubMed] 166. Sungnak, W.; Huang, N.; Bécavin, C.; Berg, M.; Barbry, P.; Brazma, A.; Desai, T.; Duong, T.E.; Eickelberg, O.; Haniﬀa, M.; et al. SARS-CoV-2 entry genes are most highly expressed in nasal goblet and ciliated cells within human airways. arXiv 2020, arXiv:2003.06122. y 167. Hui, D.S.C.; Zumla, A. Severe acute respiratory syndrome: Historical, epidemiologic, and clinical features. Infect. Dis. Clin. North. Am. 2019, 33, 869–889. [CrossRef] 168. Holshue, M.L.; DeBolt, C.; Lindquist, S.; Lofy, K.H.; Wiesman, J.; Bruce, H.; Spitters, C.; Ericson, K.; Wilkerson, S.; Tural, A.; et al. First case of 2019 novel coronavirus in the United States. N. Engl. J. Med. 2020, 382, 929–936. [CrossRef] 169. Chen, Y.; Guo, Y.; Pan, Y.; Zhao, Z.J. Structure analysis of the receptor binding of 2019-nCoV. Biochem. Biophys. Res. Commun. 2020. [CrossRef] 170. Patel, S.; Rauf, A.; Khan, H.; Abu-Izneid, T. Renin-angiotensin-aldosterone (RAAS): The ubiquitous system for homeostasis and pathologies. Biomed. Pharm. 2017, 94, 317–325. [CrossRef] 171. Burrell, L.M.; Johnston, C.I.; Tikellis, C.; Cooper, M.E. ACE2, a new regulator of the renin-angiotensin system. Trends Endocrinol. Metab. 2004, 15, 166–169. [CrossRef] 172. Gurwitz, D. Angiotensin receptor blockers as tentative SARS-CoV-2 therapeutics. Drug Dev. Res. 2020. [CrossRef] [PubMed] g [CrossRef] [PubMed] 26 of 32 26 of 32 Biomolecules 2020, 10, 1312 173. Liu, Y.; Yang, Y.; Zhang, C.; Huang, F.; Wang, F.; Yuan, J.; Wang, Z.; Li, J.; Li, J.; Feng, C.; et al. Clinical and biochemical indexes from 2019-nCoV infected patients linked to viral loads and lung injury. Sci. China. Life Sci. 2020, 63, 364–374. [CrossRef] [PubMed] 174. References Delanghe, J.R.; Speeckaert, M.M.; De Buyzere, M.L. The host’s angiotensin-converting enzyme polymorphism may explain epidemiological ﬁndings in COVID-19 infections. Clin. Chim. Acta 2020, 505, 192–193. [CrossRef] [PubMed] 175. Kuba, K.; Imai, Y.; Penninger, J.M. Angiotensin-converting enzyme 2 in lung diseases. Curr. Opin. Pharmacol. 2006, 6, 271–276. [CrossRef] 176. Hussain, M.; Jabeen, N.; Raza, F.; Shabbir, S.; Baig, A.; Amanullah, A.; Aziz, B. Structural variations in human ACE2 may inﬂuence its binding with SARS-CoV-2 spike protein. J. Med. Virol. 2020. [CrossRef] [PubMed] 177. Elrashdy, F.; Redwan, E.M.; Uversky, V.N. Intrinsic disorder perspective of an interplay between the renin-angiotensin-aldosterone system and SARS-CoV-2. Infect. Genet. Evol. 2020, 85, 104510. [CrossRef] 178. Shen, M.; Liu, C.; Xu, R.; Ruan, Z.; Zhao, S.; Zhang, H.; Wang, W.; Huang, X.; Yang, L.; Tang, Y.; et al. SARS-CoV-2 infection of cats and dogs? Preprints. 2020. 2020040116. 179. Zhai, X.; Sun, J.; Yan, Z.; Zhang, J.; Zhao, J.; Zhao, Z.; Gao, Q.; He, W.T.; Veit, M.; Su, S. Comparison of SARS-CoV-2 spike protein binding to ACE2 receptors from human, pets, farm animals, and putative intermediate hosts. J. Virol. 2020. [CrossRef] 180. Shi, J.; Wen, Z.; Zhong, G.; Yang, H.; Wang, C.; Huang, B.; Liu, R.; He, X.; Shuai, L.; Sun, Z.; et al. Susceptibility of ferrets, cats, dogs, and other domesticated animals to SARS-coronavirus 2. Science 2020. [CrossRef] 181. Patterson, E.I.; Elia, G.; Grassi, A.; Giordano, A.; Desario, C.; Medardo, M.; Smith, S.L.; Anderson, E.R.; Prince, T.; Patterson, G.T.; et al. Evidence of exposure to SARS-CoV-2 in cats and dogs from households in Italy. bioRxiv 2020. [CrossRef] 182. Abdel-Moneim, A.S.; Abdelwhab, E.M. Evidence for SARS-CoV-2 infection of animal hosts. Pathogens 2020, 9. [CrossRef] 183. Hossain, M.G.; Javed, A.; Akter, S.; Saha, S. SARS-CoV-2 host diversity: An update of natural infections and experimental evidence. J. Microbiol. Immunol. Infect. 2020. [CrossRef] [PubMed] 184. Hernandez, M.; Abad, D.; Eiros, J.M.; Rodriguez-Lazaro, D. Are animals a neglected transmission route of SARS-CoV-2? Pathogens 2020, 9. [CrossRef] [PubMed] 185. Sit, T.H.C.; Brackman, C.J.; Ip, S.M.; Tam, K.W.S.; Law, P.Y.T.; To, E.M.W.; Yu, V.Y.T.; Sims, L.D.; Tsang, D.N.C.; Chu, D.K.W.; et al. Infection of dogs with SARS-CoV-2. Nature 2020. [CrossRef] [PubMed] 186. Tiwari, R.; Dhama, K.; Sharun, K.; Iqbal Yatoo, M.; Malik, Y.S.; Singh, R.; Michalak, I.; Sah, R.; Bonilla-Aldana, D.K.; Rodriguez-Morales, A.J. COVID-19: Animals, veterinary and zoonotic links. Vet. Q. 2020, 40, 169–182. [CrossRef] [PubMed] 187. Gollakner, R.; Capua, I. References Kostaki, E.G.; Karamitros, T.; Bobkova, M.; Oikonomopoulou, M.; Magiorkinis, G.; Garcia, F.; Hatzakis, A.; Paraskevis, D. Spatiotemporal characteristics of the HIV-1 CRF02_AG/CRF63_02A1 epidemic in Russia and Central Asia. AIDS Res. Hum. Retrovir. 2018, 34, 415–420. [CrossRef] 201. Baric, R.S.; Fu, K.; Schaad, M.C.; Stohlman, S.A. Establishing a genetic recombination map for murine coronavirus strain A59 complementation groups. Virology 1990, 177, 646–656. [CrossRef] 202. Lai, M.M. RNA recombination in animal and plant viruses. Microbiol. Rev. 1992, 56, 61–79. [CrossRef] [PubMed] 203. Pyrc, K.; Dijkman, R.; Deng, L.; Jebbink, M.F.; Ross, H.A.; Berkhout, B.; van der Hoek, L. Mosaic structure of human coronavirus NL63, one thousand years of evolution. J. Mol. Biol. 2006, 364, 964–973. [CrossRef] 204. Hon, C.C.; Lam, T.Y.; Shi, Z.L.; Drummond, A.J.; Yip, C.W.; Zeng, F.; Lam, P.Y.; Leung, F.C. Evidence of the recombinant origin of a bat severe acute respiratory syndrome (SARS)-like coronavirus and its implications on the direct ancestor of SARS coronavirus. J. Virol. 2008, 82, 1819–1826. [CrossRef] 205. Sabir, J.S.; Lam, T.T.; Ahmed, M.M.; Li, L.; Shen, Y.; Abo-Aba, S.E.; Qureshi, M.I.; Abu-Zeid, M.; Zhang, Y.; Khiyami, M.A.; et al. Co-circulation of three camel coronavirus species and recombination of MERS-CoVs in Saudi Arabia. Science 2016, 351, 81–84. [CrossRef] [PubMed] 206. Bisht, H.; Roberts, A.; Vogel, L.; Bukreyev, A.; Collins, P.L.; Murphy, B.R.; Subbarao, K.; Moss, B. Severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice. Proc. Natl. Acad. Sci. USA 2004, 101, 6641–6646. [CrossRef] [PubMed] 207. Enjuanes, L.; Dediego, M.L.; Alvarez, E.; Deming, D.; Sheahan, T.; Baric, R. Vaccines to prevent severe acute respiratory syndrome coronavirus-induced disease. Virus Res. 2008, 133, 45–62. [CrossRef] 208. Jiaming, L.; Yanfeng, Y.; Yao, D.; Yawei, H.; Linlin, B.; Baoying, H.; Jinghua, Y.; Gao, G.F.; Chuan, Q.; Wenjie, T. The recombinant N-terminal domain of spike proteins is a potential vaccine against Middle East respiratory syndrome coronavirus (MERS-CoV) infection. Vaccine 2017, 35, 10–18. [CrossRef] 209. Domingo, E.; Sheldon, J.; Perales, C. Viral quasispecies evolution. Microbiol. Mol. Biol. Rev. 2012, 76, 159–216. [CrossRef] [PubMed] 210. Domingo, E.; Escarmís, C.; Menéndez-Arias, L.; Perales, C.; Herrera, M.; Novella, I.S.; Holland, J.J. CHAPTER 4—Viral quasispecies: Dynamics, interactions, and pathogenesis ** dedicated to Manfred Eigen on the occasion of his 80th birthday, for the insights that his pioneer studies have represented for virology. References Is COVID-19 the ﬁrst pandemic that evolves into a panzootic? Vet. Ital. 2020, 56, 7–8. [CrossRef] 188. Mao, L.J.; Xu, J.; Xu, Z.H.; Xia, X.P.; Li, B.; He, J.G.; Zhao, P.; Pan, J.W.; Zhang, D.; Su, Y.; et al. A child with household transmitted COVID-19. BMC Infect. Dis. 2020, 20, 329. [CrossRef] 189. Liu, Y.; Gu, Z.; Xia, S.; Shi, B.; Zhou, X.N.; Shi, Y.; Liu, J. What are the underlying transmission patterns of COVID-19 outbreak?—An age-speciﬁc social contact characterization. EClinicalMedicine 2020, 100354. [CrossRef] 190. Uversky, V.N.; Elrashdy, F.; Aljadawi, A.; Redwan, E.M. Household pets and SARS-CoV2 transmissi the light of the ACE2 intrinsic disorder status. J. Biomol. Struct. Dyn. 2020, in press. 191. Boni, M.F.; Lemey, P.; Jiang, X.; Lam, T.T.-Y.; Perry, B.; Castoe, T.; Rambaut, A.; Robertson, D.L. Evolutionary origins of the SARS-CoV-2 sarbecovirus lineage responsible for the COVID-19 pandemic. bioRxiv 2020. [CrossRef] 192. Karamitros, T.; Papadopoulou, G.; Bousali, M.; Mexias, A.; Tsiodras, S.; Mentis, A. SARS-CoV-2 exhibits intra-host genomic plasticity and low-frequency polymorphic quasispecies. bioRxiv 2020. 2020.2003.2027.009480. [CrossRef] 193. Capobianchi, M.R.; Rueca, M.; Messina, F.; Giombini, E.; Carletti, F.; Colavita, F.; Castilletti, C.; Lalle, E.; Bordi, L.; Vairo, F.; et al. Molecular characterization of SARS-CoV-2 from the ﬁrst case of COVID-19 in Italy. Clin. Microbiol. Infect. 2020. [CrossRef] 194. Altan-Bonnet, N.; Chen, Y.H. Intercellular transmission of viral populations with vesicles. J. Virol. 2015, 89, 12242–12244. [CrossRef] 27 of 32 27 of 32 Biomolecules 2020, 10, 1312 195. Borderia, A.V.; Isakov, O.; Moratorio, G.; Henningsson, R.; Aguera-Gonzalez, S.; Organtini, L.; Gnadig, N.F.; Blanc, H.; Alcover, A.; Hafenstein, S.; et al. Group selection and contribution of minority variants during virus adaptation determines virus ﬁtness and phenotype. PLoS Pathog. 2015, 11, e1004838. [CrossRef] [PubMed] 196. Domingo, E.; Baranowski, E.; Ruiz-Jarabo, C.M.; Martin-Hernandez, A.M.; Saiz, J.C.; Escarmis, C. Quasispecies structure and persistence of RNA viruses. Emerg. Infect. Dis. 1998, 4, 521–527. [CrossRef] 197. Park, D.; Huh, H.J.; Kim, Y.; Son, D.-S.; Jeon, H.-J.; Im, E.-H.; Kim, J.-W.; Lee, N.; Kang, E.-S.; Kang, C.; et al. Analysis of intra-patient heterogeneity uncovers the microevolution of Middle East respiratory syndrome coronavirus. Cold Spring Harb. Mol. Case Stud. 2016, 2, a001214. [CrossRef] p g 198. Xu, D.; Zhang, Z.; Wang, F.S. SARS-associated coronavirus quasispecies in individual patients. N. Engl. J. Med. 2004, 350, 1366–1367. [CrossRef] [PubMed] 199. Mahy, B.W.J. The evolution and emergence of RNA viruses. Emerg. Infect. Dis. 2010, 16, 899. [CrossRef] 200. References [CrossRef] 224. Sauce, D.; Larsen, M.; Fastenackels, S.; Duperrier, A.; Keller, M.; Grubeck-Loebenstein, B.; Ferrand, C.; Debre, P.; Sidi, D.; Appay, V. Evidence of premature immune aging in patients thymectomized during early childhood. J. Clin. Invest. 2009, 119, 3070–3078. [CrossRef] [PubMed] 225. Zlamy, M.; Almanzar, G.; Parson, W.; Schmidt, C.; Leierer, J.; Weinberger, B.; Jeller, V.; Unsinn, K.; Eyrich, M.; Wurzner, R.; et al. Eﬀorts of the human immune system to maintain the peripheral CD8+ T cell compartment after childhood thymectomy. Immun. Ageing 2016, 13, 3. [CrossRef] [PubMed] 226. Britanova, O.V.; Putintseva, E.V.; Shugay, M.; Merzlyak, E.M.; Turchaninova, M.A.; Staroverov, D.B.; Bolotin, D.A.; Lukyanov, S.; Bogdanova, E.A.; Mamedov, I.Z.; et al. Age-related decrease in TCR repertoire diversity measured with deep and normalized sequence proﬁling. J. Immunol. 2014, 192, 2689–2698. [CrossRef] [PubMed] 227. Sansoni, P.; Vescovini, R.; Fagnoni, F.; Biasini, C.; Zanni, F.; Zanlari, L.; Telera, A.; Lucchini, G.; Passeri, G.; Monti, D.; et al. The immune system in extreme longevity. Exp. Gerontol. 2008, 43, 61–65. [CrossRef] [PubMed] 228. Weiskopf, D.; Weinberger, B.; Grubeck-Loebenstein, B. The aging of the immune system. Transpl. Int. 2009, 22, 1041–1050. [CrossRef] [PubMed] 229. LeMaoult, J.; Messaoudi, I.; Manavalan, J.S.; Potvin, H.; Nikolich-Zugich, D.; Dyall, R.; Szabo, P.; Weksler, M.E.; Nikolich-Zugich, J. Age-related dysregulation in CD8 T cell homeostasis: Kinetics of a diversity loss. J. Immunol. 2000, 165, 2367–2373. [CrossRef] 230. Posnett, D.N.; Sinha, R.; Kabak, S.; Russo, C. Clonal populations of T cells in normal elderly humans: The T cell equivalent to “benign monoclonal gammapathy”. J. Exp. Med. 1994, 179, 609–618. [CrossRef] 231. Callahan, J.E.; Kappler, J.W.; Marrack, P. Unexpected expansions of CD8-bearing cells in old mice. J. Immunol. 1993, 151, 6657–6669. 232. Blackman, M.A.; Woodland, D.L. The narrowing of the CD8 T cell repertoire in old age. Curr. Opin. Immunol. 2011, 23, 537–542. [CrossRef] 233. Yager, E.J.; Ahmed, M.; Lanzer, K.; Randall, T.D.; Woodland, D.L.; Blackman, M.A. Age-associated decline in T cell repertoire diversity leads to holes in the repertoire and impaired immunity to inﬂuenza virus. J. Exp. Med. 2008, 205, 711–723. [CrossRef] 234. Messaoudi, I.; Lemaoult, J.; Guevara-Patino, J.A.; Metzner, B.M.; Nikolich-Zugich, J. Age-related CD8 T cell clonal expansions constrict CD8 T cell repertoire and have the potential to impair immune defense. J. Exp. Med. 2004, 200, 1347–1358. [CrossRef] [PubMed] 235. Johnson, S.A.; Cambier, J.C. Ageing, autoimmunity and arthritis: Senescence of the B cell compartment—Implications for humoral immunity. Arthritis Res. Ther. References In Origin and Evolution of Viruses, 2nd ed.; Domingo, E., Parrish, C.R., Holland, J.J., Eds.; Academic Press: London, UK, 2008; pp. 87–118. [CrossRef] 211. Saghazadeh, A.; Rezaei, N. Immune-epidemiological parameters of the novel coronavirus—A perspective. Expert Rev. Clin. Immunol. 2020, 1–6. [CrossRef] 212. Koﬀ, W.C.; Williams, M.A. Covid-19 and immunity in aging populations—A new research agenda. N. Engl. J. Med. 2020. [CrossRef] 213. Arentz, M.; Yim, E.; Klaﬀ, L.; Lokhandwala, S.; Riedo, F.X.; Chong, M.; Lee, M. Characteristics and outcomes of 21 critically ill patients with COVID-19 in Washington state. JAMA 2020. [CrossRef] 214. Zhou, F.; Yu, T.; Du, R.; Fan, G.; Liu, Y.; Liu, Z.; Xiang, J.; Wang, Y.; Song, B.; Gu, X.; et al. Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: A retrospective cohort study. Lancet 2020, 395, 1054–1062. [CrossRef] 215. Pan, Y.; Zhang, D.; Yang, P.; Poon, L.L.M.; Wang, Q. Viral load of SARS-CoV-2 in clinical samples. Lancet Infect. Dis. 2020, 20, 411–412. [CrossRef] 28 of 32 28 of 32 Biomolecules 2020, 10, 1312 216. Zou, L.; Ruan, F.; Huang, M.; Liang, L.; Huang, H.; Hong, Z.; Yu, J.; Kang, M.; Song, Y.; Xia, J.; et al. SARS-CoV-2 viral load in upper respiratory specimens of infected patients. N. Engl. J. Med. 2020, 382, 1177–1179. [CrossRef] [PubMed] 217. Liu, Y.; Yan, L.M.; Wan, L.; Xiang, T.X.; Le, A.; Liu, J.M.; Peiris, M.; Poon, L.L.M.; Zhang, W. Viral dyna mild and severe cases of COVID-19. Lancet Infect. Dis. 2020. [CrossRef] 218. Pawelec, G.; Weng, N.P. Can an eﬀective SARS-CoV-2 vaccine be developed for the older population? Immun. Ageing 2020, 17, 8. [CrossRef] 219. Weng, N.P. Aging of the immune system: How much can the adaptive immune system adapt? Immunity 2006, 24, 495–499. [CrossRef] 220. Oh, S.J.; Lee, J.K.; Shin, O.S. Aging and the immune system: The impact of immunosenescence on viral infection, immunity and vaccine immunogenicity. Immune Netw. 2019, 19, e37. [CrossRef] 221. Miyashita, N.; Kawai, Y.; Akaike, H.; Ouchi, K.; Hayashi, T.; Kurihara, T.; Okimoto, N. Inﬂuence of age on the clinical diﬀerentiation of atypical pneumonia in adults. Respirology 2012, 17, 1073–1079. [CrossRef] 222. Pawelec, G.; Akbar, A.; Caruso, C.; Solana, R.; Grubeck-Loebenstein, B.; Wikby, A. Human immunosenescence: Is it infectious? Immunol. Rev. 2005, 205, 257–268. [CrossRef] 223. Lee, J.; Yoon, S.R.; Choi, I.; Jung, H. Causes and mechanisms of hematopoietic stem cell aging. Int. J. Mol. Sci. 2019, 20. References 2004, 6, 131–139. [CrossRef] [PubMed] 236. Allman, D.; Miller, J.P. B cell development and receptor diversity during aging. Curr. Opin. Immunol. 2005, 17, 463–467. [CrossRef] 29 of 32 29 of 32 Biomolecules 2020, 10, 1312 237. Frasca, D.; Diaz, A.; Romero, M.; Blomberg, B.B. Human peripheral late/exhausted memory B cells express a senescent-associated secretory phenotype and preferentially utilize metabolic signaling pathways. Exp. Gerontol. 2017, 87, 113–120. [CrossRef] [PubMed] 238. Frasca, D.; Diaz, A.; Romero, M.; Phillips, M.; Mendez, N.V.; Landin, A.M.; Blomberg, B.B. Unique biomarkers for B-cell function predict the serum response to pandemic H1N1 inﬂuenza vaccine. Int. Immunol. 2012, 24, 175–182. [CrossRef] [PubMed] 239. Shi, Y.; Yamazaki, T.; Okubo, Y.; Uehara, Y.; Sugane, K.; Agematsu, K. Regulation of aged humoral immune defense against pneumococcal bacteria by IgM memory B cell. J. Immunol. 2005, 175, 3262–3267. [CrossRef] 239. Shi, Y.; Yamazaki, T.; Okubo, Y.; Uehara, Y.; Sugane, K.; Agematsu, K. Regulation of aged humoral immune defense against pneumococcal bacteria by IgM memory B cell. J. Immunol. 2005, 175, 3262–3267. [CrossRef] 240. Khurana, S.; Frasca, D.; Blomberg, B.; Golding, H. AID activity in B cells strongly correlates with polyclonal 240. Khurana, S.; Frasca, D.; Blomberg, B.; Golding, H. AID activity in B cells strongly correlates with polyclonal antibody aﬃnity maturation in-vivo following pandemic 2009-H1N1 vaccination in humans. PLoS Pathog. 2012, 8, e1002920. [CrossRef] 241. Frasca, D.; Landin, A.M.; Alvarez, J.P.; Blackshear, P.J.; Riley, R.L.; Blomberg, B.B. Tristetraprolin, a negative regulator of mRNA stability, is increased in old B cells and is involved in the degradation of E47 mRNA. J. Immunol. 2007, 179, 918–927. [CrossRef] 242. Muramatsu, M.; Nagaoka, H.; Shinkura, R.; Begum, N.A.; Honjo, T. Discovery of activation-induced cytidine deaminase, the engraver of antibody memory. Adv. Immunol. 2007, 94, 1–36. [CrossRef] 43. Nikolich-Zugich, J.; Li, G.; Uhrlaub, J.L.; Renkema, K.R.; Smithey, M.J. Age-related changes in CD8 T homeostasis and immunity to infection. Semin. Immunol. 2012, 24, 356–364. [CrossRef] 244. Gorina, Y.; Kelly, T.; Lubitz, J.; Hines, Z. Trends in inﬂuenza and pneumonia among older persons in the United States. Aging Trends 2008, 8, 1–11. 245. Franceschi, C.; Capri, M.; Monti, D.; Giunta, S.; Olivieri, F.; Sevini, F.; Panourgia, M.P.; Invidia, L.; Celani, L.; Scurti, M.; et al. Inﬂammaging and anti-inﬂammaging: A systemic perspective on aging and longevity emerged from studies in humans. Mech. Ageing Dev. 2007, 128, 92–105. [CrossRef] 246. Franceschi, C.; Campisi, J. References Chronic inﬂammation (inﬂammaging) and its potential contribution to age-associated diseases. J. Gerontol. A Biol. Sci. Med. Sci. 2014, 69 (Suppl. S1), S4–S9. [CrossRef] 247. Greene, M.A.; Loeser, R.F. Aging-related inﬂammation in osteoarthritis. Osteoarthr. Cartil. 2015, 23, 1966–1971. [CrossRef] [PubMed] 248. Lloyd, C.M.; Marsland, B.J. Lung homeostasis: Inﬂuence of age, microbes, and the immune system. Im 2017, 46, 549–561. [CrossRef] [PubMed] 249. Boe, D.M.; Boule, L.A.; Kovacs, E.J. Innate immune responses in the ageing lung. Clin. Exp. Immunol. 2017, 187, 16–25. [CrossRef] 250. Bahadoran, A.; Lee, S.H.; Wang, S.M.; Manikam, R.; Rajarajeswaran, J.; Raju, C.S.; Sekaran, S.D. Immune responses to inﬂuenza virus and its correlation to age and inherited factors. Front. Microbiol. 2016, 7, 1841. [CrossRef] 251. Krone, C.L.; van de Groep, K.; Trzcinski, K.; Sanders, E.A.; Bogaert, D. Immunosenescence and pneumococcal disease: An imbalance in host-pathogen interactions. Lancet Respir. Med. 2014, 2, 141–153. [CrossRef] 252. Leung, G.M.; Hedley, A.J.; Ho, L.M.; Chau, P.; Wong, I.O.; Thach, T.Q.; Ghani, A.C.; Donnelly, C.A.; Fraser, C.; Riley, S.; et al. The epidemiology of severe acute respiratory syndrome in the 2003 Hong Kong epidemic: An analysis of all 1755 patients. Ann. Intern. Med. 2004, 141, 662–673. [CrossRef] y p 253. Hakim, F.T.; Gress, R.E. Immunosenescence: Deﬁcits in adaptive immunity in the elderly. Tissue Antigens 2007, 70, 179–189. [CrossRef] 254. Agostini, L.; Martinon, F.; Burns, K.; McDermott, M.F.; Hawkins, P.N.; Tschopp, J. NALP3 forms an IL-1beta-processing inﬂammasome with increased activity in Muckle-Wells autoinﬂammatory disorder. Immunity 2004, 20, 319–325. [CrossRef] 255. Kanneganti, T.D.; Body-Malapel, M.; Amer, A.; Park, J.H.; Whitﬁeld, J.; Franchi, L.; Taraporewala, Z.F.; Miller, D.; Patton, J.T.; Inohara, N.; et al. Critical role for Cryopyrin/Nalp3 in activation of caspase-1 in response to viral infection and double-stranded RNA. J. Biol. Chem. 2006, 281, 36560–36568. [CrossRef] [PubMed] 256. Martinon, F.; Tschopp, J. Inﬂammatory caspases and inﬂammasomes: Master switches of inﬂammation. Cell Death Diﬀer. 2007, 14, 10–22. [CrossRef] [PubMed] 257. Mariathasan, S.; Weiss, D.S.; Newton, K.; McBride, J.; O’Rourke, K.; Roose-Girma, M.; Lee, W.P.; Weinrauch, Y.; Monack, D.M.; Dixit, V.M. Cryopyrin activates the inﬂammasome in response to toxins and ATP. Nature 2006, 440, 228–232. [CrossRef] 30 of 32 30 of 32 Biomolecules 2020, 10, 1312 258. Monie, T.P. The canonical inﬂammasome: A macromolecular complex driving inﬂammation. Subcell Biochem. 2017, 83, 43–73. [CrossRef] 259. Nakahira, K.; Haspel, J.A.; Rathinam, V.A.; Lee, S.J.; Dolinay, T.; Lam, H.C.; Englert, J.A.; Rabinovitch, M.; Cernadas, M.; Kim, H.P.; et al. References The role of the NLRP3 inﬂammasome in pulmonary 264. Shi, C.S.; Nabar, N.R.; Huang, N.N.; Kehrl, J.H. SARS-coronavirus open reading frame-8b triggers intracellular stress pathways and activates NLRP3 inﬂammasomes. Cell Death Discov. 2019, 5, 101. [CrossRef] [PubMed] stress pathways and activates NLRP3 inﬂammasomes. Cell Death Discov. 2019, 5, 101. [CrossRef] [PubMed] 265. Hosseinian, N.; Cho, Y.; Lockey, R.F.; Kolliputi, N. The role of the NLRP3 inﬂammasome in pulmonary diseases. Ther. Adv. Respir. Dis. 2015, 9, 188–197. [CrossRef] [PubMed] 265. Hosseinian, N.; Cho, Y.; Lockey, R.F.; Kolliputi, N. The role of the NLRP3 inﬂammasome in pulmonary diseases. Ther. Adv. Respir. Dis. 2015, 9, 188–197. [CrossRef] [PubMed] 266. Castano-Rodriguez, C.; Honrubia, J.M.; Gutierrez-Alvarez, J.; DeDiego, M.L.; Nieto-Torres, J.L.; Jimenez-Guardeno, J.M.; Regla-Nava, J.A.; Fernandez-Delgado, R.; Verdia-Baguena, C.; Queralt-Martin, M.; et al. Role of severe acute respiratory syndrome coronavirus viroporins E, 3a, and 8a in replication and pathogenesis. mBio 2018, 9. [CrossRef] [PubMed] 267. Farag, N.S.; Breitinger, U.; Breitinger, H.G.; El Azizi, M.A. Viroporins and inﬂammasomes: A key to understand virus-induced inﬂammation. Int. J. Biochem. Cell Biol. 2020, 122, 105738. [CrossRef] 268. Siu, K.L.; Yuen, K.S.; Castano-Rodriguez, C.; Ye, Z.W.; Yeung, M.L.; Fung, S.Y.; Yuan, S.; Chan, C.P.; Yuen, K.Y.; Enjuanes, L.; et al. Severe acute respiratory syndrome coronavirus ORF3a protein activates the NLRP3 inﬂammasome by promoting TRAF3-dependent ubiquitination of ASC. FASEB J. 2019, 33, 8865–8877. [CrossRef] 269. Chan, J.F.-W.; Kok, K.-H.; Zhu, Z.; Chu, H.; To, K.K.-W.; Yuan, S.; Yuen, K.-Y. Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan. Emerg. Microbes Infect. 2020, 9, 221–236. [CrossRef] 270. Danthi, P. Viruses and the diversity of cell death. Annu. Rev. Virol. 2016, 3, 533–553. [Cross 271. Chen, C.Y.; Ping, Y.H.; Lee, H.C.; Chen, K.H.; Lee, Y.M.; Chan, Y.J.; Lien, T.C.; Jap, T.S.; Lin, C.H.; Kao, L.S.; et al. Open reading frame 8a of the human severe acute respiratory syndrome coronavirus not only promotes viral replication but also induces apoptosis. J. Infect. Dis. 2007, 196, 405–415. [CrossRef] p p p f 272. Yue, Y.; Nabar, N.R.; Shi, C.S.; Kamenyeva, O.; Xiao, X.; Hwang, I.Y.; Wang, M.; Kehrl, J.H. SARS-coronavirus open reading frame-3a drives multimodal necrotic cell death. Cell Death Dis. 2018, 9, 904. [CrossRef] 273. Zhao, C.; Zhao, W. NLRP3 Inﬂammasome—A key player in antiviral responses. Front. Immunol. 2020, 11, 211. [CrossRef] 274. References Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inﬂammasome. Nat. Immunol. 2011, 12, 222–230. [CrossRef] 260. Petrilli, V.; Papin, S.; Dostert, C.; Mayor, A.; Martinon, F.; Tschopp, J. Activation of the NALP3 inﬂammasome is triggered by low intracellular potassium concentration. Cell Death Diﬀer. 2007, 14, 1583–1589. [CrossRef] 260. Petrilli, V.; Papin, S.; Dostert, C.; Mayor, A.; Martinon, F.; Tschopp, J. Activation of the NALP3 inﬂammasome is triggered by low intracellular potassium concentration. Cell Death Diﬀer. 2007, 14, 1583–1589. [CrossRef] 261. Cho, S.J.; Plataki, M.; Mitzel, D.; Lowry, G.; Rooney, K.; Stout-Delgado, H. Decreased NLRP3 inﬂammasome expression in aged lung may contribute to increased susceptibility to secondary Streptococcus pneumoniae 261. Cho, S.J.; Plataki, M.; Mitzel, D.; Lowry, G.; Rooney, K.; Stout-Delgado, H. Decreased NLRP3 inﬂammasome expression in aged lung may contribute to increased susceptibility to secondary Streptococcus pneumoniae infection. Exp. Gerontol. 2018, 105, 40–46. [CrossRef] 262. Hoegen, T.; Tremel, N.; Klein, M.; Angele, B.; Wagner, H.; Kirschning, C.; Pﬁster, H.W.; Fontana, A.; Hammerschmidt, S.; Koedel, U. The NLRP3 inﬂammasome contributes to brain injury in pneumococcal meningitis and is activated through ATP-dependent lysosomal cathepsin B release. J. Immunol. 2011, 187, 5440–5451. [CrossRef] 263. Deftereos, S.G.; Siasos, G.; Giannopoulos, G.; Vrachatis, D.A.; Angelidis, C.; Giotaki, S.G.; Gargalianos, P.; Giamarellou, H.; Gogos, C.; Daikos, G.; et al. The Greek study in the eﬀects of colchicine in COvid-19 complications prevention (GRECCO-19 study): Rationale and study design. Hell. J. Cardiol. 2020. [CrossRef] 264. Shi, C.S.; Nabar, N.R.; Huang, N.N.; Kehrl, J.H. SARS-coronavirus open reading frame-8b triggers intracellular stress pathways and activates NLRP3 inﬂammasomes. Cell Death Discov. 2019, 5, 101. [CrossRef] [PubMed] 265 H i i N Ch Y L k R F K lli ti N Th l f th NLRP3 i ﬂ i l 263. Deftereos, S.G.; Siasos, G.; Giannopoulos, G.; Vrachatis, D.A.; Angelidis, C.; Giotaki, S.G.; Gargalianos, P.; Giamarellou, H.; Gogos, C.; Daikos, G.; et al. The Greek study in the eﬀects of colchicine in COvid-19 complications prevention (GRECCO-19 study): Rationale and study design. Hell. J. Cardiol. 2020. [CrossRef] complications prevention (GRECCO-19 study): Rationale and study design. Hell. J. Cardiol. 2020. [CrossRef] 264. Shi, C.S.; Nabar, N.R.; Huang, N.N.; Kehrl, J.H. SARS-coronavirus open reading frame-8b triggers intracellular stress pathways and activates NLRP3 inﬂammasomes. Cell Death Discov. 2019, 5, 101. [CrossRef] [PubMed] 265. Hosseinian, N.; Cho, Y.; Lockey, R.F.; Kolliputi, N. References Lung Surfactants: Basic Science and Clinical Applications; Marcel Dekker: New York, NY, USA, 2000. 287. Reynolds, H.Y. Lung inﬂammation: Normal host defense or a complication of some diseases? Annu. Rev. Med. 1987, 38, 295–323. [CrossRef] 288. Fagiolo, U.; Cossarizza, A.; Santacaterina, S.; Ortolani, C.; Monti, D.; Paganelli, R.; Franceschi, C. Increased cytokine production by peripheral blood mononuclear cells from healthy elderly people. Ann. N. Y. Acad. Sci. 1992, 663, 490–493. [CrossRef] [PubMed] 289. Moliva, J.I.; Rajaram, M.V.; Sidiki, S.; Sasindran, S.J.; Guirado, E.; Pan, X.J.; Wang, S.H.; Ross, P., Jr.; Lafuse, W.P.; Schlesinger, L.S.; et al. Molecular composition of the alveolar lining ﬂuid in the aging lung. Age 2014, 36, 9633. [CrossRef] [PubMed] 290. Usman, M.S.; Siddiqi, T.J.; Khan, M.S.; Patel, U.K.; Shahid, I.; Ahmed, J.; Kalra, A.; Michos, E.D. Is there a smoker’s paradox in COVID-19? BMJ Evid. Based Med. 2020. [CrossRef] [PubMed] 291. Jiang, C.; Chen, Q.; Xie, M. Smoking increases the risk of infectious diseases: A narrative review. Tob. Induc. Dis. 2020, 18, 60. [CrossRef] 292. Kaur, G.; Lungarella, G.; Rahman, I. SARS-CoV-2 COVID-19 susceptibility and lung inﬂammatory storm by smoking and vaping. J. Inﬂamm. 2020, 17, 21. [CrossRef] 293. Liu, W.; Tao, Z.W.; Wang, L.; Yuan, M.L.; Liu, K.; Zhou, L.; Wei, S.; Deng, Y.; Liu, J.; Liu, H.G.; et al. Analysis of factors associated with disease outcomes in hospitalized patients with 2019 novel coronavirus disease. Chin. Med. J. 2020, 133, 1032–1038. [CrossRef] 294. Lippi, G.; Henry, B.M. Active smoking is not associated with severity of coronavirus disease 2019 (COVID-19). Eur. J. Intern. Med. 2020, 75, 107–108. [CrossRef] 295. Chakladar, J.; Shende, N.; Li, W.T.; Rajasekaran, M.; Chang, E.Y.; Ongkeko, W.M. Smoking-mediated upregulation of the androgen pathway leads to increased SARS-CoV-2 susceptibility. Int. J. Mol. Sci. 2020, 21. [CrossRef] [PubMed] 296. Wambier, C.G.; Goren, A. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is likely to be androgen mediated. J. Am. Acad. Derm. 2020, 83, 308–309. [CrossRef] 297. Lucas, J.M.; Heinlein, C.; Kim, T.; Hernandez, S.A.; Malik, M.S.; True, L.D.; Morrissey, C.; Corey, E.; Montgomery, B.; Mostaghel, E.; et al. The androgen-regulated protease TMPRSS2 activates a proteolytic cascade involving components of the tumor microenvironment and promotes prostate cancer metastasis. Cancer Discov. 2014, 4, 1310–1325. [CrossRef] 298. Penna, C.; Mercurio, V.; Tocchetti, C.G.; Pagliaro, P. Sex-related diﬀerences in COVID-19 lethality. Br. J. Pharmacol. 2020. [CrossRef] [PubMed] 299. Papadopoulos, V.; Li, L.; Samplaski, M. References Wei, L.; Sun, S.; Xu, C.H.; Zhang, J.; Xu, Y.; Zhu, H.; Peh, S.C.; Korteweg, C.; McNutt, M.A.; Gu, J. Pathology of the thyroid in severe acute respiratory syndrome. Hum. Pathol. 2007, 38, 95–102. [CrossRef] [PubMed] 275. Zhang, Q.L.; Ding, Y.Q.; He, L.; Wang, W.; Zhang, J.H.; Wang, H.J.; Cai, J.J.; Geng, J.; Lu, Y.D.; Luo, Y.L. Detection of cell apoptosis in the pathological tissues of patients with SARS and its signiﬁcance. Di Yi Jun Yi Da Xue Xue Bao 2003, 23, 770–773. [PubMed] 276. Bordi, L.; Castilletti, C.; Falasca, L.; Ciccosanti, F.; Calcaterra, S.; Rozera, G.; Di Caro, A.; Zaniratti, S.; Rinaldi, A.; Ippolito, G.; et al. Bcl-2 inhibits the caspase-dependent apoptosis induced by SARS-CoV without aﬀecting virus replication kinetics. Arch. Virol. 2006, 151, 369–377. [CrossRef] [PubMed] 277. Ren, L.; Yang, R.; Guo, L.; Qu, J.; Wang, J.; Hung, T. Apoptosis induced by the SARS-associated coronavirus in vero cells is replication-dependent and involves caspase. DNA Cell Biol. 2005, 24, 496–502. [CrossRef] 31 of 32 31 of 32 Biomolecules 2020, 10, 1312 278. Krahling, V.; Stein, D.A.; Spiegel, M.; Weber, F.; Muhlberger, E. Severe acute respiratory syndrome coronavirus triggers apoptosis via protein kinase R but is resistant to its antiviral activity. J. Virol. 2009, 83, 2298–2309. [CrossRef] Lim, Y.X.; Ng, Y.L.; Tam, J.P.; Liu, D.X. Human coronaviruses: A review of virus-host interactions. Diseases 2016, 4. [CrossRef] 280. Chu, H.; Zhou, J.; Wong, B.H.; Li, C.; Chan, J.F.; Cheng, Z.S.; Yang, D.; Wang, D.; Lee, A.C.; Li, C.; et al. Middle East respiratory syndrome coronavirus eﬃciently infects human primary T lymphocytes and activates the extrinsic and intrinsic apoptosis pathways. J. Infect. Dis. 2016, 213, 904–914. [CrossRef] 281. Jiang, Y.; Li, J.; Teng, Y.; Sun, H.; Tian, G.; He, L.; Li, P.; Chen, Y.; Guo, Y.; Li, J.; et al. Complement receptor C5aR1 inhibition reduces pyroptosis in hDPP4-transgenic mice infected with MERS-CoV. Viruses 2019, 11. [CrossRef] 282. Vaz Fragoso, C.A. Epidemiology of lung disease in older persons. Clin. Geriatr. Med. 2017, 33, 491–501. [CrossRef] 283. Dyer, C. The interaction of ageing and lung disease. Chron. Respir. Dis. 2012, 9, 63–67. [CrossRef] 284. Akgun, K.M.; Crothers, K.; Pisani, M. Epidemiology and management of common pulmonary d older persons. J. Gerontol. Ser. A Biol. Sci. Med. Sci. 2012, 67, 276–291. [CrossRef] [PubMed] 85. Olloquequi, J. COVID-19 Susceptibility in chronic obstructive pulmonary disease. Eur. J. Clin. Invest. 2 [CrossRef] [PubMed] 286. Notter, R.H. References Why does COVID-19 kill more elderly men than women? Is there a role for testosterone? Andrology 2020. [CrossRef] 32 of 32 Biomolecules 2020, 10, 1312 32 of 32 300. Li, Y.; Jerkic, M.; Slutsky, A.S.; Zhang, H. Molecular mechanisms of sex bias diﬀerences in COVID-19 mortality. Crit. Care 2020, 24, 405. [CrossRef] [PubMed] 301. Patel, V.G.; Zhong, X.; Liaw, B.; Tremblay, D.; Tsao, C.K.; Galsky, M.D.; Oh, W.K. Does androgen deprivation therapy protect against severe complications from COVID-19? Ann. Oncol. 2020. [CrossRef] 302. Caﬀo, O.; Zagonel, V.; Baldessari, C.; Berruti, A.; Bortolus, R.; Buti, S.; Ceresoli, G.L.; Donini, M.; Ermacora, P.; Fornarini, G.; et al. On the relationship between androgen-deprivation therapy for prostate cancer and risk of infection by SARS-CoV-2. Ann. Oncol. 2020. [CrossRef] 303. Montopoli, M.; Zumerle, S.; Vettor, R.; Rugge, M.; Zorzi, M.; Catapano, C.V.; Carbone, G.M.; Cavalli, A.; Pagano, F.; Ragazzi, E.; et al. Androgen-deprivation therapies for prostate cancer and risk of infection by SARS-CoV-2: A population-based study (N = 4532). Ann. Oncol. 2020, 31, 1040–1045. [CrossRef] 305. Marban, C.; Suzanne, S.; Dequiedt, F.; de Walque, S.; Redel, L.; Van Lint, C.; Aunis, D.; Rohr, O. Recruitment of chromatin-modifying enzymes by CTIP2 promotes HIV-1 transcriptional silencing. EMBO J. 2007, 26, 412–423. [CrossRef] [PubMed] 306. Taylor, T.J.; Knipe, D.M. Proteomics of herpes simplex virus replication compartments: Association of cellular DNA replication, repair, recombination, and chromatin remodeling proteins with ICP8. J. Virol. 2004, 78, 5856–5866. [CrossRef] [PubMed] 307. Mehta, S.; Jeﬀrey, K.L. Beyond receptors and signaling: Epigenetic factors in the regulation of innate immunity. Immunol. Cell Biol. 2015, 93, 233–244. [CrossRef] 308. Gomez-Diaz, E.; Jorda, M.; Peinado, M.A.; Rivero, A. Epigenetics of host-pathogen interactions: The road ahead and the road behind. PLoS Pathog. 2012, 8, e1003007. [CrossRef] [PubMed] 309. Marazzi, I.; Ho, J.S.; Kim, J.; Manicassamy, B.; Dewell, S.; Albrecht, R.A.; Seibert, C.W.; Schaefer, U.; Jeﬀrey, K.L.; Prinjha, R.K.; et al. Suppression of the antiviral response by an inﬂuenza histone mimic. Nature 2012, 483, 428–433. [CrossRef] 310. Hale, B.G.; Randall, R.E.; Ortin, J.; Jackson, D. The multifunctional NS1 protein of inﬂuenza A viruses. J. Gen. Virol. 2008, 89, 2359–2376. [CrossRef] 311. Menachery, V.D.; Eisfeld, A.J.; Schafer, A.; Josset, L.; Sims, A.C.; Proll, S.; Fan, S.; Li, C.; Neumann, G.; Tilton, S.C.; et al. Pathogenic inﬂuenza viruses and coronaviruses utilize similar and contrasting approaches to control interferon-stimulated gene responses. mBio 2014, 5, e01114–e01174. [CrossRef] 312. References Sawalha, A.H.; Zhao, M.; Coit, P.; Lu, Q. Epigenetic dysregulation of ACE2 and interferon-regulated genes might suggest increased COVID-19 susceptibility and severity in lupus patients. Clin. Immunol. 2020, 215, 108410. [CrossRef] © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W4390344548	https://jpionline.org/storage/2023/12/IntJPharmInvestigation-14-1-30.pdf	English	null	Unlocking the Clinical Significance of Cytochrome P450 Enzymes	International journal of pharmaceutical investigation	2,023	cc-by	8,980	ABSTRACT ABSTRACT Aim/Background: The Cytochrome P450 (CYP) enzyme family, consisting of 57 distinct genes, plays a pivotal role in various biological processes. These genes encode enzymes with multifaceted functions, impacting vital processes like arachidonic acid metabolism, xenobiotic detoxification, eicosanoids production, and drug metabolism. Moreover, CYP enzymes are indispensable for the synthesis of bile acids, steroids, and numerous metabolic pathways. They also participate in the hydroxylation of retinoic acid, illustrating their remarkable versatility. While some CYP enzymes still have undiscovered functions, their role continues to captivate researchers across diverse domains. Additionally, mutations in CYP genes can give rise to inborn metabolic disorders, leading to clinically significant diseases, underscoring the importance of CYP enzymes in maintaining metabolic equilibrium and overall well-being. Beyond their initial association with hepatic drug detoxification, recent research has unveiled a broader spectrum of enzymatic processes undertaken by cytochrome P450. The complexity of CYP enzymes is progressively unfolding, emphasizing their clinical significance and opening new avenues for drug development, precision medicine, and patient care. methodology: This abstract presents a comprehensive overview of the literature on cytochrome P450 enzymes and their diverse functions. The information was gathered from a wide range of sources, including scientific articles, textbooks, and research publications. The analysis covers the genetic basis of CYP enzymes, their roles in various metabolic pathways, and the clinical implications of CYP gene mutations. Additionally, the abstract highlights the recent advances in our understanding of CYP enzymes and their expanding role in maintaining human life. Results: The results of this study showcase the remarkable diversity of functions performed by cytochrome P450 enzymes. They are intricately involved in processes critical to human health, ranging from drug metabolism to the synthesis of vital biomolecules. The study underlines the clinical significance of CYP enzymes, as mutations in these genes can lead to severe metabolic disorders and diseases. Furthermore, it emphasizes the evolving understanding of CYP enzyme complexity and its implications for drug development, precision medicine, and patient care. Conclusion: In conclusion, this abstract sheds light on the exceptional versatility and clinical importance of cytochrome P450 enzymes. Their diverse functions have far-reaching consequences for human health, making them a focal point of research in pharmacology and medicine. The evolving understanding of CYP enzyme complexity paves the way for future advancements in drug development and personalized patient care, offering new possibilities for enhancing the well-being of individuals and populations. Int. J. Pharm. Investigation, 2024; 14(1):30-38. https://www.jpionline.org Int. J. Pharm. Investigation, 2024; 14(1):30-38. https://www.jpionline.org Review Article ABSTRACT This research underscores the significance of CYP enzymes and their potential to revolutionize the fields of medicine and pharmacology. j Assistant Professor, Department of Pharmacy Practice, Sumandeep Vidyapeeth (Deemed to be University), Vadodara-391760, Gujarat, INDIA. Email: docrajesh.hadia@gmail.com Received: 29-09-2023; Revised: 19-10-2023; Accepted: 11-05-2023. Keywords: Cytochrome P450, Drug Metabolism, Clinical Significance. Unlocking the Clinical Significance of Cytochrome P450 Enzymes Rajesh Hadia, Vishal Singh, Nidhi Solanki, Shiwanand Sharma, Varunsingh Saggu, Cyril Sajan, Rahul Trivedi, Sunil Kardani, Sunil Baile, Hemraj Singh Rajput, Rajesh Maheshwari Department of Pharmacy, Sumandeep Vidyapeeth (Deemed to be University), Vadodara, Gujarat, INDIA. Received: 29-09-2023; Revised: 19-10-2023; Accepted: 11-05-2023. Correspondence: Dr. Rajesh Hadia Assistant Professor, Department of Pharmacy Practice, Sumandeep Vidyapeeth (Deemed to be University), Vadodara-391760, Gujarat, INDIA. Email: docrajesh.hadia@gmail.com International Journal of Pharmaceutical Investigation, Vol 14, Issue 1, Jan-Mar, 2024 INTRODUCTION In addition to confirming the existence of numerous CYP enzymes, later cloning research also revealed the enormous diversity present in this family of proteins. Fascinating discoveries were made through the comparative examination of their sequences, which showed startling parallels between the cytochromes P450 present in humans and bacteria. These similarities suggested the existence of a shared ancestor gene that dates back around three billion years.2 Such findings highlighted the crucial function of CYP enzymes in fundamental biological processes and its deep evolutionary significance. A systematic classification system was created more than 15 years ago in response to the growing understanding of the complexity of the CYP family. This naming approach, which was founded on the ideas of divergent evolution, intended to scientifically classify and name the different CYP enzymes. This nomenclature system is dynamic and is constantly being improved and expanded through online platforms, guaranteeing that our knowledge of CYP enzymes keeps up with the rapidly expanding field of molecular biology.3,4 These advancements not only reflect important turning points in the study of CYP but also demonstrate how collaborative and dynamic scientific research is as it reveals the mysteries of this amazing enzyme family. This nomenclature system is dynamic and is constantly being improved and expanded through online platforms, guaranteeing that our knowledge of CYP enzymes keeps up with the rapidly expanding field of molecular biology. These advancements not only reflect important turning points in the study of CYP but also demonstrate how collaborative and dynamic scientific research is as it reveals the mysteries of this amazing enzyme family. identity are categorized into distinct families, each of which is denoted by an Arabic number. This classification makes it easier to comprehend the evolutionary connections and functional traits that enzymes in the same family share. In addition, subfamilies are formed for enzymes that have a stronger degree of sequence similarity, often at a threshold of 55% or higher. This level of categorization granularity enables a more exact discrimination between closely related enzymes, frequently revealing subtle functional variations. The Cytochrome P450 nomenclature organizes the vast and complicated world of CYP proteins in a systematic and hierarchical manner, simplifying the complex terrain of these enzymes while also giving researchers and scientists a framework to work within. This strategy emphasizes the value of sequence-based classification in helping us better understand the roles and purposes of these crucial proteins. INTRODUCTION Since its original identification in 1961, Cytochrome P450 (CYP) has emerged as a pivotal milestone in the history of scientific research. The peculiar designation P450 was given to this cellular chromophore because it exhibits a spectacular 450 nm spectral peak when examined in a reduced state when bound to carbon monoxide. As research developed until the middle of the 1960s, CYP's real complexity started to emerge, demonstrating its crucial function in drug metabolism and steroid production. CYP was initially thought of as a single enzyme during the early 1960s. The late 1970s saw intriguing hypotheses of the existence of numerous P450 enzymes emerge as the scientific community dug deeper into the mysteries of CYP. However, revealing the entire fabric of this enzyme system proved to be a difficult task. The hydrophobic nature of CYP, which is embedded in cellular membranes and hinders straightforward purification, makes it Since its original identification in 1961, Cytochrome P450 (CYP) has emerged as a pivotal milestone in the history of scientific research. The peculiar designation P450 was given to this cellular chromophore because it exhibits a spectacular 450 nm spectral DOI: 10.5530/ijpi.14.1.5 Copyright Information : Copyright Author (s) 2024 Distributed under Creative Commons CC-BY 4.0 Publishing Partner : EManuscript Tech. [www.emanuscript.in] International Journal of Pharmaceutical Investigation, Vol 14, Issue 1, Jan-Mar, 2024 30 Hadia, et al.: Clinical Significance of Cytochrome P450 Enzymes difficult to determine the specific number of proteins involved. This process of learning, characterized by early excitement, progressive revelation, and continuing hurdles, illustrates the ongoing intrigue and significance of Cytochrome P450 in the field of biology and medicine. In this introduction tale, we begin a historical investigation of CYP, a protein whose intricate nature continues to fascinate researchers and holds the potential to provide deep new understandings of fundamental biochemical processes and their clinical ramifications. A critical period in the study of cytochrome P450 (CYP) occurred in the early 1980s, which helped to spark important developments in the discipline of molecular biology. Gonzalez and his colleagues successfully isolated the first complete cDNA encoding a cytochrome P450 protein during this transformative era, which contributed to the advancement of mRNA purification techniques.1 This accomplishment paved the way for a deeper investigation of the CYP family and opened the door to a new world of opportunities. International Journal of Pharmaceutical Investigation, Vol 14, Issue 1, Jan-Mar, 2024 INTRODUCTION Our understanding of P450 enzymes has been fundamentally altered by advances in molecular biology and genetics, which cast doubt on the idea that these enzymes are principally involved in the liver's metabolism of drugs. First of all, by catalyzing a variety of reactions on a wide range of endogenous substrates, these enzymes have demonstrated amazing adaptability. A wide variety of substances, including fatty acids, eicosanoids, sterols, steroids, bile acids, retinoids, and uroporphyrinogens, are transformed by these enzymes through oxidative, peroxidative, and reductive processes. Furthermore, P450 enzymes demonstrate the ability to metabolize a wide range of exogenous substances, including medications, contaminants, and natural chemicals obtained from plants, demonstrating their adaptability to varied molecular configurations.4,5 P450 enzymes can also produce potentially hazardous by-products, which can increase the risk of cancer, birth abnormalities, and other toxic effects, even though they frequently detoxify foreign substances. The buildup of particular substrates can also cause the expression of a variety of P450 enzymes. For instance, relevant P450 enzymes are activated to effectively digest and remove the extra substrate when certain chemicals build up in the liver.8,9 These discoveries deepen our understanding of P450 enzymes and demonstrate the diverse functions they play in both health and sickness. These results demonstrate the diversity of cytochrome P450 enzymes and the importance of their role in the metabolism of a wide variety of exogenous and endogenous chemicals. Understanding the functions of these enzymes in both typical physiological processes and pathological situations is crucial because of the complicated interactions that these enzymes have with the numerous substrates that they bind to. This wide viewpoint is crucial for understanding the complexity of biological systems and developing targeted treatments for both health and illness. The cytochrome P450 enzymes play a crucial role in the metabolism of eicosanoids, arachidonic acid, and exogenous compounds. Foreign chemicals, also known as xenobiotics, include drugs, secondary metabolites from plants or fungi consumed through food, as well as different environmental pollutants like halogenated hydrocarbons, polycyclic aromatic hydrocarbons, arylamines, combustion by-products, industrial mixtures, herbicides, and pesticides. The CYP1, CYP2, CYP3, and, to a lesser extent, CYP4 family of cytochrome P450 enzymes are principally responsible for metabolizing these foreign substances within the human body. It's important to note that each of these gene families has a large number of allelic variants, which results in pharmacogenetic variation between people. INTRODUCTION Enzymes that display at least 40% sequence International Journal of Pharmaceutical Investigation, Vol 14, Issue 1, Jan-Mar, 2024 31 Hadia, et al.: Clinical Significance of Cytochrome P450 Enzymes been found in equal numbers in the rice genome.4 However; the human genome only has 57 CYP genes and 33 pseudogenes, which is a striking difference. There are 42 subfamilies and 18 families in which these genes fall. Unless new active members of the CYP2G and CYP2T subfamilies are discovered, which could potentially increase the existing number; it is important to note that large changes to this count are unlikely. This discrepancy in CYP gene representation levels between species emphasizes the distinctive genetic traits and functional requirements inherent to each creature.7 The CYP gene families' extreme diversity and complexity illustrate the fascinating connection between genetics and the myriad biological mechanisms that control many animals. We learn more about the processes governing metabolism, adaptability, and the evolution of life on Earth as we delve deeper into these varied genetic landscapes. medications that depend on CYP3A enzymes for their breakdown to be eliminated from the body more quickly. Rifampicin also affects a number of CYP2C enzymes, which causes the elimination of drugs processed by these enzymes to happen more quickly. When one P450 substrate affects the concentrations and metabolism of another, the phenomenon known as drug interactions results, complicating treatment regimens.10 Individual cytochrome P450s have a variety of biological and clinical roles, and the availability of cloned genes and the subsequent creation of biochemical and immunochemical probes derived from these cDNAs have shed light on their different biological and clinical functions. The metabolism of endogenous substrates and the synthesis of hydrophobic lipids including cholesterol, bile acids, steroid hormones, and fatty acids are both crucially dependent on these enzymes. We give a brief review of the physiological functions performed by numerous human cytochrome P450s below, along with how those functions may affect clinical outcomes. This information sheds light on the complex interactions that exist between these enzymes and physiological functions in the body, which ultimately affect how diseases develop, how drugs work, and how patients fare as a whole. It is essential to comprehend these intricate relationships in order to improve patient care and therapeutic approaches. INTRODUCTION Let's look at two well-known examples to show how useful this classification method is: the sterol 27-hydroxylase and Vitamin D3 24-hydroxylase enzymes. These enzymes belong to the same CYP27 family because they share a considerable amount of sequence similarity-at least 40%. However, by identifying separate subfamily names based on the level of sequence similarity, the approach improves precision. Due to less than 55% commonality in their protein sequences, vitamin D3 24-hydroxylase and sterol 27-hydroxylase are classified as CYP27A and CYP27B, respectively. This naming scheme is particularly strong since it is flexible enough to accommodate new discoveries. If, for example, another enzyme were to appear that shared at least 55% of the sequence with sterol 27-hydroxylase, it would naturally be given the name CYP27A2, maintaining a steady and orderly progression. This methodological approach has successfully reduced the naming-related ambiguity that is frequently present in gene families and super families. In addition to fostering clarity and precision in communication, establishing a well-structured system for the classification of Cytochrome P450 enzymes also makes it easier to grasp their various roles within the scientific community. As a result, the nomenclature system has evolved into a crucial instrument in the field of molecular biology, facilitating the classification and identification of these vital proteins.1,2 There are already more than 270 different Cytochrome P450 (CYP) gene families, 18 of which have been shown to exist in mammals, making the landscape of CYP gene families nothing short of comprehensive.4 Given the enormous variety of tiny molecules present in the world of plants, this astounding diversity should not come as a surprise. It was anticipated that plants would have a large number of cytochrome P450 enzymes,5,6 and when the genome of the little mustard plant Arabidopsis thaliana was examined, this prediction was verified. A startling total of 249 active CYP genes and 24 non-functional pseudogenes were found in Arabidopsis thaliana, accounting for an amazing 1% of the plant's total number of genes. At least 324 functional CYP genes have also Based mostly on the degree of amino acid sequence similarity, the classification of Cytochrome P450 proteins into separate families and subfamilies offers a highly effective and methodical methodology. This approach helps to categorize these enzymes thoroughly by making the naming system more organized and approachable. International Journal of Pharmaceutical Investigation, Vol 14, Issue 1, Jan-Mar, 2024 INTRODUCTION These genetic variants alter the efficacy and safety profiles of medications by influencing how individuals metabolize and react to xenobiotics. Cytochrome P450 enzymes are essential for the metabolism of arachidonic acid and eicosanoids in addition to their function in the metabolism of foreign chemicals. These lipid mediators are essential for controlling physiological processes like inflammation, vascular homeostasis, and others. Arachidonic acid is metabolized by cytochromes P450 of the CYP2 and CYP4 families, which produce eicosanoids such prostaglandins and leukotrienes that control a variety of cellular functions. The intricacy of inter individual variations in medication response, vulnerability to environmental toxins, and overall health outcomes is highlighted by the occurrence of allelic variants within the gene families implicated in metabolizing foreign compounds and lipid mediators. Understanding the effects of genetic differences in cytochrome P450 enzymes is essential for The expression of human CYP3A enzymes can be induced by the consumption of medications like rifampicin, which is frequently administered for bacterial infections. In addition to speeding up the metabolism of rifampicin, this induction also causes other 32 Hadia, et al.: Clinical Significance of Cytochrome P450 Enzymes directing personalized medicine techniques, enabling customized pharmacological regimens, and minimizing potential side effects. An substantial database of human CYP gene allelic variations is available online, offering a consistent classification scheme to aid in this understanding.11 Both scholars and doctors can benefit from this database as a beneficial tool. The consensus or reference sequence, designated as the (1 allele) within this classification scheme, often reflects an efficient-metabolism phenotype. The poor metabolism phenotype, on the other hand, is typically encoded by variant alleles and is characterized by reduced or absent enzyme activity toward particular medicines. A variant genotype may occasionally represent an ultra-metabolism phenotype with extremely high enzyme activity as a result of gene duplications. It is critical to understand that individual variations in these genes can have a big impact on how drugs work and how they are metabolized. The rates of ambient chemical detoxification and metabolic activation can also range significantly amongst people with various CYP haplotypes. Particularly among people with an efficient metabolism or high activity phenotype, some CYP enzymes, including CYP1A1, CYP1A2, CYP1B1, CYP2D6, CYP2E1, CYP3A4, and CYP3A5, have been related to an increased risk of certain types of cancer or harmful consequences. INTRODUCTION These effects can also be made worse by the co-inheritance of other polymorphic enzymes that are involved in the same metabolic pathway as the medication or chemical.12-18 Despite the strong evidence provided by animal research, it is still extremely difficult to confirm these correlations in clinical populations. The crucial function of cytochrome P450 enzymes in regulating individual reactions to medications and environmental exposures is nevertheless highlighted by these studies, opening the door for more specialized and efficient medicinal interventions. Additionally, CYP1A1 has the ability to inactivate prostaglandin G2, whereas CYP1A2 and CYP1B1 play different roles de the hydroxylation of estrogen at the carbon-2 and carbon-4 locations, respectively. Additionally, CYP1A2 participates in the metabolism of melatonin and helps to oxidize uroporphyrinogen.20-22 Comparatively, CYP1A1 and CYP1B1 are not predominantly drug-metabolizing enzymes, but CYP1A2 demonstrates metabolic activity toward about 10-20 distinct medications. Although the causes of the higher expression of CYP1B1 in some solid tumors are yet unknown,23 this finding may present prospects for the development of chemotherapeutic therapies. The ability of all three CYP1 enzymes to detoxify or activate different environmental carcinogens is significant. Understanding the complex roles and controls of the CYP1 gene family can help us better understand how PAHs, arylamines, endogenous ligands, and other substrates are metabolized. Understanding the mechanisms underlying their participation in solid tumors offers up the possibility of using these enzymes for specialized treatment approaches. The CYP1 enzymes' extensive detoxifying and activation capacities show their importance in protecting human health by underscoring their critical role in managing environmental toxins. Intriguing results have been obtained from extensive research with animals lacking the Cyp1a1, Cyp1a2, and Cyp1b1 genes. These mice continue to live despite changes in drug and carcinogen metabolism, indicating functional redundancy or non-essential functions for these enzymes in the metabolization of endogenous substances.24-26 The precise roles and contributions of these enzymes in typical physiological processes are called into doubt by these discoveries in compelling ways. Contrarily, primary congenital glaucoma, also known as buphthalmos, has been linked to mutations in the human CYP1B1 gene.27 This clinical finding shows that CYP1B1's metabolism of an essential endogenous substrate is necessary for the correct development of the anterior chamber of the eye during embryogenesis. Given that CYP1B1 is involved in the synthesis and breakdown of retinoic acid,28 it is conceivable to assume that this route contributes to the genesis of primary congenital glaucoma. International Journal of Pharmaceutical Investigation, Vol 14, Issue 1, Jan-Mar, 2024 INTRODUCTION More than 75 medications are believed to be metabolized by CYP2D6 according to in vitro research.15 Different medications are also metabolized by other family members, including CYP2A6, CYP2A13, CYP2B6, CYP2E1, CYP2F1, and CYP2J2.30,31 The roles of other members, including CYP2A7, CYP2R1, CYP2S1, CYP2U1, and CYP2W1, are currently unknown. While the CYP2C subfamily of enzymes are predominantly involved in the metabolism of drugs, there is evidence that a CYP2C enzyme, stimulated by -naphthoflavone, contributes to the vascular endothelium's synthesis of the vasodilator 11,12-epoxyeicosatrienoic acid.32 The majority of CYP gene products in vertebrates presumably initially developed for critical life tasks before obtaining the capacity to breakdown plant compounds and metabolize medicines, according to this study that emphasizes a recurrent topic in P450 research.8 The CYP2G and CYP2T subfamilies appear to be pseudogenes in humans, despite the fact that they encode functional genes in rodents. This suggests that whatever purposes these genes may have served during the mammalian radiation some 80 million years ago are no longer required in humans. Notably, mice lacking the Cyp2e1 gene appear normal on the outside but have remarkable resistance to benzene toxicity,26 highlighting the role of this subfamily in xenobiotic metabolism. These findings highlight the CYP2 gene family's amazing diversity and complexity, with its considerable contributions to drug metabolism and potential significance in a number of physiological processes. The particular roles of the uncharacterized members must be revealed, and their consequences for human health and disease must be better clarified, through ongoing research. (PXR), a transcription factor that is ligand-activated and a member of the nuclear hormone receptor superfamily, mediates this induction. PXR interacts with particular DNA patterns or response elements in the regulatory regions of CYP3A genes to bind to small molecules and stimulate the transcription of those genes. The capacity of a substance to engage in interaction with the ligand-binding region of the PXR receptor can be used to explain differences in the induction of CYP3A enzymes between species.38,39 The ability of some medications to shield organisms from the hazardous effects of other chemicals is explained by this regulatory mechanism and its pharmacological features.40 For instance, pregnenolone derivatives can reduce the hepatotoxicity brought on by consuming drugs like indomethacin and digitoxin. These pregnenolone-related chemicals function as PXR ligands, causing the creation of CYP3A enzymes, which then render dangerous drugs inactive. The active ingredient in St. INTRODUCTION John's wort, hypericum, activates the PXR and CYP3A genes in a manner similar to this, enhancing the metabolism of a variety of substances, including prescription medicines and the negative consequences linked to them.41 In addition to PXR, the Constitutive Androstane Receptor (CAR), a different member of the nuclear hormone receptor superfamily, can also stimulate several CYP2B and CYP3A genes. It has been found that CAR can activate CYP3A genes through PXR response elements, and PXR can control CYP2B genes through the CAR or phenobarbital response element, despite the regulatory elements or DNA patterns in the regulatory regions of these genes being different for CAR and PXR.42,43 This interaction of receptor transcription factors provides an additional line of defence against the negative effects of hazardous substances, including those present in plants. Predicting drug-drug interactions and developing new therapies can both benefit greatly from an understanding of the mechanisms behind drug metabolism and the regulatory pathways including PXR and CAR. PXR and its target genes, including the CYP3A enzymes, can be used in screening techniques that could help us better understand possible drug interactions and create treatments that work better. The four members of the CYP3 gene family are CYP3A4, CYP3A5, CYP3A7, and CYP3A43. These include the highly expressed CYP3A4 and CYP3A5, which are essential for the metabolism of over 120 regularly prescribed medications12-17 as well as endogenous substrates such steroids and bile acids.33,34 They play an important clinical role in the metabolism of some antifungal and immunosuppressive medications. When given the recommended amount, those with a poor-metabolizer phenotype might experience excessive drug concentrations, whereas people with an extensive-metabolizer phenotype might have poor metabolism of these medications, resulting in inadequate drug levels. Further research on the involvement of hepatic CYP3A43 in drug metabolism is necessary because its function is not fully understood. While CYP3A7 is expressed in the uterine endometrium and fetal liver, its precise roles in these tissues are still understood. A key route controls the expression of CYP3A enzymes in the liver and gut.35 The expression of members of the CYP3A family has been demonstrated to be induced by a number of medications, and the degree of induction by a specific molecule can differ between species.36,37 Pregnane X Receptor CYP4A11, CYP4B1, CYP4F2, CYP4F3, CYP4F8, CYP4F11, CYP4F12, CYP4F22, CYP4F22, CYP4A20, CYP4A22, CYP4V2, and CYP4X1 are the 12 members of the CYP4 gene family. INTRODUCTION It is still unclear how the overlapping substrate specificities of CYP1A1 and CYP1A2 interact with the retinoic acid pathway. The complicated relationships within this biological network are highlighted by the fact that mice with a defective Ahr gene, relevant to this debate, show liver retinoid buildup and impaired retinoic acid metabolism. These fascinating findings raise questions about the specific roles and interactions of the CYP1 gene family in healthy physiology as well as disease processes. Further investigations are essential to elucidate the specific roles of these enzymes in various pathways, including retinoic acid metabolism, and to gain a comprehensive understanding of their significance in human health and development.29 The largest P450 family in mammals and home to the widest In order to implement personalized medicine strategies, it is essential to comprehend the effects of genetic polymorphisms in the Cytochrome P450 (CYP) genes. This will help medical professionals choose the best drugs, alter their dosages, and assess the likelihood of adverse responses. The intricate interactions between genetic diversity, medication metabolism, and individual responses are gradually becoming clearer because to ongoing research and advancements in genomic medicine, which ultimately improve patient care and treatment outcomes. A member of the CYP gene family, CYP1 is notable for being predominantly controlled by the polycyclic aromatic hydrocarbons (PAHs)-activated aryl hydrocarbon receptor, a transcription factor. Smoke from cigarettes, charcoal-grilled meals, and industrial incineration products are all common sources of these PAHs. While CYP1A2 primarily metabolizes arylamines and N-heterocyclics, CYP1A1 and CYP1B1 of the CYP1 gene family show variable expression levels across organs and effectively metabolize PAHs. Interestingly, an unexplained endogenous ligand for the aryl hydrocarbon receptor is metabolized by CYP1A1, CYP1A2, and potentially CYP1B1.19 The largest P450 family in mammals and home to the widest variety of enzymes with a variety of roles is the CYP2 gene family. In particular, numerous members of this family of human International Journal of Pharmaceutical Investigation, Vol 14, Issue 1, Jan-Mar, 2024 33 Hadia, et al.: Clinical Significance of Cytochrome P450 Enzymes enzymes, including CYP2C8, CYP2C9, CYP2C18, and CYP2C19, play crucial roles in the metabolism of more than half of the medications that are often prescribed, as well as other compounds including arachidonic acid and certain steroids. International Journal of Pharmaceutical Investigation, Vol 14, Issue 1, Jan-Mar, 2024 INTRODUCTION important to note that human kidney enzymes like CYP4A11 and CYP4F2 have also been identified to convert arachidonic acid to 20-HETE,45 even though research on blood pressure regulation has mostly been done in rats. In addition to the cytochrome P450 enzymes' roles in the metabolism of pharmaceuticals and foreign substances, arachidonic acid serves as a substrate for a number of these enzymes.8 Prostaglandins D2, F2, and E2 as well as Epoxyeicosatrienoic acids (EETs), Hydroxyeicosatetraenoic acids (HETEs), and Hydroperoxyeicosatetraenoic acids (HPETEs) are among the over 100 eicosanoids metabolites produced as a result of arachidonic acid metabolism. These eicosanoids play a role in a variety of physiological processes, including the contraction of smooth muscle, edema formation, intestinal vasodilation, allergic reactions, chemotaxis, inhibition of platelet aggregation, bone resorption, the production of fever, the mobilization of intracellular calcium, the modulation of sodium and potassium ATPase, egg formation, and angiogenesis.46,47 It is very likely that there are disorders caused specifically by mutations in the P450 enzymes involved in the metabolism of arachidonic acid, even if these diseases have not been thoroughly characterized. Notably, thromboxane A2 synthase (CYP5A1) and prostacyclin synthase (CYP8A1), two distinct P450 enzymes, perform opposite roles in blood clotting. Thromboxane A2, which is made by CYP5A1, lowers the level of cyclic AMP in platelets and encourages platelet aggregation. Prostaglandin I2 (prostacyclin), on the other hand, is produced by CYP8A1 and increases intracellular cyclic AMP levels while preventing platelet aggregation. Therefore, it is expected that mutations in the CYP5A1 or CYP8A1 genes may cause clotting and inflammatory disorders, including illnesses like coronary artery disease and pulmonary hypertension.48,49 Several cytochrome P450 enzymes work in concert to synthesize and process steroids, particularly in the early embryonic phases of sexual differentiation. The nuclear hormone receptor gene family member steroid-factor-1, a transcription factor, is essential for upregulating P450 genes, which include those from the CYP11, CYP17, CYP19, and CYP21 families and are involved in the manufacture of steroid hormones.59 A trio of enzymes called CYP11A1, CYP11B1, and CYP11B2 are found in the mitochondria. Cortisol, testosterone, and estrogen are all produced by the enzyme CYP17A1, while androgenic precursors are changed into estrogens by the enzyme CYP19A1. The endoplasmic reticulum contains the genes CYP17A1 and CYP19A1. CYP17A1 is a dual-purpose enzyme that can catalyse both the cleavage and oxidation of the side chains of steroid substrates as well as the 17-hydroxylation of those same substrates. INTRODUCTION Even though some of these genes play a part in the metabolism of drugs, their main function is in the metabolism of fatty acids, such as arachidonic acid, leukotrienes, prostaglandins, Epoxyeicosatrienoic acids (EETs), Hydroxyeicosatetraenoic acids (HETEs), and Hydroperoxyeicosatetraenoic acids (HPETEs). Particularly, CYP4F8, CYP4F11, CYP4F12, and CYP4F22 are connected to the metabolism of fatty acids and arachidonic acid (7). CYP4A20, CYP4A22, CYP4V2, and CYP4X1 are currently thought to have uncertain roles (43). The distal convoluted tubules of the kidney express a number of CYP4A and CYP4B enzymes. Alterations in salt metabolism, water balance, and arterial blood pressure can result from defects in particular CYP4 genes.44 It's 34 Hadia, et al.: Clinical Significance of Cytochrome P450 Enzymes to the carbon-7 of the B ring in the substrates cholesterol and oxysterol. A nuclear hormone receptor called the farnesoid X receptor (FXR) controls the CYP7A1 gene. Bile acids, cholesterol, triglycerides, and proatherogenic serum lipoproteins are all increased in Fxr-deficient mice.52 The major bile acid cholate is produced by the sterol 12-hydroxylase CYP8B1.53 Involved in the production of oxysterol and the oxidation of the sterol side-chain, CYP27A1 functions as a sterol 27-/26-hydroxylase.54 Additionally, CYP46A1 takes involvement in the synthesis of oxysterols.55 Because it is primarily expressed in central nervous system neurons, CYP46A1 stands apart within the P450 superfamily. This enzyme changes cholesterol into a particular oxysterol called 24S-hydroxycholesterol, which, unlike cholesterol itself, may easily pass the blood-brain barrier. Because it is transformed by the liver into bile acids, 24S-hydroxycholesterol production and excretion into the bloodstream are essential for maintaining healthy levels of cholesterol in the brain. They also help with reverse cholesterol transport. Multiple P450 genes involved in the production of bile acids have had mutations discovered and characterized. Hypercholesterolemia and medication resistance to cholesterol-lowering statins are caused by CYP7A1 gene dysfunction.56,57 A male infant's severe hyperoxysterolemia was shown to be caused by a CYP7B1 gene mutation.58 Additionally, cerebrotendinous xanthomatosis, a hereditary condition marked by accelerated atherosclerosis and severe neurological impairment, has been linked to approximately 50 distinct mutations in the CYP27A1 gene. Cholic acid, which aids in restoring the bile acid pool and preventing the formation of harmful sterol intermediates within the bile acid pathway, can be used to treat CYP27A1 deficiency. International Journal of Pharmaceutical Investigation, Vol 14, Issue 1, Jan-Mar, 2024 INTRODUCTION Deficits in the generation of glucocorticoids and sex hormones are caused by mutations in CYP17A1 that affect these enzyme functions. However, shortages in sex hormones alone are caused by mutations that specifically stop oxidation and side chain shortening. Lipoid adrenal hyperplasia is brought on by CYP11A1 mutations, whereas 11-hydroxylase insufficiency is brought on by CYP11B1 errors. Corticosterone methyl oxidase Several cytochrome P450 enzymes work together to metabolize cholesterol and produce bile acids. At least seven, and maybe nine, P450 enzymes are involved in this complex process that turns acetate into sterols and bile acids. The CYP51A1 gene encodes lanosterol 14-demethylase, an essential enzyme in the production of cholesterol. By using oxidative processes to remove two methyl groups from the intermediate molecule lanosterol, this enzyme is essential for the creation of cholesterol. Notably, antifungal medications like ketoconazole target lanosterol 14-demethylase. Surprisingly, this enzyme is highly conserved in a wide range of species, including animals, fungi, plants, and even the prokaryote Mycobacterium TB. Due to its extensive distribution, it has been hypothesized that this enzyme may be the ancestor of all eukaryotic cytochrome P450 enzymes.4,50,51 A important catabolic mechanism for cholesterol removal in animals is the formation of bile acids from cholesterol. The metabolic transformations are catalysed by a variety of cytochrome P450 enzymes from distinct families, including CYP3, CYP7, CYP8, CYP27, CYP39, and CYP46. For instance, CYP7A1, CYP7B1, and CYP39A1 start the production of bile acids by adding a hydroxyl group International Journal of Pharmaceutical Investigation, Vol 14, Issue 1, Jan-Mar, 2024 35 Hadia, et al.: Clinical Significance of Cytochrome P450 Enzymes type I deficiency or corticosterone methyl oxidase type II deficiency are caused by allele-specific mutations in CYP11B2. Glucocorticoid-remediable aldosteronism is brought on by recombination of the closely related CYP11B1 and CYP11B2 genes on chromosome 8, which encode functional chimeric enzymes.60,61 By aromatizing the A ring of androgenic steroid substrates, CYP19A1 produces estrogen. The importance of estrogens in bone development is highlighted by the possibility that loss-of-function mutations in CYP19A1 can cause or manifest skeletal problems. Males who have uncommon gain-of- function mutations in CYP19A1 may develop gynecomastia.62 A important stage in the production of glucocorticoids and mineralocorticoids is the hydroxylation of steroid precursors at carbon-21, which is carried out by the enzyme CYP21A2. Over 90% of cases of congenital adrenal hyperplasia, a common genetic condition, are caused by disruptions in the 21-hydroxylation process. INTRODUCTION Males who have uncommon gain-of- function mutations in CYP19A1 may develop gynecomastia.62 A important stage in the production of glucocorticoids and mineralocorticoids is the hydroxylation of steroid precursors at carbon-21, which is carried out by the enzyme CYP21A2. Over 90% of cases of congenital adrenal hyperplasia, a common genetic condition, are caused by disruptions in the 21-hydroxylation process. Classic congenital adrenal hyperplasia, simple virilizing congenital adrenal hyperplasia, and non-classic congenital adrenal hyperplasia are the three main classifications of this disorder based on clinical presentation and severity. While mild virilizing congenital adrenal hyperplasia largely causes excessive virilization without the severe salt-wasting symptoms, classic congenital adrenal hyperplasia manifests with life-threatening symptoms such salt wasting and masculinization of females. In non-classical congenital adrenal hyperplasia, CYP21 activity is only slightly impaired. Congenital adrenal hyperplasia can also result from mutations in other steroidogenesis-related genes, such as CYP11A1, CYP11B1, CYP11B2, CYP17A1, and CYP19A1. Each of these genes is essential for the production of different steroid hormones. Congenital adrenal hyperplasia can be developed as a result of disruptions in their function, which can change hormone production.63-66 The CYP26 gene family, which comprises of three genes from distinct subfamilies and suggests a shared history going back at least 150–200 million years, mediates the essential process of retinoic acid hydroxylation. Each of these genes produces the enzymes needed to hydroxylate retinoic acid, a kind of vitamin A. One of these, CYP26A1, is only able to metabolize all-trans retinoic acid and is not capable of doing so with 9-cis or 13-cis retinoic acid. During the development of vertebrates, retinoic acid functions as a crucial morphogen, acting through a variety of retinoic acid receptors and retinoid X receptors. CYP26A1 may act as a catabolic enzyme for vitamin A, similar to many other cytochrome P450 enzymes involved in drug metabolism. CYP26A1 may control and reduce the powerful developmental signals mediated by retinoids by degrading the ligand for retinoic acid receptors. It is yet unclear exactly how CYP26B1 and CYP26C1 contribute to the metabolism of retinoic acid or the CONCLUSION The CYP gene families, which comprise CYP2, CYP3, CYP4, and CYP26, are essential to human biology since they function in several aspects such as drug metabolism, fatty acid processing, steroid production, and control of retinoic acid. Significant variability in CYP2 enzymes affects individual responses, and these enzymes are essential for the metabolism of drugs and other substances. Members of the CYP3 family, such as CYP3A4 and CYP3A5, are critical for drug metabolism; however, different genetic variants can result in different pharmacological effects. The main physiological function of CYP4 genes is the metabolism of fatty acids, which helps control blood pressure and other bodily functions. Development is impacted by the CYP26 family’s involvement in the metabolism of retinoic acid. Additional roles and connections between CYP gene variations and genetic illnesses, environmental reactions, and complex diseases are anticipated to be uncovered by future study. These findings enable customized treatments based on individual genetic profiles, which has significant implications for personalized medicine. These gene families’ genetic diversity reveals the complex interactions between chemicals, genes, and human health, opening up new and intriguing possibilities for our knowledge of human biology and the development of therapeutic interventions. The CYP26 gene family, which comprises of three genes from distinct subfamilies and suggests a shared history going back at least 150–200 million years, mediates the essential process of retinoic acid hydroxylation. Each of these genes produces the enzymes needed to hydroxylate retinoic acid, a kind of vitamin A. One of these, CYP26A1, is only able to metabolize all-trans retinoic acid and is not capable of doing so with 9-cis or 13-cis retinoic acid. During the development of vertebrates, retinoic acid functions as a crucial morphogen, acting through a variety of retinoic acid receptors and retinoid X receptors. CYP26A1 may act as a catabolic enzyme for vitamin A, similar to many other cytochrome P450 enzymes involved in drug metabolism. CYP26A1 may control and reduce the powerful developmental signals mediated by retinoids by degrading the ligand for retinoic acid receptors. It is yet unclear exactly how CYP26B1 and CYP26C1 contribute to the metabolism of retinoic acid or the processing of its products. To understand the precise biological actions these enzymes take in relation to retinoic acid metabolism, further study is required.7 International Journal of Pharmaceutical Investigation, Vol 14, Issue 1, Jan-Mar, 2024 INTRODUCTION Classic congenital adrenal hyperplasia, simple virilizing congenital adrenal hyperplasia, and non-classic congenital adrenal hyperplasia are the three main classifications of this disorder based on clinical presentation and severity. While mild virilizing congenital adrenal hyperplasia largely causes excessive virilization without the severe salt-wasting symptoms, classic congenital adrenal hyperplasia manifests with life-threatening symptoms such salt wasting and masculinization of females. In non-classical congenital adrenal hyperplasia, CYP21 activity is only slightly impaired. Congenital adrenal hyperplasia can also result from mutations in other steroidogenesis-related genes, such as CYP11A1, CYP11B1, CYP11B2, CYP17A1, and CYP19A1. Each of these genes is essential for the production of different steroid hormones. Congenital adrenal hyperplasia can be developed as a result of disruptions in their function, which can change hormone production.63-66 Future research on CYP gene products is expected to reveal a growing number of functions that they have in many biological systems. High levels of polymorphism are present in the CYP superfamily genes, which is a trait shared by the majority of genes in the human genome. Inter-individual changes in phenotype caused by this genetic variability within the P450 enzymes will have a profound impact on medicine and treatment modalities. Numerous investigations will soon reveal links between particular CYP variant alleles and a variety of genetic disorders, environmental toxicities, cancer, and other complicated diseases, it is predicted. These studies will help us comprehend the complex relationship between genetic variables and illness vulnerability better. Such discoveries have enormous potential for personalized medicine since they can lead to customized treatment plans based on each person's particular genetic makeup. It is critical to recognize that the area of genetics is always evolving and that new developments and discoveries will probably keep happening. Therefore, new knowledge about the wide and varied functions of CYP enzymes will surely become available in the future, influencing our understanding of human health and directing potential therapeutic approaches. g y recombination of the closely related CYP11B1 and CYP11B2 genes on chromosome 8, which encode functional chimeric enzymes.60,61 By aromatizing the A ring of androgenic steroid substrates, CYP19A1 produces estrogen. The importance of estrogens in bone development is highlighted by the possibility that loss-of-function mutations in CYP19A1 can cause or manifest skeletal problems. REFERENCES 26. Gonzalez FJ, Kimura S. Understanding the role of xenobiotic metabolism in chemical carcinogenesis using gene knockout mice. Mutat Res. 2001;477(1-2):79-87. doi: 10.1 016/s0027-5107(01)00109-9, PMID 11376689. 1. Gonzalez FJ, Mackenzie PI, Kimura S, Nebert DW. Isolation and characterization of mouse full-length cDNA and genomic clones of 3-methylcholanthrene-inducible cytochrome P1-450 and P3-450. Gene. 1984;29(3):281-92. doi: 10.1016/0378-1119( 84)90057-x, PMID 6548461. 27. Stoilov I, Akarsu AN, Sarfarazi M. Identification of three different truncating mutations in the cytochrome P450 1B1 (CYP1B1) gene as the principal cause of primary congenital glaucoma (buphthalmos) in families linked to the GLC3A locus on chromosome 2p21. Hum Mol Genet. 1997;6(4):641-47. doi: 10.1093/hmg/6.4.64 1, PMID 9097971. 2. Nebert DW, Gonzalez FJ. P450 genes: structure, evolution and regulation. Annu Rev Biochem. 1987;56:945-93. doi: 10.1146/annurev.bi.56.070187.004501, PMID 3304150. 2. Nebert DW, Gonzalez FJ. P450 genes: structure, evolution and regulation. Annu Rev Biochem. 1987;56:945-93. doi: 10.1146/annurev.bi.56.070187.004501, PMID 3304150. 28. Stoilov I. Cytochromes P450: coupling development and environment. Trends Genet. 2001;17(11):629-32. doi: 10.1016/s0168-9525(01)02444-1, PMID 11672862. 3. Nebert DW, Adesnik M, Coon MJ, Estabrook RW, Gonzalez FJ, Guengerich FP, et al. The P450 gene superfamily: recommended nomenclature. DNA. 1987;6(1):1-11. doi: 10.1 089/dna.1987.6.1, PMID 3829886. 29. Andreola F, Fernandez-Salguero PM, Chiantore MV, Petkovich MP, Gonzalez FJ, De Luca LM. Aryl hydrocarbon receptor Ahr(-/-) knockout mice exhibit liver retinoid accumulation and reduced retinoic acid metabolism. Cancer Res. 1997;57(14):2835-38. PMID 9230184. 4. Nelson DR. Cytochrome P450 gene superfamily [online] [cited Jul 10, 2020]. Available from: http://drnelson.utmem.edu/cytochromeP450.html. 5. Gonzalez FJ, Nebert DW. Evolution of the P450 gene superfamily: animal-plant ”warfare,” molecular drive, and human genetic differences in drug oxidation. Trends Genet. 1990;6(6):182-86. doi: 10.1016/0168-9525(90)90174-5, PMID 2196721. 30. Meyer UA, Zanger UM. Molecular mechanisms of genetic polymorphisms of drug metabolism. Annu Rev Pharmacol Toxicol. 1997;37:269-96. doi: 10.1146/annurev.pha rmtox.37.1.269, PMID 9131254. 6. Nelson DR, Kamataki T, Waxman DJ, Guengerich FP, Estabrook RW, Feyereisen R, et al. The P450 superfamily: update on new sequences, gene mapping, accession numbers, early trivial names, and nomenclature. DNA Cell Biol. 1993;12(1):1-51. doi: 10.1089/dna.1993.12.1, PMID 7678494. 31. Guengerich FP, Hosea NA, Parikh A, Bell-Parikh LC, Johnson WW, Gillam EM, et al. Twenty years of biochemistry of human P450s: purification, expression, mechanism, and relevance to drugs. Drug Metab Dispos. 1998;26(12):1175-78. PMID 9860923. 32. Fisslthaler B, Popp R, Kiss L, Potente M, Harder DR, Fleming I, et al. Cytochrome P450 2C is an EDHF synthase in coronary arteries. Nature. 1999;401(6752):493-97. doi: 10. 1038/46816, PMID 10519554. 7. Nebert DW, Nelson DR. Hadia, et al.: Clinical Significance of Cytochrome P450 Enzymes commitment to fostering research and academic excellence, which has greatly contributed to the success of our work. 12. Meyer UA. Pharmacogenetics and adverse drug reactions. Lancet. 2000;356(9242):1667-71. doi: 10.1016/S0140-6736(00)03167-6, PMID 11089838. 13. Evans WE, Johnson JA. Pharmacogenomics: the inherited basis for interindividual differences in drug response. Annu Rev Genomics Hum Genet. 2001;2:9-39. doi: 10.1 146/annurev.genom.2.1.9, PMID 11701642. ACKNOWLEDGEMENT We would like to express our sincere appreciation for the support and resources provided by the Department of Pharmacy at Sumandeep Vidyapeeth (Deemed to be University) in Vadodara. Your assistance has been invaluable in the preparation and completion of our recent article. We are grateful for your International Journal of Pharmaceutical Investigation, Vol 14, Issue 1, Jan-Mar, 2024 36 ABBREVIATIONS 16. Clapper ML. Genetic polymorphism and cancer risk. Curr Oncol Rep. 2000;2(3):251-56. doi: 10.1007/s11912-000-0075-z, PMID 11122850. CYP: Cytochrome P450; DNA: Deoxyribonucleic Acid; RNA: Ribonucleic Acid; mRNA: Messenger Ribonucleic Acid; cDNA: Complementary DNA; CYP2G: Cytochrome P450 2G; CYP2T: Cytochrome P450 2T; PAHs: Polycyclic Aromatic Hydrocarbons; N-heterocyclics: N-heterocyclic compounds; CYP1A1: Cytochrome P450 1A1; CYP1A2: Cytochrome P450 1A2; CYP1B1: Cytochrome P450 1B1; Ahr: Aryl Hydrocarbon Receptor; HETEs: Hydroxyeicosatetraenoic Acids; EETs: Epoxyeicosatrienoic Acids; HPETEs: Hydroperoxyeicosatetraenoic Acids; FXR: Farnesoid X Receptor; PXR: Pregnane X Receptor. 17. Ingelman-Sundberg M. Genetic susceptibility to adverse effects of drugs and environmental toxicants: the role of the CYP family of enzymes. Mutat Res. 2001;482(1-2):11-9. doi: 10.1016/s0027-5107(01)00205-6, PMID 11535244. 18. Nebert DW. Pharmacogenetics and pharmacogenomics: why is this relevant to the clinical geneticist? Clin Genet. 1999;56(4):247-58. doi: 10.1034/j.1399-0004.1999.560 401.x, PMID 10636440. 19. Nebert DW, Roe AL, Dieter MZ, Solis WA, Yang Y, Dalton TP. Role of the aromatic hydrocarbon receptor and [Ah] gene battery in the oxidative stress response, cell cycle control, and apoptosis. Biochem Pharmacol. 2000;59(1):65-85. doi: 10.1016/ s0006-2952(99)00310-x, PMID 10605936. 20. Plastaras JP, Guengerich FP, Nebert DW, Marnett LJ. Xenobiotic metabolizing cytochromes P450 convert prostaglandin endoperoxide to hydroxyheptadecatrienoic acid and the mutagen, malondialdehyde. J Biol Chem. 2000;275(16):11784-90. doi: 1 0.1074/jbc.275.16.11784, PMID 10766802. 21. Smith AG, Clothier B, Carthew P, Childs NL, Sinclair PR, Nebert DW, et al. Protection of the Cyp1a2(-/-) null mouse against uroporphyria and hepatic injury following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol Appl Pharmacol. 2001;173(2):89-98. doi: 10.1006/taap.2001.9167, PMID 11384210. The authors declare that there is no conflict of interest. The authors declare that there is no conflict of interest. The authors declare that there is no conflict of interest. 15. Nebert DW, Jorge-Nebert LF. Pharmacogenetics and pharmacogenomics. In: Rimoin DL, Connor JM, Pyeritz RE, Korf BR, editors. Emery and Rimoin’s principles and practice of medical genetics. 4th ed. Edinburgh: Harcourt Brace; 2002. p. 590-631. SUMMARY 22. Härtter S, Wang X, Weigmann H, Friedberg T, Arand M, Oesch F, et al. Differential effects of fluvoxamine and other antidepressants on the biotransformation of melatonin. J Clin Psychopharmacol. 2001;21(2):167-74. doi: 10.1097/00004714-200104000-0000 8, PMID 11270913. Cytochrome P450 (CYP) enzymes, with 57 distinct genes, are crucial for various biological functions, including drug metabolism, synthesis of essential compounds, and maintaining metabolic balance. Mutations in CYP genes can lead to serious health issues. Recent research has unveiled their broader roles, expanding possibilities for drug development and personalized medicine. Understanding CYP enzyme complexity is revolutionizing pharmacology and medicine. 23. Murray GI, Taylor MC, McFadyen MC, McKay JA, Greenlee WF, Burke MD, et al. Tumor-specific expression of cytochrome P450 CYP1B1. Cancer Res. 1997;57(14):3026-31. PMID 9230218. 24. Dalton TP, Dieter MZ, Matlib RS, Childs NL, Shertzer HG, Genter MB, et al. Targeted knockout in the Cyp1a1 gene does not alter hepatic constitutive expression of other genes in the mouse [Ah] battery. Biochem Biophys Res Commun. 2000;267(1):184-89. doi: 10.1006/bbrc.1999.1913, PMID 10623596. 25. Liang HCL, Li H, McKinnon RA, Duffy JJ, Potter SS, Puga A, et al. Cyp1a2(-/-) null mutant mice develop normally, but show deficient drug metabolism. Proc Natl Acad Sci U S A. 1996;93(4):1671-76. doi: 10.1073/pnas.93.4.1671, PMID 8643688. CONFLICT OF INTEREST 14. Phillips KA, Veenstra DL, Oren E, Lee JK, Sadee W. Potential role of pharmacogenomics in reducing adverse drug reactions: a systematic review. JAMA. 2001;286(18):2270-79. doi: 10.1001/jama.286.18.2270, PMID 11710893. REFERENCES Cytochrome P450 (CYP) superfamily. In: London: Nature Publishing Group:article 667. Encyclopaedia of the human genome [online] Cooper DN, editor. Available from: http://www.naturereference.com/ehg/ehg.htm. 33. Waxman DJ, Lapenson DP, Aoyama T, Gelboin HV, Gonzalez FJ, Korzekwa K. Steroid hormone hydroxylase specificities of eleven cDNA expressed human cytochrome P450s. Arch Biochem Biophys. 1991;290(1):160-66. doi: 10.1016/0003-9861(91) 90602-f, PMID 1898086. 8. Nebert DW, Dieter MZ. The evolution of drug metabolism. Pharmacology. 2000;61(3):124-35. doi: 10.1159/000028393, PMID 10971198. 9. Schuetz EG. Induction of cytochromes P450. Curr Drug Metab. 2001;2(2):139-47. doi: 10.2174/1389200013338595, PMID 11469722. 34. Araya Z, Wikvall K. 6-Hydroxylation of taurochenodeoxycholic acid and lithocholic acid by CYP3A4 in human liver microsomes. Biochim Biophys Acta. 1999;1438(1):47-54. doi: 10.1016/s1388-1981(99)00031-1, PMID 10216279. 10. Tang W, Stearns RA. Heterotropic cooperativity of cytochrome P4503A4 and potential drug-drug interactions. Curr Drug Metab. 2001;2(2):185-98. doi: 10.2174/ 1389200013338658, PMID 11469725. 35. Goodwin B, Redinbo MR, Kliewer SA. Regulation of CYP3A gene transcription by the pregnane X receptor. Annu Rev Pharmacol Toxicol. 2002;42:1-23. doi: 10.1146/annur ev.pharmtox.42.111901.111051, PMID 11807162. 11. Oscarson M, Ingelman-Sundberg M, Daly AK, Nebert DW. Human cytochrome P450 (CYP) alleles [cited Jul 10, 2002]. Available from: http://www.imm.ki.se/CYPalleles/. Hadia, et al.: Clinical Significance of Cytochrome P450 Enzymes 36. Lu AYH, Somogyi A, West S, Kuntzman R, Conney AH. Pregnenolone-16-carbonitrile: a new type of inducer of drug metabolizing enzymes. Arch Biochem Biophys. 1972;152(2):457-62. doi: 10.1016/0003-9861(72)90239-1, PMID 4344124. 51. Yoshida Y, Aoyama Y, Noshiro M, Gotoh O. Sterol 14-demethylase P450 (CYP51) provides a breakthrough for the discussion on the evolution of cytochrome P450 gene superfamily. Biochem Biophys Res Commun. 2000;273(3):799-804. doi: 10.1006 /bbrc.2000.3030, PMID 10891326. 37. Barwick JL, Quattrochi LC, Mills AS, Potenza C, Tukey RH, Guzelian PS. Trans-species gene transfer for analysis of glucocorticoid-inducible transcriptional activation of transiently expressed human CYP3A4 and rabbit CYP3A6 in primary cultures of adult rat and rabbit hepatocytes. Mol Pharmacol. 1996;50(1):10-6. PMID 8700101. 52. Sinal CJ, Tohkin M, Miyata M, Ward JM, Lambert G, Gonzalez FJ. Targeted disruption of the nuclear receptor FXR/BAR impairs bile acid and lipid homeostasis. Cell. 2000;102(6):731-44. doi: 10.1016/s0092-8674(00)00062-3, PMID 11030617. 53. Eggertsen G, Olin M, Andersson U, Ishida H, Kubota S, Hellman U, et al. Molecular cloning and expression of rabbit sterol 12-hydroxylase. J Biol Chem. 1996;271(50):32269-75. doi: 10.1074/jbc.271.50.32269, PMID 8943286. 38. Xie W, Barwick JL, Downes M, Blumberg B, Simon CM, Nelson MC, et al. Humanized xenobiotic response in mice expressing nuclear receptor SXR. Nature. 2000;406(6794):435-39. doi: 10.1038/35019116, PMID 10935643. 54. Andersson S, Davis DL, Dahlbäck H, Jörnvall H, Russell DW. Cloning, structure, and expression of the mitochondrial cytochrome P450 sterol 27-hydroxylase, a bile acid biosynthetic enzyme. J Biol Chem. 1989;264(14):8222-29. doi: 10.1016/S0021-9258( 18)83172-6, PMID 2722778. 39. Watkins RE, Wisely GB, Moore LB, Collins JL, Lambert MH, Williams SP, et al. The human nuclear xenobiotic receptor PXR: structural determinants of directed promiscuity. Science. 2001;292(5525):2329-33. doi: 10.1126/science.1060762, PMID 11408620. 40. Selye H. Hormones and resistance. J Pharm Sci. 1971;60(1):1-28. doi: 10.1002/jps.26 00600102, PMID 4926765. 55. Lund EG, Guileyardo JM, Russell DW. cDNA cloning of cholesterol 24-hydroxylase, a mediator of cholesterol homeostasis in the brain. Proc Natl Acad Sci U S A. 1999;96(13):7238-43. doi: 10.1073/pnas.96.13.7238, PMID 10377398. 41. Moore LB, Goodwin B, Jones SA, Wisely GB, Serabjit-Singh CJ, Willson TM, et al. St John’s wort induces hepatic drug metabolism through activation of the pregnane X receptor. Proc Natl Acad Sci U S A. 2000;97(13):7500-02. doi: 10.1073/pnas.1301550 97, PMID 10852961. 56. Bjorkhem I, Boberg KM, Leitersdort F. Inborn errors in bile acid biosynthesis and storage of sterols other than cholesterol. In: Scriver CR, Beaudet A, Sly WS, et al. The metabolic and molecular bases of inherited disease. Hadia, et al.: Clinical Significance of Cytochrome P450 Enzymes New York: McGraw-Hill; 2001. p. 2981-88. 42. Xie W, Barwick JL, Simon CM, Pierce AM, Safe S, Blumberg B, et al. Reciprocal activation of xenobiotic response genes by nuclear receptors SXR/PXR and CAR. Genes Dev. 2000;14(23):3014-23. doi: 10.1101/gad.846800, PMID 11114890. 57. Pullinger CR, Eng C, Salen G, Shefer S, Batta AK, Erickson SK, et al. Human cholesterol 7-hydroxylase (CYP7A1) deficiency has a hypercholesterolemic phenotype. J Clin Invest. 2002;110(1):109-17. doi: 10.1172/JCI15387, PMID 12093894. 43. Chawla A, Repa JJ, Evans RM, Mangelsdorf DJ. Nuclear receptors and lipid physiology: opening the X-files. Science. 2001;294(5548):1866-70. doi: 10.1126/science.294.5548. 1866, PMID 11729302. 58. Setchell KD, Schwarz M, O’Connell NC, Lund EG, Davis DL, Lathe R, et al. Identification of a new inborn error in bile acid synthesis: mutation of the oxysterol 7-hydroxylase gene causes severe neonatal liver disease. J Clin Invest. 1998;102(9):1690-703. doi: 1 0.1172/JCI2962, PMID 9802883. 44. Simpson AE. The cytochrome P450-4 (CYP4) family. Gen Pharmacol. 1997;28(3):351-59. doi: 10.1016/s0306-3623(96)00246-7, PMID 9068972. 45. Lasker JM, Chen WB, Wolf I, Bloswick BP, Wilson PD, Powell PK. Formation of 20-hydroxyeicosatetraenoic acid, a vasoactive and natriuretic eicosanoid, in human kidney: role of CYP4F2 and CYP4A11. J Biol Chem. 2000;275(6):4118-26. doi: 10.1074 /jbc.275.6.4118, PMID 10660572. 59. Ryner LC, Swain A. Sex in the ’90s. Cell. 1995;81(4):483-93. doi: 10.1016/0092-8674(9 5)90069-1, PMID 7758104. 60. Miller WL, Geller DH, Auchus RJ. The molecular basis of isolated 17,20-lyase deficiency. Endocr Res. 1998;24(3-4):817-25. doi: 10.3109/07435809809032692, PMID 9888582. 46. Nebert DW. Drug-metabolizing enzymes in ligand-modulated transcription. Biochem Pharmacol. 1994;47(1):25-37. doi: 10.1016/0006-2952(94)90434-0, PMID 8311842. 61. Nebert DW, McKinnon RA. Cytochrome P450: evolution and functional diversity. Prog Liver Dis. 1994;12:63-97. PMID 7746878. 62. Simpson ER. Genetic mutations resulting in loss of aromatase activity in humans and mice. J Soc Gynecol Investig. 2000; 7(1);Suppl:S18-21. doi: 10.1177/1071557600007 001S07, PMID 10732324. 47. Funk CD. Prostaglandins and leukotrienes: advances in eicosanoid biology. Science. 2001;294(5548):1871-75. doi: 10.1126/science.294.5548.1871, PMID 11729303. 48. Hennan JK, Huang J, Barrett TD, Driscoll EM, Willens DE, Park AM, et al. Effects of selective cyclooxygenase-2 inhibition on vascular responses and thrombosis in canine coronary arteries. Circulation. 2001;104(7):820-25. doi: 10.1161/hc3301.0927 90, PMID 11502709. 63. White PC. Congenital adrenal hyperplasias. Best Pract Res Clin Endocrinol Metab. 2001;15(1):17-41. doi: 10.1053/beem.2000.0117, PMID 11469809. 64. Stoilov I, Jansson I, Sarfarazi M, Schenkman JB. Roles of cytochrome P450 in development. Drug Metab Drug Interact. 2001;18(1):33-55. doi: 10.1515/dmdi.200 1.18.1.33, PMID 11522124. 49. Tuder RM, Cool CD, Yeager M, Taraseviciene-Stewart L, Bull TM, Voelkel NF. International Journal of Pharmaceutical Investigation, Vol 14, Issue 1, Jan-Mar, 2024 37 International Journal of Pharmaceutical Investigation, Vol 14, Issue 1, Jan-Mar, 2024 Hadia, et al.: Clinical Significance of Cytochrome P450 Enzymes International Journal of Pharmaceutical Investigation, Vol 14, Issue 1, Jan-Mar, 2024 Cite this article: Hadia R, Singh V, Solanki N, Sharma S, Saggu V, Sajan C, et al. Unlocking the Clinical Significance of Cytochrome P450 Enzymes. Int. J. Pharm. Investigation. 2024;14(1):30-8. Hadia, et al.: Clinical Significance of Cytochrome P450 Enzymes The patho-biology of pulmonary hypertension: the endothelium. Clin Chest Med. 2001;22(3):405-18. doi: 10.1016/s0272-5231(05)70280-x, PMID 11590837. 65. Nebert DW. Suggestions for the nomenclature of human alleles: relevance to ecogenetics, pharmacogenetics, and molecular epidemiology. Pharmacogenetics. 2000;10(4):279-90. doi: 10.1097/00008571-200006000-00001, PMID 10862518. 50. Nelson DR, Koymans L, Kamataki T, Stegeman JJ, Feyereisen R, Waxman DJ, et al. Cytochrome P450 superfamily: update on new sequences, gene mapping, accession numbers, and nomenclature. Pharmacogenetics. 1996;6(1):1-42. doi: 10.1097/ 00008571-199602000-00002, PMID 8845856. 66. Kruglyak L, Nickerson DA. Variation is the spice of life. Nat Genet. 2001;27(3):234-6. doi: 10.1038/85776, PMID 11242096. Cite this article: Hadia R, Singh V, Solanki N, Sharma S, Saggu V, Sajan C, et al. Unlocking the Clinical Significance of Cytochrome P450 Enzymes. Int. J. Pharm. Investigation. 2024;14(1):30-8. Cite this article: Hadia R, Singh V, Solanki N, Sharma S, Saggu V, Sajan C, et al. Unlocking the Clinical Significance of Cytochrome P450 Enzymes. Int. J. Pharm. Investigation. 2024;14(1):30-8. 38
https://openalex.org/W3099796214	https://hal.archives-ouvertes.fr/hal-01897138/document	English	null	Turbulent and wind-driven accretion in dwarf novae threaded by a large-scale magnetic field	Astronomy & astrophysics	2,018	cc-by	15,986	Turbulent and wind-driven accretion in dwarf novae threaded by a large scale magnetic field Nicolas Scepi, Geoffroy Lesur, Guillaume Dubus, Mario Flock Nicolas Scepi, Geoffroy Lesur, Guillaume Dubus, Mario Flock To cite this version: Nicolas Scepi, Geoffroy Lesur, Guillaume Dubus, Mario Flock. Turbulent and wind-driven accretion in dwarf novae threaded by a large scale magnetic field. Astronomy and Astrophysics - A&A, 2018, 620, pp.A49. 10.1051/0004-6361/201833921. hal-01897138 1. Introduction the hot or cold state has a higher or lower accretion rate than the mass accretion rate coming from the companion, which causes the disk to ﬁll up or empty. This leads to an hysteresis cycle in luminosity. The timescales involved are related to the ability for the disk to eﬃciently extract its angular momentum, providing an observational handle on α. Outburst-decay timescales imply α ≈0.1 in the hot state (Kotko & Lasota 2012), and recurrence timescales gives α ≈0.01 in the cold state (Cannizzo et al. 1988, 2012). It is unclear why this change in α occurs and if it is related to the properties of turbulence. Nevertheless, we can use these observational constraints as a benchmark to distinguish between diﬀerent transport mechanisms for DNe. The missing link between accretion theory and observations is the mechanism transporting angular momentum. Turbu- lent transport is one candidate and is often parameterized by the dimensionless parameter α, ratio of the ﬂuid stress to the local thermal pressure (Shakura & Sunyaev 1973). The- ory of thin viscous α-disks has been widely applied to var- ious systems such as protoplanetary disks (Sano et al. 2000; Papaloizou & Terquem 1999), soft X-ray transients (SXT; King & Ritter 1998; Tetarenko et al. 2018), dwarf novae (DNe; Smak 1984), or even active galactic nuclei (AGN; Starling et al. 2004; see, however, Hameury et al. 2009). However, in soft X-ray transient sources and dwarf novae, which are the focus of this paper, the best estimators of α are often found because their nature is time dependent (King et al. 2007). Consensus appears to be reached that the magneto-rotational instability (MRI; Balbus & Hawley 1991) is the main driver of turbulent motions as it requires only a subthermal magnetic ﬁeld threading the disk and a radially decreasing angular velocity; these are quasi-omnipresent conditions in astrophysical disks. However, magneto-hydrodynamical processes require coupling between gas and magnetic ﬁeld, and this can be problematic in cold regions of protoplanetary disks (Gammie 1996), but also for dwarf novae in quiescence (Gammie & Menou 1998; Scepi et al. 2018, hereafter S18). DNe are compact binary systems in which matter is trans- ferred by Roche-lobe overﬂow from a solar-type star to a white dwarf. DNe have been observed over decades, and their light curves show periodic outbursts with amplitudes of 2–3 magni- tudes (Warner 2003). 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. HAL Id: hal-01897138 https://hal.science/hal-01897138v1 Submitted on 18 Nov 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Astronomy & Astrophysics A&A 620, A49 (2018) https://doi.org/10.1051/0004-6361/201833921 c⃝ESO 2018 ABSTRACT Dwarf novae (DNe) are accreting white dwarfs that show eruptions caused by a thermal-viscous instability in the accretion disk. The outburst timescales constrain α, the ratio of the viscous stress to the thermal pressure, which phenomenologically connects to the mechanism of angular momentum transport. The eruptive state has α ≈0.1 while the quiescent state has α ≈0.03. Turbulent transport that is due to the magneto-rotational instability (MRI) is generally considered to be the source of angular momentum transport in DNe. The presence of a large-scale poloidal ﬁeld threading the disk is known to enhance MRI-driven transport. Here, we perform 3D local magnetohydrodynamic (MHD) shearing-box simulations including vertical stratiﬁcation, radiative transfer, and a net constant vertical magnetic ﬂux to investigate how transport changes between the outburst and quiescent states of DNe. We ﬁnd that a net vertical constant magnetic ﬁeld, as could be provided by the white dwarf or by its stellar companion, provides a higher α in quiescence than in outburst, in opposition to what is expected. Including resistivity quenches MRI turbulence in quiescence, suppressing transport, unless the magnetic ﬁeld is high enough, which again leads to α ≈0.1. A major diﬀerence between simulations with a net poloidal ﬂux and simulations without a net ﬂux is that angular momentum transport in the former is shared between turbulent radial transport and wind-driven vertical transport. We ﬁnd that wind-driven transport dominates in quiescence even for moderately low magnetic ﬁelds ∼1 G. This can have a great impact on observational signatures since wind-driven transport does not heat the disk. Furthermore, wind transport cannot be reduced to an α prescription. We provide ﬁts to the dependence of α with β, the ratio of thermal to magnetic pressure, and Teﬀ, the eﬀective temperature of the disk, as well as a prescription for the wind torque as a function of β that is in agreement with both local and global simulations. We conclude that the evolution of the thermal-viscous instability, and its consequences on the outburst cycles of CVs, needs to be thoroughly revised to take into account that most of the accretion energy may be carried away by a wind instead of being locally dissipated. ey words. accretion, accretion disks – magnetohydrodynamics (MHD) – turbulence – convection – stars: dwarf nova Turbulent and wind-driven accretion in dwarf novae threaded by a large-scale magnetic ﬁeld N. Scepi1, G. Lesur1, G. Dubus1, and M. Flock2 1 Univ. Grenoble Alpes, CNRS, Institut de Planétologie et d’Astrophysique de Grenoble (IPAG), 38000 Grenoble, France e-mail: nicolas.scepi@gmail.com 2 g 2 Max Planck Institute for Astronomy, Königstuhl 17, 69117 Heidelberg, Germany 2 Max Planck Institute for Astronomy, Königstuhl 17, 69117 Heidelberg, Germany Received 21 July 2018 / Accepted 30 September 2018 2.1. Basic equations Radiative transfer is treated separately from the MHD step using an implicit time-stepping following the implementation of Flock et al. (2013). In this step, we solve the coupled matter- radiation equations in the ﬂux-limited diﬀusion approximation ∂ER ∂t −∇∇∇cλ(R) κRρ ∇∇∇ER = κPρc(aRT 4 −ER) ∂ϵ ∂t = −κPρc(aRT 4 −ER), ∂ER ∂t −∇∇∇cλ(R) κRρ ∇∇∇ER = κPρc(aRT 4 −ER) ∂ϵ ∂t = −κPρc(aRT 4 −ER), where ρ is the density, vvv the ﬂuid velocity vector, P the ther- mal pressure, B the magnetic ﬁeld vector, Φ = Ω2(R0)(−3x2 + z2)/2 is the gravitational potential in the corotating frame, η the Ohmic resistivity, J = (c/4π)∇∇∇× B the current density vector, E = ϵ + 0.5ρvvv2 + B2/8π the total energy, ϵ the internal energy, Pt = P + B2/8π the thermal pressure plus the magnetic pres- sure, c the speed of light, aR = (4σ/c) the radiation constant with σ the Stefan–Boltzmann constant, T the temperature, ER the radiation density energy, κP the Planck opacity, and κR the Rosseland opacity. The radiative energy ﬂux in the ﬂux-diﬀusion approximation is Frad = (cλ(R)/κR(T)ρ)∇∇∇ER. The ﬂux limiter is deﬁned as λ(R) ≡(2 + R)/(6 + 3R + R2) with R ≡\|∇E\|/(κRρE) (Turner & Stone 2001). We performed two simulations with the limiter from Minerbo (1978) and found no notable diﬀerences. We did not take into account radiation pressure as it is negligible for the temperatures reached by DNe; Hirose et al. (2014) found that it contributes 6% of the gas+radiation pressure at most. q gg y Hence, the simple application of ZNF MRI to the case of DNe leads to inconsistencies with the DIM model and the obser- vations. ZNF is a common assumption since the magnetic con- ﬁguration is unknown in DNe. Moreover, large-scale magnetic ﬁelds leads to higher Alfvén speeds in the atmosphere and thus are more computationally demanding. They also require a robust solver when the magnetization is high. It is well known, how- ever, that α depends on the ratio of the thermal to magnetic pressure, β (Hawley et al. 1996), which can lead to α ≳0.1 if the disk is threaded by a strong enough large-scale magnetic ﬁeld. Additionally, it is expected that a large magnetic ﬁeld will sustain MRI turbulence to lower ionization levels than in ZNF (Fleming et al. 2000). This readmits MRI as a possible candidate on the cold branch. 2.1. Basic equations Curvature terms are not taken into account in the shearing-box approximation, and the diﬀerential Keplerian velocity is mod- eled as a linear shear ﬂow v0 y = −(3/2)Ωx, where x, y, and z correspond to the radial, azimuthal, and vertical directions, respectively. The set of equations in the corotating frame is ∂ρ ∂t + ∇∇∇· ( ρvvv) = 0, (1) ρ∂vvv ∂t + ( ρvvv · ∇∇∇)vvv = −∇∇∇ P + B2 8π ! + B 4π · ∇∇∇ ! B + ρ(−2Ωˆzˆzˆz × vvv + 3Ω2xˆxˆxˆx −Ω2zˆzˆzˆz, (2) ∂E ∂t + ∇∇∇· [(E + Pt)vvv −(vvv · B)B] = −ρvvv · ∇∇∇Φ −κPρc(aRT 4 −ER), (3) ∂B ∂t = ∇∇∇× vvv × B −4π c ηJ ! . (4) (1) Isothermal simulations of MRI with ZNF are known to give a universal value of α ≈0.01 (Hawley et al. 1996; Simon et al. 2012) that is characteristic of the quiescent state of DNe. Using an approximate function of cooling, Latter & Papaloizou (2012) were able to retrieve thermal equilibrium curves of DNe from ZNF MRI simulations, showing the two expected stable branches, the hot and the cold state, which correspond to the eruptive and the quiescent states, respectively. However, their simulations exhibit a value of α ≈0.01 regardless of the branch. By including vertical stratiﬁcation, a realistic equation of state and radiative transfer in the regime of DNe, Hirose et al. (2014) found for the ﬁrst time that ZNF MRI can lead to α ≈0.1 in the low-density part of the hot branch. The actual interpretation is that there exists a non-linear coupling with convection which enhances α in this regime. This result was conﬁrmed in S18 with a diﬀerent code and in Coleman et al. (2018) in a diﬀerent regime of opacities. However, this enhanced α at the tip of the hot branch fails to reproduce the light curves of DNe by inducing reﬂares, which is inconsistent with observations (Coleman et al. 2016). Moreover, S18 found that in ZNF the cold branch was too resistive to sustain magnetic turbulence, leading to α ≈0 in quiescence, as suggested by Gammie & Menou (1998). (3) (4) (3) (3) (4) The last three terms of Eq. (1) represent the Coriolis force, the tidal force, and the vertical component of the gravitational force, respectively; ˆxˆxˆx and ˆzˆzˆz are the unit vectors in the x and z directions. 1. Introduction Their eruptive behavior is well explained by the disk instability model (DIM; Lasota 2001; Dubus et al. 2018), in which a thermal instability caused by the ionization of hydrogen is responsible for a hysteresis cycle. In this system, Given the complexity of MRI saturation, numerical simula- tions have been extensively used to study the characteristics of A49, page 1 of 13 A49, page 1 of 13 A&A 620, A49 (2018) the resulting turbulence and transport. The properties of MRI- driven turbulence are known to depend on the magnetic conﬁgu- ration, especially the amplitude of the local net magnetic ﬁeld (Hawley et al. 1995), which is largely unconstrained in DNe disks. A self-sustaining version of the MRI turbulence also exists in the absence of an externally imposed magnetic ﬁeld, known as zero net-ﬂux conﬁguration (ZNF), and it is known to be inde- pendent of the initial magnetic seed (Hawley et al. 1996). Hence, ZNF simulations provide a minimum level of angular momen- tum transport by the MRI that does not require a large-scale magnetic ﬁeld. We add the caveat that the convergence of ZNF simulations is still debated (see Ryan et al. 2017 for more infor- mation on the subject). 2.1. Basic equations The purpose of this paper is twofold: to study the local thermal equilibrium of a disk in realistic conditions of DNe threaded by a large-scale magnetic ﬁeld, and to determine whether this ﬁeld could resolve the discrepancies between MRI transport and observations. To close our set of equations, we used the following equation of state (EOS) and internal energy function: P = ρ µ( ρ, T)kBT ϵ = ϵ(ρ, T), P = ρ µ( ρ, T)kBT 2.2. Boundary conditions We used shear-periodic boundary conditions in the x-direction and periodic boundary conditions in the y-direction. We showed in S18 that the choice of vertical boundary conditions only slightly change the ﬁnal thermal equilibrium with zero net- ﬂux. Since periodic boundary conditions are much more com- putationally consuming than modiﬁed outﬂow boundaries, we chose to use the latter. The modiﬁed outﬂow boundaries (Brandenburg et al. 1995) assume a zero-gradient extrapolation for all hydrodynamic quantities leaving the box and prevent mat- ter from outside from entering the simulation box. The diﬀerence with pure outﬂow conditions is that they impose the magnetic ﬁeld to be vertical at the z-boundary. We did not investigate other boundary conditions. We started with an isothermal layer and assumed hydro- static equilibrium to ﬁx the initial vertical density proﬁle. We let the MRI develop, then triggered radiative transfer after 32 local orbits (time was normalized by the quantity 1/Ω0, thus one orbital period is equivalent to 2π in code units) and let the disk equilibrate and reach a quasi-steady state (if there was one). For simulations with β ≥105, the isothermal temperature Tc0 was set to be approximately the mid-plane temperature found using the vertical structure code of Hameury et al. (1998) for a given surface density Σ0 and a given eﬀective temperature Teﬀ, as in the case of ZNF in S18. This code solves the vertical structure equations assuming an α-prescription for the angular momentum transport and associated heating rate. It also uses radiative trans- fer in the diﬀusion approximation, and convection described by mixing length theory with a mixing coeﬃcient αml = 1.5 (based on models of the Sun). However, when β < 105, to avoid inap- propriate box sizes that are due to the inﬂuence of the magnetic ﬁeld, we evaluated the expected mid-plane temperature from test simulations. y The mass in the shearing box may decrease as a result of the outﬂow boundaries. To avoid this, we normalized the total mass to the initial mass at each time step by multiplying the den- sity by a corrective factor. For simulations with β ≈103, outﬂows become important. In the simulation 439F with β ≈103, the disk would be empty after seven orbits if no normalization were used. When β ≈102, the disk expands considerably because of mag- netic pressure and the location of the photosphere is outside of the box. 2.5. Runs and diagnostics Table 1 lists the runs that we performed. We partly adopted the notation of Hirose et al. (2014) to label the runs. Σ0 is the initial surface density and H ≡cs(Tc0)/Ωis the pressure scale-height (with cs the sound speed). The horizontal extent of the box was ±6H for the hot branch and ±3H for the cold and middle branch. Lx, Ly, and Lz follow the ratio 1:4:8 on the hot branch and 1:4:4 on the cold branch. The resolution was 32 × 128 × 256, except for test simulations. We chose to use smaller vertical boxes on the cold branch to avoid high Alfvén velocities near the boundaries, resulting in an enhanced numerical diﬀusion and thus numerical heating. 2.2. Boundary conditions When losses of matter and energy are important, we normalized the pressure by the same factor as for the density to account for the energy loss of the disk through vertical bound- aries. When the pressure was normalized, we observed a change of 40% in the midplane temperature for β ≈102 compared to only 7% when β ≈103. We ran two simulations with β as low as ≈102 and a normalized pressure, but tried to avoid this conﬁguration otherwise. 2. Methods We adopted the local shearing-box approximation (Hawley et al. 1995) to simulate a vertically stratiﬁed patch of an accretion disk located at a distance R0 = 1.315 × 1010 cm from a 0.6 M⊙white dwarf, giving an angular velocity Ω(R0) = 5.931 × 10−3 s−1 to facilitate comparisons with Hirose et al. (2014). The simulations include radiative transport in the ﬂux-limited diﬀusion approxi- mation and thermodynamic quantities appropriate to the temper- ature and density regime sampled by DNe. where µ is the mean molecular weight. To compute µ and ϵ, we used precalculated tables and interpolated linearly between the table values. Tables were computed from the Saha equations assuming ionization equilibrium for the solar composition of Grevesse & Sauval (1998) with a hydrogen abundance X = 0.7 and a metallicity Z = 0.02. The adiabatic index Γ, the entropy, and the thermal capacity Cv were computed similarly. A49, page 2 of 13 N. Scepi et al.: Turbulent and wind-driven accretion in dwarf novae threaded by a large-scale magnetic ﬁeld N. Scepi et al.: Turbulent and wind-driven accretion in dwarf novae threaded by a large-scale magnetic ﬁeld N. Scepi et al.: Turbulent and wind-driven accretion in dwarf novae threaded by a large-scale magnetic ﬁeld We used the opacity tables of Ferguson et al. (2005), which cover the low-temperature region from 2.7 < log(T) < 4.5, and OPAL (Iglesias & Rogers 1996), which covers the high- temperature region from 3.75 < log(T) < 8.7. We used a linear interpolation to connect the two and extended the resulting table, where necessary, using a zero-gradient extrapolation. 2.4. Initial conditions The initial ﬂow was Keplerian with a starting constant vertical magnetic ﬁeld Bz 0 of variable intensity. We chose the intensity of the magnetic ﬁelds to be 8 G, 2 G, or 0.8 G. This is consistent with a white dwarf with a radius of 109 cm and a dipolar surface magnetic ﬁeld of 20 000, 5000, and 2000 G, respectively, or a stellar companion of ≈1 R⊙with a dipolar surface magnetic ﬁeld of 60, 15, and 6 G, respectively, at a binary separation of 1011 cm. The choice of our boundary conditions in x and y ensures that the net ﬂux of magnetic ﬁeld crossing the equatorial plane is constant throughout our simulation. 2.3. Numerical method We solved the MHD equations on a 3D Cartesian grid with the conservative Godunov-type code PLUTO (Mignone 2009). We chose a second-order Runge-Kutta time-integration method. Constrained transport ensures that ∇· B = 0 up to machine pre- cision. We used the HLLD solver to solve the Riemann problem. The HLLD solver can be used with a general EOS where Γ is not a constant. However, when we computed the maximum and minimum propagation speed waves, we used the Davis estimate (Davis 1988) and assumed that Γ is constant on each side of the propagating waves. The Riemann solver is HLLD except where the pressure diﬀerence between two adjacent cells exceeds ﬁve times the local pressure, the local ratio of thermal pressure over magnetic pressure is lower than 10−3 or the Alfvénic speed is greater than 30, in which case we used the more diﬀusive solver HLL. In our simulations each cell spent less than 10% of its time in HLL. To solve the radiative transfer equations, we followed the same implicit scheme as Flock et al. (2013), except that we used the biconjugate gradient solver KSPIBCGS (Yang & Brent 2002) as implemented in PETSC (Balay et al. 2016), which we ﬁnd to provide stabler, faster convergence than BiCGSTAB for our application. In order to avoid very small time steps, we used ﬂoors of 10−6 and 5 × 10−2 of the initial mid-plane values for the density and temperature, respectively. g In the following, we use several averaged quantities denoted in the following way: ⟨X⟩ρ ≡ 1 ΣLxLy Z Lz/2 −Lz/2 Z Ly/2 −Ly/2 Z Lx/2 −Lx/2 ρX dxdydz ⟨X⟩x,y,z ≡ 1 LxLyLz Z Lz/2 −Lz/2 Z Ly/2 −Ly/2 Z Lx/2 −Lx/2 X dxdydz ⟨X⟩≡ 1 LxLy Z Ly/2 −Ly/2 Z Lx/2 −Lx/2 X dxdy, where where Σ = Z Lz/2 −Lz/2 ⟨ρ⟩x,y dz. We use square brackets to denote spatial averages and curly brackets to indicate an average over a time tavg performed when the simulation reached a quasi-steady state. The averag- ing timescale tavg is indicated for each run in the last column of A49, page 3 of 13 A&A 620, A49 (2018) Table 1. Initial parameters and results for our ideal MHD simulations. Table 1. Initial parameters and results for our ideal MHD simulations. 2.3. Numerical method p Run Σ0 Tc0 {Tmid} ± σTmid {Teﬀ} ± σTeﬀ {β} α ± σα qsurface ζ fconv ˙MRφ ˙MZφ H/R tavg Net Flux Simulations B0 = 8 G Upper branch 439F 540 75000 76708 ± 3497 11869 ± 1121 120079 0.046 ± 0.014 81 2.07E-4 0.003 4.78E17 1.38E17 3.46E-2 700 437F 174 50000 48009 ± 1578 8218 ± 424 30236 0.055 ± 0.009 48 2.97E-4 0.012 1.10E17 8.26E16 2.74E-2 600 442F 113 34500 38085 ± 3389 7520 ± 339 17177 0.078 ± 0.017 52 6.43E-4 0.10 7.56E16 8.90E16 2.43E-2 600 456F 80 35000 26024 ± 4367 6989 ± 310 9571 0.144 ± 0.040 81 8.47E-4 0.28 5.83E16 1.39E17 2.02E-2 600 457F 70 30000 R R R R R R R R R R 200 Lower branch 401F 200 11000 R R R R R R R R R R 600 435F 191 5000 13751 ± 638 5656 ± 302 14202 0.067 ± 0.013 70 1.89E-3 0.59 2.80E16 1.21E17 1.46E-2 600 436F 150 9000 9238 ± 573 4544 ± 274 7817 0.089 ± 0.025 37 3.74E-3 0.21 1.37E16 6.33E16 1.20E-2 600 465F 116 6000 3925 ± 300 3681 ± 228 3839 0.156 ± 0.027 34 4.28E-3 0.22 8.06E15 5.84E16 7.83E-3 600 434F 93 5000 3290 ± 163 3240 ± 183 2563 0.186± 0.034 31 4.37E-3 0.26 5.52E15 5.40E16 7.17E-3 600 477F 70 3000 3005 ± 172 3044 ± 181 1591 0.275 ± 0.063 31 1.01E-2 0.32 5.15E15 5.37E16 6.81E-3 600 476F 50 2500 2416 ± 121 2653 ± 150 939 0.354 ± 0.060 30 1.25E-2 0.25 3.33E15 5.22E16 6.12E-3 600 Net Flux Simulations B0 = 2 G Upper branch 439F 540 75000 75531 ± 2228 11541 ± 785 1954988 0.040 ± 0.009 377 1.28E-4 0.003 4.10E17 3.93E16 3.44E-2 600 437F 174 50000 44748 ± 3496 7965 ± 384 478899 0.047 ± 0.015 256 1.21E-4 0.08 8.60E16 1.81E16 2.64E-2 600 442F 113 34500 34349 ± 5792 7386 ± 327 263340 0.078 ± 0.027 264 2.66E-4 0.20 6.67E16 2.75E16 2.32E-2 600 452F 100 30000 28530 ± 4463 7064 ± 293 207469 0.097 ± 0.025 322 3.06E-4 0.37 5.62E16 3.37E16 2.11E-2 600 454F 90 28000 R R R R R R R R R R 150 Middle branch 415F 360 14000 14194 ± 298 5913 ± 402 552927 0.046 ± 0.018 X X 0.81 4.32E16 X 1.49E-2 600 413F 340 10000 12259 ± 127 5230 ± 243 380814 0.041 ± 0.011 X X 0.61 2.50E16 X 1.38E-2 600 411F 320 4500 9840 ± 113 4140 ± 419 291836 0.037 ± 0.012 X X 0.74 1.39E16 X 1.24E-2 600 Lower branch 409F 300 4000 4973 ± 842 3497 ± 156 187517 0.028 ± 0.010 74 8.84E-4 0.31 4.69E15 7.72E15 8.78E-3 1500 407F 280 4000 3450 ± 71 2996 ± 137 128410 0.028 ± 0.009 63 2.75E-4 0.16 2.46E15 6.61E15 7.31E-3 600 404F 230 3500 3465 ± 92 3053 ± 147 106544 0.039 ± 0.014 85 6.70E-4 0.15 2.93E15 8.85E15 7.32E-3 600 403F 220 3500 3230 ± 70 2925 ± 135 94363 0.038 ± 0.011 67 4.44E-4 0.09 2.31E15 6.99E15 7.07E-3 600 401F 200 4000 2728 ± 142 2672 ± 137 74292 0.035 ± 0.009 49 1.94E-4 0.03 1.43E15 5.10E15 6.51E-3 600 465F 116 3000 2176 ± 98 2371 ± 130 37650 0.051 ± 0.011 50 3.91E-4 0.03 9.97E14 5.20E15 5.80E-3 600 434F 93 2500 2021 ± 107 2243 ± 144 28981 0.055 ± 0.014 41 4.96E-4 0.03 7.69E14 4.34E15 5.60E-3 600 477F 70 2000 1885 ± 44 2097 ± 125 21167 0.065 ± 0.012 45 9.96E-4 0.03 6.47E14 4.78E15 5.42E-3 600 Net Flux Simulations B0 = 0, 8 G Upper branch 439F 540 75000 73173 ± 3826 10731 ± 1269 12025687 0.032 ± 0.013 2116 8.09E-5 0.001 3.17E17 3.53E16 3.38E-2 600 437F 174 50000 47560 ± 3365 8367 ± 528 3068497 0.054 ± 0.017 1121 1.52E-4 0.019 1.06E17 1.87E16 2.73E-2 900 442F 113 35000 31312 ± 6879 7273 ± 316 1561523 0.082 ± 0.025 1099 2.51E-4 0.27 6.22E16 1.83E16 2.21E-2 800 452F 100 30000 R R R R R R R R R R R Middle branch 415F 360 5000 10123 ± 245 4259 ± 187 2117377 0.031 ± 0.009 X X 0.83 1.41E16 X 1.26E-2 600 Lower branch 411F 320 4000 3480 ± 101 3002 ± 112 1140102 0.024 ± 0.008 148 1.89E-4 0.15 2.71E15 2.48E15 7.35E-3 1200 409F 300 3500 3623 ± 54 3093 ± 135 911231 0.029 ± 0.009 202 4.41E-4 0.18 3.08E15 3.38E15 7.48E-3 600 407F 280 3000 3508 ± 92 3026 ± 113 814922 0.032 ± 0.011 212 6.30E-4 0.19 2.93E15 3.54E15 7.37E-3 1000 405F 250 2500 3209 ± 131 2864 ± 155 665472 0.038 ± 0.017 338 8.89E-4 0.14 2.60E15 5.64E15 7.07E-3 600 404F 230 2000 2810 ± 118 2626 ± 131 540606 0.035 ± 0.010 152 7.82E-4 0.1 1.72E15 2.55E15 6.62E-3 600 403F 220 3500 2788 ± 512 2689 ± 312 531776 0.035 ± 0.014 165 2.51E-4 0.05 1.73E15 2.75E15 6.57E-3 1500 401F 200 3500 2716 ± 92 2667 ± 129 462249 0.037 ± 0.010 155 2.35E-4 0.04 1.55E15 2.60E15 6.48E-3 600 465F 116 3000 1954 ± 92 2170 ± 135 224681 0.035 ± 0.009 93 1.57E-4 0.01 6.01E14 1.56E15 5.50E-3 600 434F 93 2000 1878 ± 45 2037 ± 146 175993 0.039 ± 0.011 99 3.59E-4 0.02 5.16E14 1.65E15 5.41E-3 600 477F 70 2000 1806 ± 18 1877 ± 106 130298 0.041 ± 0.007 98 3.74E-4 0.02 3.81E14 1.63E15 5.31E-3 600 Net Flux Simulations β ≈104 Upper branch 439F 540 79000 82633 ± 3629 13426 ± 1143 12166 0.077 ± 0.020 33 X 0.01 8.53E17 5.73E17 3.59E-2 950 442F 113 34500 39219 ± 2659 7767 ± 444 12732 0.086 ± 0.018 46 X 0.07 8.83E16 1.10E17 2.48E-2 600 452F 100 34500 37777 ± 2428 7523 ± 388 12122 0.093 ± 0.021 55 X 0.11 7.72E16 1.15E17 2.4E-2 600 454F 90 34500 30721 ± 6055 7200 ± 369 10793 0.110 ± 0.034 55 X 0.17 6.48E16 1.05E17 2.18E-2 600 456F 80 34500 29261 ± 4841 7221 ± 346 10470 0.128 ± 0.033 71 X 0.20 6.18E16 1.21E17 2.14E-2 550 Lower branch 434F 93 1976 2392 ± 70 2608 ± 116 7168 0.099 ± 0.015 34 X 0.05 1.68E15 1.58E16 6.11E-3 600 434F_β12000 93 1976 2109 ± 38 2376 ± 101 13450 0.067 ± 0.009 46 X 0.03 9.94E14 1.07E16 5.74E-3 600 477F 70 1976 3065 ± 193 2967 ± 148 9063 0.104 ± 0.019 73 X 0.05 1.96E15 2.56E16 6.92E-3 600 Net Flux Simulations β ≈103 Upper branch 439F 540 79000 93180 ± 13750 19550 2791 0.424 ± 0.126 36 X 0.06 5.35E18 6.56E18 3.82E-2 300 442F 113 50000 49332 ± 6163 12031 ± 1274 637 0.444 ± 0.082 44 X 0.04 6.02E17 1.23E18 2.78E-2 520 452F 100 45000 45917 ± 4835 11569 ± 1167 490 0.469 ± 0.101 45 X 0.07 5.27E17 1.09E18 2.68E-2 380 Lower branch 434F 93 4500 11915 ± 4413 5993 ± 529 1051 0.315 ± 0.097 61 X 0.88 4.84E16 2.98E17 1.36E-2 500 Net Flux Simulations β ≈102 Upper branch 442F 113 34500 31015 ± 9461 10821 ± 2124 101.9 0.850 ± 0.191 32 X X 8.03E17 3.78E18 2.20E-2 200 Lower branch 434F 93 2000 5926 ± 2007 4609 ± 662 153.1 0.774 ± 0.280 32 X X 5.32E16 7.41E17 9.62E-3 200 tes. 2.3. Numerical method Horizontal dotted gray lines show the value of α in ZNF simulations from S18 in a highly convective simulation of the hot branch (upper line) and a typical cold branch case (bottom line). The linear ﬁt is made using data from the cold branch simulations with β ≲2 × 104. vz is the vertical velocity, and the quantity ϵvz represents the advective ﬂux of internal energy Fadv. vz is the vertical velocity, and the quantity ϵvz represents the advective ﬂux of internal energy Fadv. We deﬁned α, the ratio of stress to pressure, as α ≡ {⟨Wxy⟩x,y,z} {⟨P⟩x,y,z} , α ≡ {⟨Wxy⟩x,y,z} {⟨P⟩x,y,z} , (top), and for the purely radiative run 434O (bottom) in S18. α ranges between 0.275 and 0.026. We ﬁnd that α scales as β−0.56 (with β deﬁned in Eq. (6)) when we only include the simulations from the cold branch. This is consistent with previous works (Hawley et al. 1995; Salvesen et al. 2016). and β, the ratio of the time-averaged midplane pressure to the magnetic pressure due to the mean vertical ﬁeld as follows: and β, the ratio of the time-averaged midplane pressure to the magnetic pressure due to the mean vertical ﬁeld as follows: β = 8π{⟨Pthermal mid⟩} ⟨Bz⟩2 · (6) β = 8π{⟨Pthermal mid⟩} ⟨Bz⟩2 · (6) As in ZNF, convection plays a role in determining the value of α (see Hirose et al. 2014 and S18). For β > 105, simulations from both the hot and cold branch depart from the β−0.56 rela- tion and tend toward the ZNF case. Moreover, Fig. 1 shows for β ≈104 that convective simulations from the hot branch leave the relation, whereas cold branch simulations do not. We note an increasing deviation from the trend with fconv for these con- vective simulations. However, in simulations with a high level of magnetization, the disk is thicker because of the magnetic pres- sure in the midplane and thus pushes the zone where convection could set in out of the box. We doubled the vertical size and res- olution of the disk in 456F_bigbox_β3, but only observed one convection episode. The eﬀect on α is not clear from this single event. All we can say is that the vertical equilibrium is domi- nated by magnetic pressure, hence it would seem reasonable that convection plays a lesser role than that for higher β. 2.3. Numerical method Σ0 is the initial surface density in g cm−2, Tc0 the initial midplane temperature in K, and H/R0 where H is the corresponding scale hei ackets {} are for quantities averaged over tavg (given in local orbits) with σ the associated standard deviation. R signals simulations with runa i li A49, page 4 of 13 N. Scepi et al.: Turbulent and wind-driven accretion in dwarf novae threaded by a large-scale magnetic ﬁeld N. Scepi et al.: Turbulent and wind-driven accretion in dwarf novae threaded by a large-scale magnetic ﬁeld N. Scepi et al.: Turbulent and wind-driven accretion in dwarf novae threaded by a large-scale magnetic ﬁeld 102 103 104 105 106 107 β 10−2 10−1 100 α log10(α) = −0.56 × log10(β) + 1.18 0.00 0.04 0.08 0.12 0.16 0.20 0.24 0.28 0.32 0.36 0.40 fconv Fig. 1. Square markers denote cold branch simulations, and circle mark- ers denote hot branch simulations. The color indicates the value of fconv. Horizontal dotted gray lines show the value of α in ZNF simulations from S18 in a highly convective simulation of the hot branch (upper line) and a typical cold branch case (bottom line). The linear ﬁt is made using data from the cold branch simulations with β ≲2 × 104. Table 1. We typically averaged over 100 orbits. {Σ}, {Tmid}, and {Teﬀ} are thus the time-averaged values of the surface density, midplane temperature, and eﬀective temperature, respectively. σTmid, σTeﬀ, and σα are the standard deviations of the ﬂuctuations with time of these quantities. 102 103 104 105 106 107 β 10−2 10−1 100 α log10(α) = −0.56 × log10(β) + 1.18 0.00 0.04 0.08 0.12 0.16 0.20 0.24 0.28 0.32 0.36 0.40 fconv q We computed Teﬀas We computed Teﬀas T 4 eﬀ= 1 2σB (F+ rad z −F− rad z), T 4 eﬀ= 1 2σB (F+ rad z −F− rad z), where F+/− rad z is the radiative ﬂux in the vertical direction on the upper or lower boundary of our simulation box. We also deﬁned ⟨Q−⟩x,y the local cooling rate as ⟨Q−⟩x,y ≡d dz⟨Frad z⟩x,y + d dz⟨ϵvz⟩x,y. (5) (5) Fig. 1. Square markers denote cold branch simulations, and circle mark- ers denote hot branch simulations. The color indicates the value of fconv. 2.3. Numerical method We add the caveat that, in this particular simulation, pressure is normalized. This may have an impact on the onset of convection. The instantaneous stress-to-pressure ratio is ˜α = ⟨Wxy⟩x,y,z/⟨P⟩x,y,z. The turbulent stress Wxy is ρ(uxuy −vAxvAy), where uuu = vvv + 3 2Ωxˆyˆyˆy, and vA is the Alfvén velocity computed from the mean ﬁeld. 3. Ideal MHD runs First, we gather all of our simulations and discuss the inﬂuence of a net vertical magnetic ﬁeld on the local properties of MRI in Sect. 3.1 and the properties of MRI-driven outﬂows in Sect. 3.2. In Sect. 3.3, we discuss the impact of a large-scale poloidal mag- netic ﬁeld with three diﬀerent magnitudes on the S curves. In Sect. 3.4, we investigate the relative importance of viscous and wind transport of angular momentum on the ensuing accretion rate. Finally, in Sect. 3.5, we discuss the impact of the wind- driven angular momentum transport on observables. S18 showed that α is not correlated with the convective frac- tion. This result was also conﬁrmed by Coleman et al. (2018), who observed that α appears to be more correlated with the verti- cal Mach number. However, they added that the speed of the con- vective eddies is only 10% of the turbulent speed. This indicates that convection is weaker than turbulence and does not signiﬁ- cantly drive additional motion that could feed the dynamo. All these characteristics are also true for the net ﬂux. In the absence of any deep insight on how convective MRI leads to an enhance- ment of α, we plot in Fig. 2, a 2D map of α in the Teﬀ, β space. We ﬁt our data with Eq. (7), which is similar to Coleman et al. (2016) with an additional term for the dependence in β based on Fig. 1, 3.1. Local turbulent transport They are deﬁned as q = [⟨ρuφuz⟩−⟨BφBz⟩/4π]surface+ surface− 2⟨Pthermal mid⟩ β (9) and ζ = ⟨ρuz⟩Lz/2 −Lz/2 2⟨ρmidcs mid⟩· (10) q = [⟨ρuφuz⟩−⟨BφBz⟩/4π]surface+ surface− 2⟨Pthermal mid⟩ β (9) (9) and ζ = ⟨ρuz⟩Lz/2 −Lz/2 2⟨ρmidcs mid⟩· (10) ζ = ⟨ρuz⟩Lz/2 −Lz/2 2⟨ρmidcs mid⟩· ζ = ⟨ρuz⟩Lz/2 −Lz/2 2⟨ρmidcs mid⟩· (10) Shearing box simulations are very useful for studying the local properties of MRI turbulence. However, they suﬀer lim- itations concerning the global geometry of the outﬂows. The shearing-box symmetries do not ensure a physical conﬁgura- tion of the magnetic ﬁeld. For example, they can allow a sur- face torque of the same sign on both sides of the disk; this con- ﬁguration does not extract angular momentum. Additionally, it is a well-known result that the sign of ⟨By⟩ﬂips regularly in a shearing box (Brandenburg et al. 1995). To cope with these lim- itations, we deﬁned a function S that gives the sign of ⟨By⟩in each hemisphere. We multiplied the wind torque by S in our time average. This allowed us to compute wind torques that do not depend on the geometry picked up by the shearing box at a given time. Fig. 2. Value of log(α) in our simulations is shown by color. Simulations at β = 109 are ZNF simulations taken from S18 with additional simu- lations from this article. The background color map shows the result of the ﬁt of α as a function of Teﬀand β from Eq. (7). in the simulations with β < 104. We plot the ﬁt on the back- ground of Fig. 2. The best-ﬁt parameters are A = 5.35 × 10−2, T0 = 6866 K, σ = 853 K, B = 1.65 × 10−3, C = 3.65 × 10−2, D = 1.37, and E = 0.58. This prescription will be useful in a DIM model or any global model that does not resolve the turbu- lent transport. A surface for the disk needs to be deﬁned so that q can be estimated. We took the height where the maximum value of the wind torque is reached as the deﬁnition of the disk surface because above this surface, magnetic energy is transferred to kinetic energy to accelerate the outﬂow. qsurface = max(S (⟨ρvφvz⟩+ ⟨BφBz⟩)(z+) + S (⟨ρvφvz⟩+ ⟨BφBz⟩)(z−)) 2⟨Pthermal mid⟩ β (11) )β. )β. (11) We plot qsurface in Fig. 3 as a function of β for all simula- tions. We ﬁnd that qsurface is roughly constant for β ≲104 with an approximate value of qsurface ≈40. For low β, the wind torque is dominated by the magnetic part. This means that, remarkably, across almost three orders of magnitude in β, the MRI gives a quasi-constant ratio Bφ/Bz. This value agrees with local simu- lations from Fromang et al. (2013) and Simon et al. (2013) as well as with the global simulation of Zhu & Stone (2018), where q ≈10, according to their Figs. 6 and 7. Bai & Stone (2013a) found a lower value of q, but they measured the wind torque at the external boundaries; our results again agree when we adopt the same deﬁnition. When β exceeds 105, we tend toward the ZNF case where the magnetic ﬁeld becomes mainly azimuthal, and we expect q ∝β0.5. We ﬁnd that a dependence in β0.6 ﬁts our data well. We propose in Fig. 3 a function that allows a smooth transition between the two asymptotic regimes. Such outﬂows extract matter, angular momentum, and energy away from the disk. Since the classical DIM only includes transport of angular momentum through anormalous diﬀusive transport, we need to quantify the transport that is due to the outﬂows to include them in a more sophisticated DIM. Following Balbus & Papaloizou (1999), we derived the conservation of angular momentum of an axisymmetric disk in cylindrical coordinates including the surface terms. After some algebra, we can write the surface density equation of evolution in cylindrical coordinates in the following form: ∂tΣ −2 R0 ∂R0 R1/2 0 ∂R0 Σ Ω0 R1/2 0 α⟨c2 s⟩ρ ! + 2q β ⟨Pthermal mid⟩R0 Ω0 ! + ζ⟨ρmidcs mid⟩= 0. (8) (8) The amplitude of the wind torque normalized by thermal pressure is given by 2qsurface/β. It varies as β−1 for β ≲104 and as β−0.4 for lower magnetization; angular momentum is more eﬃciently removed by the wind at low β, as we expected (see Sect. 3.4). Equation (8) shows three ways for the surface density to change: through radial accretion due to a transport of angu- lar momentum either through turbulence or outﬂows, and loss of matter from the vertical boundaries. 3.1. Local turbulent transport We add the caveat that our vertically modiﬁed outﬂow boundary conditions impose a purely vertical magnetic ﬁeld that constrains the geometry of the magnetic ﬁeld lines and therefore imposes q = 0 at the bound- aries. Numerically, we deﬁned q as A49, page 6 of 13 3.1. Local turbulent transport Local MRI turbulent transport is well known to depend on the magnetization parameter β (Hawley et al. 1995). We plot in Fig. 1 α as a function of β for all of our simulations. Squares are used for cold branch simulations and circles for the hot branch (see Sect. 3.3 for details on the cold and hot branch). The color of the circles gives fconv, which is deﬁned as fconv ≡1 2 max 0, max (Fadv(z > 0) F+ rad z )!! + max 0, max (Fadv(z < 0) F− rad z )!!! , α =  A exp(−(Teﬀ−T0)2 2σ2 ) + B tanh( Teﬀ−T0 σ ) + C + Dβ−E, if β < 5 × 104 A exp(−(Teﬀ−T0)2 2σ2 ) + B tanh( Teﬀ−T0 σ ) + C, if β > 5 × 104 . (7) where Fadv = ⟨evz⟩. fconv is a dimensionless measure of the impor- tance of convective transport compared to radiative transport. (7) We chose to constrain A, T0, σ, B, and C using only simu- lations with β > 5 × 104. Then, we ﬁt the remaining parameters The gray dashed lines in Fig. 1 correspond to the value of α for the convective run 452O, which shows an enhanced α A&A 620, A49 (2018) 102 103 104 105 106 107 108 109 β 103 104 Teﬀ −2.0 −1.8 −1.6 −1.4 −1.2 −1.0 −0.8 −0.6 −0.4 −0.2 0.0 α 102 103 104 105 106 107 108 109 β 103 104 Teﬀ −2.0 −1.8 −1.6 −1.4 −1.2 −1.0 −0.8 −0.6 −0.4 −0.2 0.0 α Fig. 2. Value of log(α) in our simulations is shown by color. Simulations at β = 109 are ZNF simulations taken from S18 with additional simu- lations from this article. The background color map shows the result of the ﬁt of α as a function of Teﬀand β from Eq. (7). α parameter in Sect. 3.1. Outﬂows lead to the deﬁnition of two additional dimensionless parameters: q, which quantiﬁes the strength of the wind torque, and ζ, which is the ratio of the mass loss from the vertical boundaries to the sound speed times the density in the midplane. 3.2. Outﬂows and angular momentum transport Net magnetic ﬂux signiﬁcantly aﬀects the dynamic of the disk as it allows the formation of magneto-centrifugally driven out- ﬂows from MRI (Fromang et al. 2013; Lesur et al. 2013). All of our simulations with a β ≲104 clearly exhibit these magneto- centrifugally driven outﬂows. By computing the Bernoulli invari- ant along streamlines, we observe that thermal eﬀects also play a signiﬁcant role in launching the outﬂow. Our simulations repro- duce the main characteristic that have been extensively described in the literature (Blandford & Payne 1982; Ferreira & Pelletier 1995; Fromang et al. 2013; Lesur et al. 2013). qsurface = max(S (⟨ρvφvz⟩+ ⟨BφBz⟩)(z+) + S (⟨ρvφvz⟩+ ⟨BφBz⟩)(z−)) 2⟨Pthermal mid⟩ β N. Scepi et al.: Turbulent and wind-driven accretion in dwarf novae threaded by a large-scale magnetic ﬁeld conditions on the cold branch to compare this with the net ﬂux case. These additional simulations show better agreement than S18 with Hirose et al. (2014) concerning the cold branch. Returning to the net ﬂux case, we see that similarly to ZNF simulations, there are always two branches of stability: the hot branch, and the cold branch. However, as observed in Hirose (2015) and S18, there is also a middle branch, which is a contin- uation at higher temperature of the cold branch; these points are indicated as stars in Fig. 5. There is no obvious middle branch in the case of Bz0 = 8 G since the temperature on the cold branch gradually increases from 3005 K to 13751 K. For Bz0 = 0.8, 2 G and ZNF we note that the middle branch does not converge with the box size. The equilibrium temperature strongly depends on the box size. We do not know the origin of this dependence. Given the uncertainties regarding the stability of these points, we did not include these simulations in any other ﬁgure (see also Hirose 2015). We show in Fig. 4 the dependence of ζ on β. Crosses show simulations on a cold branch with an extent of 12H in the z- direction, and squares show simulations with an extent of 6H in the z-direction, as for Figs. 1 and 3. The dependence of ζ on the vertical size of the box is clear. Smaller boxes exhibit higher ζ. However, ζ scales approximately as β−1 for the two box sizes in the range β < 105. Like α and qsurface, ζ reaches the asymptotic value given by the zero net-ﬂux case as we increase β. The results are more dispersed than for α since by deﬁnition, they depend on the boundary conditions and the relative box size compared to the disk. All these results are consistent with Suzuki & Inutsuka (2009), Bai & Stone (2013a), and Fromang et al. (2013). More- over, it seems that convection enhances ζ for β > 105, whereas it does not for lower β. This is illustrated by the higher ζ of the convective hot branch simulations. The extrema of the S-curves, deﬁned by Σ+ crit/Σ− crit and T + eﬀcrit/T − eﬀcrit, are of special importance as they determine the shape of the light curves and in particular the presence of reﬂares (Lasota 2001; Coleman et al. N. Scepi et al.: Turbulent and wind-driven accretion in dwarf novae threaded by a large-scale magnetic ﬁeld Squares denote cold branch simulations, and circles denote hot branch simulations. The color indicates the value of fconv. 101 102 103 104 105 106 107 108 β 101 102 103 qsurface qsurface = 31.6 × (8 × 10−10 × β2 + 1.8)0.3 0.00 0.04 0.08 0.12 0.16 0.20 0.24 0.28 0.32 0.36 0.40 fconv Fig. 4. Squares denote cold branch simulations with a vertical extent Lz = 6H, and circles denote hot branch simulations with a vertical extent Lz = 12H. Crosses are used for cold branch simulations with the same box size as on the hot branch (results from those simulations are not reported in Table 1). The color indicates the value of fconv. Horizontal dotted gray lines show the value of ζ in ZNF simulations from S18 in a highly convective simulation of the hot branch (upper line) and a typical cold branch case (bottom line). The blue and green solid lines are linear ﬁts to the points from the cold branch simulations with β ≲2×104. This ﬁgure only reports results from simulations with Bz0 = 0.8, 2, and 8 G. Fig. 3. Squares denote cold branch simulations, and circles denote hot branch simulations. The color indicates the value of fconv. is unclear whether convection aﬀects the φz component of the stress tensor for higher magnetization. We note that the mag- netic component ⟨BφBz⟩accounts for ≈70% of the wind torque for β ≲105. For β ≳105, the Reynolds component can account for up to ≈60% of the total wind torque. Since the vertical extent of our box is only a few scale heights, part of the matter leaving the box, which in our simu- lations is deﬁnitely lost, is susceptible to falling back onto the disk. This picture is consistent with decreasing mass outﬂow with increasing box size, as observed in Fromang et al. (2013); this is also observed here. The value of ζ can therefore only be an estimate in a shearing-box simulation (Suzuki & Inutsuka 2009; Fromang et al. 2013). We computed the critical points for the simulation 434F_8G and found that the fast Alfvén point is reached at the vertical boundary exactly as in Fromang et al. (2013), suggesting a numerical artifact of the shearing box. Tak- ing this into account, we conclude that the ζ we measured are maximum values for the mass loss rate through the vertical boundaries. N. Scepi et al.: Turbulent and wind-driven accretion in dwarf novae threaded by a large-scale magnetic ﬁeld 2016). For Bz0 < 8 G, the S-curves are not very diﬀerent from the ZNF case. We ﬁnd Σ− crit ≈110 g cm−2, which is compatible with S18 given the uncertainty on the stability limit. However, for Bz0 = 8 G, we have Σ− crit = 80g cm−2, which extends the hot branch to lower densities. The same result applies to the beginning of the middle branch. However, the conclusions concerning Σ+ crit are more dif- ﬁcult to draw from our simulations as we see a box-dependent behavior of the middle branch, as reported above. In the DIM, S-curves with higher α are shifted toward lower densities. Since increasing the magnetization gave higher values of α, our results are consistent with the DIM. N. Scepi et al.: Turbulent and wind-driven accretion in dwarf novae threaded by a large-scale magnetic ﬁeld 103 104 105 106 β 10−5 10−4 10−3 10−2 ζ log10(ζ) = −0.95 × log10(β) + 1.01 0.00 0.04 0.08 0.12 0.16 0.20 0.24 0.28 0.32 0.36 0.40 fconv Fig. 4. Squares denote cold branch simulations with a vertical extent Lz = 6H, and circles denote hot branch simulations with a vertical extent Lz = 12H. Crosses are used for cold branch simulations with the same box size as on the hot branch (results from those simulations are not reported in Table 1). The color indicates the value of fconv. Horizontal dotted gray lines show the value of ζ in ZNF simulations from S18 in a highly convective simulation of the hot branch (upper line) and a typical cold branch case (bottom line). The blue and green solid lines are linear ﬁts to the points from the cold branch simulations with β ≲2×104. This ﬁgure only reports results from simulations with Bz0 = 0.8, 2, and 8 G. 101 102 103 104 105 106 107 108 β 101 102 103 qsurface qsurface = 31.6 × (8 × 10−10 × β2 + 1.8)0.3 0.00 0.04 0.08 0.12 0.16 0.20 0.24 0.28 0.32 0.36 0.40 fconv Fig. 3. Squares denote cold branch simulations, and circles denote hot branch simulations. The color indicates the value of fconv. is unclear whether convection aﬀects the φz component of the 103 104 105 106 β 10−5 10−4 10−3 10−2 ζ log10(ζ) = −0.95 × log10(β) + 1.01 0.00 0.04 0.08 0.12 0.16 0.20 0.24 0.28 0.32 0.36 0.40 fconv Fig. 4. Squares denote cold branch simulations with a vertical extent Lz = 6H, and circles denote hot branch simulations with a vertical extent Lz = 12H. Crosses are used for cold branch simulations with the same box size as on the hot branch (results from those simulations are not reported in Table 1). The color indicates the value of fconv. Horizontal dotted gray lines show the value of ζ in ZNF simulations from S18 in a highly convective simulation of the hot branch (upper line) and a typical 103 104 105 106 β 10−5 10−4 10−3 10−2 ζ log10(ζ) = −0.95 × log10(β) + 1.01 101 102 103 104 105 106 107 108 β 101 102 103 qsurface qsurface = 31.6 × (8 × 10−10 × β2 + 1.8)0.3 0.00 0.04 0.08 0.12 0.16 0.20 0.24 0.28 0.32 0.36 0.40 fconv Fig. 3. qsurface = max(S (⟨ρvφvz⟩+ ⟨BφBz⟩)(z+) + S (⟨ρvφvz⟩+ ⟨BφBz⟩)(z−)) 2⟨Pthermal mid⟩ β (11) We have studied the radial turbulent transport of angular momentum deﬁned by the Convective simulations from the hot branch have a slightly higher qsurface than cold branch simulations for β > 105. It 3.3. Thermal equilibrium curves with a constant value of Bz0 We plot in the left (right) panels of Fig. 5 the thermal equilibrium curves in the Tmid(Teﬀ) versus Σ plane, also known as S-curves, for several net magnetic ﬁelds. We emphasize that for a given S-curve, Bz0 is ﬁxed, but not β. The S-curve in ZNF is taken from S18 with additional simulations with outﬂow z-boundary The S-curve with Bz0 = 8 G has another notable feature compared to the lower net magnetic ﬁeld S-curves and S18: here alone, the maximum value of α is obtained on the cold branch. We postpone the discussion of this point to Sect. 5. For Σ < 116 g cm−2, magnetization starts to be important on the A49, page 7 of 13 A&A 620, A49 (2018) 103 104 105 Tmid (K) Zero Flux Net α=0,02 α=0,03 α=0,05 α=0,08 104 Teﬀ(K) Zero Net Flux α=0,02 α=0,03 α=0,05 α=0,08 103 104 105 Tmid (K) B0 = 0.8 G α=0,02 α=0,03 α=0,05 α=0,08 104 Teﬀ(K) B0 = 0.8 G α=0,02 α=0,03 α=0,05 α=0,08 103 104 105 Tmid (K) B0 = 2 G α=0,02 α=0,03 α=0,05 α=0,08 104 Teﬀ(K) B0 = 2 G α=0,02 α=0,03 α=0,05 α=0,08 102 103 Σ (g cm−2) 103 104 105 Tmid (K) B0 = 8 G α=0,02 α=0,03 α=0,05 α=0,08 102 103 Σ (g cm−2) 104 Teﬀ(K) B0 = 8 G α=0,02 α=0,03 α=0,05 α=0,08 0.08 0.12 0.16 0.20 0.24 0.28 0.32 α 0.08 0.12 0.16 0.20 0.24 0.28 0.32 α 0.032 0.040 0.048 0.056 0.064 0.072 0.080 0.088 0.096 α 0.032 0.040 0.048 0.056 0.064 0.072 0.080 0.088 0.096 α 0.024 0.032 0.040 0.048 0.056 0.064 0.072 0.080 α 0.024 0.032 0.040 0.048 0.056 0.064 0.072 0.080 α 0.032 0.040 0.048 0.056 0.064 0.072 0.080 α 0.032 0.040 0.048 0.056 0.064 0.072 0.080 α Fig. 5. Thermal equilibrium curves in the Σ −Tmid(−Teﬀ) plane for diﬀerent magnetic ﬁeld conﬁgurations. From top to bottom, ﬁrst panel: result of S18 (circles) with outﬂow conditions with new additional simulations using a zero net magnetic ﬁeld conﬁguration, second panel: Bz0 = 0.8G, third panel: Bz0 = 2G, last panel: Bz0 = 8G. The colors of the dot correspond to the value of α. Triangles show the instability points where the patch of disk undergoes critical cooling (downward-pointing triangle) or heating (upward). Stars indicate the simulations from the middle branch that did not converge. 3.3. Thermal equilibrium curves with a constant value of Bz0 Gray vertical lines are plotted at the end of the hot and cold branch to facilitate comparisons between the panels. Finally, the colored S-curves in solid lines shows the DIM model with convection using diﬀerent values for α. 104 Teﬀ(K) Zero Net Flux α=0,02 α=0,03 α=0,05 α=0,08 104 Teﬀ(K) B0 = 0.8 G α=0,02 α=0,03 α=0,05 α=0,08 104 Teﬀ(K) B0 = 2 G α=0,02 α=0,03 α=0,05 α=0,08 102 103 Σ (g cm−2) 104 Teﬀ(K) B0 = 8 G α=0,02 α=0,03 α=0,05 α=0,08 0.08 0.12 0.16 0.20 0.24 0.28 0.32 α 0.032 0.040 0.048 0.056 0.064 0.072 0.080 0.088 0.096 α 0.024 0.032 0.040 0.048 0.056 0.064 0.072 0.080 α 0.032 0.040 0.048 0.056 0.064 0.072 0.080 α Teﬀ(K) Teﬀ(K) 102 103 Σ (g cm−2) 104 Teﬀ(K) 0 α=0,02 α=0,03 α=0,05 α=0,08 0.08 0.12 0.16 0.20 0.24 0.28 0.32 α 10 Σ (g cm−2) 10 Σ (g cm−2) 10 Σ (g cm−2) Fig. 5. Thermal equilibrium curves in the Σ −Tmid(−Teﬀ) plane for diﬀerent magnetic ﬁeld conﬁgurations. From top to bottom, ﬁrst panel: result of S18 (circles) with outﬂow conditions with new additional simulations using a zero net magnetic ﬁeld conﬁguration, second panel: Bz0 = 0.8G, third panel: Bz0 = 2G, last panel: Bz0 = 8G. The colors of the dot correspond to the value of α. Triangles show the instability points where the patch of disk undergoes critical cooling (downward-pointing triangle) or heating (upward). Stars indicate the simulations from the middle branch that did not converge. Gray vertical lines are plotted at the end of the hot and cold branch to facilitate comparisons between the panels. Finally, the colored S-curves in solid lines shows the DIM model with convection using diﬀerent values for α. momentum transport, momentum transport, cold branch with Bz0 = 2 G and α is greater than the ZNF case. Otherwise, in the remaining S-curve and for weaker magnetic ﬁeld strengths, the values of α are similar to the ZNF case. ˙MRφ = 4π Ω0 1 R0 ∂R0(ΣR2 0α⟨c2 s⟩ρ), ˙Mzφ = 8π β q⟨Pthermal mid⟩R0 Ω0 · (12) Finally, we note that on the hot branch our simulations are well ﬁt by the S-curves from the DIM. However, simulations with the strongest magnetization, which are located on the cold branches of cases Bz0 = 2 and 8 G, clearly do not ﬁt the equi- librium curves from the DIM. 3.3. Thermal equilibrium curves with a constant value of Bz0 As the disk instability model is a purely hydrodynamic code, this is not surprising. (12) Because the curvature of the shearing box is absent, the term ˙MRφ measured in our simulation is zero. However, by making a few assumptions on the radial structure of the disk, we can have an estimate of ˙MRφ. We assumed the following scalings: Σ ≈Rp and ⟨c2 s⟩ρ ≈Rs. Equation (12) then becomes Because the curvature of the shearing box is absent, the term ˙MRφ measured in our simulation is zero. However, by making a few assumptions on the radial structure of the disk, we can have an estimate of ˙MRφ. We assumed the following scalings: Σ ≈Rp and ⟨c2 s⟩ρ ≈Rs. Equation (12) then becomes 3.5. Stability criterion in a disk-wind model Squares indicates cold branch simulations and circles hot branch simulations. Dashed lines show the limit ˙MRφ/ ˙Mzφ = 1 from the empirical formulas given by Eq. (13). σT 4 eﬀ= 3GM ˙M 4πR3 0 1 − Rin R0 !1/2 − 1 2 ˙MR1/2 0 Z R0 Rin ˙Mzφr−1/2dr (15) = 3GM 8πR7/2 0 Z R0 Rin ˙MRφr−1/2dr. (16) (16) in our simulations. This means that ˙MRφ ˙Mzφ ≈Cα × β 2q H R0 ! We can rewrite this equation as σT 4 eﬀ= 3GM ˙ Mvis 4πR3 0 1 − Rin R0 !1/2, (17) We can use the empirical relations from Figs. 1 and 3, and we ﬁnd We can use the empirical relations from Figs. 1 and 3, and we ﬁnd (17) ˙MRφ ˙Mzφ ≈  0.19C × β0.44 H R0 , if β < 104 0.30C × β0.4 H R0 , if β > 104 . (13) ˙MRφ ˙Mzφ ≈  0.19C × β0.44 H R0 , if β < 104 0.30C × β0.4 H R0 , if β > 104 . (13) where ˙ Mvis corresponds to the viscous mass transfer rate, which a purely viscous disk would have to match the dissipation rate of a turbulent, wind-driven disk. (13) The values of ˙MRφ and ˙Mzφ can be found in Table 1. We plot in Fig. 6 the ratio of the radial to vertical accretion rate as a func- tion of β and H/R. Dashed lines show the limit ˙MRφ/ ˙Mzφ = 1 from the empirical formulas given by Eq. (13). Simulations from the hot and cold branch are represented by circles and squares, respectively. Figure 5 shows that the cold branch is entirely dom- inated by accretion that is due to vertical transport of angular momentum. Only simulations from the denser and hotter part of the hot branch are dominated by viscous radial transport, and even in this case, vertical transport remains non-negligible. This shows that the vision of a purely viscous α-disk is inappropriate to describe an DNe disk if a large-scale poloidal ﬁeld is present. In such a case, the role of the wind torque in removing angular momentum must be taken into account when computing the disk evolution. This can be done using the ﬁts we provide for qsurface (see Fig. 3). 3.5. Stability criterion in a disk-wind model 10−2 H/R 102 103 104 105 106 107 β Viscously driven Wind-driven C = 6, β < 104 C = 3.5, β < 104 C = 2, β > 104 −1.2 −0.9 −0.6 −0.3 0.0 0.3 0.6 0.9 1.2 l ( ˙MRφ) The essence of the DIM is the thermal instability around ion- ization of hydrogen. In the DIM, the eﬀective temperature is directly related to the mass accretion rate in the disk by σT 4 eﬀ= 3GM ˙M 4πR3 0 1 − Rin R0 !1/2. (14) (14) In a stationary system, the mass accretion rate is imposed by the mass transfer rate from the companion. Hence, in a range of ˙M, part of the disk is ionizing hydrogen and thus unstable; this corresponds to DNe. If the accretion rate is high enough for the annulus at the external radius to be fully ionized, then the disk is stable; this a novae-like system. This simple argument provides an observational test of the DIM. Dubus et al. (2018) systematically examined the relation between ˙M and the stability of the system for ≈100 CVs and found that predictions based on the DIM are robust. In a stationary system, the mass accretion rate is imposed by the mass transfer rate from the companion. Hence, in a range of ˙M, part of the disk is ionizing hydrogen and thus unstable; this corresponds to DNe. If the accretion rate is high enough for the annulus at the external radius to be fully ionized, then the disk is stable; this a novae-like system. This simple argument provides an observational test of the DIM. Dubus et al. (2018) systematically examined the relation between ˙M and the stability of the system for ≈100 CVs and found that predictions based on the DIM are robust. We ﬁnd that Teﬀcrit−does not change much between ZNF and net ﬂux simulations. It is determined by the ionization temper- ature of hydrogen and is ≈7000 K. However, when we include a contribution from the wind in the conservation of energy and angular momentum, we ﬁnd that the classical relation between ˙M and Teﬀin the disk changes to H/R Fig. 6. Ratio of the accretion rate due to radial transport of angular momentum ˙MRφ to the accretion rate due to vertical transport of angular momentum ˙Mzφ as a function of β and H/R. The color shows the value of log( ˙MRφ/ ˙Mzφ). 3.4. Vertical and radial transport of angular momentum In a disk, the removal of angular momentum from turbulent MRI and surface torque leads to an inward ﬂow of matter along the radial direction. We deﬁne the mass accretion rate in the disk ˙M as ˙MRφ = p + s + 2 Ω0 4πΣα⟨c2 s⟩ρ. ˙MRφ = p + s + 2 Ω0 4πΣα⟨c2 s⟩ρ. Classical viscous disk theory predicts Σ ∝R−3/4 and Teﬀ∝R−3/4 (Frank et al. 2002). This means that (p + s + 2) is not far from unity. The ratio of the radial to vertical accretion rate is then Classical viscous disk theory predicts Σ ∝R−3/4 and Teﬀ∝R−3/4 (Frank et al. 2002). This means that (p + s + 2) is not far from unity. The ratio of the radial to vertical accretion rate is then ˙M ≡−2πR0Σ⟨uR⟩ρ. From the conservation of angular momentum, we ﬁnd that ˙MRφ ˙Mzφ = (p + s + 2) β 2q Σ⟨c2 s⟩ρ ⟨Pthermal mid⟩ α R0 · ˙M = 4π Ω0 1 R0 ∂R0(ΣR2 0α⟨c2 s⟩ρ) + 2q β ⟨Pthermal mid⟩R0 ! From our simulations, we ﬁnd that Σ⟨c2 s⟩ρ/⟨Pthermal mid⟩≈ C(β, H) × H, where the factor C(β, H) extends from 7.4 to 1.6 Following Fromang et al. (2013), we decompose ˙M into a radial component, ˙MRφ, and a vertical component, ˙Mzφ, of angular Following Fromang et al. (2013), we decompose ˙M into a radial component, ˙MRφ, and a vertical component, ˙Mzφ, of angular A49, page 8 of 13 A49, page 8 of 13 N. Scepi et al.: Turbulent and wind-driven accretion in dwarf novae threaded by a large-scale magnetic ﬁeld 10−2 H/R 102 103 104 105 106 107 β Viscously driven Wind-driven C = 6, β < 104 C = 3.5, β < 104 C = 2, β > 104 −1.2 −0.9 −0.6 −0.3 0.0 0.3 0.6 0.9 1.2 log10( ˙MRφ ˙MZφ) Fig. 6. Ratio of the accretion rate due to radial transport of angular momentum ˙MRφ to the accretion rate due to vertical transport of angular momentum ˙Mzφ as a function of β and H/R. The color shows the value of log( ˙MRφ/ ˙Mzφ). Squares indicates cold branch simulations and circles hot branch simulations. Dashed lines show the limit ˙MRφ/ ˙Mzφ = 1 from the empirical formulas given by Eq. (13). 3.5. Stability criterion in a disk-wind model Additionally, the limit ˙MRφ/ ˙Mzφ = 1 is well repro- duced by empirical formulas from Eq. (13), providing a good estimate of the constant C. As a comparison, in the global simu- lations from Zhu & Stone (2018), where H/R = 0.1 and β ≈103, the transport is dominated by the viscous torque with a contri- bution of only 5% from the wind torque. Using our empirical formula with C = √ 2π, we ﬁnd that the wind torque accounts for ≈20% of the total torque. Again, our simple shearing-box estimates of the wind torque are on the same order of magnitude as global simulations. In the presence or absence of wind-driven accretion, heat- ing is only due to viscous accretion. This implies that WRφ must be positive since it is of the same sign as the heating, which, by deﬁnition, is positive (see Balbus & Hawley 1998). With the assumption that WRφ(Rin) = 0 in a purely viscous disk, ˙MRφ needs to be constant and positive to ensure stationarity. This is no longer true when angular momentum is partly extracted by a wind. We see from Eq. (16) that in a stationary disk, ˙MRφ does not have to be positive at all radii or constant in order to ensure positive heating. This breaks down the classical relation between the viscous mass accretion rate and eﬀective tempera- ture (Pringle 1981) and opens up the possibility for more com- plex disk structures in which the disk can be viscously excreting and still be in steady state. Equation (15) implies that a system with a high ˙M could be unstable if there is a signiﬁcant contribution to the angular momentum transport from the wind torque. The term in paren- theses on the right-hand side of Eq. (15) is positive and smaller than unity. Hence, the disk heating is weaker than would be deduced from a viscous model with the same total ˙M. The disk temperature can become very low if most of the transport is due to the wind (with no associated heating), so that even a disk with a high total ˙M could reside on the cold branch. Dubus et al. 4. Resistive MHD runs Ideal MHD is a very poor approximation on the cold branch. Previous results from S18 showed that in a zero net ﬂux con- ﬁguration, a threshold in temperature exists below which MRI can no longer sustain turbulence. This transition occurs when the magnetic Reynolds number Rm is lower than ≈5 × 103 with Rm = csh η · Fig. 7. Dots show the magnetic Reynolds number Rm, turb for which tur- bulence ceases in our simulation as a function of βturb. The oblique solid line is drawn from Eq. (36), which gives Rm, linear, the linear criterion for stability of resistive MRI. The oblique dashed line gives the Rm, cooling below which turbulence is aﬀected by resistivity and will ultimately be suppressed. In the ZNF case, the ﬂow has to sustain the ﬁeld for the MRI to operate through a dynamo feedback loop. This positive feed- back is not required in the net ﬂux case since the ﬁeld is provided by the environment, such that turbulent motions can be excited at lower Rm. Therefore the presence of a net ⟨Bz⟩reduces the threshold below which linear MRI cannot grow and oﬀers the possibility for MRI to survive down to lower ionization frac- tions. This behavior was observed in Fleming et al. (2000), who described a cyclic revival of MRI down to Rm = 50 compared to the case of ZNF, where MRI turbulence disappears for Rm < 104. However, Fleming et al. (2000) considered a uniform ﬁxed ver- tical proﬁle of resistivity with an isothermal equation of state, which can lead to an artiﬁcial quasi-steady state. We wish to determine whether by relaxing this constraint, the patch of disk can keep turbulence active to lower ionization fractions than in ZNF. agree with the linear criterion plotted as an oblique solid black line on Fig. 7. Nonetheless, we observe that turbulence is weaker than the pure ideal case even for Rm > Rm, turb. In 407F_B0.8G, for exam- ple, the Reynolds magnetic number is larger than Rm, turb for ≈100 orbits after we added resistivity. The turbulence is aﬀected by resistivity, however; α slowly decreases, causing the disk to cool until it reaches the point where Rm < Rm, turb and turbu- lence ceases. This shows that a region exists where MRI is still linearly unstable but turbulence irremediably ceases. 3.5. Stability criterion in a disk-wind model (2018) computed the mass accretion rates from optical luminosi- ties, hence they measured ˙ Mvis, that is, a measure of the viscous A49, page 9 of 13 A&A 620, A49 (2018) 102 103 104 105 106 107 βcrit 101 102 103 104 105 Rm MRI Unstable Ideal turbulence MRI Unstable Dying non ideal turbulence MRI Stable No turbulence Linear MRI threshold, Rm, turb Ideal turbulence threshold, Rm, cooling Fig. 7. Dots show the magnetic Reynolds number Rm, turb for which tur- bulence ceases in our simulation as a function of βturb. The oblique solid line is drawn from Eq. (36), which gives Rm, linear, the linear criterion for stability of resistive MRI. The oblique dashed line gives the Rm, cooling below which turbulence is aﬀected by resistivity and will ultimately be suppressed. dissipation rate, but not of the total ˙M or of ˙MRφ. The separa- tion they found between stable and unstable systems should still hold since the disk instability criterion is fundamentally based on temperature, hence on ˙ Mvis. However, there could be a large discrepancy between ˙ Mvis and the total ˙M, which could be tested if a direct estimate of the mass transfer from the companion ˙M were to be available. MRI Unstable Ideal turbulence 102 103 104 105 106 107 βcrit 101 102 103 104 Rm Ideal turbulence MRI Unstable Dying non ideal turbulence MRI Stable No turbulence Linear MRI threshold, Rm, turb Ideal turbulence threshold, Rm, cooling MRI Unstable Dying non ideal turbulence h e I 4. Resistive MHD runs The rea- son is that as T decreases, α decreases faster than Q−, leading to a thermally unstable situation. The timescale on which this occurs is inversely proportional to the initial Rm from which we started resistivity. For Rm ≈1.5 × 104 and 5 × 103, we have a time decay for the Maxwell stress of ≈100 and 20 orbits, respectively. Here, resistivity was computed as in Blaes & Balbus (1994): η = 230 nn ne ! T 1/2 cm2 s−1 with the assumption that this is dominated by electron-neutral collision. The values of η were pre-computed with the equation of state (Saha equilibrium) and saved in a table. p y Based on these observations, we deﬁne another parameter, Rm, cooling, below which turbulence is still active but not self- sustaining. Rm, cooling does not change much between ZNF where Rm, cooling = 18000 and net ﬂux simulations with Bz0 = 8 G where Rm, cooling = 6000. We summarize these results in Fig. 7, where the dashed black line shows Rm, cooling from our simula- tions. Three distinct zones are evident: for Rm > Rm, cooling, tur- bulence is similar to the ideal case; for Rm, cooling > Rm > Rm, turb, turbulence ceases and the disk cools until it reaches the zone Rm < Rm, turb, where MRI is stable. We restarted from previous ideal MHD simulations on the cold branch and added resistivity. We imposed a ﬂoor of 50 on the magnetic Reynolds number in order to maintain a reasonably large time step. A49, page 10 of 13 4.2. Transport of angular momentum from the upper ionized layers S18 have indeed shown that when resistivity is included in a ZNF conﬁguration, the cold branch becomes MRI stable and the trans- port is quenched. The same conclusion applies for this paper in the case of Bz = 0.8 and 2 G. For Bz = 8 G, the disk remains tur- bulent on most of the cold branch, but exhibits values of α ≳0.1. , Some net magnetic ﬂux is unavoidable in the disks of DNe since the white dwarf or companion are likely to have a non- zero dipolar ﬁeld. If this leads to low values of β, then the mass accretion is mostly due to the torque from the magnetic wind that appears. This will have an eﬀect on the evolution of the disk throughout an outburst cycle. Wind transport will also change the link between light-curve timescales and α. In a purely tur- bulent case, the light-curve timescales are related to the ther- mal (ttherm ∝1/αΩ) and viscous (tvis ∼R2/ν) timescales of the disk. For instance, the propagation time of a cooling front is ∼(tthermtvis)1/2 (Menou et al. 1999; Kotko & Lasota 2012). When a wind is present, however, the observed timescales will become a diﬀusion or advection time related to α, but also to q. This will have to be borne in mind in future determinations of α from observations. Following Glassgold et al. (1997a,b), we computed the ion- ization rate ζ assuming a photon ﬂux f0 = C LXR 4πR2 0kTXR , where C takes into account the irradiation geometry and the albedo A of the disk. S18 estimated that C = 10−2. From Byckling et al. (2010), we adopted a maximum X-ray ﬂux LXR of photons emitted from the white dwarf of 1031 erg cm−2 with a bremsstrahlung spectrum of temperature TXR = 10 keV. We adopted a characteristic surface density of 100 g cm−2. To build such a disk-wind model, we provided prescrip- tions for α and the wind torque q estimated from our shearing- box simulations. We compared our results with local sim- ulations from Bai & Stone (2013a), Fromang et al. (2013), and Simon et al. (2013) as well as global simulations from Zhu & Stone (2018), and we found that our estimates of the wind torque and the mass accretion rates agree with those in the liter- ature. However, an uncertainty remains concerning the depen- dence of q on H/R. 4.2. Transport of angular momentum from the upper ionized layers of the hot branch and cannot explain values of 0.1 in the high- density part of the hot branch, however. This could be obtained if β were high throughout the whole hot branch. However, a strong vertical magnetic ﬁeld also leads to high values of α on the cold branch. Obviously, the large magnetic ﬁeld is surely not con- stant throughout a hysteresis cycle of DNe. Then, it is possible that the advection of the magnetic ﬁeld leads to a higher magne- tization during the eruptive phase than in quiescence. Unfortu- nately, advection of a large-scale magnetic ﬁeld is a global phe- nomenon that cannot be modeled in the shearing-box framework (see Guilet & Ogilvie 2012, 2014 for recents developments on that topic). It is probable that even with a net magnetic ﬁeld, there are regions of the disk that are too cold to sustain MRI turbu- lence. In this last section, we therefore study the eﬀect of X-ray irradiation on the regions where turbulence has ceased. S18 showed that X-ray ionization from the central white dwarf (WD) cannot increase the ionization fraction in the midplane above the threshold required for MRI to be unstable. This is consis- tent with the picture described in Gammie (1996), where an X-ray illuminated accretion disk of DNe in quiescence was orga- nized into layers with a so-called “dead zone” in the central region. However, S18 neglected the active zones in the upper layers where the ionization fraction suﬃces to ensure coupling between the ﬁeld and the plasma. As in protoplanetary disks, this could ensure transport of angular momentum from the wind torque (Bai & Stone 2013b; Béthune et al. 2017). The ensuing supersonic accretion of the upper layers of the disk could then lead to the required accretion rates. Following the procedure developed in Sect. 3.4, we can estimate that for α ≈0.035 and Σ ≈100 g cm−2, the typical turbulent accretion rate is ˙MRφ ≈5 × 1014 g s−1. We wish to estimate the ionization fraction in the upper layers and determine whether we can obtain such an accre- tion rate. We investigated the problem of angular momentum transport on the cold branch and found that resistive MRI does not natu- rally lead to values of α ≈0.01, as required by the DIM models. 4.2. Transport of angular momentum from the upper ionized layers This problem needs to be addressed with global simulations. p y g We ﬁnd that ≈1% of the mass is ionized in the upper active layers. From now on, we assume that only the upper ionized layers of the disk accrete at a constant velocity vR. The typi- cal accretion rate on the cold branch being ˙M ≈2πRΣ⟨vR⟩ρ ≈5 × 1014 g s−1, this leads from a surface density of 100 g cm−2 to vR ≈5 × 103 cm s−1. From the equation of angular momentum we can obtain the following relation between q and the accretion speed: vR = 2qB2 z 3ΩΣ fioniz , On a more observational side, we know that there are winds in the eruptive state of DNe that are emitted by the disk (Cordova & Mason 1982), which may be hydromagnetically launched (Cannizzo & Pudritz 1988). However, only one poten- tial indirect detection of wind in quiescence has been reported by Perna et al. (2003). The detection is expected to be more diﬃcult than for a disk in eruption (Drew 1990; Perna et al. 2003), and it should not be concluded from the lack of observational evidence that winds are absent in quiescence. which shows that the minimum wind torque that could explain the accretion rate on the cold branch is qmin ≈10. From Fig. 1, we ﬁnd for β < 104 that qsurface takes a constant value of ≈40, which is above qmin. Adding the caveat that qsurface is only an estimate from a shearing box in ideal MHD, we propose sonic accretion in the upper layers as a reasonable option to explain accretion in resistive regions of DNe. Mass accretion rates computed through X-ray emission allow access to the total mass accretion rates. Several sources indicate that the X-ray ﬂux in quiescence is higher than what can be explained by the classical DIM (Collins & Wheatley 2010; Mukai 2017; Wheatley et al. 2003). One explanation is that the disk may be truncated by the magnetic ﬁeld of the WD (Wheatley & West 2003; Balman & Revnivtsev 2012). How- ever, this hypothesis is still debated, and there is also strong evi- dence of an accretion disk that even reaches the WD (Mukai 2017; Ishida et al. 2009). In view of our results, we suggest that 4.1. Impact of Bz on the resistive cold branch Rm, turb is the critical Reynolds number below which no turbu- lence is seen in our simulation. It is computed as the midplane value of Rm at the moment where the Maxwell stress ultimately decays to zero. , These results show that ideal turbulence can be maintained to slightly lower ionization levels in the presence of a large-scale magnetic ﬁeld. This eﬀect is too subtle for Bz0 = 0.8 and 2 G to maintain the cold branch, however. Only for Bz0 = 8 G is the cold branch maintained down to Σ = 70 g cm−2. Surely, the fact that Rm, cooling = 6000 helps in sustaining the cold branch to such low surface densities, but the main actor is that α takes high values, up to 0.275, because of the low β. This enhanced heating ensures a level of thermal ionization that is higher than in simulations with weaker magnetic ﬁelds. In this case, two eﬀects act together in keeping the MRI unstable: ﬁrst and foremost, the enhanced heating at high magnetization, and secondarily, the eﬀect of net magnetic ﬂux on resistive MRI. Following Jin (1996), we ﬁnd that the linear criterion for sta- bility of resistive MRI in the case of a vertical ﬁeld and no strat- iﬁcation is Rm, linear = β q 3 2π2 β −4 · We plot in Fig. 7 Rm, turb as a function of β together with its ana- lytical estimate from linear theory. As expected, the threshold for turbulence decreases with stronger magnetization. Our results N. Scepi et al.: Turbulent and wind-driven accretion in dwarf novae threaded by a large-scale magnetic ﬁeld 4.2. Transport of angular momentum from the upper ionized layers Acknowledgements. NS, GL, and GD thank Omer Blaes for fruitful discus- sions. We thank the anonymous referee for insightful comments. NS acknowl- edges ﬁnancial support from the pole PAGE of the Université Grenoble Alpes. GL, GD, and MF are grateful to the participants of the KITP 2017 program References M., & Balbus, S. A. 1994, ApJ, 421, 163 p Blandford, R. D., & Payne, D. G. 1982, MNRAS, 199, 883 Brandenburg, A., Nordlund, A., Stein, R. F., & Torkelsson, U. 1995, ApJ, 446, 741 Byckling, K., Mukai, K., Thorstensen, J., & Osborne, J. 2010, MNRAS, 408, 2298 Cannizzo, J. K., & Pudritz, R. E. 1988, ApJ, 327, 840 annizzo, J. K., Shafter, A. W., & Wheeler, J. C. 1988, ApJ, 333, 22 5. Discussion S18 have conﬁrmed that MRI leads to values of α ≈0.1 near the tip of the hot branch when coupled non-linearly with convec- tion. When we add a net magnetic ﬂux, we ﬁnd that convection also enhances α for magnetizations up to β ≈104, showing that convective MRI is a reliable mechanism for enhancing classi- cal MRI transport. The enhancement of α is localized at the tip A49, page 11 of 13 A&A 620, A49 (2018) on Confronting MHD Theories of Accretion Disks with Observations for the many useful conversations pertaining to this work (and thus this research was supported in part by the National Science Foundation under Grant No. NSF PHY-1125915). This work was granted access to the HPC resources of IDRIS under the allocation A0020402231 made by GENCI (Grand Equipement National de Calcul Intensif). Some of the computations presented in this paper were performed using the Froggy platform of the CIMENT infrastruc- ture (https://ciment.ujf-grenoble.fr), which is supported by the Rhône- Alpes region (GRANT CPER07_13 CIRA), the OSUG@2020 labex (reference ANR10 LABX56) and the Equip@Meso project (reference ANR-10-EQPX- 29-01) of the programme Investissements d’Avenir supervised by the Agence Nationale pour la Recherche. M. Flock has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No 757957). accretion in quiescence is mainly driven by the wind torque. This would reconcile the high X-ray ﬂux coming from the ﬂow of matter ˙M onto the white dwarf with the low values of ˙MRφ required by the DIM: in such a case the dissipation in the disk is related to ˙MRφ and not to ˙M, leading to a darker disk than a turbulence-driven disk with the same ˙M. on Confronting MHD Theories of Accretion Disks with Observations for the many useful conversations pertaining to this work (and thus this research was supported in part by the National Science Foundation under Grant No. NSF PHY-1125915). This work was granted access to the HPC resources of IDRIS under the allocation A0020402231 made by GENCI (Grand Equipement National de Calcul Intensif). 5. Discussion Some of the computations presented in this paper were performed using the Froggy platform of the CIMENT infrastruc- ture (https://ciment.ujf-grenoble.fr), which is supported by the Rhône- Alpes region (GRANT CPER07_13 CIRA), the OSUG@2020 labex (reference ANR10 LABX56) and the Equip@Meso project (reference ANR-10-EQPX- 29-01) of the programme Investissements d’Avenir supervised by the Agence Nationale pour la Recherche. M. Flock has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No 757957). We also propose that a plausible solution for the colder regions of the disk is accretion that is not driven by turbulence, but by removal of angular momentum by the wind torque in the ionized upper layers, much as in protoplanetary disks. We used observational constraints of the X-ray ﬂux in quiescence emitted from the central white dwarf to compute the ioniza- tion level in the upper layers. We estimate that it might be suf- ﬁcient to explain the α that are observed. A layered accretion is likely to have an eﬀect on how outbursts are triggered in the disk, notably on the conditions for which the heating front propagates outward (“inside-out outbursts”) or inward (“outside- in”). There is observational evidence for both in dwarf novae. In this context, the absence of heating from the wind is a major problem. This may prevent the disk from reaching high enough temperatures to ionize H and trigger the thermal-viscous insta- bility. The trigger may then be due to a decrease in the wind torque as matter accumulates in quiescence, a profound mod- iﬁcation to the DIM. Moreover, the lack of heating may not enable maintenance of the vertical structure, and some base level of heating is required in the poorly ionized parts of the disk. 6. Conclusion Cannizzo, J. K., Smale, A. P., Wood, M. A., Still, M. D., & Howell, S. B. 2012, ApJ, 747, 117 We have studied the impact of a large net vertical magnetic ﬁeld on the properties of transport and on the thermal equilibrium curves of DNe. We considered a large-scale magnetic ﬁeld as could arise from the dipolar component of the primary or the companion, which is constant for a given S-curve. This conﬁgu- ration naturally leads to lower values of β on the cold branch than on the hot branch. For Bz < 2 G, the S-curves, in particular the critical points, are quasi-identical to the ZNF case. For Bz ≳2 G, the S-curves are shifted toward low density, and we found higher values of α on the cold branch than on the hot branch. Coleman, M. S. B., Kotko, I., Blaes, O., Lasota, J.-P., & Hirose, S. 2016, MNRAS, 462, 3710 Coleman, M. S. B., Blaes, O., Hirose, S., & Hauschildt, P. H. 2018, ApJ, 857, 52 Collins, D. J., & Wheatley, P. J. 2010, MNRAS, 402, 1816 Cordova F A & Mason K O 1982 ApJ 260 716 Collins, D. J., & Wheatley, P. J. 2010, MNRAS, 402, 1816 Collins, D. J., & Wheatley, P. J. 2010, MNRAS, 402, 1816 Cordova, F. A., & Mason, K. O. 1982, ApJ, 260, 716 p Davis, S. 1988, SIAM J. Sci. Stat. Comput., 9, 445 Drew, J. 1990, IAU Colloq. (Cambridge: Cambridge University Press), 122, 228 Dubus, G., Otulakowska-Hypka, M., & Lasota, J.-P. 2018, A&A, 617, A26 Ferguson, J. W., Alexander, D. R., Allard, F., et al. 2005, ApJ, 623, 585 Ferreira, J., & Pelletier, G. 1995, A&A, 295, 807 Ferreira, J., & Pelletier, G. 1995, A&A, 295, 807 We investigated the problem of the transport of angular momentum on the cold resistive branch. We ﬁnd that for Bz = 0.8 and 2 G, MRI turbulence is suppressed on most of the cold branch. However, for Bz = 8 G, the turbulence is sustained to lower surface densities, but the cold branch exhibits values of α > 0.1. Fleming, T. P., Stone, J. M., & Hawley, J. F. 2000, ApJ, 530, 464 Flock, M., Fromang, S., González, M., & Commerçon, B. 2013, A&A, 560, A43 Frank, J., King, A., & Raine, D. 2002, Accretion Power in Astrophysics (Cambridge: Cambridge University Press) Fromang, S., Latter, H., Lesur, G., & Ogilvie, G. I. 6. Conclusion 2013, A&A, 552, A71 Gammie, C. F. 1996, ApJ, 457, 355 We showed that for R = 1.315 × 1010 cm, the entire cold branch is wind driven, even for local magnetic ﬁelds that are relatively weak. It is therefore essential to include wind-driven transport in the DIM and to review the eﬀect on our understand- ing of outburst cycles. Gammie, C. F., & Menou, K. 1998, ApJ, 492, L75 Glassgold, A. E., Najita, J., & Igea, J. 1997a, ApJ, 480, 344 Glassgold, A. E., Najita, J., & Igea, J. 1997b, ApJ, 485, 920 g j g p Grevesse, N., & Sauval, A. 1998, Space Sci. Rev., 85, 161 Guilet, J., & Ogilvie, G. I. 2012, MNRAS, 424, 2097 g Guilet, J., & Ogilvie, G. I. 2014, MNRAS, 441, 852 Outﬂows provide an alternative route for extracting angu- lar momentum that has a huge impact on the budget of angular momentum, but does not heat the disk. This means that DNe may be accreting much more than what we infer from the luminosity of the disk, and the high X-ray ﬂux observed in quiescence may support this theory. All in all, we arrive at a point where obser- vations and theory need to work hand in hand again if we wish to proceed in our understanding of accretion in DNe. Hameury, J.-M., Menou, K., Dubus, G., Lasota, J.-P., & Hure, J.-M. 1998, MNRAS, 298, 1048 References Bai, X.-N., & Stone, J. M. 2013a, ApJ, 767, 30 Bai, X.-N., & Stone, J. M. 2013b, ApJ, 769, 76 Balay, S., Abhyankar, S., Adams, M. F., et al. 2016, PETSc Web page, http: //www.mcs.anl.gov/petsc Bai, X.-N., & Stone, J. M. 2013a, ApJ, 767, 30 Bai, X.-N., & Stone, J. M. 2013b, ApJ, 769, 76 B l S Abh k S Ad M F l 2016 PETS W b h Bai, X.-N., & Stone, J. M. 2013a, ApJ, 767, 30 Bai, X.-N., & Stone, J. M. 2013b, ApJ, 769, 76 Balay, S., Abhyankar, S., Adams, M. F., et al. 2016, PETSc Web page, http: //www.mcs.anl.gov/petsc Balbus, S. A., & Hawley, J. F. 1991, ApJ, 376, 214 Balbus, S. A., & Hawley, J. F. 1998, Rev. Mod. Phy., 70, 1 Balbus, S. A., & Papaloizou, J. C. 1999, ApJ, 521, 650 Balman, ¸S., & Revnivtsev, M. 2012, A&A, 546, A112 Béthune, W., Lesur, G., & Ferreira, J. 2017, A&A, 600, A75 Blaes, O. M., & Balbus, S. A. 1994, ApJ, 421, 163 Blandford, R. D., & Payne, D. G. 1982, MNRAS, 199, 883 Brandenburg A Nordlund A Stein R F & Torkelsson U 1995 ApJ 446 Balay, S., Abhyankar, S., Adams, M. F., et al. 2016, PETSc Web page, http: //www.mcs.anl.gov/petsc Balbus, S. A., & Hawley, J. F. 1991, ApJ, 376, 214 Balbus, S. A., & Hawley, J. F. 1998, Rev. Mod. Phy., 70, 1 Balbus, S. A., & Papaloizou, J. C. 1999, ApJ, 521, 650 Balman, ¸S., & Revnivtsev, M. 2012, A&A, 546, A112 Béthune, W., Lesur, G., & Ferreira, J. 2017, A&A, 600, A75 Blaes, O. M., & Balbus, S. A. 1994, ApJ, 421, 163 Balbus, S. A., & Hawley, J. F. 1998, Rev. Mod. Phy., 70, 1 Balbus, S. A., & Papaloizou, J. C. 1999, ApJ, 521, 650 Balman, ¸S., & Revnivtsev, M. 2012, A&A, 546, A112 Béthune, W., Lesur, G., & Ferreira, J. 2017, A&A, 600, A75 Blaes O M & Balbus S A 1994 ApJ 421 163 Balbus, S. A., & Papaloizou, J. C. 1999, ApJ, 521, 650 Balman, ¸S., & Revnivtsev, M. 2012, A&A, 546, A112 Béthune, W., Lesur, G., & Ferreira, J. 2017, A&A, 600, A75 l lb 6 Balman, ¸S., & Revnivtsev, M. 2012, A&A, 546, A112 Béthune, W., Lesur, G., & Ferreira, J. 2017, A&A, 600, A Blaes, O. M., & Balbus, S. A. 1994, ApJ, 421, 163 Blaes, O. y MNRAS, 298, 1048 , , Hameury, J.-M., Viallet, M., & Lasota, J.-P. 2009, A&A, 496, 413 Hawley, J. F., Gammie, C. F., & Balbus, S. A. 1995, ApJ, 440, 742 Hawley, J. F., Gammie, C. F., & Balbus, S. A. 1996, ApJ, 464, 690 Hirose, S. 2015, MNRAS, 448, 3105 Hawley, J. F., Gammie, C. F., & Balbus, S. A. 1996, ApJ, 464, 690 Hi S 2015 MNRAS 448 3105 Hirose, S. 2015, MNRAS, 448, 3105 Hirose, S., Blaes, O., Krolik, J. H., Coleman, M. S. B., & Hirose, S., Blaes, O., Krolik, J. H., Coleman, M. S. B., & Sano, T. 2014, ApJ, 787 1 Hirose, S., Blaes, O., Krolik, J. H., Coleman, M. S. B., & Sano, T. 2014, ApJ, 787, 1 787, 1 Iglesias, C. A., & Rogers, F. J. 1996, ApJ, 464, 943 Ishida, M., Okada, S., Hayashi, T., et al. 2009, Publ. Astron. Soc. Jpn., 61, S77 Jin, L. 1996, ApJ, 457, 798 King, A., & Ritter, H. 1998, MNRAS, 293, L42 King, A., Pringle, J., & Livio, M. 2007, MNRAS, 376, 1740 Kotko, I., & Lasota, J.-P. 2012, A&A, 545, A115 Lasota, J.-P. 2001, New Astron. Rev., 45, 449 A49, page 12 of 13 N. Scepi et al.: Turbulent and wind-driven accretion in dwarf novae threaded by a large-scale magnetic ﬁeld Latter, H. N., & Papaloizou, J. C. 2012, MNRAS, 426, 1107 Lesur, G., Ferreira, J., & Ogilvie, G. I. 2013, A&A, 550, A61 Menou, K., Hameury, J.-M., & Stehle, R. 1999, MNRAS, 305, 79 Mignone, A. 2009, Mem. Soc. Astron. It. Supp., 13, 67 Minerbo, G. N. 1978, J. Quant. Spectr. Rad. Transf., 20, 541 Mukai, K. 2017, PASP, 129, 062001 Papaloizou, J. C., & Terquem, C. 1999, ApJ, 521, 823 Perna, R., McDowell, J., Menou, K., Raymond, J., & Medvedev, M. V. 2003, ApJ, 598, 545 Pringle, J. 1981, ARA&A, 19, 137 Ryan, B. R., Gammie, C. F., Fromang, S., & Kestener, P. 2017, ApJ, 840, 6 Salvesen, G., Simon, J. B., Armitage, P. J., & Begelman, M. C. 2016, MNRAS, 457, 857 Sano, T., Miyama, S. M., Umebayashi, T., & Nakano, T. 2000, ApJ, 543, 486 Scepi, N., Lesur, G., Dubus, G., & Flock, M. 2018, A&A, 609, A77 Shakura, N. I., & Sunyaev, R. A. 1973, A&A, 24, 337 Latter, H. N., & Papaloizou, J. C. 2012, MNRAS, 426, 1107 Simon, J. B., Beckwith, K., & Armitage, P. J. y MNRAS, 298, 1048 2012, MNRAS, 422, 2685 , , p , , , , Lesur, G., Ferreira, J., & Ogilvie, G. I. 2013, A&A, 550, A61 Simon, J. B., Bai, X.-N., Armitage, P. J., Stone, J. M., & Beckwith, K. 2013, ApJ, 775, 73 Simon, J. B., Bai, X.-N., Armitage, P. J., Stone, J. M., & Beckwith, K. 2013, ApJ, 775, 73 , , , , g , , , , Menou, K., Hameury, J.-M., & Stehle, R. 1999, MNRAS, 305, 79 Menou, K., Hameury, J.-M., & Stehle, R. 1999, MNRAS, 305, 79 y Mignone, A. 2009, Mem. Soc. Astron. It. Supp., 13, 67 Smak, J. 1984, Acta Astron., 34, 161 g pp Minerbo, G. N. 1978, J. Quant. Spectr. Rad. Transf., 20, 541 Starling, R. L., Siemiginowska, A., Uttley, P., & Soria, R. 2004, MNRAS, 347, 67 Starling, R. L., Siemiginowska, A., Uttley, P., & Soria, R. 2004, MNRAS, 347, 67 Starling, R. L., Siemiginowska, A., Uttley, P., & Soria Q p Mukai, K. 2017, PASP, 129, 062001 Suzuki, T. K., & Inutsuka, S.-I. 2009, ApJ, 691, L49 Papaloizou, J. C., & Terquem, C. 1999, ApJ, 521, 823 Tetarenko, B., Lasota, J.-P., Heinke, C., Dubus, G., & Sivakoﬀ, G. 2018, Nature, 554, 69 Tetarenko, B., Lasota, J.-P., Heinke, C., Dubus, G., & Sivakoﬀ, G. 2018, Nature, 554, 69 p q p Perna, R., McDowell, J., Menou, K., Raymond, J., & Medvedev, M. V. 2003, ApJ, 598, 545 Turner, N., & Stone, J. 2001, ApJSS, 135, 95 Pringle, J. 1981, ARA&A, 19, 137 Warner, B. 2003, Cataclysmic Variable Stars (Cambridge: Cambridge University Press), 28 Pringle, J. 1981, ARA&A, 19, 137 Warner, B. 2003, Cataclysmic Variable Stars (Cambridge: Cambridge University Press), 28 Ryan, B. R., Gammie, C. F., Fromang, S., & Kestener, P. 2017, ApJ, 840, 6 Wheatley, P. J., & West, R. G. 2003, MNRAS, 345, 1009 Salvesen, G., Simon, J. B., Armitage, P. J., & Begelman, M. C. 2016, MNRAS, 457, 857 Sano, T., Miyama, S. M., Umebayashi, T., & Nakano, T. 2000, ApJ, 543, 486 Scepi, N., Lesur, G., Dubus, G., & Flock, M. 2018, A&A, 609, A77 Shakura, N. I., & Sunyaev, R. A. 1973, A&A, 24, 337 Sano, T., Miyama, S. M., Umebayashi, T., & Nakano, T. 2000, ApJ, 543, 486 Scepi, N., Lesur, G., Dubus, G., & Flock, M. 2018, A&A, 609, A77 Yang, L. T., & Brent, R. P. 2002, Proc. y MNRAS, 298, 1048 Fifth International Conference on g Zhu, Z., & Stone, J. M. 2018, ApJ, 857, 34 A49, page 13 of 13

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